WO2000036135A2

WO2000036135A2 - Screening methods based on the use of protein kinases

Info

Publication number: WO2000036135A2
Application number: PCT/GB1999/004228
Authority: WO
Inventors: Jeremy William Thorner; Dario Renato Alessi; Pamela Diane Torrance; Antonio Casamayor
Original assignee: Medical Research Council; The Regents Of The University Of California
Priority date: 1998-12-14
Filing date: 1999-12-14
Publication date: 2000-06-22
Also published as: EP1141001A2; AU1789400A; CA2355362A1; JP2002532100A; WO2000036135A3

Abstract

A method of identifying a compound which inhibits to different extents (a) a host yeast cell protein kinase or kinases and (b) a protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is capable of expressing the said host yeast cell protein kinase or kinases and is not capable of expressing the said equivalent protein kinase and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a source other than the host yeast cell and the effect of the compound on the viability of the said yeast cells is measured, and a compound that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. The method may be useful in a screen for identifying compounds that inhibit a mammalian or fungal protein kinase. The compounds may be useful in medicine.

Description

SCREENING METHODS

The present invention relates to screening methods for drugs or lead compounds, enzymes, to polynucleotides encoding enzymes and to uses of enzymes and polynucleotides.

Protein kinase B (PKB) [1], also called RAC kinase [2] is the mammalian homologue of the viral oncogene product v-Akt [3] and has therefore also been termed c-Akt. The current interest in this enzyme stems from several observations. Firstly, it is activated within minutes in response to insulin or growth factors and activation is prevented by inhibitors of phosphatidylinositol (Ptdlns) 3-kinase [4-6]. There is increasing evidence that PKB may mediate a number of the actions of insulin, including stimulation of glucose and amino acid uptake, glycogen and protein synthesis and cardiac muscle glycolysis (reviewed in [7, 8]) as well as regulation of the transcription of specific genes [9, 10]. Secondly, the PKBβ isoform is overexpressed in a significant percentage of ovarian and pancreatic cancers [11, 12] and the PKBα isoform in some breast cancers [2]. It appears that PKB provides a survival signal that protects cells from apoptosis induced in a variety of ways (reviewed in [8, 13]). The activation of PKB by gene amplification and other mechanisms may therefore contribute to the generation of malignancies that are able to flourish in the absence of extracellular survival signals.

PKB phosphorylates proteins and peptides at serine and threonine residues that lie in Arg-Xaa-Arg,-Xaa-Xaa-Ser/Thr- sequences [14]. In insulin signal transduction two physiological substrates of PKB appear to be the protein kinase - glycogen synthase kinase-3 (GSK3) [15, 16] and the cardiac isoform of phosphofructokinase-2 (PFK2) [8, 17]. Phosphorylation by PKB inhibits GSK3 activity leading to dephosphorylation and activation of glycogen synthase and protein synthesis initiation factor eIF2B [18]. These events appear to contribute to the insulin-induced stimulation of glycogen synthesis and protein synthesis, respectively. PKB activates cardiac PFK2, which seems to underlie the insulin- induced stimulation of glycolysis in the heart. In the protection of cells against apoptosis, BAD appears to be one of the physiological substrates of PKB. This protein, in its dephosphorylated form, interacts with the Bel family member BCIXL, thereby inducing apoptosis in some cells. However, when PKB phosphorylates BAD at Serl36, it dissociates from BCIXL, interacts with 14-3-3 proteins instead, and apoptosis is prevented [19].

The activation of PKB by insulin or growth factors requires its phosphorylation at two sites [20]. These are Thr308, which is located in the "activation loop" of the catalytic domain between subdomains VII and VIII, and Ser473 which is very close to the C-terminus in a hydrophobic Phe-Xaa-Xaa-Phe-Ser-Phe motif that is present in a number of protein kinases that play important roles in signal transduction [21]. The phosphorylation of both sites is prevented by inhibitors of Ptdlns 3-kinase [20] . Thr308 is phosphorylated by 3-phosphoinositide-dependent protein kinase- 1 (PDKl) [22, 23] and Ser473 by a protein kinase(s) having PDK2 activity. PDKl may have PDK2 activity, for example when the kinase domain of PDKl interacts with a region of Protein kinase C-Related Kinase-2 (PRK2) termed the PDKl Infracting Fragment (PIF) or other polypeptide having a Phe- Xaa-Xaa-Phe motif. This converts PDKl from a form that phosphorylates PKB at Thr308 to a form that phosphorylates PKB at both Thr308 and Ser473 (Balendran et al (1999) Current Biology 9, 393-404).

Ptdlns 3-kinase converts phosphatidylinositide 4,5 bisphosphate (PtdIns[4,5]P2) to PtdIns[3,4,5]P3 which is then converted to PtdIns[3,4]P2 by one or more 5'- phosphatases. PKB can only be activated by PDKl in vitro in the presence of lipid vesicles containing PtdIns[3,4,5]P3 or PtdIns[3,4]P2 [22]. These 3- phosphoinositides bind to the pleckstrin homology (PH) domain at the N-terminus of PKB [24, 25], which is thought alters its conformation in such a way that Thr308 becomes accessible to PDKl. PDKl also possesses a PH domain C- terminal to the catalytic domain [23], that interacts with PtdIns[3,4,5]P3 and PtdIns[3,4]P2 even more strongly than does the PH domain of PKB [26, 83], The interaction of PDKl with 3-phosphoinositides enhances the activation of PKB in vitro, probably by facilitating the interaction of PDKl and PKB in lipid vesicles [27].

There is increasing evidence that PDKl activates a number of protein kinases in vivo, such as p70 S6 kinase [28] and isoforms of PKC [29], by phosphorylating threonine residues that lie in positions equivalent to Thr308 of PKB. These protein kinases also contain the hydrophobic PDK2 consensus sequence that lies 160-165 residues C-terminal to the PDKl phosphorylation site [8]. Thus other substrates of PDKl are also likely to be phosphorylated by a protein kinase with PDK2 activity.

We have characterised yeast protein kinases that appear to be required for cell growth and that may have mammalian homologues that are able to rescue yeast cells (ie restore their ability to grow) that are not able to express the endogenous yeast protein kinase or kinases. Characterisation of these yeast protein kinases that may be functional homologues of mammalian protein kinases may allow improved methods of identifying compounds that may modulate the activity of signalling pathways in which the mammalian homologues are involved, or compounds that may be useful as antifungal agents.

We have characterised non-mammalian proteins that may be functional homologues of PDKl and human serum and glucocorticoid induced protein kinase (SGK), an enzyme whose catalytic domain is 70% similar to PKB. Screening methods using yeast cells

A first aspect of the invention is a method of identifying a compound which modulates the activity (preferably inhibits) to different extents of (a) a host yeast cell protein kinase or kinases and (b) a protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is capable of expressing the said host yeast cell protein kinase or kinases and is not capable of expressing the said equivalent protein kinase and

2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said host yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a source other than the host yeast cell and the effect of the compound on the phenotype of the said yeast cells is measured, wherein either

(1) the host yeast cell is a pathogenic yeast (preferably a yeast pathogenic to a mammal, preferably a human) and the source other than the host yeast cell is any source other than the host yeast cell or

(2) the host yeast cell is any yeast and the source other than the host yeast cell is not a mammal.

It will be appreciated that the said first and second host yeast cells differ substantially only in the features indicated such that apart from these features the cells are essentially the same. This ensures that there is a reasonable expectation that the effect of the compound on the phenotype of the said yeast cells can be attributed to the compound's effect on the modulation of the activity to different extents of the protein kinases as said. Thus, the first and second host yeast cells are from the same species and have substantially the same genetic content with the exception of the features indicated ie the capability to express the said host yeast cell protein kinase or kinases and the ability to express the said equivalent protein kinase derivable from a source other than the host yeast cell. It will be appreciated that the said first and second host yeast cells may differ in genetic content relating to the generation or selection of the said first or second host yeast cells (for example, in selectable marker genes used in recombinant techniques, as well known to those skilled in the art).

Typically, a compound that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. More particularly, the compound so identified is selected for further study.

It will be appreciated that the phrase "modulates the activity (preferably inhibits) to different extents" includes the meanings that (1) the host yeast cell protein kinase or kinases is/are modulated (inhibited or activated) and the protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases is not modulated (inhibited or activated), (2) the host yeast cell protein kinase or kinases is/are not modulated (inhibited or activated) and the protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases is modulated (inhibited or activated), and (3) that both types of protein kinase are modulated (inhibited or activated), but that one type is modulated (inhibited or activated) more than or differently to the other.

It will be appreciated that the modulation is judged by the effect on the phenotype of the said first and second yeast cells. It will be appreciated that the effect of the compound on the protein kinase must be sufficient to cause a change in the phenotype being observed in order for any modulation to be detected. Preferably, the effect of a compound that modulates the said protein kinases on the phenotype of a cell is an alteration in the viability or ability of the cell to grow. Thus, it is preferred that the said host yeast cell protein kinase or kinases is desirable or essential for the viability or ability of the cell to grow.

By "protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases" (the "equivalent protein kinase") is included a protein kinase derivable from a source other than the said host yeast cell that is able to function in place of the said host yeast cell protein kinase or kinases in at least some aspect (ie in relation to at least one phenotypic characteristic), for example as judged by the viability or ability of a cell in which the host yeast cell protein kinase or kinase is replaced by the said equivalent protein kinase to grow. Thus by "equivalent protein kinase" is included a protein kinase that is able to function in place of the host yeast cell protein kinase or kinase in conferring on a host yeast cell a particular wild-type phenotype, for example the ability to grow under certain conditions.

For example, it is preferred that a yeast cell that is not able to express the said host yeast cell protein kinase or kinases is substantially not able to grow under given conditions, and that the said host yeast cell that is capable of expressing the said equivalent protein kinase is capable of growing under the same conditions. In the presence of a compound that inhibits the host yeast cell protein kinase or kinases but does not inhibit the equivalent protein kinase, the said first host yeast cell may not be substantially capable of growing but the said second host yeast cell may be capable of growing. Similarly, in the presence of a compound that inhibits the equivalent protein kinase but does not inhibit the host yeast cell protein kinase or kinases, the said second host cell may be substantially incapable of growing.

Alternatively, it is preferred that a yeast cell that is able to express the said host yeast cell protein kinase or kinases expresses the said host yeast cell protein kinase at a level too low to permit growth of the cell at wild-type rates, preferably at a level at which the said cell is substantially not able to grow under given conditions, and that the said host yeast cell that is capable of expressing the said equivalent protein kinase expresses the said equivalent protein kinase at a level too low to permit growth of the cell at wild-type rates, preferably at a level at which the said cell is substantially unable to grow under the same conditions. In the presence of a compound that activates the host yeast cell protein kinase or kinases but does not activate the equivalent protein kinase, the said first host yeast cell may be more capable of growing but the said second host yeast cell may remain substantially incapable of growing. Similarly, in the presence of a compound that activates the equivalent protein kinase but does not activate the host yeast cell protein kinase or kinases, the said second host cell may be more capable of growing and the said first host yeast cell may remain substantially incapable of growing. Thus, for example, the host yeast cell protein kinase or kinases and the equivalent protein kinase may be expressed from a regulatable promoter and the cells may be grown under conditions in which the activity of the promoter is reduced. For example, the GALl promoter may be used, in a manner analogous to that described in Example 1. The GALl promoter is repressed in the presence of glucose, as known to those skilled in the art; thus, when cells are grown in the presence of glucose, expression from the GALl promoter will be reduced, in a manner analogous to that described in Example 1.

Thus, a yeast host cell which is incapable of expressing the said host yeast cell protein kinase or kinases may be substantially incapable of growing unless the said yeast host cell is capable of expressing the said protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases at an sufficient level.

It will be appreciated that a compound that affects the phenotype, for example the viability or ability of the cell to grow, by a mechanism that does not directly involve the said protein kinase or kinases or equivalent protein kinase is unlikely to have a different effect on the first and second said host yeast cells. Thus, a compound that has a similar effect on both the first and second said host yeast cells (for example, no effect on the viability of either type of cell, or, alternatively, toxic to both types of cell) may not be selected for further study, as the compound may not be affecting the activity of the said protein kinase or kinases or equivalent protein kinase. For example, the compound may be a nonspecific cytotoxic agent affecting, for example, the integrity of the cellular membrane.

It will be appreciated that a compound that is capable of modulating the activity of the said host yeast cell protein kinase or kinases and the said equivalent kinase may have a similar effect on both said first and second host yeast cells; however, it will be appreciated that it may not be possible or convenient to attempt to distinguish such a compound from a compound that is affecting the phenotype by a mechanism that does not directly involve the said protein kinase or kinases or equivalent protein kinase.

It will be appreciated that the possibility of distinguishing compounds that may be acting directly on the said protein kinase or kinases or equivalent protein kinase from those that may be acting on other cellular components may be beneficial, for example as set out above. Thus, the method of the first aspect of the invention, which may allow such distinguishing, may be beneficial even if, for example in the context of the intended use of any compound identified, it is not considered of great importance that the compound is not able to inhibit the said host yeast cell protein kinase or kinases, so long as it is able to inhibit the equivalent protein kinase (or vice versa).

Thus, for example, if it is intended to identify a compound that is capable of inhibiting a particular human protein kinase and that therefore may be useful in treating a human with a disease or condition that may be caused by elevated activity of the said protein kinase, it may not be essential for the medical usefulness of the compound that it does not inhibit an equivalent protein kinase in yeast.

Similarly, if it is intended to identify a compound that is capable of inhibiting a particular yeast (for example a pathogenic yeast) protein kinase and that therefore may be useful in treating a human with a disease or condition that may be caused by that pathogenic yeast (which compound may be identified by a screen according to the first aspect of the invention in which the host yeast cell is a non- pathogenic yeast cell, for example S. cerevisae and the source from which the equivalent protein kinase is derivable is a yeast (for example Candida) which is capable of being a pathogen), it may not be essential for the compound to be medically useful that the compound does not inhibit an equivalent protein kinase in non pathogenic yeast, such as S. cerevisiae.

However, it will be appreciated that in other circumstances it may be desirable or essential for the medical or horticultural usefulness of the compound that the compound is (1) able to inhibit the said host yeast cell protein kinase or kinases, and (2) is not able to inhibit the equivalent protein kinase (or vice versa). For example, when the host yeast cell is a pathogenic yeast cell, a compound that inhibits the host yeast cell protein kinase but not an equivalent protein kinase, such as a human protein kinase or a plant protein kinase, may be useful, for example as an anti-yeast (or anti-fungal) drug or plant protection product or in the design of an anti-yeast drug (or anti-fungal) drug or plant protection product.

A further aspect of the invention provides a method of identifying a compound which modulates the activity to different extents of (a) a protein kinase derivable from a first source and (b) a protein kinase derivable from a second source, both said protein kinases being equivalent to the same host yeast cell protein kinase or kinases, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the first source and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the second source and the effect of the compound on the viability of the said yeast cells is measured, and a compound that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. More particularly, the compound so identified is selected for further study.

The method of this aspect of the invention may allow a compound to be identified that may have an effect on cells of the said first source but that may not have an effect on cells of the said second source, or vice versa. Thus, it may be useful if the said first source is a mammal, for example a human, and the said second source is a parasite or pathogen (including an opportunistic pathogen), for example a yeast pathogen such as Candida spp. The terms parasite, pathogen and opportunistic pathogen are well known to those skilled in the art, and include any source that may cause or contribute to a disease or condition of an organism, preferably a mammal, still more preferably a human.

It will be appreciated that the parasite or pathogen may be a parasite or pathogen of a plant; thus it may be useful if the said first source is a plant and the second source is a parasite or pathogen that may affect the said plant, for example is capable of causing rust. A compound that does not have an effect on cells of the said first source but that has an inhibitory effect on cells of the said second source may be useful in treating a plant that is affected by the said parasite or pathogen or in preventing the plant being affected by the said parasite or pathogen. Thus, it may be preferred that the first or second source are both not mammalian, or that either the first or second source is a pathogenic yeast cell.

It will be appreciated that the first and second source may both be plants; a compound that is capable of affecting cells of the first source and second source differently may be useful as a selective herbicide or a selective growth promoter. Thus, for example, the first source may be a monocotyledenous plant and the second source may be a dicotyledenous plant.

By "protein kinase derivable from" is included the meaning that the protein kinase is encoded by a nucleic acid, for example genomic DNA or mRNA, of the source. Thus, for example, a protein kinase derivable from a human may be a protein kinase encoded by a portion of a human genome or by a cDNA copied from human mRNA. It will be appreciated that the first and second source may be first and second tissue from an organism. In such a case, the nucleic acid may be the mRNA of a cell of the tissue. It is preferred that the protein kinase derivable from the first source is not identical to the protein kinase derivable from a second source. The said protein kinases may be tissue-specific or tissue- restricted isoforms, as well-known to those skilled in the art. Thus, the method of the invention may be used to identify a compound that is able to modulate/inhibit a protein kinase derivable from a first tissue of an organism (ie the first source) to a different extent to an equivalent (non-identical) protein kinase derivable from a second tissue of the same organism (ie the second source).

Preferred embodiments of the invention include methods according to any of the above aspects of the invention wherein any of the appropriate said non-host yeast cell sources is a human (ie the protein kinase is derivable from a human).

It will be appreciated that if the protein kinase from the first and second sources are closely related then it may prove difficult to identify a compound that is able to inhibit the protein kinase from the first source but not the protein kinase from the second source, or vice versa. However, unless the said protein kinases are identical it may be possible to identify such a compound.

In these and other aspects of the invention described below, exemplary genera of yeast contemplated to be useful, either as a said host yeast cell or as a said source other than a said host yeast cell, in the practice of the present invention are Pichia, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis and the like.

By pathogenic yeast is included pathogenic yeast of any one of the genera

Candida spp, Blastomyces spp, for example B. dermatitidis , Coccidioides spp, for example C. immitis, Histoplasma spp, for example H. capsulatum, Sporothrix spp, for example S. schenckii, Aspergillus spp, for example A. fumigatus, A. flavus, A. niger, Phialophora compacta (Fonsecaea compacta), P. pedrosoi (F. pedrosi), P. verrucosa, Cladosporium carrionii, Rhinocladiella aquaspersa, Cryptococcus spp, for example C. neoformans, Cephalosporium spp, Fusarium spp, Histoplasma spp, for example H. capsulatum, Pneumocystis carinii, Rhizopus spp, Rhizomucor spp, Madurella spp, for example M. mycetomatis, M. grisea, Pseudallescheria boydii, Paracoccidioides spp, for example P. brasiliensis , Prototheca spp, for example P. wickerhamii, Epidermophyton spp, Microsporum spp, Trichophyton spp, and Malassezia spp, for example M. furfur (Pityrosporum orbiculare).

It will be appreciated that by "yeast" we include "fungi" and, in particular we include the pathogenic fungi of the genera Aspergillus, including Aspergillus fumigatus, Cryptococcus, including Cryptococcus neoformans, and Histoplasma, including Histoplasma capsulatum.

It will be appreciated that Saccharomyces spp, for example S. cerevisiae are not considered pathogenic yeasts. Schizosaccharomyces spp, for example S. pombe are not considered pathogenic yeasts.

It will be appreciated that, in a simple embodiment, the host yeast cell is rendered not capable of expressing the said host yeast cell protein kinase or kinases by virtue of a mutation or mutations in the gene or genes encoding the said host yeast cell protein kinase. Typically the mutation is any mutation that prevents expression of an active protein kinase; it is particularly preferred if the mutation is one which cannot be spontaneously reversed, for example a deletion of all or part of the gene encoding the protein kinase.

The host yeast cell which is capable of expressing the said equivalent protein kinase from a non host yeast cell source is most conveniently made by introducing into a suitable yeast a genetic construct which encodes and is capable of expressing the said equivalent protein kinase.

It will be appreciated that the host yeast cell parental strain may not be wild-type. For example, particularly in relation to S. cerevisiae as the host yeast cell, mutant strains containing Ade^" or Leu^" or Ura^" mutations may be used as the parental strain to allow selection of plasmid uptake.

It will be appreciated that a constitutive or inducible promoter may be used in the expression of the non-host yeast cell protein kinase. Examples of constimtive promoters well known in the art are the adh or the SV40 promoter. As discussed above and in Example 1, the GALl promoter may be used. Alternatively, the promoter from the gene encoding the host yeast cell protein kinase may be used to express the non-host yeast cell protein kinase. For example, the promoters from the Pkhl or Pkh2 genes may be used, in a manner analogous to that described in Example 1.

It is preferred that the exogenous polynucleotide is stably maintained in the yeast. It is further preferred, though not essential, that the exogenous polynucleotide is stably integrated into the yeast genome.

Transformation of cell hosts is accomplished by well known methods that typically depend on the type of vector used and host cell. Transformation of Saccharomyces and related cells is described in Sherman et al (1986) Methods in Yeast Genetics, a Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 may also be useful. Schizosaccharomyces pombe may be transformed following LiCl treatment or by electroporation. Conveniently, a Bio-Rad Pulse Controller may be used for electroporation of S. pombe cells. The technique of electroporation of yeast is disclosed in Becker & Guarente (1990) Meth Enzymol 194, 182.

S. cerevisiae may be transformed by, for example, methods analogous to those described in Example 1.

It is particularly preferred if, in relation to the first aspect of the invention, the host yeast cell is incapable of expressing the protein kinase Pkhl and/or Pkh2.

Thus, a further preferred embodiment of the invention is a method of the invention wherein the said host yeast cell protein kinase or kinases is Pkhl and/or Pkh2, wherein Pkhl is the polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae and Pkh2 is the polypeptide encoded by open reading frame YOLlOOw of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae.

Pkhl from S. cerevisiae may have the following amino acid sequence:

GNRSLTEADHALLSKPLVPTSAEHTQTQEYPRPFVDGSNSQSGSELQAS PQGQFGEKALTSTNRFIPLANDDPGMQHEMGLDPSMRRRREE AERGAAK IVKDVVDPATGELTKHVVKMGIKDFKFGEQLGDGSYSSVVLATARDSGKK YAVKVLSKEYLIRQKKVKYVTVEKLALQKLNGTKGIFKLFFTFQDEASLY FLLEYAPHGDFLGLIKKYGSLNETCARYYASQIIDAVDSLHNIGIIHRDI KPENILLDKNMKVKLTDFGTAKILPEEPSNTADGKPYFDLYAKSKSFVGT AEYVSPELLNDNYTDSRCDIWAFGCILYQMLAGKPPFKAANEYLTFQKVM KIQYAFTAGFPQIVKDLVKKLLVRDPNDRLTIKQIKAHLFFHEVNFEDGS VWDDNPPEIQPYKINAEAMKPLQKVSESDTTVKMANLQLAGNGHADTPLQ APAATSQEHSVISMTAATAAFNKDYTSQPKLGSKSSTSVRSASNNTDREV IQKKVSKNRASVSSPSISTTSRGKDNRSRSSDAFWSRYLQNMDERVLLMK EVALSTRNLEDSPVGLENVALDYKNPLDIEPPTDSAGKFYKKMFLITNLG RALVFVKRRSLSM EEQEFELQFELELNDVEKIRFISDQVLEIDGSRTIF IGCKERAVLMKLWKLIHNGMTAKPKVVSPKSDHKMFDKFILQKRQNTKKK NQAPPVPQSNRLINGLPDRCILKTPEEGALHTKRPTSLQTRSSSNYSKLL ARSTQMRKNMTRTDEK (766 aa complete sequence)

Pkhl from S. cerevisiae may be encoded by the following nucleotide sequence: ATGGGAAATAGGTCTTTGACAGAGGCAGACCACGCCCTGCTGTCCAAGCC CTTGGTACCGACATCTGCGGAACATACACAAACGCAAGAGTATCCTCGTC CTTTCGTAGATGGCAGCAATTCTCAGAGCGGGTCTGAACTACAGGCTTCT CCACAAGGTCAGTTTGGAGAAAAGGCATTGACTAGTACTAATCGCTTCAT TCCCCTGGCAAATGATGACCCGGGTATGCAGCACGAGATGGGTCTTGATC CCTCAATGAGGCGTAGAAGAGAAGAATGGGCAGAACGTGGTGCGGCAAAA ATCGTCAAAGATGTTGTCGACCCAGCTACGGGGGAGTTAACTAAGCATGT TGTCAAGATGGGAATAAAGGACTTCAAGTTTGGAGAGCAACTCGGGGATG GATCATATTCTAGTGTTGTTCTGGCTACCGCCCGTGATTCGGGCAAGAAA TATGCAGTAAAAGTGTTGAGTAAAGAATATCTGATCCGTCAAAAAAAAGT TAAATACGTCACAGTGGAGAAATTGGCTTTGCAGAAGCTGAATGGCACCA AGGGCATATTCAAGCTTTTCTTCACTTTCCAGGACGAGGCAAGCTTGTAT TTCCTTCTAGAATATGCCCCCCACGGTGATTTCTTGGGCTTGATTAAGAA ATATGGATCTTTAAACGAGACATGTGCACGCTATTATGCGTCGCAGATCA TCGATGCCGTTGACTCCTTGCATAATATCGGCATTATTCACAGGGATATC AAGCCCGAAAACATATTGCTCGACAAAAATATGAAAGTGAAGTTGACGGA TTTTGGTACAGCCAAAATTTTACCGGAGGAACCTTCGAACACCGCAGATG GCAAGCCTTATTTCGATTTGTATGCTAAGTCGAAATCATTTGTTGGTACC GCAGAATATGTTTCTCCTGAGCTACTGAATGATAATTATACAGATTCCCG TTGTGACATTTGGGCATTTGGTTGCATATTGTACCAAATGCTTGCGGGAA AACCGCCTTTTAAAGCTGCCAATGAATATTTGACATTCCAAAAAGTAATG AAGATTCAATATGCGTTTACTGCAGGTTTTCCGCAAATAGTAAAAGATTT AGTTAAAAAACTATTAGTTAGGGATCCAAATGATAGATTGACCATAAAGC AGATCAAAGCACACCTCTTTTTCCATGAAGTCAACTTTGAAGATGGTTCT GTTTGGGATGATAATCCACCGGAGATACAGCCATATAAAATAAATGCAGA GGCGATGAAGCCCCTGCAAAAGGTTTCTGAATCTGATACCACTGTCAAAA TGGCCAACCTTCAGCTGGCTGGTAATGGACATGCAGATACTCCCCTGCAA GCACCAGCAGCGACCTCTCAAGAGCATTCTGTGATCAGTATGACTGCAGC AACCGCCGCATTCAATAAAGATTATACAAGTCAACCCAAATTGGGGAGCA AGTCAAGCACATCTGTTAGATCTGCCTCCAACAACACAGATCGCGAGGTA ATTCAAAAGAAGGTTTCAAAAAATCGCGCATCTGTATCATCTCCTTCAAT TTCTACTACATCACGGGGGAAAGATAATAGAAGTCGCTCTTCTGACGCCT TCTGGTCACGCTACTTGCAAAATATGGATGAACGTGTCTTGTTGATGAAG GAGGTAGCGCTTTCCACACGAAACTTAGAGGACTCACCTGTAGGTCTTGA GAACGTGGCTCTGGACTACAAGAACCCTCTTGATATCGAGCCTCCTACTG ATAGTGCAGGCAAATTTTACAAGAAAATGTTTCTAATAACAAACCTAGGC AGAGCACTTGTTTTTGTCAAGAGAAGAAGTCTCAGCATGTGGGAAGAACA GGAATTTGAATTGCAATTCGAACTAGAGTTGAATGACGTTGAGAAGATAC GCTTTATAAGTGATCAAGTCCTTGAAATTGACGGTTCCAGGACGATTTTC ATAGGATGCAAAGAGAGAGCAGTTTTAATGAAACTATGGAAATTAATACA TAATGGAATGACCGCCAAACCTAAAGTAGTATCGCCGAAGTCGGACCATA AAATGTTTGATAAGTTCATTCTTCAAAAAAGACAGAATACAAAAAAAAAG AATCAAGCTCCTCCCGTACCTCAATCGAATAGGCTAATAAATGGTTTACC GGACCGTTGCATCTTAAAGACACCCGAAGAAGGCGCACTTCACACAAAAC GTCCCACTTCGTTGCAGACCCGATCGTCATCTAATTACTCAAAATTGCTG GCAAGATCGACACAAATGCGGAAAAACATGACACGGACAGATGAAAAATG A (Genbank accession number 927745; 2301 bp complete sequence).

Pkh2 from S. cerevisiae may have the amino acid sequence: MYFDKDNSMSPRPLLPSDEQKLNINLLTKKEKFSHLDPHYDAKATPQRST SNRNVGDLLLEKRTAKPMIQKALTNTDNFIEMYHNQQRKNLDDDTIKEV INDENGKTVASTNDGRYDNDYDNNDINDQKTLDNIAGSPHMEKNRNKVKI EHDSSSQKPIAKESSKAQKNIIKKGIKDFKFGSVIGDGAYSTVMLATSID TKKRYAAKVLNKEYLIRQKKVKYVSIEKTALQKLNNSPSVVRLFSTFQDE SSLYFLLEYAPNGDFLSLMKKYGSLDETCARYYAAQIIDAIDYLHSNGII HRDIKPENILLDGEMKIKLTDFGTAKLLNPTNNSVSKPEYDLSTRSKSFV GTAEYVSPELLNDSFTDYRCDIWAFGCILFQMIAGKPPFKATNEYLTFQK VMKVQYAFTPGFPLIIRDLVKKILVKNLDRRLTISQIKEHHFFKDLNFKD GSVWSKTPPEIKPYKINAKSMQAMPSGSDRKLVKKSVNTLGKSHLVTQRS ASSPSVEETTHSTLYNNNTHASTESEISIKKRPTDERTAQILENARKGIN NRKNQPGKRTPSGAASAALAASAALTKKTMQSYPTSSSKSSRSSSPATTS RPGTYKRTSSTESKPFAKSPPLSASVLSSKVPMPPYTPPMSPPMTPYDTY QMTPPYTTKQQDYSDTAIAAPKPCISKQNVKNSTDSPLMNKQDIQWSFYL KNINEHVLRTEKLDFVTTNYDILEKKMLKLNGSLLDPQLFGKPRHTFLSQ VARSGGEVTGFRNDPTMTAYSKTEDTYYSKNIIDLQLLEDDYRIEGGDLS ELLTNRSGEGYKCNQNSSPMKDDDKSESNNKGSSVFSGKIKKLFHPTSAA ETLSSSDEKTKYYKRTIVMTSFGRFLVFAKRRQPNPVTNLKYELEYDINL RQQGTKIKELIIPLEMGTNHIVVIQTPYKSFLLSTDKKTTSKLFTVLKKI LNSNTNKIEKELLQRNQKVIERRTSSSGRAIPKDLPTSKSPSPKPRTHSQ SPSISKHNSFSESINSAKSNRSSRIFETFINAKEQNSKKHAAPVPLTSKL VNGLPKRQVTVGLGLNTGTNFKNSSAKSKRS (1081 aa complete sequence).

Pkh2 from S. cerevisiae may be encoded by the nucleotide sequence: ATGTATTTTGATAAGGATAATTCCATGAGCCCTAGGCCGTTATTGCCAAG TGATGAGCAGAAGCTAAACATTAATCTTCTAACGAAAAAGGAGAAATTCT CGCATTTAGACCCCCATTATGACGCAAAAGCCACTCCACAAAGAAGCACT TCGAATAGAAACGTTGGCGATTTACTTTTGGAAAAAAGAACCGCTAAGCC TATGATTCAAAAGGCCTTGACGAATACGGATAATTTCATTGAAATGTACC ATAATCAGCAGAGAAAAAATCTTGATGATGACACTATTAAAGAAGTAATG ATTAATGATGAAAACGGAAAAACTGTCGCTAGTACCAACGACGGCAGATA TGACAACGATTACGATAATAACGATATTAATGACCAAAAAACTTTGGATA ATATAGCGGGAAGTCCCCACATGGAAAAAAATCGAAACAAAGTAAAGATT GAACATGACTCTTCATCTCAAAAACCAATAGCTAAAGAGTCATCCAAAGC CCAAAAAAATATAATCAAAAAGGGAATCAAGGACTTTAAATTTGGTAGTG TAATAGGTGATGGCGCGTATTCTACTGTAATGTTAGCGACGTCGATTGAT ACCAAAAAGAGGTACGCCGCAAAAGTACTAAACAAAGAATATTTAATACG CCAGAAGAAAGTCAAATACGTCAGCATAGAAAAAACCGCCCTTCAAAAGC TCAATAATTCTCCTAGTGTTGTGCGATTATTTTCCACTTTTCAGGATGAA TCAAGCCTATACTTTCTCTTAGAGTATGCCCCCAATGGGGACTTTCTTTC TTTAATGAAAAAATACGGTTCATTAGACGAAACCTGCGCACGATATTATG CTGCGCAAATAATAGATGCCATAGACTACTTACATTCCAACGGTATTATT CATAGAGATATAAAACCAGAAAATATTCTTTTAGATGGAGAAATGAAGAT CAAACTGACTGATTTTGGTACTGCGAAGTTACTGAATCCTACAAATAATA GCGTTTCGAAACCAGAATACGATTTATCAACAAGGTCGAAATCTTTCGTT GGAACTGCAGAATACGTATCTCCAGAACTTTTAAATGACAGTTTTACAGA CTATCGTTGCGATATTTGGGCCTTCGGATGTATACTTTTCCAGATGATTG CCGGAAAACCACCATTCAAAGCTACCAATGAATACTTGACTTTCCAAAAG GTAATGAAAGTTCAGTACGCCTTTACACCAGGTTTCCCACTTATTATCAG AGATTTGGTTAAGAAAATCTTAGTAAAAAACTTAGACCGAAGATTGACGA TAAGCCAAATTAAGGAACATCATTTTTTCAAAGATTTGAATTTTAAAGAC GGCTCTGTTTGGTCAAAAACGCCTCCAGAGATCAAACCATATAAAATCAA CGCCAAATCCATGCAGGCAATGCCAAGCGGAAGCGATAGAAAACTGGTGA AGAAATCAGTCAACACACTTGGCAAATCGCATCTAGTGACTCAAAGGTCA GCTTCAAGTCCCTCTGTTGAGGAAACTACTCATTCAACCCTATACAATAA CAATACTCACGCTTCTACTGAAAGTGAAATATCAATAAAGAAGAGACCCA CTGATGAAAGAACAGCGCAGATACTTGAAAATGCAAGAAAGGGTATAAAC AATAGGAAAAATCAACCAGGCAAGAGAACACCAAGTGGTGCAGCTTCTGC TGCCCTAGCAGCTTCTGCTGCTTTAACCAAGAAAACCATGCAAAGCTATC CAACTTCTAGTTCGAAAAGTAGCAGGTCAAGCTCTCCTGCGACAACATCA AGACCAGGAACTTATAAGCGTACTTCTTCTACAGAAAGTAAACCATTTGC CAAATCTCCACCTTTGTCAGCATCAGTTTTATCGTCAAAAGTCCCAATGC CTCCATACACACCTCCAATGTCGCCCCCTATGACACCATATGATACATAT CAAATGACACCTCCCTATACGACAAAACAGCAGGATTATTCTGATACCGC AATTGCCGCACCTAAGCCTTGTATTAGTAAGCAAAATGTTAAAAATAGCA CAGATTCTCCCTTGATGAACAAGCAAGATATTCAATGGTCCTTTTACCTG AAAAACATCAACGAACATGTACTAAGGACGGAAAAACTGGATTTTGTTAC CACAAATTACGATATCTTAGAGAAGAAAATGCTTAAACTAAATGGTTCAT TGTTAGATCCTCAACTGTTTGGTAAGCCTAGACATACTTTTTTATCCCAA GTAGCTAGGAGTGGGGGAGAGGTTACAGGTTTTCGAAATGATCCAACTAT GACTGCTTATTCCAAAACAGAAGATACGTACTATTCGAAAAATATTATCG ATTTGCAGCTCTTGGAAGATGATTATCGAATTGAAGGAGGTGACTTATCG GAGTTGCTTACTAACAGAAGCGGAGAAGGGTACAAATGCAATCAAAACAG CTCACCAATGAAAGACGATGATAAATCCGAATCTAACAATAAAGGAAGCT CTGTTTTTTCTGGCAAGATTAAAAAATTATTTCACCCTACCTCAGCAGCT GAAACGCTCTCTTCCTCTGATGAAAAAACCAAGTACTATAAACGAACCAT TGTAATGACATCATTTGGAAGGTTTCTAGTATTTGCCAAGAGGAGGCAGC CAAATCCAGTTACAAATTTAAAGTATGAACTAGAATATGACATAAATTTG CGTCAACAGGGTACCAAAATAAAAGAACTAATCATTCCCTTGGAAATGGG AACTAATCATATAGTTGTGATTCAGACACCTTACAAGTCATTTCTTTTGA GCACTGATAAAAAAACCACGAGCAAATTGTTTACTGTTCTCAAAAAAATT CTTAATTCGAATACAAATAAAATAGAGAAAGAACTGTTGCAAAGAAACCA AAAGGTAATTGAAAGAAGAACATCATCATCCGGAAGAGCCATACCTAAAG ATCTTCCAACTTCCAAGTCTCCTTCGCCAAAACCCAGGACGCATAGCCAA TCTCCATCAATTTCAAAGCACAATTCGTTTTCTGAATCGATTAATAGCGC TAAGAGCAACAGATCAAGCAGAATTTTTGAAACCTTTATCAATGCCAAGG AACAAAATTCAAAAAAACACGCTGCTCCAGTACCGTTAACCAGTAAATTA GTTAACGGATTGCCAAAAAGACAAGTTACCGTGGGATTAGGTCTAAACAC AGGAACAAATTTCAAAAACTCATCTGCAAAATCGAAGAGGTCGTAAT (Genbank accession no 1419952; 3247 bp complete sequence). Equivalent open reading frames in yeast other than S. cerevisiae may be identified as such by methods well known to those skilled in the art and as described below.

The KSG1 gene (PDKl-like) from Schizosaccharomyces pombe may encode a polypeptide which comprises the following amino acid sequence: MRNTHNPNETEASEDAENDTQSESDLSFDHGSSEKLNRASLPKTQNSAIPQSNALN TTPNESTSQIDSSPKIPSAVPHISTPNPSSGASTPNIKRVSDFKFGEILGEGSYST VLTATENSTKREYAIKVLDKRHIIKEKKEKYVNIEKEALCILSKHPGFIKLFYTFQ DAHNLYFVLSLARNGELLDYINKLGRFNEICAQYYAALIVDSIDYMHGRGVIHRDL KPENILLDDNMRTKITDFGSAKILNSSHGSHEEDTHHADKPQAHSRSFVGTARYVS PEVLSDKIAGTASDIWAFGCILFQMLAGKPPFVAGNEYLTFQSILHLSYEIPPDIS DVASDLIKKLLVLDPKDRLTVDEIHQHPFFNGIKFDNTL ELPPPRLKPFGHTSVL SLSVPNASNKHENGDLTSPLGVPSMVSASTNAAPSPVGTFNRGTLLPCQSNLEEEN KE SSILQDDEKISKIGTLNVYSMSGINGNDAFRFFSSLFRKRKPRTFILTNFGRY LCVASDGEGRKTVKEEIPIKSVGMRCRMVKNNEHGWVVETPTKS SFEDPNGPASA WVELLDKASSISLPFGNHSVTSFSRSIARSAV (>gi133414881gnlI PID I el312259 protein kinase. S. pombe).

The KSG1 gene from S. pombe may comprise the following nucleotide sequence: GGTATCTTCAGTTAAATCTTGCGTTGGTTGTCCTTTAATGAAGATTTGTTCGTAAG CAAAATATATGAATTCGCTAGCAAGATATTGCCATTCATACCAACCCTTTTCTTTA TGTTATATCTCCAAAATGAATTGTTTACAACACTAAATTGAAACAAATCAAACACA CTTTGTTCCTATTAGGTGTAGTGGAAAATGCAATTCTAAACAATGGCACTTTATTT TTGATTCTGTGCTGAACAAATAATTCATATTTATAAACTGAATGGTGCATAATTTT CTCTCTCTGTAAAATTGATACGGTAAGTTGAAATTCAAGTAAAATATGTTTCCCGC ATATTCAGCTACGATTCTGGTATAAAAGCTTCTAATGCTCTTCGTATATAAACTGA GTTATTCATTAAAACCTAAGATCTTTTAAGTTATTGAAATTAAATCATATTGAATT CCACACCCGAAGGTAATGCTATTCAACCAGCCAAGACGAAAATACTTGGTTTGACA ATTTTTTTACTTTTATGTAATGATTTTCTCTACTCCATTAAGAATTTGCGTATGAC TGTGTATACTCAGCGCTGATTCACATAGTAATATAAATCATTACCTTGATGAACTA CTTCTCTAGGCTCCTTAAAAAAATAAATAAACTTATAGTTACTAAAAAAAGCATGA TCTAAATCAAATGAATGAATATTTAGTTTATCTAAAATGTCGAATTCACTATTAAT TTTCGCACTCACAACGATGCAAATCATAACCCGGATAGCAAAATCTTATATAACAA TAGTACAATAGTTGTATACTTGCAGTTATAAGCACTAATTTTACCGAATATGTGCA ATTTTCTTTTGATGACATTGCCGTCAATTCGTAGTTTACTGACACTGATTCACCTA CCCAGTACTCTCTTCACACCAGATATTGGTTATGCGAAATACGCACAATCCGAATG AAACTGAAGCATCAGAAGATGCAGAAAATGATACTCAAAGCGAATCCGACCTTAGT TTTGATCATGGATCAAGTGAAAAACTAAACCGAGCTTCATTACCAAAGACGCAAAA TAGTGCCATCCCGCAGTCTAATGCTTTAAATACTACACCAAATGAGTCAACGTCTC AGATCGATTCATCGCCGAAGATTCCCTCTGCAGTCCCACATATCTCTACTCCAAAT CCGTCTAGCGGTGCATCGACTCCTAACATAAAACGAGTTTCGGATTTTAAATTTGG CGAAATCCTTGGAGAAGGATCATACAGTACTGTATTAACAGCTACCGAAAACTCAA CAAAAAGAGAATATGCTATTAAGGTGTTGGATAAAAGACATATTATCAAGGAAAAA AAGGAAAAATACGTCAACATTGAAAAAGAAGCTTTGTGCATCCTCTCTAAGCATCC TGGATTTATTAAGCTATTTTATACTTTCCAAGATGCTCACAATTTGTATTTTGTTT TGAGTCTTGCTCGAAATGGTGAACTGTTGGATTATATCAATAAGGTATGTATAAAT AATGTTCATGCAGCTTGATGATGGATAGCTTACTGATTATTACAATCTTGTCTTTT TTCTTATTAAATGTTTTATTTTTTGCTTACACATAATTTAGTTGGGACGATTTAAC GAAATATGTGCACAATATTATGCTGCACTTATCGTTGACTCAATAGACTATATGCA TGGACGAGGAGTGATTCACCGTGACTTGAAGCCCGAAAATATTTTGCTGGATGATA ATATGCGTACAAAAATTACCGACTTTGGTTCAGCAAAAATATTAAACTCTTCACAT GGCAGTCATGAAGAAGACACACACCATGCTGACAAACCACAGGCTCATTCTCGATC GTTTGTAGGAACTGCTCGTTATGTATCTCCTGAAGTTTTAAG

TGATAAAATTGCTGGTACGGCTTCCGATATTTGGGCGTTTGGTTGCATTCTTTTCC AAATGCTAGCTGGCAAACCCCCTTTTGTAGCAGGAAATGAATATTTGACATTCCAA AGTATTCTTCATTTGAGTTATGAGATACCACCTGACATATCAGATGTCGCTTCTGA TTTGATTAAAAAACTGCTAGTTCTAGATCCTAAAGATCGCTTAACTGTTGATGAAA TACACCAGCATCCGTTTTTTAATGGTATCAAATTTGATAATACCCTTTGGGAACTT CCTCCTCCCCGTCTTAAACCTTTTGGTCATACTAGCGTCCTCAGCCTTTCCGTTCC TAATGCATCTAACAAACACGAAAATGGTGATTTGACCTCGCCTCTTGGTGTTCCAT CGATGGTTTCAGCATCCACCAATGCTGCACCCTCTCCGGTTGGTACTTTTAACCGA GGCACTCTATTGCCGTGTCAATCCAACCTTGAGGAGGAAAACAAGGAATGGTCGAG TATTCTTCAAGACGATGAAAAAATCTCAAAAATTGGAACCCTCAATGTTTATAGTA TGTCTGGTATCAATGGAAATGATGCCTTTCGCTTTTTCTCCAGTCTTTTTCGAAAA AGGAAACCTCGGACCTTTATACTTACAAATTTTGGTCGATACCTGTGTGTTGCCTC GGACGGTGAAGGGCGAAAAACAGTTAAAGAAGAAATACCTATTAAGAGTGTAGGCA TGCGTTGTCGGATGGTAAAAAACAATGAACATGGCTGGGTAGTTGAGACTCCAACA AAATCCTGGTCGTTTGAAGACCCGAATGGACCGGCTTCTGCTTGGGTTGAGCTTCT AGATAAGGCTAGCTCTATTTCTCTTCCATTCGGTAATCATTCTGTTACCAGCTTTT CAAGAAGCATTGCTAGAAGTGCTGTCTAATCTATCCTTTATGTCTATGAACACTTC CATCCATCCCCTCACTTTTTATTCTATCACTCTTTTTTCTGTTCTGTTCTGCATTA ATTTTCTTACGACCTTATTGATGTTTCAAGAAATTCTTTTTAAGTTTTTCTCTTAC GTTCGTTGTTTAATTCGAAAAAAAATCTTTATATCCGTGATCCTTCAGTGTGATTA CAAAATTAGATTGCCGTATGATTTCTGTATTTTGTTATTAACGGTCAATTGATTTT ATTTGCTTATTCTTATTATGGTCTATTTATTTTATATTTTTTACCTTAGCCTTAAA ATGTTAAAATGGTAAAAGTTATCGATCCAAGAACATTTCACCGTTTACTAGTTAAT ATACCTCTACCTACGTAGTAAAAAAATTTAAAGAAATTATTCCCAAAAGCCAGTTG TACATTCTCTCAATTTATGATTAATCTACAGATTAAGTTAATTATGCAAGTATGAC TTGTACATAAAAATTAGGTTTCGCTTAATATCAGAAAAAAAAACTTCAAGCATCAA TTTACTAGCATGATAAATAATGAATTACTTCTCCAAGTATGTTTTTGCGACACGAT C

(>gi133414871emb|X992801 SPKSG1 S.pombe KSG1 gene (PDKl-like))

A second PDKl-like gene (SPBC4C3.il) of S. pombe may encode a polypeptide which includes the following amino acid sequence:

MDLEHKRISRSTLPDYADPDYFEARGERNPVKPQSSNVVPGTSHIGSIKSPADYVF

GDIIGDGSFSKVRRATDKKSWKEYAIKVLDKKYIVKENKVKYVNIERDSMMRLNGF PGISRLFHTFQDDLKLYYVLELAPNGELLQYIKKYRFLDENCVRFYAAEILSSIEY MHSCGIIHRDLKPENILFDGNMHVKITDFGTAKILPPKYVNSPDYTTFPSSFVGTA EYVAPELLSRQVVSKSSDLWAFACVVYQMIVGSPPFHGSNPNNIFKKIMSLEYELP KLLPPDIVPLFSHLFRIQPSDRSTTQQIKQFPFFATITWDNLWTQDPPPMQSFRPN YNIAIPNAPAYYRSNVTAAAAANAAAAFASASIVKHQETARRQELPTVNRFTAPTA HYGYASLRSHQMPVDRLYYKLVPSSESI

( > gi 128328921 gnl | PID | e 1249768 hypothetical protein kinase phosphorylase).

The second PDKl-like gene (SPBC4C3.i l) of S. pombe may include the following nucleotide sequence: acaacttcat ttttcatgaa aacctctttt ataaatgatg gatctggagc ataaacgcat tagccgaagt acattgccgg attatgcgga tcccgattac ttcgaggcta gaggtgaaag aaatccggta aaacctcagt cttccaacgt agtaccagga acaagtcata taggatcgat caaatctccg gcggattacg tttttggtga cattatagga gatggatcat tctcaaaggt aagttgatac attgcttctc agttcagaag tttttcaacc gcaggtcgat taacctatgg accttcgatt actgacgaat aagttgtcta atattcgtta ggtgagaaga gcaactgata aaaagagttg gaaggagtac gctatcaaag tccttgataa aaaatatatt gtcaaggaaa ataaggttaa gtatgtgaat atagagagag attctatgat gagacttaat gggtttcctg gtatctctcg tcttttccat acatttcagg atgatttaaa actttattat gtgcttgaac ttgcacccaa tggtgaactt ttgcaataca tcaaaaaggt atattttttc attagtctat tcatttttcc tttattaact aagctttggt agtatcgttt tcttgatgag aattgtgtgc gcttttatgc tgctgagatt ttatcaagta tcgagtatat gcactcctgc ggtataattc acagagatct caagccagaa aagtatgttt gagtagtggt cattaaatgt tcgtttcctt ttcctaattc taacctattt tttagcattt tatttgatgg aaatatgcat gtaaaaatta ccgatttcgg cacagccaaa atcctacccc ctaaatatgt aaatagccct gattacacta cctttccaag ctcctttgtt ggcactgcgg aatatgttgc tcctgaacta ttgtctagac aagttgtttc aaaatcgtaa gaaaacctat tatcccagtc tatttttttt ctgacaaata tttaagttcc gatttatggg cttttgcgtg tgttgtttat caaatgattg ttggttcccc tccttttcat ggcagcaatc ctaataatat tttcaaaaag ataatgagcc tggaatatga gcttccaaag ctcttaccac ctgatatcgt tcctttgttt agccatcttt tccgtattca gccatctgat cgatctacaa cccaacaaat aaaacaattt cctttttttg ctactattac ttgggacaat ttatggactc aagatcctcc tcctatgcag tcattccggc ctaattataa catagccatt cctaatgctc ctgcttatta tcgctcaaat gtgacagccg cagctgctgc taatgctgcc gcggcatttg cttctgcatc cattgtaaag catcaggaaa ctgctcgacg tcaggagctt cctacggtaa atcgtttcac tgctccaact gctcattatg gctatgcttc acttcgaagc catcagatgc ctgttgacag actttattac aagttggttc catcgtctga gtcgatc

The terms Pkhl and Pkh2 may represent PKB-activating Kinase Homologues 1 and 2, respectively. Yeast, for example S. cerevisiae, cells that are capable of expressing either Pkhl or Pkh2 or both are capable of growing, but yeast, for example S. cerevisiae, cells that are not capable of expressing either Pkhl or Pkh2 are not capable of growing, as described in Example 1.

A protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, for example Pkhl and/or Pkh2, may be PDKl, for example mammalian, preferably human PDKl. As described in Example 1, yeast, for example S. cerevisiae, cells that are not capable of expressing either Pkhl or Pkh2 may be capable of growing if they are capable of expressing human PDKl.

The PDKl may be plant PDKl . An example of plant PDKl may be a polypeptide which comprises the following amino acid sequence:

MLAMEKEFDSKLVLQGNSSNGANVSRSKSFSFKAPQENFTSHDFEFGKIY GVGSYSKVVRAKKKETGTVYALKIMDKKFITKENKTAYVKLERIVLDQLE HPGIIKLYFTFQDTSSLYMALESCEGGELFDQITRKGRLSEDEARFYTAE VVDALEYIHSMGLIHRDIKPENLLLTSDGHIKIADFGSVKPMQDSQITVL PNAASDDKACTFVGTAAYVPPEVLNSSPATFGNDLWALGCTLYQMLSGTS PFKDASEWLIFQRIIARDIKFPNHFSEAARDLIDRLLDTEPSRRPGAGSE GYVALKRHPFFNGVDWKDLRSQTPPKLAPDPASQTASPERDDTHGSPWNL THIGDSLATQNEGHSAPPTSSESSGSITRLASIDSFDSRWQQFLEPGESV LMISAVKKLQKITSKKVQLILTNKPKLIYVDPSKLVVKGNIIWSDNSNDL NVVVTSPSHFKICTPKKVLSFEDAKQRASWKKAIETLQNR* (492 aa complete sequence.)

It will be appreciated that a further aspect of the invention is a substantially pure polypeptide comprising the above amino acid sequence or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative

The said amino acid sequence may be encoded by the following nucleic acid sequence:

ATGTTGGCAATGGAGAAAGAATTTGATTCAAAGCTTGTTCTTCAAGGGAA CTCATCCAACGGTGCTAATGTTTCTAGAAGCAAAAGCTTCTCCTTTAAAG CTCCTCAAGAAAATTTCACCAGCCATGATTTCGAATTTGGCAAGATCTAT GGTGTTGGTTCTTACTCTAAGGTTGTTAGGGCAAAGAAGAAGGAAACTGG AACTGTGTATGCTTTAAAGATTATGGACAAAAAGTTTATCACCAAGGAGA ATAAAACTGCTTATGTGAAATTGGAAAGGATTGTTCTTGATCAACTTGAA CATCCTGGGATCATTAAACTTTACTTCACGTTTCAAGACACATCCTCACT ATATATGGCACTTGAATCTTGTGAGGGTGGCGAGCTTTTCGACCAAATAA CCAGAAAAGGTCGGCTATCGGAGGATGAAGCTCGGTTCTACACTGCAGAA GTTGTGGATGCTCTTGAGTATATACATAGTATGGGACTGATTCATCGAGA TATTAAGCCGGAGAATCTGTTGCTGACTTCAGATGGACACATTAAGATTG CGGATTTTGGAAGTGTAAAGCCGATGCAGGATAGCCAGATCACAGTTCTA CCTAATGCAGCTTCTGACGATAAGGCGTGCACTTTTGTCGGGACTGCTGC ATATGTTCCTCCAGAAGTTCTCAACTCCTCTCCCGCAACTTTCGGGAATG ATCTTTGGGCTCTCGGCTGCACTCTCTATCAGATGCTTTCGGGGACTTCC CCATTTAAAGATGCAAGTGAATGGCTGATTTTCCAAAGAATTATAGCCAG AGATATAAAGTTCCCAAATCATTTTTCAGAAGCAGCAAGAGACCTCATCG ACCGGTTGCTGGATACCGAGCCAAGCAGAAGGCCAGGTGCTGGCTCAGAA GGTTATGTTGCTCTTAAGAGACATCCTTTCTTTAATGGAGTTGACTGGAA GGATCTAAGGTCCCAGACTCCTCCAAAACTAGCTCCAGATCCTGCGTCTC AGACAGCATCTCCCGAGAGGGATGACACACATGGTTCTCCATGGAACCTG ACACATATTGGAGATTCTTTAGCCACACAGAACGAGGGGCACAGTGCTCC TCCTACATCTTCTGAATCATCGGGTTCCATAACTCGACTTGCTTCAATAG ACTCTTTTGATTCAAGATGGCAACAGTTTTTAGAGCCAGGAGAATCGGTT CTGATGATATCAGCGGTGAAGAAGCTTCAGAAAATAACGAGCAAGAAGGT GCAGCTAATACTCACCAACAAACCCAAGCTGATATATGTCGACCCGTCAA AACTAGTTGTGAAAGGAAACATTATATGGTCTGATAACTCGAATGACCTC AACGTTGTAGTCACTAGCCCTTCACATTTCAAGATTTGCACGCCAAAGAA GGTTTTATCATTTGAAGACGCAAAACAGAGAGCTTCAGTGTGGAAAAAGG CAATCGAGACTCTTCAGAACCGCTGA (1476 bp complete sequence)

It will be appreciated that a further aspect of the invention is a recombinant polynucleotide comprising the above nucleotide sequence, or a recombinant polynucleotide encoding or suitable for expressing the above polypeptide.

A still further embodiment of the invention is a method of the invention wherein the said host yeast cell protein kinase or kinases is Ypkl and/or Ykr2. The identification of the Ypkl and Ykr2 genes is described in references 41 to 43.

Ypkl from S. cerevisiae may comprise the following amino acid sequence: YSWKSKFKFGKSKEEKEAKHSGFFHSSKKEEQQNNQATAGEHDASITRS SLDRKGTINPSNSSVVPVRVSYDASSSTSTVRDSNGGNSENTNSSQNLDE TANIGSTGTPNDATSSSGMMTIKVYNGDDFILPFPITSSEQILNKLLASG VPPPHKEISKEVDALIAQLSRVQIKNQGPADEDLISSESAAKFIPSTIML PGSSTLNPLLYFTIEFDNTVATIEAEYGTIAKPGFNKISTFDVTRKLPYL KIDVFARIPSILLPSKT QQEMGLQDEKLQTIFDKINSNQDIHLDSFHLP INLSFDSAASIRLYNHHWITLDNGLGKINISIDYKPSRNKPLSIDDFDLL KVIGKGSFGKVMQVRKKDTQKVYALKAIRKSYIVSKSEVTHTLAERTVLA RVDCPFIVPLKFSFQSPEKLYFVLAFINGGELFYHLQKEGRFDLSRARFY TAELLCALDNLHKLDVVYRDLKPENILLDYQGHIALCDFGLCKLNMKDDD KTDTFCGTPEYLAPELLLGLGYTKAVDWWTLGVLLYEMLTGLPPYYDEDV PKMYKKILQEPLVFPDGFDRDAKDLLIGLLSRDPTRRLGYNGADEIRNHP FFSQLSWKRLLMKGYIPPYKPAVSNSMDTSNFDEEFTREKPIDSVVDEYL SESVQKQFGGWTYVGNEQLGSSMVQGRSIR (680 aa complete sequence).

Ypkl from S. cerevisiae may be encoded by the following nucleotide sequence: ATGTATTCTTGGAAGTCAAAGTTTAAGTTTGGAAAATCTAAAGAAGAAAA AGAAGCCAAACATAGTGGGTTTTTTCACTCCTCTAAAAAGGAAGAACAGC AGAATAATCAAGCAACTGCGGGGGAACATGATGCTTCAATTACTCGTTCA TCTTTAGACAGGAAGGGCACGATAAATCCATCAAATTCATCAGTGGTCCC GGTTCGTGTTTCATACGATGCATCCTCATCGACCTCTACTGTACGAGATT CAAATGGTGGTAATTCAGAAAACACGAACTCATCTCAAAATTTAGATGAA ACAGCAAATATTGGTTCTACAGGGACACCCAATGATGCCACCTCAAGTTC AGGAATGATGACTATCAAAGTATATAATGGTGATGACTTTATTTTACCTT TTCCGATTACCTCAAGCGAGCAAATATTGAATAAACTATTGGCCTCGGGT GTTCCACCGCCACATAAGGAGATTAGTAAAGAAGTAGATGCTTTAATTGC ACAACTGTCCCGTGTTCAAATAAAGAACCAAGGTCCAGCTGATGAAGATT TGATCTCTAGCGAGTCAGCAGCAAAGTTCATCCCATCTACTATCATGTTA CCAGGGTCTTCGACCTTAAATCCCTTACTATATTTCACCATTGAATTTGA TAATACAGTTGCAACTATTGAAGCAGAGTATGGTACGATCGCTAAACCTG GTTTCAATAAGATATCTACCTTTGATGTGACAAGGAAATTACCTTATTTA AAGATTGATGTATTTGCAAGAATTCCGTCCATTCTTTTGCCCTCGAAGAC ATGGCAACAGGAGATGGGTTTGCAAGACGAGAAGCTACAGACCATTTTTG ATAAAATAAATTCCAATCAAGATATACATTTAGATTCTTTCCATTTGCCC ATCAACTTAAGTTTTGACTCTGCAGCTAGTATTAGACTATATAATCACCA CTGGATCACATTGGATAATGGCTTGGGTAAGATCAATATTAGTATTGATT ATAAACCTTCCAGAAATAAACCTTTGTCCATCGATGATTTCGATCTTTTG AAAGTTATCGGTAAGGGTTCGTTTGGTAAAGTGATGCAAGTCAGAAAGAA AGATACACAAAAAGTATACGCTTTGAAGGCAATCAGAAAATCATACATTG TCTCTAAATCCGAAGTCACGCATACTTTGGCAGAAAGAACCGTTCTAGCA CGTGTTGATTGTCCATTTATTGTACCTTTGAAGTTTTCTTTCCAATCACC GGAAAAATTATACTTTGTTTTAGCGTTTATCAATGGTGGTGAGTTGTTTT ATCATCTACAAAAGGAAGGAAGGTTTGATTTATCACGTGCCAGATTTTAT ACCGCAGAATTGTTATGTGCGCTAGACAACTTGCATAAACTAGATGTTGT CTATCGTGATTTGAAACCAGAGAATATTTTATTGGATTATCAAGGCCACA TTGCCCTTTGTGATTTCGGGCTATGCAAATTGAATATGAAGGATGATGAT AAGACAGATACTTTTTGTGGGACCCCAGAATACTTGGCACCAGAACTATT GCTAGGTTTAGGCTATACAAAGGCAGTAGATTGGTGGACATTGGGAGTCT TGTTATACGAGATGCTCACAGGTCTTCCTCCTTATTATGATGAAGATGTT CCAAAAATGTATAAGAAGATTCTACAGGAGCCACTAGTTTTCCCAGATGG ATTTGATAGAGATGCAAAGGATCTATTGATTGGATTATTGAGCCGTGATC CGACAAGAAGATTGGGCTACAATGGTGCCGACGAAATTCGGAACCATCCT TTTTTCAGCCAATTATCATGGAAGCGCCTGTTGATGAAGGGTTATATTCC ACCATATAAACCAGCTGTTAGTAATTCCATGGATACTAGTAATTTCGATG AGGAATTCACTAGGGAGAAGCCAATTGATAGTGTGGTAGATGAATACTTG AGTGAAAGTGTTCAAAAGCAATTTGGTGGCTGGACATACGTTGGAAATGA ACAGCTAGGTAGCTCAATGGTGCAAGGTAGAAGCATTAGATAG (Genbank accession no 486213; 2043 bp complete sequence)

Ykr2 (also known as Ypk2) from S. cerevisiae may have the following amino acid sequence: MHSWRISKFKLGRSKEDDGSSEDENEKSWGNGLFHFHHGEKHHDGSPKNH NHEHEHHIRKINTNETLPSSLSSPKLRNDASFKNPSGIGNDNSKASERKA SQSSTETQGPSSESGLMTVKVYSGKDFTLPFPITSNSTILQKLLSSGILT SSSNDASEVAAIMRQLPRYKRVDQDSAGEGLIDRAFATKFIPSSILLPGS TNSSPLLYFTIEFDNSITTISPDMGTMEQPVFNKISTFDVTRKLRFLKID VFARIPSLLLPSKN QQEIGEQDEVLKEILKKINTNQDIHLDSFHLPLNL KIDSAAQIRLYNHHWISLERGYGKLNITVDYKPSKNKPLSIDDFDLLKVI

GKGSFGKVMQVRKKDTQKIYALKALRKAYIVSKCEVTHTLAERTVLARVD

CPFIVPLKFSFQSPEKLYLVLAFINGGELFYHLQHEGRFSLARSRFYIAE

LLCALDSLHKLDVIYRDLKPENILLDYQGHIALCDFGLCKLNMKDNDKTD

TFCGTPEYLAPEILLGQGYTKTVDWWTLGILLYEMMTGLPPYYDENVPVM

YKKILQQPLLFPDGFDPAAKDLLIGLLSRDPSRRLGVNGTDEIRNHPFFK

DIS KKLLLKGYIPPYKPIVKSEIDTANFDQEFTKEKPIDSVVDEYLSAS

IQKQFGGWTYIGDEQLGDSPSQGRSIS

(677 aa complete sequence).

Ykr2 (also known as Ypk2) from S. cerevisiae may be encoded by the following nucleotide sequence:

ATGCATTCCTGGCGAATATCCAAGTTTAAGTTAGGAAGGTCCAAAGAAGA TGATGGGAGTAGTGAAGATGAAAATGAAAAATCGTGGGGTAATGGCCTGT TTCATTTCCACCATGGAGAAAAACATCACGATGGTAGCCCGAAGAATCAT AATCATGAACACGAACACCATATAAGAAAGATCAATACAAATGAGACTCT CCCAAGTTCCTTAAGTTCTCCAAAATTACGTAATGATGCATCCTTCAAGA ATCCATCGGGGATAGGAAATGACAATTCTAAGGCTTCCGAAAGGAAAGCT AGTCAGTCGTCTACTGAGACGCAGGGACCGAGTTCGGAATCCGGACTAAT GACAGTGAAGGTGTATTCTGGTAAAGATTTTACTCTTCCCTTCCCTATCA CCTCTAACTCTACTATTTTACAAAAACTACTAAGTTCCGGCATCCTTACT TCATCATCCAATGACGCTTCCGAAGTTGCAGCCATAATGCGGCAGCTACC ACGATACAAGAGAGTGGATCAAGATTCAGCAGGGGAAGGCTTGATAGATA GAGCTTTTGCCACTAAATTCATTCCTTCCTCTATATTGTTACCTGGGTCA ACAAATTCAAGCCCATTACTTTATTTTACAATTGAATTTGATAATTCTAT TACTACTATTAGTCCAGATATGGGAACGATGGAGCAACCAGTGTTTAACA AAATATCGACATTTGATGTAACAAGAAAATTACGATTTTTAAAAATCGAT GTCTTTGCAAGGATTCCATCCCTACTTTTACCCTCTAAAAACTGGCAACA GGAGATTGGCGAGCAGGACGAAGTACTGAAGGAGATTTTAAAAAAAATCA ATACAAATCAGGATATCCATTTGGACTCCTTCCATTTACCTTTGAATTTA AAAATCGATTCTGCAGCCCAAATAAGACTATACAATCACCATTGGATTTC TTTAGAAAGGGGATATGGTAAATTAAATATCACGGTGGACTACAAACCTT CTAAGAACAAGCCTCTCTCCATTGATGACTTTGATCTATTGAAGGTTATC GGGAAGGGTTCGTTCGGCAAAGTGATGCAAGTAAGGAAAAAAGATACCCA AAAGATTTACGCTTTGAAGGCTCTGAGAAAAGCATATATTGTATCGAAAT GTGAAGTGACACATACTTTAGCGGAGAGGACTGTCCTAGCAAGAGTTGAC TGCCCCTTTATTGTTCCGTTGAAGTTCTCATTCCAATCTCCGGAGAAGTT GTACCTAGTATTAGCTTTCATTAATGGCGGTGAACTGTTCTACCATTTAC AACACGAGGGACGATTCAGTCTAGCACGCTCCCGTTTTTATATTGCAGAA CTATTATGTGCTCTCGATTCATTACACAAACTTGACGTCATTTATCGTGA CCTAAAGCCTGAAAACATTCTATTGGATTACCAAGGACATATTGCACTGT GTGATTTTGGGCTTTGCAAGCTGAACATGAAGGATAATGACAAAACAGAC ACTTTCTGTGGTACTCCCGAATATTTGGCACCAGAAATCTTGTTGGGGCA GGGCTATACTAAAACAGTTGACTGGTGGACATTAGGTATCTTACTGTATG AGATGATGACAGGGCTGCCACCATACTATGATGAGAACGTTCCTGTTATG TACAAGAAAATTCTGCAGCAACCGCTACTATTTCCTGATGGATTTGACCC TGCGGCAAAAGACCTATTAATTGGCCTCTTAAGCAGAGACCCAAGCAGAA GACTCGGCGTTAACGGTACAGATGAAATTCGTAACCATCCTTTCTTTAAA GACATCTCATGGAAAAAGCTACTTTTGAAGGGCTATATTCCGCCTTACAA GCCAATTGTAAAGAGTGAAATAGATACTGCAAATTTTGATCAAGAGTTCA CTAAGGAAAAACCGATCGATAGTGTAGTGGACGAGTACTTAAGTGCAAGT ATTCAAAAGCAGTTTGGTGGGTGGACGTACATTGGTGACGAACAGTTGGG TGATTCTCCTTCGCAGGGGAGAAGCATTAGTTAG (Genbank accession no.817862; 2034 bp complete sequence)

Yeast, for example S. cerevisiae, cells that are capable of expressing either Ypkl or Ykr2 or both are capable of growing, but yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypkl or Ykr2 are not capable of growing under certain conditions, as described in Example 1. A protein kinase derivable from an source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, for example Ypkl or Ykr2, may be serum and glucocorticoid induced protein kinase (SGK), for example mammalian, preferably human SGK. As described in Example 1, yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypkl or Ykr2 may be capable of growing if they are capable of expressing human SGK .

Rat SGK may have the nucleotide and amino acid sequences shown in Figures 12 and 13, respectively. The term SGK encompasses isoforms of SGK, for example SGK 1, 2 and 3 as described in Webster et al (1993) Mol. Cell. Biol. 13, 1031- 2040 and Kobayashi et al (1999) Biochem J 344(Pt 1), 189-197 and in EMBL database records with Accession Nos AAD41091, AF169034 and AF169035.

PKB may also have some activity that is equivalent to Ypkl or Ykr2 as yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypkl or Ykr2 may be capable of growing if they are capable of expressing high levels of PKB, ie if the cells have a high accumulation of a plasmid capable of expressing PKB, as described in Example 1. Alternatively, the ability to grow of cells that are capable of expressing PKB at a lower level may be increased by growing the cells in the presence of an activator of PKB activity.

A further aspect of the invention is a method of identifying a compound that modulates (inhibits or increases) the activity of PDKl derivable from a first source, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of human PDKl (which may be Pkhl and Pkh2) and (b) is capable of expressing PDKl derivable from the said first source and optionally

2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide that is a functional equivalent of human PDKl (which may be Pkhl and/or Pkh2) and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified.

A further aspect of the invention is a method of identifying a compound that modulates (inhibits or increases) the activity of a functional equivalent of Ypkl and/or Ykr2 derivable from a first source, for example SGK or PKB, preferably human SGK or human PKB, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of Ypkl and/or Yrk2 and (b) is capable of expressing a functional equivalent of Ypkl and/or Ykr2 (for example SGK) derivable from the said first source and optionally

2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide (for example, an endogenous polypeptide) that is a functional equivalent of Ypkl and/or Yrk2 and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. As in the first aspect of the invention, it will be appreciated that the said first and second host yeast cells differ substantially only in the features indicated such that apart from these features the cells are essentially the same. This ensures that there is a reasonable expectation that the effect of the compound on the phenotype of the said yeast cells can be attributed to the compound's effect on the modulation of the activity to different extents of the protein kinases as said. Thus, the first and second host yeast cells are from the same species and have substantially the same genetic content with the exception of the features indicated ie the capability to express the said host yeast cell protein kinase or kinases and the ability to express the said equivalent protein kinase derivable from a source other than the host yeast cell. It will be appreciated that the said first and second host yeast cells may differ in genetic content relating to the generation or selection of the said first or second host yeast cells (for example, in selectable marker genes used in recombinant techniques, as well known to those skilled in the art).

It will be appreciated that the PDKl or SGK or PKB that the said first cell is capable of expressing may not be full-length PDKl or SGK or PKB; for example, it may be a truncated PDKl or SGK as appropriate. It will be appreciated that it is preferred that any PDKl or SGK or PKB (either activated or not activated) that is not full length PDKl or SGK or PKB may have at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the enzymatic activity of full length PDKl or SGK or PKB (either activated or not activated), as herein described.

However, it will be understood that in the methods of the invention described above it will be desirable to identify compounds that may modulate (including activate or inhibit), the activity of the relevant protein kinase or kinases, for example PDKl or SGK, in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between the said protein kinase and its substrate or substrates and any regulating protein/substance are substantially the same as in vivo.

It will be appreciated that these methods of the invention may be used to identify an activator of, for example, PDKl, SGK or PKB in a manner similar to that described above for the first and second aspects of the invention. Thus, the said first yeast cell may, under the growth conditions used in the method, express the said functional equivalent of Ypkl, Ykr2, Pkhl and/or Pkh2 (for example, PDKl , SGK or PKB) at a level below that required for the said yeast cell to be capable of growing at the level of the wild-type cell, preferably at a level below that required for the said yeast cell to be substantially capable of growing. In the presence of an activator of the said functional equivalent (for example, PDKl, SGK or PKB), the level of activity of the said functional equivalent may be sufficient for the said yeast cell to be more capable, preferentially substantially capable of growing, under the same growth conditions. As discussed above and in Example 1, the said functional equivalent may expressed from a regulatable promoter, for example the GALl promoter that is repressed in the presence of glucose.

The optional said second yeast cell may similarly be substantially unable to grow unless an activator of the said yeast polypeptide (for example, an endogenous polypeptide) is present. It will be appreciated that the yeast polypeptide may be expressed from a heterologous promoter, for example the GALl promoter, as discussed above and in Example 1.

It will be appreciated that when identifying compounds on the basis of the ability to increase growth (as opposed to reducing growth) of the target cell that it may be less important to make use of the optional second yeast cell in the methods above as it may be less likely that a compound that is not acting on the "target" polypeptide (for example, the said functional equivalent of Ypkl, Ykr2, Pkhl and/or Pkh2, which may be PDKl, SGK or PKB) would have the desired effect of increasing cell growth. Compounds acting through a "non-specific" mechanism, as discussed above, more frequently act to reduce cell growth.

Preferences regarding the host yeast cell and sources of the equivalent protein kinases may be as given in regard to the earlier aspects of the invention.

It will be appreciated that in the methods described herein, which may be drug screening methods, a term well known to those skilled in the art, the compound may be a drug-like compound or lead compound for the development of a druglike compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water- soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

The compound may be suitable for use as a plant protection product or may be a lead compound for the development of a compound suitable for use as a plant protection product. It will be appreciated that such compound may have characteristics similar to those described above for drug like compounds. It will be appreciated that compounds that may be identified by methods or screens performed with cells may be capable of interacting with or entering cells.

Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of a functional equivalent of Ypkl, Ykr2, Pkhl and/or Pkh2 for example, PDKl, SGK or PKB. Aliquots of an expression library in a suitable vector, as described below, may be tested for the ability to increase the growth of a first yeast cell that, under the growth conditions used in the method, expresses the said functional equivalent of Ypkl, Ykr2, Pkhl and/or Pkh2 (for example, PDKl , SGK or PKB) at a level below that required for the said yeast cell to be capable of growing at the level of the wild-type cell, preferably at a level below that required for the said yeast cell to be substantially capable of growing. In the presence of a polynucleotide expressing a polypeptide activator of the said functional equivalent (for example, PDKl, SGK or PKB), the level of activity of the said functional equivalent may be sufficient for the said yeast cell to be more capable, preferentially substantially capable of growing, under the same growth conditions. As discussed above and in Example 1, the said functional equivalent may expressed from a regulatable promoter, for example the GALl promoter that is repressed in the presence of glucose. It will be appreciated that several cycles of identifying pools of polynucleotides comprising a polynucleotide having the required property and then rescreening those polynucleotides may be required in order to identify a single species of polynucleotide with the required property.

It will be appreciated that further tests and/or sequence analysis may be required in order to distinguish polynucleotides encoding a functional equivalent of Ypkl , Ykr2, Pkhl and/or Pkh2 from polynucleotides encoding an activator of a said functional equivalent.

Methods of introducing a polynucleotide into a host yeast cell are well known to those skilled in the art. Methods of preparing a suitable expression library for screening are well known to those skilled in the art. The library may preferably be from the same source as the said functional equivalent that is expressed in the said host yeast cell ie a human expression library may be screened for effects on yeast cells expressing human PDKl, SGK or PKB at levels below the threshold required for cell growth.

The above compound/library screening methods may conveniently be carried out in a 96-well microtitre plate format. A yeast cell, such as a Saccharomyces cerevisiae cell that has the properties of a host yeast cell for use in the method of the invention may be picked and put into liquid culture in minimal medium supplemented as necessary (as described, for example, in Example 1). The culture is grown up and may then be diluted to an optical density (OD) at 595 nm of 0.01 to 0.1. The diluted culmres are then aliquoted into wells of a sterile 96 well microtitre plate containing individual test compounds. The growth of the cells is monitored over time until the OD595 reached is about ~ 0.8 for control cultures (ie those cultured in the absence of test compound). The OD595 is assessed using a microtitre plate reader. It will be appreciated that a control culture may contain an aliquot of the solvent used to dissolve the test compound. Thus, for example, the test compound may be dissolved in dimethylsulfoxide (DMSO) and both test and control cultures may therefore contain DMSO, for example at between 0.1 and 5%, preferably at about 1 % to 2% by volume.

Alternatively, the cells may be grown on solid medium and the presence, asbence, number and/or size (or other characteristic) of colonies detected. It will be appreciated that this may be performed in a high-throughput format, for example in microtitre plates. The presence, absence, number and/or size (or other characteristic) of the colonies may be detected by visual inspection, either manually or by automated techniques, including computer image analysis.

It will be appreciated that dose response measurements, as well known to those skilled in the art, may be made for selected compounds.

If appropriate, duplicate plates of compounds may be exposed to aliquots (including solid cultures) of a particular yeast culture, or may be exposed to aliquots of different yeast culmres. For example, one such plate may be exposed to wild-type (with respect to Pkhl, Pkh2, Ypkl, Yrkl as appropriate) yeast cells, and a further such plate may be exposed to cells which are mutant with respect to Pkhl, Pkh2, Ypkl, Yrk2 as appropriate but which express, for example, human equivalents (PDKl is a human equivalent of Pkhl and Pkh2, and SGK and PKB are human equivalents of Ypkl and Yrk2), as described in the methods of the invention.

Yeast cells A further aspect of the invention is a yeast cell that is not capable of expressing Pkhl and Pkh2 or any functional equivalent thereof. It will be appreciated that such a cell may not be capable of growing ie may be of limited viability.

A further aspect of the invention is a yeast cell that is not capable of expressing endogenous Pkhl and/or Pkh2. The yeast cell may be capable of expressing a functional equivalent of Pkhl and/or Pkh2 that is not the Pkhl or Pkh2 endogenous to a wild-type yeast cell from which the said yeast cell is derived. The functional equivalent may be human PDKl or a variant, fusion or derivative thereof, for example PDK1-ΔPH as described in Example 1. PDKl or PDK1- ΔPH may be capable of phosphorylating the same target substrates essential for the viability of yeast cells as Pkhl and Pkh2, as discussed in Example 1. It will be appreciated that the cell may be capable under given conditions of expressing the said functional equivalent of Pkhl and/or Pkh2 at a level below that necessary full wild- type viability of the cell, for example at a level at which the cell is substantially unable to grow under given conditions.

Thus, the yeast cell may be a S. cerevisiae cell that is not capable of expressing Pkhl from S. cerevisiae or a S. cerevisiae cell that is not capable of expressing Pkh2 from S. cerevisiae, or a S. cerevisiae cell that is not capable of expressing Pkhl and Pkh2 from S. cerevisiae. Alternatively, the yeast cell may be a cell of a different yeast species that is not capable of expressing a Pkhl that is expressed in wild type cells of that yeast species (which may be an endogenous functional equivalent of Pkhl from S. cerevisiae), or that is not capable of expressing a Pkh2 that is expressed in wild type cells of that yeast species (which may be an endogenous functional equivalent of Pkh2 from S. cerevisiae), or that is not capable of expressing any Pkhl and Pkh2 (ie not capable of expressing any endogenous functional equivalent of Pkhl and Pkh2 of S. cerevisiae) that are expressed in wild type cells of that yeast species. A yeast cell not capable of expressing Pkhl or Pkh2 or endogenous functional equivalents may be made by methods known in the art. For example, the open reading frame encoding, for example, either Pkhl or Pkh2 may be disrupted by insertion of a selectable marker, for example as described in Example 1.

A further aspect of the invention is a yeast cell wherein one or more genes encoding a functional equivalent of PDKl, for example human PDKl, are mutated such that the yeast cell is not capable of expressing the said functional equivalent of (human) PDKl. Each such gene encoding a functional equivalent of human PDKl may be mutated such that the yeast cell is not capable of expressing a functional equivalent of (human) PDKl. Such a functional equivalent of (human) PDKl may be Pkhl or Pkh2 in, for example, a S. cerevisiae cell.

It will be appreciated that cells of a yeast species other than S. cerevisiae may express one, two, three, four or more polypeptides that are at least partially functionally equivalent to Pkhl and/or Pkh2 from S. cerevisiae. It will further be appreciated that the yeast cell of the invention encompasses a cell of such a yeast species that is not capable of expressing one, two, three, four or more or all of the said polypeptides.

It will be appreciated that Pkhl, Pkh2 and any polypeptide that is a functional equivalent of Pkhl and/or Pkh2 may be a functional equivalent of mammalian, preferably human, PDKl. Thus, the invention encompasses a yeast cell wherein one or more gene that is present in a wild-type yeast cell that encodes a functional equivalent of human PDKl is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human PDKl. Each such gene present in a wild-type yeast cell that encodes a functional equivalent of human PDKl is mutated such that the yeast cell is not capable of expressing a functional equivalent of human PDKl.

The yeast cell of this aspect of the invention encompasses a yeast cell that is not capable of expressing a polypeptide with the sequence of Pkhl or a functional equivalent thereof and/or Pkh2 or a functional equivalent thereof. It will be appreciated that a said yeast cell that is not capable of expressing any functional equivalent of Pkhl or Pkh2 may not be as capable of cell growth and/or division as a wild-type yeast cell or a yeast cell that is capable of expressing a functional equivalent of Pkhl and/or Pkh2. A functional equivalent of Pkhl and/or Pkh2 may be capable of phosphorylating Ypkl and/or Ykr2. PDKl or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment that is capable of phosphorylating Ypkl and/or Ykr2 may be a functional equivalent of Pkhl and/or Pkh2.

A further aspect of the invention is a said yeast cell capable of expressing a functional equivalent of Pkhl and/or Pkh2 that is not Pkhl or Pkh2 derivable from the wild-type yeast cell ie is an exogenous functional equivalent of Pkhl and/or Pkh2. The said functional equivalent of Pkhl and/or Pkh2 may be mammalian, preferably human, PDKl or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment that may be capable of phosphorylating Ypkl and/or Ykr2. Such a yeast cell is described in Example 1. The said functional equivalent of Pkhl or Pkh2 may be Pkhl or Pkh2 from a different yeast species, for example a pathogemc yeast species; thus the yeast cell may be a S. cerevisiae cell that is (1) not capable of expressing S. cerevisiae Pkhl or S. cerevisiae Pkh2 and (2) is capable of expressing Candida Pkhl or Candida Pkh2. Thus, a yeast cell of the invention may be a yeast cell wherein the wild-type gene encoding a functional equivalent of Pkhl or Pkh2 is altered such that it is not capable of expressing a functional equivalent of the polypeptide Pkhl or Pkh2. Thus, the yeast cell may be a S. cerevisiae cell wherein the polypeptide encoded by open reading frame YDR490c in wt yeast cells is not capable of being expressed and/or the polypeptide encoded by open reading frame YOLlOOw in wt yeast is not capable of being expressed.

The gene may be altered by the open reading frame of the gene being disrupted by insertion of a selectable marker. The marker may be TRPl or HIS3, as described in Example 1.

A further aspect of the invention is a method of identifying a compound that modulates (including increases or inhibits) the activity of PDKl, for example mammalian, preferably human, PDKl, wherein a yeast cell according to the invention is used.

A further aspect of the invention is the use of a yeast cell of the invention in a method of identifying a compound that modulates (activates or inhibits) the activity of PDKl.

By PDKl is included mammalian, preferably human, PDKl, and a functional equivalent of PDKl derivable from another source, preferably a source that may be pathogemc (including an opportunistic pathogen) to a mammal, preferably a human. Thus, a functional equivalent of PDKl from a pathogemc yeast, for example Candida is included. Such a functional equivalent may be a polypeptide that is an equivalent of the Pkhl or Pkh2 polypeptide identified in S. cerevisiae. Thus, such a functional equivalent may be the Pkhl or Pkh2 identified in Candida, as discussed in Example 4, or in the plant Arabidopsis thaliana, as discussed below.

A further aspect of the invention is a yeast, for example S. cerevisiae or Candida, or other yeast as listed above, cell wherein one or more endogenous gene(s) encoding a functional equivalent of human SGK or PKB is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human SGK or PKB. The said gene may be the Ypkl or Yrk2 gene. Each such endogenous gene encoding a functional equivalent of human SGK or PKB may be mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or PKB.

A further aspect of the invention is a yeast, for example S. cerevisiae or Candida, or other yeast/fungus, as listed above, cell wherein one or more endogenous genes encoding a functional equivalent of human SGK is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human SGK.

As discussed in Example 4, the following nucleotide sequences may encode polypeptides related to S. cerevisiae Ypkl and Yrk2 polypeptides: 384362El l .sl.seq and 384286E10.sl.seq.

The said gene may be a gene from which Ypkl may be expressed or a gene from which Ykr2 may be expressed.

A further aspect of the invention is a said yeast cell wherein each such endogenous gene encoding a functional equivalent of human SGK or Ypkl or Yrk2 is mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or Ypkl or Yrk2. A further aspect of the invention is a method of identifying a compound (including a polypeptide or polynucleotide encoding a polypeptide) that modulates (activates or inhibits) the activity of SGK, for example fungal or mammalian, preferably human, SGK, wherein a yeast cell according to the above aspect of the invention is used.

A further aspect of the invention is the use of a yeast cell according to the above aspect of the invention in a method of identifying a compound that modulates (activates or inhibits) the activity of SGK.

Ypkl and Ykr2

A further aspect of the invention is a protein kinase derivable from yeast capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa- Arg-Xaa-Xaa-Ser/Thr-Hyd, for example the polypeptide Crosstide (which has the amino acid sequence GRPRTSSFAEG) or polypeptide 2, 8 or 11 shown in Table 2 of Example 1.

A further aspect of the invention is a protein kinase derivable from yeast capable of being phosphorylated by Pkhl or Pkh2 or mammalian, preferably human, PDKl.

The yeast may be S. cerevisiae. The protein kinase may share functional characteristics with mammalian PKB and/or SGK. It was not previously known that yeast, for example S. cerevisiae, may have a protein kinase with similar functional properties to mammalian PKBα or SGK.

The protein kinase may be Ypkl from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp or Ykr2 from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp. Alternatively and less preferably, it may be a related polypeptide pkcl or sch9 or other AGC (protein kinase A/protein kinase G/protein kinase C) family member.

A further aspect of the invention is a variant, derivative, fragment or fusion thereof or a fusion of a variant, derivative or fragment of a protein kinase according to the previous aspect of the invention that is capable of being phosphorylated by Pkhl or Pkh2 or mammalian, preferably human, PDKl and/or capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa- Arg-Xaa-Xaa-Ser/Thr-Hyd.

The protein kinase may be the polypeptide encoded by the YPK1 gene [41] or the YKR2/YPK2 gene [42].

Pkhl and Pkh2

A further aspect of the invention is a substantially pure polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative or a substantially pure polypeptide encoded by open reading frame YOLlOOw of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae. or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative wherein the polypeptide does not comprise the amino acid sequence of human PDKl or Drosophila PDKl (DSTPK61). The said polypeptides are considered to be protein kinases, in particular protein kinases that are capable of phosphorylating PKBα, particularly human PKBα, in particular on residue Thr308. They are further considered to be capable of phosphorylating the yeast polypeptides known as Ypkl and Ykr2, as described above, human SGK or a polypeptide with the consensus sequence Thr-Phe-Cys- Gly-Thr-X-Glu-Tyr (which may be present with the "activation loop" of the catalytic domain between conserved subdomains VII and VIII of a protein kinase).

The polypeptides described above are herein referred to as Pkhl (PKB-activating kinase homologue 1 ; encoded by open reading frame YDR490c of S. cerevisiae, for example) or Pkh2 (PKB-activating kinase homologue 2; encoded by open reading frame YOLlOOw of S. cerevisiae, for example) [42, 43].

Partial amino acid sequences of Pkhl and Pkh2 are also shown in Figure 1.

Example 4 describes the identification of Candida albicans nucleotide sequences that may encode polypeptides related to S. cerevisiae Pkhl and Pkh2. The method given in Example 4 may be applicable to identifying related open reading frames in other organisms, for example other yeasts.

The following nucleotide sequences may encode polypeptides related to S. cerevisiae Pkhl and Pkh2: 384194F08.sl .seq and 396076E03.s2.seq

By "substantially pure" we mean that the said polypeptide is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide. Thus, the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.

The invention also includes the substantially pure said polypeptide when combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found. It will be appreciated that the said substantially pure polypeptide may be obtained by expression from a recombinant nucleic acid, for example in a prokaryotic or eukaryotic cell, as discussed further below.

Variants (whether naturally-occurring or otherwise) may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below.

By "fragment of said polypeptide" we include any fragment which retains activity, for example a protein kinase activity, for example as described above, or which is useful in some other way, for example, for use in raising antibodies or in a binding assay.

By "fusion of said polypeptide" we include said polypeptide fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions to GST are given in Example 1. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as Hisδ or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention.

By "variants" of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide (for example, the ability to phosphorylate PKBα, particularly human PKBα, in particular on residue Thr308). Variants of Pkhl or Pkh2 do not include polypeptides which have the amino acid sequence of Schizosaccharomyces pombe, human PDKl or Drosophila PDKl (DSTPK61); see Figure 1.

It will be appreciated that a variant that comprises substantially full-length Pkhl or Pkh2 may be particularly useful. By "substantially full-length" is meant comprising at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the sequence of the full length polypeptide.

By "conservative substitutions" is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.

It is particularly preferred if the polypeptide variant has an amino acid sequence which has at least 65 % identity with the amino acid sequence given above, more preferably at least 75%, still more preferably at least 80%, yet more preferably at least 90%, and most preferably at least 95% or 99% identity with the amino acid sequence given above.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (Thompson et al (1994) Nucl Acid Res 22, 4673-4680). The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

Thus, using these parameters, the catalytic domain of Pkhl and Pkh2 may have 50% and 49% identity respectively with the catalytic domain of human PDKl and 41 % identity with the catalytic domain of Drosophila PDKl. There appears to be no significant identity between the non-catalytic domains of human or Drosophila PDKl and either Pkhl or Pkh2.

A particular embodiment of the invention provides a substantially pure yeast Pkhl polypeptide or naturally occurring allelic variants thereof. A partial amino acid sequence is also shown in Figure 1.

A further particular embodiment of the invention provides a substantially pure Pkh2 polypeptide or naturally occurring allelic variants thereof.

It is particularly preferred, although not essential, that the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of Pkhl or Pkh2, respectively, with respect to the phosphorylation (or activation) of PKBα (particularly human PKBα) or other polypeptide comprising the consensus sequence B-T-F-C-G-T-P/I-D/E-Y-L/I/M-A-P-E, for example a protein kinase belonging to the AGC subfamily (protein kinase A/G/C subfamily), in particular SGK, p70 S6 Kinase or PKCς, as discussed in Example 1 and shown in Figure 6. It is more preferred if the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 50 % , preferably at least 70 % and more preferably at least 90 % of the enzyme activity of Pkhl or Pkh2 with respect to the phosphorylation of PKBα or any one of the alternatives described above. However, it will be appreciated that variants or fusions or derivatives or fragments which are devoid of enzymatic activity may nevertheless be useful, for example by interacting with another polypeptide, or as antigens in raising antibodies.

A further aspect of the invention provides a recombinant polynucleotide encoding a said polypeptide of the invention (for example, Pkhl or Pkh2 from S. cerevisiae) or encoding a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the said polynucleotide variant are the same as given above.

A further aspect of the invention provides a recombinant polynucleotide suitable for expressing a polypeptide of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the said polynucleotide variant are the same as in the first aspect of the invention.

By "suitable for expressing" is mean that the polynucleotide is a polynucleotide that may be translated to form the polypeptide, for example RNA, or that the polynucleotide (which is preferably DNA) encoding the polypeptide of the invention is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. The polynucleotide may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by any desired host; such controls may be incorporated in the expression vector.

Thus a further aspect of the invention is a replicable vector suitable for expressing a polypeptide of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the said polynucleotide variant are the same as for the polypeptide of the invention. For example, the replicable vector may be suitable for expressing a fusion of the polypeptide of the invention, in particular a GST fusion, for example as described in Example 1. It will be appreciated that a construct is not a recombinant polynucleotide as defined above if it lacks sequences necessary for the translation and therefore expression of the encoded polypeptide of the invention.

A further aspect of the invention is a polynucleotide encoding a fusion of the polypeptide of the invention, or a fusion of a variant or fragment or derivative, in particular a GST fusion. A still further aspect is a vector suitable for replication in a mammalian/eukaryotic cell, comprising a polynucleotide encoding the polypeptide, or a variant or fragment or derivative or a fusion of the polypeptide, as defined in the first aspect of the invention, or a fusion of a variant or fragment or derivative, in particular a GST fusion.

Characteristics of vectors suitable for replication in mammalian/eukaryotic cells are well known to those skilled in the art, and examples are given below. It will be appreciated that a vector may be suitable for replication in both prokaryotic and eukaryotic cells. A polynucleotide comprising a fragment of the recombinant polynucleotide encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful. Preferably, the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length. Such polynucleotides are useful as PCR primers. A polynucleotide complementary to the polynucleotide (or a fragment thereof) encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful. Such complementary polynucleotides are well known to those skilled in the art as antisense polynucleotides.

The polynucleotide or recombinant polynucleotide of the invention may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.

A "variation" of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis" Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.

Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, the polynucleotide of the invention includes polynucleotide that shows at least 60%, preferably 70%, and more preferably at least 80% and most preferably at least 90% homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful. Such a polypeptide may be a functional homologue of the polypeptide of the invention. The polypeptide may, for example, have a similar enzymatic activity to the polypeptide of the invention or may be able to substitute for the polypeptide of the invention in a cell, as discussed above.

It will be appreciated that such a method may be used in the identification of a functional homologue of Ypkl or Ykr2, as discussed above.

It is preferred that the polynucleotide is derivable from a yeast, for example a yeast other than S. cerevisiae or Candida. It is particularly preferred that the polynucleotide is derivable from a pathogenic yeast or a yeast that may be useful as the host cell in a screening assay as described below.

Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group. DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1XSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By "high stringency" we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.

The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.

For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25:FD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

Methods and vectors for transformation of Candida spp are described in, for example, Zhou P, et al "A system for gene cloning and manipulation in the yeast Candida glabrata." Gene. 1994 May 3; 142(1): 135-40; Mehra RK, et al "Cloning system for Candida glabrata using elements from the metallothionein-IIa- encoding gene that confer autonomous replication." Gene. 1992 Apr 1;113(1): 119-24; Ohkuma M, et al "Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, adel, ura3) as a useful host for the genetic engineering of Candida maltosa. " Curr Genet. 1993 Mar;23(3):205-10; Sakai Y, et al "Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5' -phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation." J Bacteriol. 1991 Dec; 173(23) :7458-63; Haas LO, et al "Development of an integrative DNA transformation system for the yeast Candida tropicalis." J Bacteriol. 1990 Aug;172(8):4571-7; Sakai Y, et al "High- frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae. " J Bacteriol. 1993 Jun; 175(11):3556-62; Kitada K, et al "Cloning of the Candida glabrata TRPl and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation." Gene. 1995 Nov 20;165(2):203- 6; Macreadie IG, et al "Heterologous gene expression and protein secretion from Candida glabrata. " Biotechnol Appl Biochem. 1994 Jun; 19 ( Pt 3):265-9. Hsu WH, et al "Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis." J Bacteriol. 1983 Jun; 154(3): 1033-9; Hanic- Joyce PJ, et al "A high-copy-number ADE2-bearing plasmid for transformation of Candida glabrata. " Gene. 1998 May 12;211(2):395-400.

Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below. In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.

Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culmre of those cells, preferably a monoclonal (clonally homogeneous) culmre, or a culture derived from a monoclonal culture, in a nutrient medium.

A further aspect of the invention provides a method of making the polypeptide of the invention or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative from said host cell. Methods of cultivating host cells and isolating recombinant proteins are well known in the art.

The invention also includes a polypeptide, or a variant, fragment, derivative or fusion thereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention.

A still further aspect of the invention provides an antibody reactive towards a polypeptide of the invention. Examples of such antibodies and of methods of preparing such antibodies are given in Example 1. Antibodies reactive towards the said polypeptide of the invention may be made by methods well known in the art. In particular, the antibodies may be polyclonal or monoclonal.

Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in " Monoclonal Antibodies: A manual of techniques" , H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982).

In a preferred embodiment the antibody is raised using any suitable peptide sequence obtainable from the amino acid sequence of Pkhl or Pkh2 as appropriate. It is preferred if polyclonal antipeptide antibodies are made.

It is particularly preferred if the antibody does not react substantially with another protein kinase, in particular another yeast protein kinase. Accordingly, it may be preferred if pep tides based on the Pkhl or Pkh2 sequence are used which vary significantly from any peptides found in any other protein kinase, particularly a yeast protein kinase. It may also be preferred that an antibody reacts with Pkhl but does not react substantially with Pkh2, and vice versa.

Techniques for preparing antibodies are well known to those skilled in the art, for example as described in Harlow, ED & Lane, D "Antibodies: a laboratory manual" (1988) New York Cold Spring Harbor Laboratory.

Other methods

A further aspect of the invention provides a method of identifying a drug-like compound or lead compound for the development of a drug-like compound that modulates the activity of the polypeptide Pkhl, Pkh2, Ypkl or Ykr2, the method comprising contacting a compound with the polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the protein kinase activity of the said polypeptide is changed compared to the activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.

The terms "drug-like compound" and "lead compound" are well known to those skilled in the art, as discussed above.

The compound may act by interacting with the said polypeptide and modulating, for example inhibiting, its activation by an "upstream activator". The compound may act by interacting with Ypkl or Ykr2 and modulating, for example inhibiting, its activation by an "upstream activator", for example Pkhl or Pkh2.

It will be understood that it will be desirable to identify compounds that may modulate the activity of the polypeptide in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between the said polypeptide and its substrate are substantially the same as between yeast, for example S. cerevisiae Pkhl, Pkh2, Ypkl or Ykr2 and their substrate or substrates in vivo. An example of a substrate of said Pkhl or Pkh2 polypeptide is Ypkl or Ykr2.

In one embodiment, the compound decreases the activity of said polypeptide. For example, the compound may bind substantially reversibly or substantially irreversibly to the active site of said polypeptide. In a further example, the compound may bind to a portion of said polypeptide that is not the active site so as to interfere with the binding of the said polypeptide to its substrate. In a still further example, the compound may bind to a portion of said polypeptide so as to decrease said polypeptide's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity, for example in the activation of the said polypeptide by an "upstream activator", such as Pkhl or Pkh2 for Ypkl and Ykr2.

In a further embodiment, the compound increases the activity of said polypeptide. For example, the compound may bind to a portion of said polypeptide that is not the active site so as to aid the binding of the said polypeptide to its substrate. In a still further example, the compound may bind to a portion of said polypeptide so as to increase said polypeptide's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity for example in the activation of the said polypeptide by an "upstream activator", such as Pkhl or Pkh2 for Ypkl and Ykr2.

Conveniently, the method makes use of the fact that Pkhl or Pkh2 phosphorylates, for example Ypkl or Ykr2 as described in Example 1, or that Ypkl or Ykr2 phosphorylate peptides as shown in Table 2 and discussed in Example 1, but any suitable substrate may be used. Thus the phosphorylation of Ypkl or Ykr2 or peptide substrate of Ypkl or Ykr2 may be measured using techniques well known to those skilled in the art. Conveniently, the method makes use of an assay which may be substantially the same as that described in Example 1 or which may be adapted to render the assay more convenient for high-throughput screening, as well known to those skilled in the art. For example, a scintillation proximity assay (SPA; Amersham) may be useful. It is preferred that the polypeptide (for example, Pkhl, Pkh2, Ypkl or Ykr2) is recombinant. It is preferred that the substrate is recombinant or synthetic. Alternatively, a change in the activity of the substrate may be measured. For example, the protein kinase activity of Ypkl or Ykr2 or a substrate of Ypkl or Ykr2 may be measured, as described above. This may be done in a whole cell system or using purified or partially purified components. Expression of an protein encoded by an RNA transcribed from a promoter regulated by a susbtrate of Ypkl or Ykr2 may be measured. The protein may be one that is physiologically regulated by a substrate of Ypkl or Ykr2 or may be a "reporter" protein, as well known to those skilled in the art (ie a recombinant construct may be used). A reporter protein may be one whose activity may easily be assayed, for example β-galactosidase, chloramphenicol acetyltransferase or luciferase (see, for example, Tan et al (1996)).

A further aspect of the invention is a method of identifying a compound which modulates, for example blocks, the activation of a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase(or is derivable from a non-mammalian source) by an interacting polypeptide, for example Pkhl, Pkh2 or PDKl, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase (or is derivable from a non-mammalian source) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase (or is derivable from a non-mammalian source) or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof. It will be understood that it will be desirable to identify compounds that may modulate the activity of the polypeptide in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between the said polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase (or is derivable from a non- mammalian source) and the interacting polypeptide, for example Pkhl, Pkh2, PDKl and/or a protein kinase with PDK2 activity are substantially the same as between a said namrally occuring polypeptide that is a functional equivalent of Ypkl and/or Ykr2 and a namrally occuring interacting polypeptide in vivo. PDKl may be a polypeptide with PDK2 activity, for example when bound to PIF or a related polypeptide, as discussed above.

It will be appreciated that the said suitable variant, fragment, derivative or fusion of a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase (or is derivable from a non-mammalian source), or a fusion of a said fragment, derivative or fusion is not SGK, PKBα or p70S6 kinase.

A further aspect of the invention is the use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDKl to phosphorylate and/or activate a polypeptide that is Ypkl and/or Ykr2 or SGK or PKBα or a functional equivalent thereof or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion.

A further aspect of the invention is the use of PDKl or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof to phosphorylate and/or activate a polypeptide that is Ypkl and/or Ykr2 or SGK or a functional equivalent thereof that is not PKBα or p70S6 kinase or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion.

A further aspect of the invention is a method of identifying a compound which binds to Ypkl or Ykr2 or SGK (or other substrate of Pkhl, Pkh2 or PDKl) and either enhances or prevents its activation by Pkhl, Pkh2 or PDKl, the method comprising determining whether a compound enhances or prevents the interaction of Ypkl or Ykr2 or SGK (or other substrate of Pkhl, Pkh2 or PDKl) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with Pkhl, Pkh2 or PDKl or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative or determining whether the compound substantially blocks activation of Ypkl or Ykr2 or SGK (or other substrate Pkhl, Pkh2 or PDKl) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative by Pkhl, Pkh2 or PDKl.

Suitable assays may be similar to those described above.

A further aspect of the invention is a method of identifying a polypeptide that interacts with Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDKl, the method comprising 1) contacting a) the said polypeptide with b) a composition that may contain such an interacting polypeptide, 2) detecting the presence of a complex containing the said polypeptide and an interacting polypeptide, and optionally 3) identifying any interacting polypeptide bound to the said polypeptide. In one embodiment, the composition may comprise material from cells. In particular, the cells may be selected from the following types: (1) cells which have Pkhl or Pkh2 activity after exposure to a stimulus, but which have not been so exposed and (2) cells of type 1 after exposure to the stimulus. Polypeptides that are found in one only of types 1 or 2 are of particular interest and may be characterised further. Such a peptide may be an activator of Pkhl or Pkh2. Alternatively, it may be an inactivator of Pkhl or Pkh2.

It will be appreciated that the method may be performed within a cell, for example using the yeast two hybrid system as is well known in the art. In this example, cDNAs copied from mRNA from the two cell types described above would be used.

It will further be appreciated that a recombinant yeast cell in which a Pkhl or Pkh2 gene is altered and/or a recombinant polynucleotide capable of expressing Pkhl or Pkh2 is present, may be useful in, for example, identifying a substrate of Pkhl or Pkh2, as described in Example 1.

A further aspect of the invention is a polypeptide identifiable by the said method.

A still further aspect of the invention provides a method of identifying a compound which modulates the activation of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDKl by an "upstream activator". By "upstream activator" is meant a molecule that interacts with the polypeptide of the invention with the result that the protein kinase activity of the polypeptide of the invention is increased. It may be a polypeptide. Preferably, it is a physiological activator of native Pkhl or Pkh2. Such an activator may be identified by the method given above. A further aspect of the invention is the use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof for the activation of Ypkl or Ykr2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion that is not SGK or PKBα.

A further aspect of the invention is a method of identifying a compound which blocks the activation of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof by a said interacting polypeptide identifiable by the above method, the method comprising determining whether a compound enhances or disrupts the interaction between (a) the said Pkhl or Pkh2 and (b) said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide as defined in the first aspect of the invention by said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof.

It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase, as described above and ³²P-ATP or ³³P-ATP and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for ³²P or ³³P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.

Medical aspects

A further aspect of the invention is a compound identified or identifiable by any appropriate one of the above methods of the invention.

A still further aspect of the invention is a said compound for use in medicine.

The compound may be a compound that inhibits the activity mammalian, preferably human PDKl or mammalian, preferably human SGK. Alternatively, the compound may inhibit a functional homologue of PDKl or SGK in a pathogen or opportunistic pathogen, for example of a mammal, preferably a human. For example, the compound may inhibit a functional homologue of PDKl (which may be Pkhl or Pkh2) or SGK (which may be Ypkl or Yrk2) in Candida species or any other pathogenic yeast species as listed above. Such a compound may be useful as an antifungal agent.

It will be appreciated that the pathogen or opportunistic pathogen may affect a plant, which may be a monocotyledenous plant or a dicotyledenous plant. For example, the pathogen may be a member of the Uredinales group of fungi that may cause the plant disease rust, for example Puccinia graminis which may cause black rust of cereals, or may be Phtophthora infestans which may cause potato blight. Thus, a compound that may inhibit a functional homologue of PDKl (which may be Pkhl or Pkh2) or SGK (which may be Ypkl or Yrk2) in a yeast that is pathogemc for a plant may be useful as an antifungal agent for use as a plant protection product ie for treating or preventing a fungal infection of a plant.

It will be appreciated that a compound that inhibits mammalian, for example human PDKl may be useful in medicine. Thus, a further aspect of the invention is a method of modulating in a cell the activity of PDKl (which may include its interactions with protein kinase B or SGK), the method comprising exposing the cell to a compound of the invention. It will be appreciated that the method may be used in vitro or in vivo. It is preferred that the compound of the invention is capable of entering the cell and that it enters the cell.

A still further aspect of the invention is a method of treating a patient in need of modulation, preferably inhibition, of the activity of PDKl or its interactions with protein kinase B the method comprising administering to the patient an effective amount of a compound of the invention that inhibits mammalian, for example human, PDKl.

Compounds identifiable in the screening methods of the invention that inhibit PKB, PDKl or the activation of PKB by PDKl are believed to be useful in treating cancer. PKB is the cellular homologue of v-akt which is involved in leukaemias. Two isoforms of PKB are overexpressed in ovarian, pancratic and breast cancers. It is believed that PKB mediates protection of cells to apoptosis mediated, for example, by IGF-1. Overexpression of PKB may allow cancer cells to proliferate by stopping apoptosis. Promotion of apoptosis may be beneficial in the resolution of inflammation.

A compound that results in activation of PKB or PDKl may be useful in the treatment of diabetes or obesity. Such a compound may also be useful in the treatment of patient before, after or during heart surgery. Such a compound may also be useful in reducing apoptosis; thus, such a compound may be useful in treating a patient in need of protection against apoptosis. Reducing apoptosis may be useful following ischaemic injury, for example stroke or myocardial infarction, and in tissue repair.

A compound of the invention that inhibits a fungal functional homologue of PDKl (which may be Pkhl or Pkh2) or SKG (which may be Ypkl or Yrk2) may be useful in medicine, for example as an antifungal agent. It may be useful in treating a Candida infection, for example thrush. Thus, a further aspect of the invention is the use of a compound of the invention that inhibits a fungal functional homologue of PDKl or SGK in the manufacture of a medicament for treating or preventing a fungal infection, for example thrush.

Further examples of fungal infections that may be treated by a compound of the invention include infections that may be caused by a pathogenic fungus listed above. Still further examples of infections or conditions that may be treated or prevented by a compound of the invention are listed below. It will be appreciated that it may be preferred that the compound of the invention has been selected, for example using a method of the invention as described above, for an effect on a protein kinase of the yeast genus or species indicated.

Thus, a compound of the invention may be used in the manufacture of a medicament for the treatment or prevention of an infections that may be caused by a pathogenic fungus listed above or an infection or condition listed below. Aspergillosis may be caused by fungi of the genus Aspergillus, usually A. fumigatus. Blastomycosis is caused by the fungus Blastomyces dermatitidis . Infection may take place through the lungs but the infection may become widely disseminated, with the skin, skeleton and genito-urinary system becoming infected.

Candidiasis is caused by Candida spp, particularly C. albicans, and usually requires a predisposing factor, such as antibacterial therapy, diabetes, pregnancy or immunodeficiency. Mucocutaneous candidiasis is known as thrush. Chromoblasomycosis may be caused by opportunistic pathogens including Phialophohra compacta, P. pedrosoi, P. verrucosa, Cladosporium carrionii and Rhinocladiella aquaspersa following skin trauma, particularly in tropical and subtropical climates.

Coccidioidomycosis (valley fever, desert fever, desert rheumatism) may be caused by inhalation of spores of Coccidioides immitis and occurs particularly in arid and semi-desert areas of North, Central and South America. Cryptococcosis is caused by the fungus Cryptococcus neoformans. It is rare in normal individuals, but important in immunocompromised patients, often occurring as cryptococcal meningitis.

Endocarditis may be caused by fungi such as Aspergillus or Candida, as may eye infections. Cephalosporium, Fusarium, Blastomyces, Cryptococcus and Sporothrix may also cause eye infections.

Histoplasmosis may be caused by Histoplasma capsulatum, found in soil and in bird and bat excrement. It is a sytemic infection that is endemic in central USA and central Africa (mainly H. capsulatum var duboisii. Mucormycosis is caused by Rhizopus or Rhizomucor species and usually affects only immunocompromised individuals. Mycetoma is a tropical and subtropical disease caused by Madurella mycetomatis, M. grisea or Pseudallescheria boydii which enter the tissue through skin trauma. Paracoccidioidomycosis is a disease principally of Central and South America caused by Paracoccidioides brasiliensis.

Pneumocystis carinii pneumonia is caused by Pneumocystis carinii, which is thought to be a fungus. It is of particular significance in immunocompromised patients, particularly those with AIDS.

Skin infections include the dermatophytoses, pityriasis versicolor and candidiasis (discussed above), as well as forms of other fungal infections discussed above, such as apergillosis. Dermatophytoses include ringworm and tinea (including athlete's foot), and are caused by Epidermophyton, Microsporum and Trichophyton spp. Pityriasis versicolor (tinea versicolor) is caused by Mάlassezia furfur and is more common in tropical than temperate climates and exposure to the sun may trigger the infection.

Sporotrichosis is caused by Sporothrix schenckii and is seen mainly in the Americas and Africa. It occurs in cutaneous and extracutaneous forms.

Further information regarding fungal infections for which the compounds of the invention may be useful may be found in, for example, the Martindale Pharmacopaeia, 32^nd edition.

It will be appreciated that fungal infections may be more serious in immunocompromised hosts, for example a human with AIDS (acquired immunodeficiency syndrome); thus, yeast/fungi that are of low pathogenicity in otherwise healthy individuals may be capable of causing more severe disease in an immunocompromised individual.

Kit of parts

A further aspect of the invention is a kit of parts comprising means useful for carrying out a screening method of the invention.

Thus, a kit of parts of the invention may comprise a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a first source and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from an source other than the first source.

Figure Legends.

Figure 1. Comparison of the primary structures of Pkhl and Pkh2 with PDKl. (a) Alignment of the deduced amino acid sequences of yeast Pkhl and Pkh2 with the catalytic domains of human PDKl and its Drosophila homologue, DSTPK61 [27], carried out using the CLUSTAL W program. Identical residues are denoted by white-on-black letters, and similar residues by grey boxes. In our work, five independent PKH1 clones generated by PCR all differed from the sequence deposited in the Saccharomyces Genome Database, and indicated that Phel87 (TTC) should actually be He (ATC). (b) Schematic diagrams of the structures of PDKl -related proteins. Blue boxes indicate the catalytic domain in each protein kinase and green boxes indicate PH domains.

Figure 2. Deletion of both PKHl and PKH2 produces non-viable cells. (a) Growth of spores from a representative tetratype tetrad from strain AC306 (MATalMATa PKHllpkhlΔ::HIS3 PKH2lpkh2Δ::TRPl). The deduced pkhl A pkh2Δ spore is inviable. (b) Verification by PCR using the appropriate primers indicated the presence of the expected deletion-substitution alleles. Presence of the HIS3 and TRPl markers inserted into PKHl and PKH2, respectively, are denoted by a shift of the size of the resulting product from about 0.5 kbp in the intact loci, to 1.2 kbp (pkhlΔ::HIS) or 1.3 kbp (pkh2Δ::TRPl).

Figure 3. Genetic evidence that PKHl and PKH2 are functional homologues.

(a) Strain AC306 (MATalMATa PKHl /pkhl Δ::HIS3 PKH2lpkh2Δ::TRPl), transformed with plasmid pYES2-PKHl, which is marked with URA3, was sporulated. Verification by PCR of the genotype of two derived haploids, PKHl PKH2 [pYES2-PKHl] (spore A) and pkhlΔ pkh2Δ [pYES2-PKHl] (spore B).

(b) Four spores derived from a tetratype ascus of the diploid described in (a), all carrying pYES2-PKHl , were plated either on medium lacking uracil (right) to select for the presence of the plasmid, or on medium containing 5-FOA (left) to select for loss of the plasmid [40] . In the absence of the plasmid-borne copy of PKHl, pkhlΔpkh2Δ cells are unable to continue vegetative proliferation.

Figure 4. Intact human PDKl or PDK1-ΔPH rescue the inviability of pkhlΔ pkh2Δ cells.

(a) Derivatives of strain AC306 (MATalMATa PKHl /pkhl Δ::HIS3 PKH2lpkh2Δ::TRPl), transformed with either YEplacl95-PDKl, YEplacl95- PDK1-ΔPH, or YEplacl95-P£H7, all marked with URA3, were sporulated. Verification by PCR of the genotype of two derived double mutant spores maintained by expression of either authentic yeast PKHl (spore A) or human PDKl (spore B). As expected, only the haploid carrying YEplacl95-PDKl possesses a PDKl -derived sequence, (b) Four spores derived from tetratype asci of the diploids described in (a), carrying either YEplacl95-PDKl or YEplacl95- PDK1-ΔPH, as indicated, were plated either on medium lacking uracil (top left) to select for the presence of the plasmids (only one representative plate is shown), or on medium containing 5-FOA (top right and bottom right, respectively) to select for loss of the plasmids. The pkhlΔ pkh2Δ cells are able to grow only when the plasmids expressing human PDKl or PDK1-ΔPH are present.

Figure 5. Both yeast Pkhl and human PDKl phosphorylate and activate human

PKB in a PtdIns[3,4,5]P3- or PtdIns(3,4)P2 -dependent manner.

Purified GST-PKB was incubated for 30 min at 30°C with either GST-Pkhl (a) or GST-PDKl (c) in the presence of 100 μM ATP and phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and the various Ptdlns lipids indicated, all at a final concentration of 10 μM. Reactions were terminated by adjusting the mixtures to a final concentration of 1 % (by vol) Triton X-100 [17] to dissolve the lipid vesicles, and the resulting increase in specific activity (U/mg) of GST-PKB was then determined in the presence of [γ-³²P] ATP, as described in Materials and Methods. The increase in specific activity given is that relative to control incubations in which GST-Pkhl or GST-PDKl were omitted. To measure the amount of incorporation into GST-PKB catalysed by either GST- Pkhl (b) or GST-PDKl (d), reactions were conducted in the presence of 100 μM [γ-³²P]ATP and terminated by adjusting the solutions to a final concentration of 1 % SDS. The resulting denatured samples were then subjected to SDS-PAGE. Extent of phosphorylation was assessed by autoradiography of the Coomassie blue-stained band corresponding to GST-PKBα. Abbreviations: SA-PI[3,4,5]P3 is srt-1-stearoyl, 2-arachidonyl D-PtdIns[3,4,5]P3; DP-PI[3,4,5]P3 is sn-1,2- dipalmitoyl D-Ptdlns [3,4,5]Ps; DP-PI[3,4]P2 is s«-l,2-dipalmitoyl D- PtdIns[3,4]P₂; DP-PI[3]P is -m-l,2-dipalmitoyl D-Ptdlns-3P. PI[4,5]P2 was purified from brain extract.

Figure 6. Consensus sites for PDKl- and PDK2-dependent phosphorylation, and compari-son of the primary structures of Ypkl and Ykr2 with their closest human homologues.

(a) Conserved motifs predicted to be phosphorylated by PDKl-like and PDK2- like enzymes are indicated with the residue phosphorylated shown in boldface type. In all cases, the putative PDK2 site is located 157-166 residues C-terminal to the PDKl site. GenBank accession numbers: .PKBα: X65687; p70S6Kα: M60725; SGK: Y10032; PKCς: L07032; Ypkl: P12688; Ykr2: P18961; Pkcl: M32491; and Sch9: U00029. (b) In the alignment of Ypkl and Ykr2 to each other and to mammalian SGK, PKBα, p70 S6 kinase and βARK, identities to Ypkl are indicated by white-on-black letters.

Figure 7. Expression of either Ypkl or Ykr2 is required for viability.

Strain YES7 (MATalMATa YPKllypklΔ::HIS3 YKR2lykr2Δ::TRPl), transformed with the LEU2-mzxksά plasmid, pGAL-YKR2, were sporulated under conditions (galactose-containing media) that induce expression of YKR2 from the plasmid. The four spores of a tetratype ascus derived from this diploid were recovered on medium lacking leucine to demand the presence of the plasmid. When subsequently streaked out on the same medium containing glucose as the carbon source, which represses expression of the plasmid-borne YKR2 gene, the ypklΔ ykr2Δ double mutant cells are unable to continue vegetative proliferation. In contrast, the cells expressing either YPK1 or YKR2 (or both) from their normal chromosomal loci remain viable. Figure 8. SGK, a PKB-related enzyme, rescues the inviability of ypkl Δ ykr2Δ cells.

(a) Strain YPT28 (MATa ypklΔ ykr2Δ) carrying pYKR2, a URA3- aτked plasmid that expresses YKR2 from its authentic promoter, was generated as described in Materials and methods and then transformed with either empty LEU2-maτked vectors (pAD4M or YEp351GAL) or the same vectors expressing from either the ADH1 or the GALl promoters, as indicated, either YPK1, YKR2, SGK, PKBα, p70 S6 kinase or βARK. When streaked onto medium selective for the presence of both plasmids (left), all of the transformants grew well. However, when plated on the same medium containing 5-FOA (right), to demand loss of the URA3-maτked plasmid expressing YKR2, only cells expressing SGK were able to sustain normal growth of the ypkl A ypklΔ cells, and did so as efficiently as authentic YPK1 or YKR2. Survival of a few colonies was reproducibly observed for cells expressing PKBα, suggesting that only very high levels of PKBα expression can support the continued growth of Ypkl- and Ykr2-deficient cells. (b) The temperature-conditional strain YPT40 (MATa ypkl-l'^s ykr2Δ) was constructed as described in Materials and methods and then transformed with the indicated plasmids, described in (a). The transformants isolated at permissive temperature (left) were then tested for their ability to continue to grow at the restrictive temperature (right). Again, SGK was able to substitute for Ypkl and Ykr2 function and permit growth just as well as YPK1 itself. Weak complementation by PKB was again observed, in that microcolonies indicative of very slow growth were reproducibly recovered at the non-permissive temperature.

Figure 9. Pkhl phosphorylates and activates yeast Ypkl and human SGK in a 3- phospho-inositide-independent manner.

Either GST- Ypkl (a) or GST-SGK (c) were incubated for 30 min at 30°C with GST-Pkhl or GST-PDKl, as indicated, in the presence of 100 μM ATP, with and without phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer and 10 μM of the D-enantiomer of sw-l-stearoyl, 2-arachidonyl D- PtdIns[3,4,5]P3 ("PIP3"). Reactions were terminated and the degree of activation assessed as indicated in the legend to Fig. 5. To measure the amount of incorporation into GST- Ypkl (b) or GST-SGK (d) catalysed by either GST- Pkhl or GST-PDKl, as indicated, reactions were carried out in the presence of 100 M [γ-³²P]ATP and the products were analysed by SDS-PAGE followed by autoradiography, as described in the legend to Fig. 5.

Figure 10. Phosphorylation of Thr504 in Ypkl is essential for its activation by Pkhl.

(a) GST- Ypkl, GST-Ypkl(T504D), GST- Ypkl (T662D) or GST- Ypkl (T504D T662D) were incubated with either GST-Pkhl or a catalytically-inactive derivative, GST-Pkhl(KD), as indicated, for 30 min at 30°C with ATP (100 μM). Reactions were terminated and the degree of activation assessed as indicated in the legend to Fig. 5. To follow the amount of incorporation into GST- Ypkl and its various derivatives (b), reactions were carried out in the presence of 100 μM [γ-³²P]ATP and the products were analysed by SDS-PAGE followed by autoradiography, as described in the legend to Fig. 5.

Figure 11. Parallels between PDKl -dependent signaling pathways in animal cells and Pkhl- and Pkh2-dependent signaling in yeast. See text for further details.

Figure 12. Nucleotide sequence of rat SGK.

Figure 13. Amino acid sequence of rat SGK (gi 14770981 pir | |A48094 serum and glucocorticoid-regulated kinase - rat). Example 1: Functional homologues of mammalian 3-phosphoinositide- dependent protein kinase-1 (PDKl) and protein kinase B (PKB/c-Akt) are components of a signaling pathway in the yeast Saccharomyces cerevisiae

Background: In animal cells, recruitment and activation of phosphatidylinositol 3-kinase by the binding of growth factors to their cognate receptors generates 3- phosphoinositides. These lipids induce 3-phosphoinositide-dependent protein kinase-1 (PDKl) to phosphorylate and activate a variety of target protein kinases, including protein kinase B (PKB/c-Akt) and p70 S6 kinase, that subsequently mediate appropriate physiological responses.

Results: The Saccharomyces cerevisiae genome encodes two protein kinases (PKHl and PKH2 gene products) whose catalytic domains are 72% identical to each other and 50% identical to human or Drosophila PDKl. Both pkhl Δ and pkh2Δ single mutants are viable, but a pkhlΔ pkh2Δ double mutant is inviable, indicating that Pkhl and Pkh2 have essential, but overlapping, functions.

Expression of human PDKl permits growth of pkhl Δ pkh2Δ cells. The yeast genome also encodes two other protein kinases (YPKl and YKR2 gene products) whose catalytic domains are 88% identical to each other and 58% identical to serum- and glucocorticoid-induced protein kinase (SGK), an enzyme closely related to PKB (56% identity). Either a ypkl A or a ykr2Δ single mutant is viable, but a ypklΔ ykr2Δ double mutant is inviable, indicating that Ypkl and Ykr2 also have essential, but overlapping, functions. Growth of ypklΔ ykr2Δ cells is fully rescued by expression of rat SGK, weakly by mouse PKB, and not at all by rat p70 S6 kinase. Pkhl, expressed in 293 cells as a fusion to glutathione S-transferase (GST), phosphorylates and activates mammalian PKB and SGK. Likewise, in vitro Pkhl activates Ypkl by phosphorylating the residue (T504) in the kinase domain equivalent to those in PKB and SGK that are phosphorylated by PDKl. Activation of Ypkl by Pkhl does not require phosphatidylinositol-3,4,5-traphosphate (PtdIns[3,4,5]P3), consistent with the lack of pleckstrin homology (PH) domains in these yeast proteins, whereas activation of human PKB by Pkhl is dependent on PtdIns[3,4,5]P3. The minimum consensus sequence for phosphorylation by Ypkl is Arg-X-Arg-X-X- Ser-Hyd (where Hyd is a bulky hydrophobic residue), as found for PKB.

Conclusions: These results demonstrate that Pkhl and Pkh2 are functional homologues of PDKl, that Ypkl and Ykr2 are functional homologues of SGK (and perhaps PKB), and that, as in animal cells, these enzymes are components of a protein kinase cascade that is required for cell growth and viability.

Background Protein kinase B (PKB) [1], also called RAC (for "Related to PKA and PKC") kinase [2], is the mammalian homologue of a retroviral oncogene product, v-Akt [3], and is, therefore, also designated c-Akt. There are several reasons for the current interest in this enzyme and its function. First, PKB is activated within minutes in response to insulin and other growth factors, and activation is prevented by inhibitors of phosphatidylinositol (Ptdlns) 3-kinase [4-6]. Second, increasing evidence indicates that PKB mediates a number of the actions of insulin, including stimulation of glucose and amino acid uptake, glycogen and protein synthesis and, in cardiac muscle, glycolysis (reviewed in [7, 8]), as well as induction of the transcription of specific genes [9, 10]. Third, the PKB isoform is overexpressed in a significant percentage of ovarian and pancreatic cancers [11 , 12], and the PKB isoform is elevated in some breast cancers [13]. Fourth, PKB action provides a survival signal that protects cells from apoptosis induced in a variety of ways [14, 15]. Hence, activation of PKB by gene amplification and other mechanisms may contribute to the generation of malignant cells that are able to proliferate independently of extracellular growth and survival signals.

PKB phosphorylates proteins and peptides at serine or threonine residues that lie in an -Arg-X-Arg-X-X-Ser/Thr- motif [16]. In insulin signal transduction, two physiological substrates of PKB appear to be the protein kinase, glycogen synthase kinase-3 (GSK3) [17, 18], and the cardiac isoform of PFK2 [7, 19]. Phosphorylation by PKB inhibits GSK3 activity, leading to dephosphorylation and activation of glycogen synthase and protein synthesis initiation factor eIF-2B [20]. These events presumably contribute to the insulin-induced stimulation of glycogen synthesis and protein synthesis, respectively. PKB activates cardiac PFK2, which seems to underlie the insulin-induced stimulation of glycolysis observed in the heart. In the protection of cells against apoptosis, BAD appears to be one of the physiological substrates of PKB [14, 15]. In its dephosphorylated form, BAD interacts with the Bel family member, BclXL, thereby inducing apoptosis in some cells. However, when phosphorylated by PKB at Serl36, BAD dissociates from BclXL, interacts with 14-3-3 proteins instead, and apoptosis is prevented [21].

Activation of PKB in response to insulin or growth factors requires phosphorylation [22, 23], which occurs at two sites [23]. These are Thr308, located in a -Thr-Phe-Cys-Gly-Thr-X-Glu-Tyr- motif within the "activation loop" of the catalytic domain between conserved subdomains VII and VIII, and Ser473, situated in the hydrophobic motif -Phe-X-X-Phe-Ser-Phe- , close to the C- terminus. Both sequence motifs are present in a number of protein kinases that play important roles in signal transduction [7, 24, 25]. Phosphorylation of both sites in PKB is prevented by inhibitors of Ptdlns 3-kinase [22]. Thr308 is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDKl) [26, 27] and Ser473 by a distinct protein kinase activity, termed PDK2 activity, which may be possessed by PDKl (Balendran et al (1999) Current Biology 9, 393-404).

PKB can only be activated by PDKl in vitro in the presence of lipid vesicles containing PtdIns[3,4,5]P3 or PtdIns[3,4]P2 [26]. The former lipid is generated from PtdIns[4,5]P2 by Ptdlns 3-kinase, and can be converted to the latter by one of several different classes of 5-phosphoinositide 5-phosphatases [28, 29]. The 3- phosphoinositides bind to a pleckstrin homology (PH) domain at the N-terminus of PKB [30, 31], presumably inducing thereby a conformational change that makes Thr308 accessible to PDKl. PDKl also possesses a PH domain, C- terminal to the catalytic domain [27], that binds PtdIns[3,4,5]P3 and PtdIns[3,4]P2 even more tightly than does the PH domain of PKB [32, 33]. The interaction of PDKl with 3-phosphoinositides enhances activation of PKB in vitro, probably by facilitating encounter of PDKl and PKB via their mutual binding to lipid vesicles [33]. There is increasing evidence that, in addition to PKB, PDKl activates a number of other protein kinases in vivo, such as p70 S6 kinase [25, 34], certain PKC isoforms [35] and, more recently, SGK [36], by phosphorylating a threonine residue that lie at an equivalent position and within the same sequence motif as Thr308 of PKB. Like PKB, these enzymes also contain the second, hydrophobic, phosphorylation consensus sequence, situated 160-165 residues C- terminal to the PDKl -dependent phosphorylation site [7] and, hence, are likely to be substrates for PDK2 protein kinase activity.

If enzymes of the PDKl and PKB class perform functions vital to signaling in all eukaryotic cells, then these molecules should be evolutionarily conserved. We have shown previously that this pathway is apparently present in the fruit fly, Drosophila melanogaster [27]. Here we demonstrate both genetically in vivo and biochemically in vitro that PDKl-like and PKB-like protein kinases exist even in the unicellular eukaryotic microbe, Saccharomyces cerevisiae (baker's yeast). Moreover, we show that these enzymes are indispensible for cell growth and viability. Additional findings suggests that, as in animal cells, the PDKl-like gene products are likely to play a role in activating other classes of protein kinases, in addition to the PKB-like enzymes.

Results

PKHl and PKH2 encode homologues of mammalian PDKl

The genome of the yeast Saccharomyces cerevisiae contains two, previously uncharacterised open reading frames (YDR490c and YOLlOOw) that encode protein kinases whose catalytic domains share 50% amino acid sequence identity with either human or Drosophila PDKl and are 72% identical to each other (Fig. 1A). Hence, these loci were designated PKHl and PKH2, respectively (for "PKB-activating Kinase Homologues 1 and 2"). PKHl is located on the right arm of chromosome IV [37] and encodes a 766-residue protein (86 kDa); PKH2 is situated on the left arm of chromosome XV [38] and encodes a 1,081-residue protein (121 kDa). The Pkhl and Pkh2 polypeptides also contain N-terminal extensions and long C-terminal extensions (Fig. IB) and are much less similar to each another in these regions (27% identity). In these domains, there is no significiant homology with the non-catalytic regions of PDKl and no significant homology with other known proteins. In particular Pkhl and Pkh2 lack the PH domain that is found near the C-termini of both the human and Drosophila PDKl.

PKHl and PKH2 are essential genes that are functionally redundant As a means to determine if PKHl and PKH2 are expressed genes, the phenotypic effect of loss-of-function mutations in these loci was examined. For this purpose, each open reading frame was deleted and replaced with a selectable marker. The resulting alleles, pkhlΔ::TRPl and pkh2Δ::HIS3, were used to replace the normal chromosomal loci by homologous recombination. Both a haploid pkhlΔ::TRPl mutant (AC301) and a haploid pkh2A::HIS3 mutant (AC303) grew normally and indistinguishably from congenic PKH1 + and PKH2+ haploids isolated from the same tetrad. In addition, the pkhlΔ and pkhlΔ single mutants displayed no apparent phenotype when the cells were challenged under various conditions, including high concentrations of salt or caffeine, different carbon sources, and different temperatures, or when subjected to heat shock.

To determine if PKHl and PKH2 might share a common function, strain AC301

(MAT pkhlΔ::TRPl) was crossed with strain AC303 (MATa pkh2Δ::H!S3). Upon sporulation of the resulting doubly-heterozygous diploid (AC306), the majority of the 30 tetrads dissected, yielded three viable and one non-viable spore

(Fig. 2A). The viable spores were analysed both by plating on appropriate selective media and by PCR (Fig 2B). None of the viable haploid cells were

Trp+ and His + , and none carried both the pkhl A and the pkh2Δ mutations. Microscopic observation of the non-viable spores revealed that most germinated and underwent two or three cycles of cell division before ceasing to grow.

Hence, pkhlΔ pkh2Δ double mutants are inviable, indicating that PKHl and

PKH2 encode genes that are functionally redundant and that share some role that is essential for cell growth and survival.

To confirm that the lethality of pkhl Δ pkh2Δ cells was due solely to the absence of PKHl or PKH2 function, AC306 was transformed with either YEplacl95- PKH1, a C/ft 5-marked plasmid expressing PKHl from its endogenous promoter, or the empty vector (YEplacl95), and the resulting Ura+ transformants were subjected to sporulation and tetrad dissection. It was possible to identify many Trpl + His+ and Ura+ spore clones from the diploid transformed with YE l95- PKH1, but not from that transformed with the empty vector (data not shown). Thus, the pkhlΔ pkh2Δ double mutant was able to survive if it expressed PKHl from a plasmid. Likewise, when AC306 was transformed with a C/Λ45-marked plasmid (pYES2) expressing either PKHl (Fig. 3 A) or PKH2 (data not shown) from the GALl promoter, it was possible to obtain viable pkhlΔ pkh2Δ spores, even when the cells were propagated on glucose (presumably because the particular constructs used are not efficiently repressed on this carbon source [39]). Moreover, when the pkhlΔ pkh2Δ cells harboring either pYES2-PKHl (Fig. 3B) or pYES2-PKH2 (data not shown) were plated on medium containing 5-fluoro-orotic acid (5-FOA), which selects for cells that lack a functional URA3 gene [40] and, hence, for loss of the £/& 5-marked pYES2 plasmid, the pkhlΔ pkh2Δ cells were no longer capable of growing (Fig 3B).

Human PDKl is a functional homologue of Pkhl and Pkh2

To determine if Pkhl and Pkh2 are homologous in function, as well as in sequence, to PDKl, AC306 was transformed with YEplacl95 expressing either full-length human PDKl or human PDKl lacking its C-terminal PH domain (PDKl-ΔPH), under control of the authentic PKHl promoter. Expression of either PDK or PDKl-ΔPH from this vector permitted the recovery of viable pkhlΔ pkh2Δ spores (Fig. 4B). Moreover, selection against these C/ft 5-marked plasmids by plating on 5-FOA medium prevented the growth of the phklΔ pkh2Δ double mutants, but not a pkhlΔ or a pkhlΔ single mutant carrying the same plasmids (Fig. 4B). Viable phklΔ pkh2Δ double mutant spores could also be recovered when AC306 was transformed with a vector (pYES2) expressing either PDKl or PDKl-ΔPH from the GALl promoter, even when the cells were germinated and grown on glucose-containing medium, presumably because the particular constructs used are not efficiently repressed on this carbon source (data not shown). These results demonstrate that the catalytic domain of PDKl is sufficient to substitute for the function of Pkhl and Pkh2 and suggests that PDKl is able to phosphorylate the same essential target substrates in yeast cells as Pkhl and Pkh2. Purification and characterisation of Pkhl kinase activity

The PKHl coding region was expressed, as a fusion to GST, in human 293 cells and purified from cell lysates by affinity chromatography on gluthatione- Sepharose. The material obtained showed a single Coomassie blue-staining band on SDS-PAGE that migrated as a 112 kDa species, in agreement with the molecular mass expected for the GST-Pkhl fusion. A catalytically-inactive ("kinase-dead") mutant altered in a conserved residue known to be critical for binding the Mg²⁺-ATP substrate, Pkhl(D267A), was also expressed and purified. This GST-KD-Pkhl construct served as a control.

The activity of GST-Pkhl was assessed via its ability to phosphorylate and activate GST-PKB, a known substrate of human PDKl . Phosphorylation of PKB was assessed by the incorporation of label in reactions containing [γ-³²P]ATP. Activity of PKB was measured via its ability, after its incubation with GST- Pkhl, to phosphorylate a specific peptide substrate, Crosstide [17]. Yeast Pkhl was able to activate human PKB (Fig. 5A), provided that lipid vesicles containing 3-phosphoinositides that are known to interact with the PH domain of PKB were present. There was an excellent correlation between the Pkhl- dependent phosphorylation of PKB (Fig. 5B) and the degree of activation observed (Fig. 5A). No activation or phosphorylation of PKB was observed if PtdIns[3,4,5]P3 or PtdIns[3,4]P2 were omitted, or replaced by PtdIns[4,5]P2 or Ptdlns-3P. As observed with human PDKl (Fig. 5C and 5D), Pkhl phosphorylated and activated PKB more efficiently in the presence of the naturally-occurring stearoyl-arachidonyl derivative of PtdIns[3,4,5]P3 than in the presence of the dipalmitoyl derivative. GST-KD-Pkhl did not activate or phosphorylate PKB under any condition tested (data not shown). To determine what residue in PKB was phosphorylated by Pkhl, GST-PKB was phosphorylated to completion by prolonged incubation in the presence of [γ-

³²P]ATP, cleaved with trypsin, and the resulting digest resolved by HPLC on an

RP-C18 column. Only one major labelled phosphopeptide was observed (data not shown). The elution position of this species was congruent with that of the phosphopeptide (residues 308-328) obtained by tryptic digestion of PKB phosphorylated by human PDKl . No ³²P-labelled material eluted at the position corresponding to the PKB peptide that contains Ser473. The peptide labelled by

Pkhl contained phosphothreonine (and no other phosphoamino acid) and all the radioactivity was released after one cycle of Edman degradation (data not shown). This analysis establishes that yeast Pkhl phosphorylates PKB at

Thr308, the same residue phosphorylated by human PDKl. Hence, the yeast and human enzymes display the same specificity in vitro using this substrate.

YPKl and YKR2 encode homologues of mammalian PKB

PKB is one of the founding members of the so-called AGC sub-family of protein kinases. Moreover, PDKl is known to phosphorylate PKB and several other members of this sub-family at a specific motif that lies in the so-called "activation loop" situated between conserved elements VII and VIII in the catalytic domains of these enzymes (Fig. 6A). In addition, all of these targets also share a different consensus sequence at a C-terminal phosphorylation site for another protein kinase activity (PDK2). The S. cerevisiae genome encodes four, previously characterised protein kinases that possess both of these same motifs (Fig. 6A). The YPKl gene was originally identified by screening a yeast genomic library using a cDNA encoding the catalytic subunit of mammalian cyclic-dependent protein kinase as the probe [41]. Similarly, the highly-related YKR2 gene, also called YPK2 [42], was first identified by library screening with a mammalian PKC cDNA probe [43]. The SCH9 gene was identified by its ability, when overexpressed, to rescue the lethality of cells defective in the activation of Ras [44]. Finally, the PKC1 gene was identified as a bona fide yeast homologue of major mammalian PKC isotypes [45]. However, among these four gene products, the catalytic domains of Ypkl and Ykr2 share 88% identity, as well as extensive similarities across their N- and C-terminal extensions. Most significantly, among all other protein kinases in available databases, the catalytic domains of Ypkl and Ykr2 share greatest similarity to members of the AGC subfamily (Fig. 6B), including mammalian SGK (58% identity) [36, 46], PKB (54% identity) and p70 S6 kinase (52% identity). ARK (50% identity) [47] is another AGC subfamily member but does not have consensus phosphorylation PDKl motifs. This sequence homology raised the possibility that Ypkl and Ykr2 may be physiological substrates for Pkhl and Pkh2.

YPKl and YKR2 are essential genes that are functionally redundant

It has been reported previously that cells lacking either Ypkl or Ykr2 are viable, whereas cells lacking both Ypkl and Ykr2 are inviable [42]. To confirm this observation, and to generate a strain in which the ability of potential mammalian homologues to rescue this inviability could be readily tested, otherwise isogenic ypklΔ::HIS3 and ykr2Δ::TRPl haploid strains (YES5 and YES1, respectively) were constructed and crossed to form a doubly heterozygous diploid (YES7). This diploid was transformed with a LEU2-maτked plasmid expressing the YKR2 gene under the tight control of the GALl promoter, and a Leu-f transformant was subjected to sporulation and tetrad dissection on galactose-containing medium.

Under these conditions, most of the tetrads yielded four viable spores, and His+

Trp+ Leu+ isolates were readily recovered; whereas, when the Leu+ transformant was sporulated on glucose, no His+ Trp+ Leu+ spores could be recovered (data not shown). Moreover, as expected if the absence of both YPKl and YKR2 function is lethal, when the ypkl A ykr2Δ double mutant carrying pGAL-YKR2, previously maintained on galactose medium, was streaked on glucose medium, the cells failed to grow, whereas otherwise isogenic wild-type cells or ypklΔ and ykr2Δ single mutants carrying the same plasmid grew well on glucose (Fig. 7). These results demonstrate that YPKl and YKR2 encode genes that are functionally redundant and that share some role that is essential for cell growth and survival. In our hands, however, a ypklΔ mutant grows detectably more slowly that otherwise isogenic wild-type cells or a ykr2Δ mutant, either in liquid culture or on solid agar medium (data not shown).

PKB-related enzyme, SGK, is a functional homologue of Ypkl and Ykr2

To test the ability of mammalian protein kinases to rescue the lethality of the ypklΔ ykr2Δ double mutant, diploid YES7 was transformed with a low-copy- number (CEN) plasmid marked with URA3 and expressing YKR2 from its endogenous promoter. A Ura-I- transformant was sporulated, dissected, and a His+ Tφ+ Ura+ spore, representing a ypklΔ ykr2Δ haploid maintained by expression of YKR2 from the plasmid (strain YPT28), was recovered. YPT28 was then transformed with either empty LEU2-maτked, high-copy-number (2 μm DNA) vectors (pAD4M or YEp351GAL) or the same vectors expressing (from either the GALl promoter or the constimtive ADH1 promoter [48]) YPKl, YKR2, or cDNAs encoding rat SGK, mouse PKB/c-AKT, rat p70 S6 kinase, or bovine βARK. The nucleotide and amino acid sequences of rat SGK are shown in Figures 12 and 13, respectively. All of these strains are able to grow on galactose medium due to the presence of the plasmid expressing YKR2, which also demonstrated that expression of none of the heterologous protein kinases tested was deleterious to yeast cell growth (Fig. 8A). In contrast, when plated on the same medium containing 5-FOA, thereby demanding loss of the pYKR2(URA3) plasmid, the ypklΔ ykr2Δ cells carrying the empty ZJsC/2-marked vectors were unable to grow, whereas those harboring plasmids expressing either YPKl or YKR2 remained viable, as expected. Likewise, any other protein kinase that is able to perform the essential function of Ypkl and Ykr2 should also permit growth on 5-FOA. Of the four mammalian cDNAs tested, only SGK displayed efficient complementation of the ypkl A ypklΔ double mutant (Fig. 8A). However, we also reproducibly observed weak complementation by PKB, in that a small percentage of the colonies were able to survive (presumably representing cells expressing exceedingly high levels of PKB resulting from accumulation of the 2 μm DNA plasmid due to its notoriously poor segregation efficiency [49]). All of the mammalian protein kinases were produced at high levels in yeast (grown on SCGal-Leu), as judged by immunoblotting with appropriate antibodies, and were active, as judged by assaying extracts of the yeast cells with appropriate specific substrates (data not shown); hence, the failure of p70 S6 kinase and βARK to complement was not due to their lack of expression. Because p70 S6 kinase, in particular, is under such complex regulation in animal cells [50], truncations of the N-, C-, and both N- and C-termini [51] were also tested in the same way; although all were expressed, none was able to rescue the lethality of the ypklΔ ykr2Δ cells (data not shown).

To confirm these results by an independent method for assessing the ability of the mammalian protein kinases to complement, the same plasmids described above were introduced into a yeast strain (YPT40) that displays temperature-conditional growth because it carries a null mutation (ykr2Δ) in YKR2 and a temperature- sensitive (ts) mutation (ypkl-l^ts) in YPKl, the latter of which was generated as described in detail in Materials and Methods. As expected, all of the transformants were able to grow at the permissive temperature (26°C), whereas the strain carrying an empty vector (YEp351GAL) was unable to survive at the restrictive temperature (35°C), but the strain expressing YPKl from the same vector grew well (Fig. 8B). As observed before, the cells expressing SGK also were able to grow well at 35°C, and the cells expressing PKB were able to grow weakly, in that microcolonies were observed outside of the heavy initial streak, whereas cells expressing p70 S6 kinase or βARK did not grow (Fig. 8B). Ypkl and mammalian SGK are efficient substrates for Pkhl

Based on the observations described in the preceding sections, Ypkl (and/or Ykr2) should be physiological substrates of Pkhl (and/or Pkh2). In addition, yeast Pkhl (and/or Pkh2) should be able to phosphorylate and activate mammalian SGK in vitro, as it has previously been shown that PDKl is capable of phosphorylating and activating SGK [36]. To test these hypotheses, Ypkl and SGK lacking the N-Terminal 60 amino acids [36] were expressed as GST fusion proteins in 293 cells and purified. Each purified protein yielded a single Coomassie blue-stained band on SDS-PAGE with an apparent mobility in good agreement with its expected molecular mass (data not shown). In the absence of any other factor, purified GST- Ypkl displayed no detectable activity toward the peptide substrate (Crosstide); however, after pre-incubation with purified GST- Pkhl, GST- Ypkl was activated and catalysed a readily detectable level of incorporation into the substrate in the presence or absence of lipid vesicles containing PtdIns[3,4,5]P3 (Fig. 9A). Consistent with activation resulting from Pkhl -dependent phosphorylation, when [γ-³²P]ATP was included in the reaction, incoφoration of label into GST- Ypkl was readily detected (Fig. 9B) and was present exclusively as phosphothreonine (data not shown). A "kinase-dead" derivative, GST-KD-Ypkl(D488A) (see Materials and methods), was phosphorylated by GST-Pkhl but, as expected, was not catalytically-active (data not shown). Also, as predicted, GST-SGK was both phosphorylated and activated upon incubation in vitro with GST-Pkhl , even more efficiently than by GST-PDKl (Fig. 9C and 9D).

As one means to map the Pkhl phosphorylation site in Ypkl, a GST- Ypkl(T504D) mutant was expressed in 293 cells and purified. The altered residue is equivalent to the PDKl target residue (Thr308) in PKBα. As expected, GST- Ypkl (T504D) was not detectably phosphorylated by Pkhl (Fig. 10), consistent with the conclusion that Pkhl phosphorylates Ypkl at this residue. Since GST- Ypkl (T504D) was neither constitutively active nor activated by Pkhl, Asp at this position cannot substitute for phosphothreonine to activate this protein kinase. Ypkl also contains a putative consensus site (Thr662) for PDK2 phosphorylation (Fig. 6A), equivalent to Ser473 in PKBα. GST-Ypkl(T662D) expressed and purified from 293 cells was also inactive; however, this protein was phosphorylated and activated by Pkhl in a manner identical to GST- Ypkl itself (Fig. 10). A form of Ypkl in which both Thr504 and Thr662 were mutated to Asp was also generated. This GST- Ypkl (T504D T662D) mutant also displayed no detectable activity, before or after incubation with Mg²⁺-ATP and GST-Pkhl , demonstrating that, unlike PKBα, mutation of these residues to Asp cannot substitute for phosphothreonine to produce a constitutively-active enzyme.

Substrate specificity of Ypkl is similar to PKB

PKBα phosphorylates its substrates at the minimal consensus sequence Arg-X- Arg-X-X-Ser-Hyd, where Hyd is a bulky hydrophobic residue [16]. Using several series of peptide substrates of related sequence, efficiency of phosphorylation by Pkhl -activated Ypkl was observed to be quite similar to that observed for PDKl -activated PKB (Table 2). Moreover, the effect of alterations on the rate of phosphorylation showed nearly identical trends. For example, as seen for PKBα, changing either Arg to Lys, or absence of the P-5 Arg, or absence of the P+ l hydrophobic residue, all drastically reduced or abolished phosphorylation by Ypkl .

Discussion Here we have shown that S. cerevisiae contains protein kinases that are homologues of mammalian PDKl and PKB family members, in terms of sequence, physiological function in vivo, and biochemical specificity in vitro. Specifically, we demonstrated, first, that two previously uncharacterised open reading frames, now designated the PKHl and PKH2 genes, encode PDKl-like protein kinases. The function(s) of Pkhl and Pkh2 appear to overlap because loss of either enzyme produces no obvious phenotype, whereas a cell deficient in both enzymes is inviable. Expression of the N-terminal catalytic domain of mammalian PDKl alone is sufficient to overcome the lethality of pkhl A pkh2Δ cells. Consistent with this finding, purified Pkhl phosphorylates and activates known substrates of mammalian PDKl, including PKB and SGK and, where analysed, phosphorylates the same residue (Thr308 in PKBα) as PDKl.

Next, we demonstrated that the YPKl and YKR2 gene products are PKB-like protein kinases. In agreement with a previous report [42], we found that loss of Ykr2 produces no discernable phenotype, that absence of Ypkl results in slower cell growth, and that a ypklΔ ykr2Δ double mutant is inviable, indicating that these protein kinases also have some functional redundancy. Expression of SGK, a close relative of PKBα, that lacks an obvious N-terminal PH domain, rescues efficiently the inviability of ypklΔ ykr2Δ cells. We observed that PKB itself was able to sustain only weakly the growth of the ypklΔ ykr2Δ double mutant. This latter result is consistent with the prediction that PKB will exist in yeast largely in the inactive state because Saccharomyces cerevisiae lacks any enzymic machinery capable of generating either PtdIns[3,4,5]P3 or PtdIns[3,4]P2 [52-54]. Binding of these phospholipids is apparently necessary to relieve conformational constraints in PKB and thereby allow it to be an efficient substrate either for mammalian PDKl or, as we have shown, for yeast Pkhl. This situation probably also explains why all four of the yeast proteins (Pkhl, Pkh2, Ypkl and Ykr2) lack discernible PH domains and why Pkhl -dependent activation of Ypkl is not influenced by the presence or absence of such 3-phosphoinositides. Indeed, Pkhl does not bind PtdIns[3,4,5]P3 under conditions where this phospholipid binds tightly to PDKl (results not shown). Therefore, the fact that Pkhl -mediated activation of PKB only occurs efficiently in the presence of lipid vesicles containing PtdIns[3,4,5]P3 or PtdIns[3,4]P2 confirms unequivocally that these 3- phosphoinositides exert their effects solely by interacting with the PH domain of PKBα. The signals that lead to activation of Pkhl and Pkh2, or mobilize recruitment of these enzymes to particular subcellular locations, is not yet known. Likewise, the signals that promote encounter of Ypkl and Ykr2 with Pkhl and Pkh2 are also unknown.

Despite the fact that the catalytic domains of Ypkl and Ykr2 are also rather similar to other AGC sub-family protein kinases from animal cells, including p70 S6 kinase and βARK, these enzymes, although expressed in active form in yeast, were unable to support any detectable growth of cells deficient in Ypkl and Ykr2. In addition to functional complementation in vivo, other results also indicate that Ypkl and Ykr2 represent PKB-like enzymes. First, like its ability to phosphorylate and activate PKB and SGK, yeast Pkhl is able to phosphorylate and activate Ypkl in vitro. Second, mutagenesis studies indicated that Pkhl phosphorylates Ypkl at a residue (Thr504) that is equivalent to the position in PKB (Thr308) that is phosphorylated by PDKl and by Pkhl. Finally, using a series of synthetic peptide substrates, we found that Pkhl -activated Ypkl has a substrate specificity in vitro nearly identical to that displayed by PDKl -activated PKB [16].

Specifically, Ypkl was able to phosphorylate Ser and Thr residues that lie in an Arg-X-Arg-X-X-Ser/Thr-Hyd motif. Thus, some of the physiological substrates of Ypkl and Ykr2 may contain this consensus sequence. However, the yeast homologues of some of the known substrates of mammalian PKBα do not contain any perfect matches to this canonical site. For example, none of the four GSK3 homologues encoded in the S. cerevisiae genome [55] possess the Arg-X- Arg-X-X-Ser motif that is present at the N-terminus of the GSK3 isoforms targeted by mammalian PKBs. Also, yeast PFK2 lacks the two-C-terminal Arg- X-Arg-X-X-Ser motifs found in the cardiac isoform of PFK2. Hence, the physiologically relevant substrates of Ypkl and Ykr2 remain to be determined.

Taken together, these results indicate that Ypkl (and, most likely, Ykr2) lie down-stream of Pkhl (and/or Phk2) in a protein kinase cascade that is essential for both the growth and survival of yeast cells. In mammalian cells, PDKl appears to be responsible for the activation of a number of different protein kinases of the AGC sub-family, in addition to PKB , including p70 S6 kinase, certain PKC isotypes and, most recently SGK [36], in agreement with results presented here. Are Ypkl and Ykr2 the only substrates of Pkhl and Pkh2 that are essential for yeast cell viability? Several observations indicate that, as for PDKl in animal cells, Pkhl and Pkh2 have multiple targets. First, human PKB can be partially activated by a T308D mutation (in the PDKl site) or by a S473D mutation (in the PDK2 site), and almost fully activated by the simultaneous presence of both mutations [22]; nonetheless, expression of such a PKB (T308D S473D) mutant is unable to support vegetative growth of a pkhlΔ pkh2Δ double mutant (although, following germination of a pkhlΔ pkh2Δ spore, expression of such a construct does allow many more cycles of cell division before cessation of growth ensues than expression of PKB itself, yielding a microcolony visible under the microscope(results not shown)). Second, if Ypkl or Ykr2 are the sole essential targets of Pkhl and Pkh2, and possess any basal activity in the absence of Pkhl- and/or Pkh2-dependent phosphorylation, then rampant overexpression of these enzymes might bypass the need for Pkhl and Pkh2 function; however, gross oveφroduction (approximately 50-fold) of either Ypkl or Ypk2 from the strong GALl promoter on multi-copy plasmids also does not rescue the inviability of pkhl Δ pkh2Δ cells (results not shown). In contrast to mammalian PKBα, it has not yet proved possible to generate constitutively-active variants of yeast Ypkl or human SGK [36] by mutating to Asp the residues equivalent to Thr308 and Ser473 in PKBα. Hence, we have been unable to test whether constitutively- active forms of Ypkl (or SGK) would be able to rescue the lethality of a pkhlΔ pkh2Δ double mutant. Third, one other potential Pkhl and Pkh2 substrate, Pkcl, is known to be essential for yeast cell viability under standard growth conditions [45] because it sits at the head of a protein kinase cascade that controls a MAP kinase (Slt2/Mpkl) required for proper cell wall biosynthesis and survival under hypertonic stress (reviewed in [56]). However, again as expected if Pkhl and Pkh2 have multiple essential targets, attempts to rescue the lethality of pkhl Δ pkh2Δ cells by overexpression of PKC1 were also unsuccessful (results not shown). Yet another potential Pkhl and Pkh2 target, Sch9, is thought, on the basis of genetic results, to serve as an effector enzyme in a pathway that is parallel to, and largely redundant in function with, the three yeast cyclic- AMP- dependent protein kinase catalytic subunits [44]. Indeed, additional genetic results suggest that activation of the α subunit of a heterotrimeric G protein, Gpa2, but not activation of Ras2, leads to activation of Sch9 [57]. Like the absence of Ypkl alone, loss of Sch9 causes slow growth, apparently due to prolongation of the GI phase of the cell cycle [44].

Intriguingly, like mammalian PKBα, p70 S6 kinase, and SGK, the known and suspected substrates for yeast Pkhl and Pkh2, including Ypkl, Ykr2, Pkcl and Sch9, all also contain the consensus sequence (Phe-X-X-Phe/Tyr-Ser/Thr- Phe/Tyr) for phosphorylation by PDK2 protein kinase activity and at the same position (160-165 residues C-terminal) relative to the Thr known (or suspected) to be phosphorylated by Pkhl (and, presumbly, Pkh2). This conservation strongly suggests, first, that the S. cerevisiae genome encodes one (or more) protein kinases with a PDK2-like function and, second, that this enzyme(s) is likely to be or act coordinately with Pkhl and/or Pkh2 to control activation of a number of target protein kinases, as observed for PDKl and PDK2 activity in animal cells (Fig. 11). Conclusions

We have demonstrated here that the yeast, S. cerevisiae, possesses functional homologues of two effector enzymes, PDKl and PKB, which are vital to the proper metabolic regulation and survival of mammalian cells in response to a variety of extracellular stimuli. This conservation suggests that the physiological roles of these two classes of protein kinase must be evolutionarily quite ancient and central to the growth and viability of all eukaryotic cells. The presence of these proteins in yeast should allow the application of genetic approaches to identify both upstream activators of the PDKl-like enzymes, Pkhl and Pkh2, and downstream substrates for the PKB-like enzymes, Ypkl and Ykr2. Such information may, in mm, shed light on previously unsuspected modes of regulation of mammalian PDKl and previously unknown potential targets of PKB. In addition, analysis in yeast may assist in revealing the nature of the as yet uncharacterised PDK2 enzyme that is required, along with PDKl, for full activation of PKB in animal cells, and also presumably required for full activation of Ypkl and Ykr2 in yeast.

Materials and methods

Cell culture S. cerevisiae strains used in this study are described in Table 1, and were isogenic derivatives of either AYS927 (W303 background) or YPH499 (S288C background). Yeast cells were grown at 30°C in a rich medium (YPD) containing 1 % yeast extract, 2% peptone (Difco) and 2% glucose, or in a synthetic minimal medium (S), containing either galactose (Gal) or glucose (Glc, or D, for dextrose) as the carbon source and supplemented with nutrients appropriate for maintaining selection for markers and plasmids [58]. Standard methods [58, 59] for genetic manipulation of yeast were used. E. coli cells (typically DH5 ) were grown at 37°C in Luria-Bertani medium [60] containing (when needed) 50 μg/ml ampicillin for plasmid selection. Recombinant DNA techniques

Restriction enzyme digests, DNA ligations and other recombinant DNA procedures were performed as described [60]. Transformation of bacterial cells was achieved using electroporation. Transformation of yeast cells was accomplished by minor modifications of the lithium acetate method [61, 62]. The PKHl and PKH2 genes used in this study, and the YPKl gene used in some constructions, were recovered from genomic DNA of strain AYS927 using PCR amplification with the Expand High Fidelity PCR System (Boehringer Mannheim), gel purified using the Kristal gelex kit (Cambridge Molecular Technologies, Cambridge, UK), and cloned first into the pCR2.1-TOPO vector using the TOPO system (Invitrogen). Site-directed mutagenesis was performed using the QuikChange Kit (Strategene) following instructions provided by the manufacturer. All DNA constructs were verified by automatic DNA sequencing using an automated DNA Sequencer (Model 373; Applied Biosystems).

PCR amplification of yeast genomic DNA

The primers used to amplify the PKH2 coding region were 5'-CG GGA TCC

GCC ACC ATG GAG CAG AAG CTG ATC TCT GAA GAG GAC TTG TAT TTG ATA AGG ATA ATT CCA TG-3' (forward) and 5' -ATA AGA AT GCG GCC GC TTA CGA CCT CTT CGA TTT TGC AG-3' (reverse), and incoφorated BamHI and Notl sites, respectively (indicated by italics). The primers used to amplify the PKHl coding region were 5' -ATA AGA AT GCG GCC GC TGC CAC C ATG GAG CAG AAC CTG TCT CTG AAG AGG ACT TG GGA AAT AGG TCT TGA CAG AGG-3^* (forward) and 5'-ATA AGA AT GCG GCC GC TCA TTT TTC ATC TGT CCG TGT C-3' (reverse), and incoφorated Notl sites (indicated in italics). Both 5 '-primers also contained a sequence encoding a 10-residue c-Myc epitope tag (underlined). The primers used to amplify the YPKl gene were: 5' -GGA TCC GCC ACC ATG TAC CCA TAC GAT GTG CCA GAT TAC GCC TAT TCT TGG AAG TTT AAG-3' (forward) and 5'-GGT ACC CTA TCT AAT GCT TCT ACC TTG C-3^* (reverse), and incoφorated BamHI and Kpnl restriction sites, respectively (italics). The initiator or termination codons in all these primers are also indicated (boldface type).

Gene disruptions and strain constructions

A PCR-based method [63] was used for disruption of the PKHl and PKH2 genes. To generate the pkh2Δ::HIS3 mutation, HIS3 was amplified from pRS313 [64] using as primers 5' -AAG TAA CAT CTT GAT GAA CCG AGA AGC CAC TAA CTA GTT TT GTG CAC CAT AAT TTT CCG-3' (forward), where the underlined sequence corresponds to nucleotides -93 to 53 from the PKH2 initiator codon and the remainder of the primer corresponds to nucleotides -326 to -311 from the initiator codon of HIS3, and 5' -TAA GTA GCT TGA TGA AAA CAT TAG ATA AAA TTA CTA A TTA CCG TCG AGT TCA AGA G- 3', where the underlined sequence corresponds to the nucleotides immediately after the PKH2 stop codon (in boldface type) and the remainder corresponds to nucleotides 204 to 189 after the HIS3 stop codon. The resulting 3.3 kb product was used for DNA-mediated transformation of a diploid strain (AYS927). Transformants were selected on SD-His plates, and disruption was verified by PCR analysis of from one of the His+ isolates using appropriate primers. This heterozygous PKH2/pkh2Δ: :HIS3 diploid (AC200) was sporulated, and the resulting tetrads were dissected. Spore clones were analysed by plating on selective medium and confirmed by PCR to identify a haploid containing the pkh2Δ::HIS3 disruption (AC303). To generate the pkhlΔ::TRPl mutation, the TRPl marker in pRS314 [64] was amplified using 5'-GCA CGT GTA CTT GCT TGA ATA CTG CTA CTA TAT CAT TAA T ATG GTA CTG AGA GTG CAC C-3' (forward), where the underlined sequence corresponds to nucleotides immediately upstream of the initiator codon (boldface type) and the remainder corresponds to nucleotides situated -300 to 285 nucleotides from the initiator codon of TRPl, and 5'-TAT TAT GCA TTA CAC TTT CCC CTT CAC CAT GTC TTA CAT ATG CAT CCG CAG GCA AGT GCA C-3' (reverse), where the underlined nucleotides correspond to positions +69 to 25 after the PKHl stop codon and the remainder of the primer corresponds to the region situated +51 to 36 nucleotides after the TRPl stop codon. The resulting 2.4 kb product was used for transformation of AYS927, and transformants were selected on SD- Tφ plates. Disruption was verified by PCR analysis from one of the Tφ+ isolates using appropriate primers. This heterozygous diploid PKHl /pkhl Δ:. -TRPl (AC201) was sporulated, dissected, analysed, and a haploid spore containing the pkhl A:. -TRPl mutation (AC301) was identified.

In both the ypkl-Δl::HIS3 allele and the ykr2-Δl::TRPl alleles [65], the coding sequences for the entire catalytic domains of both enzymes have been deleted and replaced with the indicated markers. To generate a ypklΔ ykr2Δ double mutant, a MATa ypklΔ strain (YES5) was mated to a MATa ykr2Δ strain (YES1) carrying pYKR2(URA3) (see below). The resulting diploid was subjected to sporulation and tetrad dissection, and a His+ Tφ+ Ura+ spore, representing a MATa ypklΔ ykr2Δ cell kept alive with the plasmid-borne YKR2 gene, was designated YPT28. To create the temperature-conditional ypkl-T^s ykr2Δ strain (YPT40), a temperature-sensitive allele of YPKl was first generated, as follows. A genomic insert containing the YPKl gene [41], cloned into the Xbαl site in the vector, pGEM3™ (Promega), and generously provided by Richard A. Maurer (then at the University of Iowa, Iowa City, IA), was excised as a 4.1 kb Xbαl-SαH fragment and inserted into the EC/2-containing vector, pRS315 [64], yielding pRS315- YPK1. The sequence encoding the catalytic domain of YPKl was then amplified under moderately error-prone conditions using AmpliTaq™ DNA polymerase (Perkin Elmer Cetus), with pRS315-YPKl as the template and a 5 '-primer (PI), 5'-TGC CCT CGA AGA CAT GGC-3', corresponding to a sequence beginning at nucleotide 788 (where the first base of the ATG start codon is +1) and a 3'- primer (P2), 5'-CTT GAA CAC AGT AAG TAA CGG-3', corresponding to the flanking genomic sequence commencing 68-bp downstream of the stop codon. The resulting 1350-bp linear PCR product was gel purified and co-transformed into YPT28 along with a ^" 9 kb linear fragment of pRS315-7PA7, that had been generated by digestion with Pstl and Ncol, and gel purified. Transformants were selected on SCD-Leu plates at 26°C. This procedure allows for replacement of the corresponding sequence in the parent vector with potentially mutant sequences, and regeneration of circular plasmids, via in vivo repair of the gapped plasmid by recombination with homologous sequences present at each end of the PCR product [66, 67]. To determine which /fC/2-containing plasmids expressed functional YPKl at 26°C, the Leu+ transformants were subsequently replica- plated onto -Leu plates containing 5-fluoro-orotic acid (5-FOA) [39], which selects for loss of the pYKR2(URA3) plasmid initially present in the recipient ypklΔ ykr2Δ strain. To determine which of the YPKl- and -ιt/2-containing plasmids harbored a temperature-sensitive allele of YPKl, the Leu+ Ura- cells were tested by replica-plating for their ability to grow on SCD-Leu plates at 37°C. One transformant was identified that reproducibly failed to grow at this temperature. The £'i72-containing plasmid carried by this strain (pypklTS ) was recovered [68], and direct nucleotide sequence analysis of the YPKl open reading frame in the plasmid revealed the presence of two amino acid substitutions (I484T and Y536C). Sub-cloning and re-transformation confirmed that these mutations were sufficient to confer the temperature-sensitive (ts) phenotype, and this allele was designated ypkl-1¹⁵. The ypkl-l'^s allele was used to transplace the normal YPKl chromosomal locus in the ykr2Δ strain (YESl), as follows. First, PCR was used to generate a customized DNA fragment containing BαmHl and Smαl restriction sites 96 and 108 bp, respectively, downstream from the stop codon of the YPKl coding sequence in pypklTS plasmid, and this fragment was substituted for the corresponding segment of the 3 '-flanking region, yielding pINT. A 2.6 kb Scal-BamHl fragment containing the HIS3 gene, excised from vector, pRS303

[64] , was gel-purified and inserted into pINT that had been digested with BamHI and Smal, yielding pINT-HIS, which was able to confer both leucine and histidine prototrophy to a leu2 his3 strain, YPH499 [64]. Finally, a 4.6 kb Clal- Xhol fragment from pINT-HIS, containing the ypkl -l'^s allele, the HIS3 gene, and additional genomic DNA from the YPKl locus flanking the HIS3 gene to its 3'- side, was gel purified and used for transformation of YESl . His+ transformants were selected at 26°C, and then the presence of the integrated ypkl -V^s allele (and the absence of the normal YPKl locus) was confirmed by PCR analysis of DNA isolated from the transformants and by demonstrating that such cells were unable to grow at 37°C. One such isolate that met all of these criteria was designated strain YPT40.

Plasmids For expression of PKHl, YPKl as GST fusions in mammalian 293 cells, the corresponding coding sequences were excised from the appropriate pCR2.1- TOPO derivative and inserted into the mammalian expression vector, pEBG-2T [69]. PKB [26], PDKl [27] and SGK lacking the N-terminal 60 residues [36] were subcloned into the pEBG-2T vector as described elsewhere. For expression of PKHl in yeast, a 2.1 kb fragment, from an internal Smal site (simated about 250 bp downstream from the initiator codon) to an EcoRI site in the pCR2.1- TOPO vector, was first inserted into the 2 μm DNA vector, YEplacl95 [70], that had been digested with Smal and EcoRI, yielding YEplacl95-2.1 £H7. To restore the 5'-end, a 1 kb fragment was amplified by PCR using the primers 5'- GCT TGA CTC AAT TAA GGC GAC-3^* (forward), corresponding to nucleotides 628-634 upstream of the initiator codon, and 5'-ACA TGC TTA GTT AAC TCC-3' (reverse), corresponding to the region located 350 bp downstream of the initiator codon. The resulting product was first cloned into pCR2.1- TOPO, which was then digested with Smal and Sphl to liberate a 0.9 kb fragment that was inserted into the YEplacl95-2.1 JΩ77 construct that had been digested with Sphl and Smal, yielding YEplacl95-P£H7, which contains the complete coding region of the PKHl gene plus 0.5 kb of its promoter region and carries the JJRA3 gene as the selectable marker. To express PKHl under control of the GALl promoter, a 2.3 kb Notl-Notl fragment containing the myc-tagged version of the entire PKHl coding sequence was inserted into the C/&45-marked, 2 μm DNA-containing vector, pYES2 [71], yielding pYES2-PKHl. Likewise, to express PKH2 under GALl promoter control, a 3.3 kb BamHl-Notl fragment containing the entire PKH2 open reading frame was inserted into pYES2, yielding pYES2-PKH2.

To express mammalian PDKl in yeast under control of the PKHl promoter, first, a 0.7 kb fragment corresponding to the PKHl promoter region was amplified by PCR from the YEplacl95-P H construct using the primers 5'-GG GGT ACC GCT TGA CTC AAT TAA GGC GAC-3' (forward) and 5'-CTT CAG AGA TCA GCT TCT GCT CCA T ATT AAT GAT ATA GTA-3' (reverse), corresponding to the start of the PDKl coding sequence (underlined). Second, a 1.4 kb fragment comprising the N-terminal sequence of PDKl was amplified from a human PDKl cDNA using as primers the 0.7 PCR amplification product (forward) and 5'-ACA CGA TCT CAG CCG TGT AAA A-3' (reverse), corresponding to residues 190-184 of PDKl. The 1.4 kb product was cleaved at the Kpnl site (italics) and also with Hindϊll, which cleaves at an internal site in the PDKl coding sequence. The resulting Kpήl-Hindlϊl fragment was used to replace a 0.5 kb segment encoding the N-terminal end of the PDKl protein either in a construct containing either the complete PDKl coding sequence, yielding YEplacl95-PDKl, or in a construct containing just the first 404 residues of the PDKl, corresponding to its catalytic domain and lacking its C-terminal PH domain, generating YEplacl95-PDKl-ΔPH. To express human PDKl under control of the GALl promoter, a 2.0 kb Bglϊl-Xbal fragment containing the complete PDKl coding sequence was inserted into pYES2, yielding pYES2-PDKl. Similarly, a 1.4 kb BgHl-Xbal fragment containing the kinase domain of PDKl, but lacking the PH domain, was inserted into pYES2, creating pYES2- PDKl-ΔPH.

To express YKR2 in yeast from a .it/2-marked, high-copy-number (2 μm DNA- based) plasmid under control of the GALl promoter, a 2.4 kb Xhol-Hindlll fragment of genomic DNA containing the entire YKR2 open reading frame [43] was excised from an insert in pUC18 (generously provided by Shigeo Ohno, Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) and ligated into the vector, YEp351GAL [72], that had been linearized by digestion with Sail and Hindlll, yielding pGAL-YKR2. An essentially identical approach was used to express YPKl, excised from a genomic DNA fragment (see above), yielding pGAL-YPKl. Alternatively, a 2.1 kb BamHl-Notl fragment encoding an HA-tagged version of the YPKl coding sequence, generated by PCR, was inserted into pYES2, yielding pYES2-YPKl. To place the YKR2 gene under control of its endogenous promoter on a low-copy- number (CEN) plasmid carrying the URA3 gene, a 2.5 kb EcoRl-EcoRI fragment of the original 7^R2-containing insert in pUClδ was ligated into the vector, pRS316 [64], that had been linearized with EcoRI, generating pYKR2(URA3). To generate a version of Ypkl tagged at its C-terminal end with the c-Myc epitope recognized by the monoclonal antibody 9E10 [73], a PCR-based method for precise gene fusion [74] was performed using the YPKl sequence cloned in pGEM3 as one template and, as the other template, a sequence encoding the 16- residue version of the Myc epitope followed by a (His)ό tag cloned in pBluescript (Stratagene), and three appropriate synthetic oligonucleotide primers: PI; T3 (Stratagene), 5'-AAT TAA CCC TCA CTA AAG GG-3', corresponding to sequences in the pBluescript vector; and, a "joiner" primer (P3), 5'-TTC AGA AAT CAA CTT TTG TTC TCT AAT GCT TCT ACC TTG C-3', corresponding to the 3 '-end of the YPKl coding sequence and the first several residues of the c-Myc epitope. A 2 kb Clal-SaH fragment of the resulting product was used to replace the corresponding segment in the original YPK1- containing pGEM3 vector, yielding pYPKlmyc. A 1.2 kb Ncόl-Hindlll fragment of pYPKlmyc was used to replace the corresponding segment of p GAL-YPK1, yielding pGAL-YPKlmyc.

To express SGK in yeast, a 1.3 kb Ncol-EcoRI fragment encoding a rat SGK cDNA (Webster et al (1993) Mol Cell Biol 13(4), 2031-2040) was converted to blunt ends by treatment with the Klenow fragment of E. coli DNA polymerase I in the presence of dNTPs and inserted behind the ADH1 promoter in the vector pAD4M [75] that had been linearized with Smal. Correct orientation of the fragment was confirmed by appropriate restriction enzyme digests. An essentially identical approach was used to express bovine βARK, yielding pADH- βARK, which was constructed by Henrik Dohlman (Thorner laboratory). To express PKB in yeast, a 1.5 kb BamHl-BamHl fragment encoding mouse c-Akt was excised from an insert in a two-hybrid bait vector, pASIIA (supplied by Zhou Songyang, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA), and ligated into YEp351GAL that had been linearized by digestion with BamHI, yielding pGAL-PKB. Alternatively, a 2.5 kb EcoRl-Xbal fragment encoding a human PKB cDNA [1] was inserted into pYES2, generating pYES2-PKBα. In addition, a 1.5 kb EcoRl-Xbal insert expressing a constitutively-active mutant version of PKBα [22], in which Thr308 and Ser473 have been replaced by Asp, was inserted into pYES2, creating pYES2-DD- PKBα. To express p70 S6 kinase in yeast, a 1.6 kb Xbal-SaH fragment encoding rat p70 S6 kinase [76] was excised from p2B4 (provided by George Thomas, Friedrich Miescher Institute, Basel, Switzerland) and inserted into YEp351GAL that had been linearized by digestion with Xbal-Sall, yielding pGAL-S6K. In addition, an N-terminal truncation, a C-terminal truncation, and a double N- and C-terminal truncation of p70 S6 kinase (generously provided by John Blenis, Department of Cell Biology, Harvard Medical School, Boston, MA), whose constructions are described in detail elsewhere [51], were also inserted into a yeast expression vector, YEp352 [78], each under control of the methionine- repressible MET3 promoter [78], using essentially identical methods.

Site-directed mutagenesis

To generate a catalytically-inactive ("kinase-dead") version of Pkhl (KD-Pkhl),

Asp276 (GAT) was changed to Ala (GCT). This position corresponds to a conserved residue critical for recognition of the Mg²⁺-ATP substrate in all protein kinases [79]. Likewise, a catalytically-inactive Ypkl derivative (KD- Ypkl) was generated by changing Asp488 (GAT) to Ala (GCT). To attempt to generate a constitutively-active YPKl derivative, the Phkl -dependent phosphorylation site (Thr504) and another presumptive phosphorylation site (Thr662) that matches the consensus for phosphorylation by PDK2 protein kinase activity were both replaced by Asp codons.

Expression and purification of GST-Pkhl, GST-Ypkl and GST-SGK

The human embryonic kidney cell line, 293, was cultured on forty 10-cm dishes, and each dish was transfected with 20 μg of the appropriate expression construct using a modified calcium phosphate method [60] . At 24 h after transfection, each dish of cells was harvested and the cells ruptured in 1 ml of ice-cold lysis buffer, which contained 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 % (by vol) Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium β-glycerol- phosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 μM microcystin-LR, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 10 μg/ml leupeptin and 0.1 % (by vol) 2-mercaptoethanol. The forty lysates were pooled, clarified by centrifugation at 4°C for 10 min at 13,000 x g, and the GST-fusion proteins were purified by affinity chromatography on glutathione-Sepharose [26]. Approximately 0.5 mg of each purified GST-fusion protein was obtained, snap frozen in aliquots in liquid N2, and stored at -80°C.

Measurement of Ypkl, SGK and PKB activities Assay of Ypkl was carried out in two stages. First (Stage 1), GST- Ypkl was activated by incubation with GST-Pkhl and Mg²⁺-ATP, as follows. A reaction mixture (18 μl) containing 2.5 μM PKI, 1 μM microcystin-LR, 10 mM Mg- acetate, 100 μM unlabelled ATP and 0.6 μM GST- Ypkl was prepared in Buffer A [50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, and 0.1 % (by vol) 2- mercaptoethanol]. Reaction was initiated by addition of 2 μl of 50 nM GST-Pkhl in Buffer A containing 1 mg/ml bovine serum albumin, and was incubated at 30°C for 30 min. Second (Stage 2), activated Ypkl was assayed by adding 30 μl of a mixture in Buffer A containing 2.5 μM PKI, 1 μM microcystin-LR, 10 mM Mg-acetate, 100 μM [γ³²P]ATP (200-400 cpm/pmol) and 100 μM Crosstide (GRPRTSSFAEG) [17], a peptide phosphoacceptor substrate. After incubation for 15 min at 30°C, reaction was terminated by spotting a portion (45 μl) of each reaction mixture onto small squares of phosphocellulose paper (Whatman P81), which were washed and analysed as described [80]. Control reactions omitted either GST- Ypkl or GST-Pkhl and resulted in incoφoration of less than 5 % of the radioactivity measured in the presence of both of these proteins. One unit of GST- Ypkl activity was defined as that amount required to catalyse phosphorylation of 1 nmole of Crosstide in 1 min. Assay of SGK and PKB activities were carried out in identical manner, except that GST-SGK and GST- PKBα replaced GST- Ypkl in the first stage of the assay.

Phosphorylation of GST-Ypkl, GST-PKB and GST-SGK by Pkhl Incubations were identical to Stage 1 of the Ypkl assay described above, except that [γ-³²P]ATP (500-1000 cpm/pmol) was used instead of unlabelled ATP, and reactions were terminated by adding SDS to a final concentration of 1 % . The resulting samples were resolved on 7.5% SDS-polyacrylamide gels and, after staining with Coomassie blue, analysed by autoradiography. Also, the stained band corresponding to the GST-fusion protein of interest was excised and the amount of radioactivity incoφorated was quantified by liquid scintillation counting. Ability of GST-Pkhl to phosphorylate and activate human PKBα was examined using methods identical to those described immediately above for the phosphorylation and activation of yeast Ypkl by GST-Pkhl, except that reactions were performed in the presence of lipid vesicles containing various 3- phosphoinositides [26], as described in Results.

Determination of Ypkl substrate specificity

GST- Ypkl was activated with Pkhl in vitro and incubated under standard assays conditions, as described above, except that Crosstide was replaced by 100 μM of the peptides discussed in detail in Results. GST-PKB, derived from transfected IGF 1 -stimulated 293 cells [26], was assayed in parallel.

References

1. Coffer & Woodgett Eur J Biochem. 1991 , 201:475-481.

2. Jones et al (1991) Proc Natl Acad Sci. USA 88:4171-4175.

3. Belacossa et al (1991) Science 254:244-247.

4. Franke et al (1995) Cell 81:727-736.

5. Burgering et al (1995) Nature 376:599-602. 6. Kohn et al (1995) EMBO J 14:4288-4295.

7. Alessi & Cohen (1998) Curr Opin Genet Dev 8:55-62.

8. Downward (1998) Curr Opin Cell Biol 10:262-267.

9. Barthel et al (1997) Endocrinol 183:3559-3562.

10. Cichy et al (1998) J Biol Chem 273:6482-6487. 11. Cheng et al (1992) Proc Natl Acad Sci USA 89 : 9267-9271.

12. Cheng et al (1996) Proc Natl Acad Sci USA 93: 3636-3641.

13. Bellacosa et al (1995) Intl. J. Cancer 1995, 64: 280-285.

14. del Peso et al (1997) Science 278:687-689. 15. Dudek et al (1997) Science 275: 661-665.

16. Alessi et al (1996) FEBS Lett 399: 333-338.

17. Cross et al (1995) Nature 378:785-789.

18. van Weeren et al (1998) J Biol Chem 273 : 13150-13156.

19. Deprez et al (1997) J Biol Chem 111: 17269-17275. 20. Welsh et al (1998) FEBS Lett. 421:125-130.

21. Datta et al (1997) Cell 91:231-241.

22. Alessi et al (1996) EMBO J 15:6541-6551.

23. Kohn et al (1996) J Biol Chem 271: 21920-21926.

24. Pearson et al (1995) EMBO J 14: 5278-5287. 25. Alessi et al ( 1998) Curr Biol 8:69-81.

26. Alessi et al (1997) Curr Biol 7: 261-269.

27. Alessi et al (1997) Cur Biol 7:776-789.

28. Kavanaugh et al (1996) Curr Biol 6:438-445.

29. LeGood et al (1998) Science 281:2042-2045. 30. James et al ( 996) Biochem J 315:709-713.

31. Freeh et al (1996) J Biol Chem 272:8474-8478.

32. Stephens et al (1988) Science 279:710-714.

33. Currie et al (1999) Biochem J 337( Pt 3), 575-83

34. Pullen et al (1998) Science 279:707-710. 35. Le Good et al (1998) Science 281(5385), 2042-5

36. Kobayashi & Cohen P (1999) Biochem J 339 ( Pt 2), 319-28

37. Jacq et al (1997) Nature 387SS:75-78.

38. Dujon et al (1997) Nature 387SS: 98-102. 39. Mumberg et al (1994) Nucleic Acid Res 22:5767-5768.

40. Boeke et al (1986) Mol Gen Genet 197:345-346

41. Maurer et al (1988) DNA 7:469-474.

42. Chen et al (1993) Mol Gen Genet 236:443-447. 43. Kubo et al (1989) Gene 76:177-180.

44. Toda et al (1988) Genes Dev 2:517-527.

45. Levin et al (1990) Cell 62:213-224.

46. Webster et al (1993) Mol Cell Biol 13:2031-2040.

47. Benovic et al (1991) J Biol Chem 266: 14939-14946. 48. Bennetzen et al (1982) J Biol Chem 257:3018-3025.

49. Rose et al (1990) Methods Enzymol 185:234-279.

50. Proud et al (1996) Trends Biochem Sci 21:181-185.

51. Cheatham et al (1995) Proc. Natl. Acad. Sci. USA, 92:1696-1700.

52. Hawkins et al (1993) J Biol Chem 268:3374-3383. 53. Dove et al (1997) Nature 390:187-192.

54. DeWald et al (1997) Biochem Soc Trans 25:1141-1146.

55. Hunter & Plowman (1997) Trends Biochem Sci 22:18-22.

56. Mellor & Parker (1998) Biochem J 332:281-292.

57. Xue et al (1998) EMBO J 17:1996-2007. 58. Sherman F, Fink GR, Hicks JB: Methods in Yeast Genetics. Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press; 1986

59. Guthrie C, Fink GR (Editors): Guide to Yeast Genetics and Molecular Biology, New York NY: Academic Press, Inc.; 1991

60. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual, Cold Spring Harbor NY: Cold Spring Harbor Laboratory Press; 1989

61. Schiestl & Gietz (1989) Curr. Genet. 16:339-346.

62. Gietz et al (1992) Nucleic Acid Res 20:1425.

63. Lorenz et al (1995) Gene 158:113-117. 64. Sikorski & Hieter (1989) Genetics 122:19-27.

65. Schnieders, EA: Ypkl and Ykr2. Appendix, Doctoral Dissertation, University of California, Berkeley, CA, 1996, pp.158-185.

66. Muhlrad et al (1992) Yeast 8:79-82. 67. Staples & Dieckmann (1993) Genetics 135:981-991.

68. Hoffman & Winston (1987) Gene 57:267-272.

69. Sanchez et al (1994) Nature 212:194-19%.

70. Gietz & Sugino (1988) Gene 74:527-534.

71. Ramer et al (1992) Proc Natl Acad Sci USA 89: 11589-11593. 72. Benton et al (1994) J Cell Biol 127:623-639.

73. Evan et al (1985) Mol Cell Biol 5:3610-3616.

74. Yon & Fried (1989) Nucleic Acids Res 17: 4895.

75. Martin et al (1990) Cell 63:843-849.

76. Kozma et al (1990) Proc Natl Acad Sci USA 87:7365-7369. 77. Hill et al (1986) Yeast 2:163-167.

78. Cherest et al (1987) Mol. Gen. Genetics 210:307-313.

79. Taylor SS & Radzio-Andzelm (1994) Structure 2:345-355.

80. Alessi et al (1995) Methods Enzymol 255:279-290.

81. Julius et al (1984) Cell 36:309-318. 82. Ma et al (1995) Mol Biol Cell 6: 889-909.

83. Currie et al (1999) Biochem J 337( Pt 3), 575-83

Table 1. Saccharomyces cerevisiae strains used.

Strain Genotype Source AYS927 MATalMATa ade2-l/ade2-l his3-ll,15/his3-l 1,15 M. J.R. Stark Ieu2-3,112/leu2-3,112 trpl-l/trpl-1 ura3-l/ura3-l canl-lOO/canl-100 ssdl-d2/ssdl-d2

AC200 AYS927 PKH2lpkh2Δ::HIS3 This study AC201 AYS927 PKHllpkhlΔ::TRPl This study

AC303 MATa pkh2Δ::HlS3 (derived from AC200) This study

AC301 MAT pkhlΔ::TRPl (derived from AC201) This study MATalMATa PKHl /pkhl Δ::TRP1 PKH2lpkh2Δ::HlS3 This study AC306 (AC303 X AC301)

YPH499 MATa αde2-101^oc his3-Δ200 Ieu2-Δl lys2-801^αm [64] trpl-Δl urα3-52

YPH500 MATα otherwise isogenic to YPH499 [64]

YESl YPH499 ykr2-Δl r.TRPl This study

YES5 YRH500ypkl-Δl::HIS3 This study MATalMATa YPKl /ypkl Δ::HIS3 YKR2/ykr2Δ::TRPl This study YES7

(YESl X YES5)

YPT28 MATa ypklΔ::HIS3 ykr2Δ::TRPl [pYKR2(URA3)] This study

YPT40 MATa ypkl-l'^s ykr2Δ This study

Table 2. Substrate selectivity of yeast Ypkl and mammalian PKBα. Peptide Relative Rate of Phosphorylation

(at 0 .1 mM) GST-Ypkl GST-PKBa

1 GRPRTSSFAEG [100] [ 100 ]

2 RPRTSSF 92 139

3 KPRTSSF 11 27

4 RPKTSSF 5 30

5 RPRTSAF 0 0*

6 PRTSSF 0 0*

7 RPRTSS 0 2 *

8 KKRNRTLSVA 133 157

9 KKKNRTLSVA 28 13

10 KKRNKTLSVA 14 25

11 RPRTSSF 92 139

12 RPRTSSV 24 108

13 RPRTSSL 31 130

14 RPRTSSA 9 70

15 RPRTSSK 14 67 *

16 RPRTSSE 6 63

*Data taken from Ref. 16.

Example 2: screening assay for inhibitors of human PDKl

The effect of compounds on parental (wild-type with respect to Pkhl and/or Pkh2) S. cerevisiae cells and S. cerevisiae cells in which the endogenous Pkhl and Pkh2 genes have been inactivated and in which human PDKl is expressed under control of the Pkhl promoter, are compared. Compounds that affect the growth of the PDKl -expressing cells but not the Pkhl/Pkh2 expressing (wild- type) cells are selected and may be used to design further compounds for manufacture and test, in order to develop a structure-activity relationship (SAR).

The compounds for test may be selected on the basis of known kinase inhibitory activity or other known property, or may be part of a library of synthetic or natural molecules that may be screened in a "lead generation" screening project.

Example 3: screening assay for inhibitors of PDKl from a pathogenic source

The effect of compounds on parental (wild-type with respect to Pkhl and/or Pkh2) S. cerevisiae cells and S. cerevisiae cells in which the endogenous Pkhl and Pkh2 genes have been inactivated and in which Candida Pkhl or Pkh2 is expressed under control of the Pkhl promoter, are compared. Compounds that affect the growth of the Candida Pkhl or Pkh2-expressing cells but not the wild- type Pkhl/Pkh2 expressing cells are selected and may be used to design further compounds for manufacture and test, in order to develop a structure-activity relationship (SAR).

Example 4: Identification of Candida genes related to S. cerevisiae Pkhl, Pkh2, Ypkl and Ykr2 genes.

The Candida sequence database only allows DNA searches of the Candida data. Therefore, for each of the 4 S. cerevisiae sequences identified and characterised, as described in example 1 (Pkhl, Pkh2, Ypkl, Ykr2), the coding sequence for each S. cerevisiae gene was used to search the Candida database.

The best matches in each case were selected and compared with proteins in the Genbank database. With one exception, this comparison identified the two related S. cerevisiae genes (ie Pkhl/Pkh2 or Ypkl/Tkr2) as the most closely related proteins.

The Candida albicans database entries selected for further study on the basis of the four coding sequence searches were:

384230A10.s4.seq (search 1)

384362Ell.sl.seq (search 1 and search 2)

384286E10.sl.seq (search 1 and search 2)

396076E03.s2.seq (search 3 and search 4) 384194F08.sl.seq (search 3 and search 4)

There appear to be fragments of three Candida genes in the database related very closely to S. cerevisiae Pkhl, Pkh2, Ypkl and Ykr2.

384194F08.sl.seq and 396076E03.s2.seq appear to be closely related to Pkhl and Pkh2.

384362Ell.sl.seq and 384286E10.sl.seq appear to be closely related to Ypkl and Ykr2/Ypk2.

Full-length coding sequences may be determined for the genes from which the above nucleotide sequences are derived by well known techniques, which may include further database interrogation and/or techniques of molecular biology, which may include PCR or library-based cloning techniques. Search 1

Blast Results

Smallest

Sum

High

Probability

Sequences producing High-scoring Segment Pairs: Score P(N) N

384230A10.s4.seq 635 3 .2e-43 1

384362Ell.sl.seq 595 3 .8e-40 1

CAPKC1 C. albicans gene for protein kinase C. 490 5 .2e-37 2

384286E10.sl.seq 411 1 .5e-24 1

384201A03.s2.seq 363 1 .4e-20 1

385002F05.xl.seq 350 1 .9e-19 1

396143F08.s3.seq 307 7 .le-16 1

CAU73457 Candida albicans Cst20p (CST20) gene, complete . 240 4 .4e-10 1

YSASTPK Candida albicans serine/threonine protein kinase. 240 4 .4e-10 1

265251G07.xl.seq 233 1 .4e-09 1

265251H07.xl.seq 233 1, .4e-09 1

385040C04.xl.seq 229 2. .9e-09 1

386003B12.xl.seq 218 2. ,5e-08 1

396076E03. s2. seq 189 7. .le-06 1

384281A03.Sl.seq 174 0. ,00012 1

384194F08.sl.seq 172 0. .00017 1

384253B09.sl.seq 165 0. .00069 1

384329A05.sl.seq 164 0. 00084 1

265251H06.xl.seq 162 0. 0012 1

265273Hll.yl.seq 157 0. 0032 1

265266H04.xl.seq 155 0. 0049 1

384068G08.sl.seq 152 0. 0069 1

396170G09.sl.seq 151 0. 0096 1

Search 2

Blast Results

Smallest

Sum

High

Probability

Sequences producing High-scoring Segment Pairs: Score P(N) N

384362Ell.sl.seq 586 2.2e-49 2

384286E10.sl.seq 497 8.7e-32 1

CAU73457 Candida albicans Cst20p (CST20) gene, complete . 287 5.3e-14 1

YSASTPK Candida albicans serine/threonine protein kinase. 287 5.3e-14 1

265251G07.xl.seq 264 3.6e-12 1

265251H07.xl.seq 262 5.3e-12 1

265251H06.xl.seq 242 2.4e-10 1

265266H04.xl.seq 226 5.6e-09 1

385040C04.xl.seq 219 2.0e-08 1

YSASTKIN Candida albicans (clone pKB66) serine/threonine. 185 1.6e-05 1 CAU72980 Candida albicans Map kinase kinase (STE7) gene, 185 1.7e-05 1

386003B12.xl.seq 181 3.2e-05 1

385081C04.xl.seq 164 0.00079 1

396039B07.sl.seq 159 0.0021 1

384281A03.sl.seq 156 0.0039 1

Search 3

Blast Results

Smallest

Sum

High

Probability

Sequences producing High-scoring Segment Pairs: Score P(N) N

396076E03.s2.seq 636 6.9e-85 2

384194F08.sl.seq 674 1.5e-46 1

384254G10.sl.seq 266 3.8e-12 1

385051D09.xl.seq 244 2.1e-10 1

384022B04. sl.seq BCK1 244 2.6e-10 1

CAU73457 Candida albicans Cst20p (CST20) gene, complete . 229 5.9e-09 1

YSASTPK Candida albicans serine/threonine protein kinase. 229 5.9e-09 1

384201A03.s2.seq 220 2.5e-08 1

384168E05.s2.seq 216 3.1e-08 1

CAU87996 Candida albicans C A4 protein kinase homolog ge . 218 4.8e-08 1

396039B07. sl.seq 195 3.3e-06 1

384282H11. sl.seq 187 1.5e-05 1

385066D07.xl.seq 171 0.00033 1

265235H07.yl.seq 169 0.00047 1

396039H02. sl.seq 162 0.0012 1

396256F10. sl.seq 164 0.0013 1

384102B12. sl.seq 163 0.0016 1

265091D04.xl.seq PAK1 159 0.0035 1

Unp-Fl-F17E2 -Reverse (-) Assignment :KIN3 156 0.0061 1

385009G05.xl.seq 154 0.0091 1

384179F07. sl.seq 153 0.011 1

265001Ell.r2.seq STE11 150 0.020 1

Search 4

Blast Results

Smallest

Sum

High

Probability

Sequences producing High- scoring Segment Pairs: Score P(N) N

396076E03.s2.seq 636 6.9e-85 2

384194F08. sl.seq 674 1.5e-46 1

384254G10. sl.seq 266 3.8e-12 1

385051D09.xl.seq 244 2.1e-10 1

384022B04. sl.seq BCK1 244 2.6e-10 1

CAU73457 Candida albicans Cst20p (CST20) gene, complete 229 5.9e-09 1 YSASTPK Candida albicans serine/threonine protein kinase. 229 5.9e-09 1

384201A03.s2.seq 220 2 .5e-08 1

384168E05.s2.seq 216 3 .le-08 1

CAU87996 Candida albicans CLA4 protein kinase homolog ge . 218 4 -8e-08 1

396039B07. sl.seq 195 3 .3e-06 1

384282H11. sl.seq 187 1 .5e-05 1

385066D07.xl.seq 171 0 00033 1

265235H07.yl.seq 169 0 00047 1

396039H02. sl.seq 162 0 0012 1

396256F10. sl.seq 164 0 0013 1

384102B12. sl.seq 163 0 0016 1

265091D04.xl.seq PAK1 159 0 0035 1

Unp-Fl-F17E2-Reverse (- ) Assignment : KIN3 156 0 0061 1

385009G05.xl.seq 154 0. 0091 1

384179F07. sl.seq 153 0. 011 1

265001Ell.r2.seq STE11 150 0. 020 1

Genbank search results using 384194F08. sl.seq (search 3 and search 4)

Blast Results

Smallest Sum Reading High Probability

Sequences producing High- scoring Segment Pairs: Frame Score P(N) N

.|663254 (Z48149) probable protein kinase [Saccharom.. . -3 452 .0e-55 1

|1419952 (Z74842) ORF YOLlOOw [Saccharomyces cerevis.. . -3 452 4.0e-55 1

. j 1431588 (X99280) protein kinase [Schizosaccharomyce .. . -3 441 1.7e-54 1

|927745 (U33050) Ydr490cp; CAI : 0.11 [Saccharomyces.. . -3 446 2.1e-54 1

J2407613 (AF017995) 3 -phosphoinositide dependent pro.. . -3 232 4.0e-48 2

. j 2505936 (Y15056) PkB kinase [Homo sapiens] -3 232 4.0e-48 2

. j 2665356 (Y15748) PkB kinase [Rattus norvegicus] -3 232 4.1e-48 2

.12832892 (AL021730) hypothetical protein kinase phos.. . -3 393 1.8e-47 1

. j 927730 (U33050) Ydr466wp; CAI: 0.11 [Saccharomyces.. . -3 250 3.5e-47 3

|2980851 (Y07908) serine/threonine protein kinase [D.. . -3 351 3.3e-41 1

.11000069 (U15210) rac-alpha serine/threonine kinase .. . -3 184 2.0e-31 2

. j 1673493 (Z81140) W10G6.2 [Caenorhabditis elegans] -3 277 4.Oe-31 1

. I 294637 (L01624) serine/threonine protein kinase [R.. . -3 277 4.1e-31 1

11834511 (Y10032) serine/threonine protein kinase [H.. . -3 276 5.6e-31 1

,|2463201 (AJ000512) serine/threonine protein kinase .. . -3 276 5.6e-31 1

.1189967 (M80335) protein kinase A-alpha [Homo sapiens] -3 156 5.9e-29 2

,|2632252 (Y12464) serine/threonine kinase [Sorghum b.. . -3 161 2.3e-28 2

.11401040 (D49836) RAC- PK gamma [Rattus norvegicus] -3 167 2.5e-28 2

.J1890142 (D83380) catalytic subunit of cAMP-dependen.. . -3 167 4.1e-28 2

. I 773642 (U23730) vinclozolin resistance protein [Us.. . -3 172 6.5e-28 2

. j 2213667 (U83459) Riml5p [Saccharomyces cerevisiae] -3 255 8.5e-28 1

. j 836721 (D50617) YFL033C [Saccharomyces cerevisiae] -3 255 8.5e-28 1

. I 465398 (D28577) Protein kinase C [Mus musculus] -3 253 1.2e-27 1

.1432274 (L18964) protein kinase C iota [Homo sapiens] -3 253 1.2e-27 1

.1598225 (L33881) protein kinase C iota [Homo sapiens] -3 253 1.2e-27 1

. I 507141 (M81709) cAMP-dependent protein kinase cata.. -3 166 1.2e-27 2

|2253148 (Z82096) ZK909.2a [Caenorhabditis elegans] -3 162 1.2e-27 2

.J304273 (L17008) cAMP-dependent protein kinase [Bla.. -3 166 1.4e-27 2

.|2253149 (Z82096) ZK909.2b [Caenorhabditis elegans] -3 162 1.5e-27 2

.J853791 (Z34989) Ndr protein kinase [Caenorhabditis.. -3 178 1.7e-27 2

|2113796 (X94399) cAMP-dependent protein kinase cata.. -3 159 1.9e-27 2

. I 56916 (X68400) protein kinase [Rattus norvegicus] -3 251 2.4e-27 1

. j 220527 (D90242) nPKC-eta [Mus musculus] -3 251 2.4e-27 1 gi I 665540 (U15983) cAMP-dependent protein kinase cata.. , -3 160 3.1e-27 2 gi j 189989 (M55284) protein kinase C-L [Homo sapiens] -3 250 3.3e-27 1 gi j 806542 (Z49233) calcium-stimulated protein kinase ... -3 249 4.2e-27 1 gi|473894 (U08622) cAMP-dependent protein kinase [Sch... -3 169 4.3e-27 2 gi |484305 (D23667) catalytic subunit of the cAMP-depe... -3 169 4.3e-27 2 gij 853820 (Z35103) Ndr protein kinase [Drosophila mel ... -3 164 4.5Θ-27 2 gi |2632254 (Y12465) serine/threonine kinase [Sorghum b... -3 163 5.6e-27 2 gij 1911 (X05998) C-beta subunit (338 AA) [Sus scrofa] -3 155 6.5e-27 2 gij 516040 (U12335) cAMP-dependent protein kinase cata... -3 159 6.6e-27 2 gi J35479 (X07767) protein kinase catalytic subunit t... -3 156 7.5e-27 2 gi J1487920 (Z75953) F57F5.5 [Caenorhabditis elegans] -3 247 7.6e-27 1 giJ3005054 (AF041843) protein kinase Ukclp [Ustilago ... -3 177 8.4e-27 2 giJ392435 (U00181) protein kinase C [Caenorhabditis e... -3 247 8.7e-27 1 gi J2304746 (A52140) HUMAN NDR [unidentified] -3 172 8.9e-27 2 gij 854170 (Z35102) Ndr protein kinase [Homo sapiens] -3 172 8.9e-27 2 gij 173011 (M17073) cAMP-dependent protein kinase subu... -3 160 9.7e-27 2 gi j 4625 (Y00694) put. kinase (AA 1-380) [Saccharomy... -3 160 9.7e-27 2 gij 1370422 (Z73559) ORF YPL203w [Saccharomyces cerevis... -3 160 9.7e-27 2 gi I 191175 (M63311) cAMP-dependent protein kinase alph... -3 156 1.0e-26 2 gij 156233 (M37120) cAMP-dependent protein kinase cata... -3 156 1.3e-26 2 gi j 1086654 (U41016) coded for by C. elegans cDNA CEESC... -3 178 1.6e-26 2 gi J288120 (X53261) cAMP-dependent protein kinase cata... -3 153 1.7e-26 2 gi J2760821 (L06196) serine/threonine protein kinase [E... -3 174 1.7e-26 2 gi J1050791 (X92517) N1727 gene product [Saccharomyces ... -3 175 1.7e-26 2 gij 1302128 (Z71437) ORF YNLlδlw [Saccharomyces cerevis... -3 175 1.7e-26 2 gi J3043598 (AB011109) KIAA0537 protein [Homo sapiens] -3 196 1.9e-26 2 gi j 1853976 (D31773) protein kinase [Schizosaccharomyce... -3 245 2.0e-26 1 gi J200367 (M12303) cAMP-dependent protein kinase cata... -3 158 2.0e-26 2 gi |387513 (M19960) cAMP-dependent protein kinase alph... -3 158 2.0e-26 2 gij 155792 (M94884) protein kinase C [Aplysia californ... -3 244 2.3e-26 1 gij 178326 (M95936) protein serine/threonine kinase [H... -3 160 2.5e-26 2 gi j 191177 (M63312) cAMP-dependent protein kinase beta... -3 152 2.7e-26 2 gi|200387 (J02626) cAMP-dependent protein kinase beta... -3 152 2.7e-26 2 gi J220704 (D10770) cAMP-dependent protein kinase cata... -3 152 2.7e-26 2 gi j 163532 (J02647) protein kinase beta-catalytic subu... -3 151 2.7e-26 2 gij 189983 (M34181) cAMP-dependent protein kinase cata... -3 151 2.7e-26 2 gi J337491 (M77198) rac protein kinase-beta [Homo sapi... -3 160 3.0e-26 2 gi J2911458 (AF046921) cAMP- ependent protein kinase ca... -3 164 3.1e-26 2 gi J2244814 (Z97336) protein kinase [Arabidopsis thaliana] -3 242 3.1e-26 1 gi |56912 (X57986) cAMP-dependent protein kinase subu... -3 156 3.8e-26 2 gij 162787 (M60482) cAMP-dependent protein kinase Il-b... -3 151 4.0e-26 2 gij 165563 (M20014) protein kinase C [Oryctolagus cuni ... -3 242 4.4e-26 1 giJ35495 (X65293) protein kinase C epsilon [Homo sap... -3 242 4.4e-26 1 gi J2605804 (AF028009) protein kinase C epsilon [Mus mu... -3 242 4.4Θ-26 1 gij 633 (X67154) protein kinase [Bos taurus] -3 156 5.2e-26 2 gij 942578 (U22445) serine/threonine kinase [Mus muscu... -3 157 6.5e-26 2 gij 157052 (M18655) cAMP-dependent protein kinase cata... -3 155 7.3e-26 2 gij 7807 (X16969) catalytic subunit [Drosophila mela... -3 155 7.3e-26 2

Genbank results using 384230A10. s4.seq (search 1)

Blast Results

Smallest Sum Reading High Probability

Sequences producing High-scoring Segment Pairs: Frame Score P (N) N gi 11050791 (X92517) N1727 gene product [Saccharomyces . . +3 921 8.3e-137 3 gij 1302128 (Z71437) ORF YNL161w [Saccharomyces cerevis. +3 921 8.3e-137 3 gij 3005054 (AF041843) protein kinase Ukclp [Ustilago m. +3 817 3.0e-117 2 gij 2443511 (AF009512) protein kinase Orb6p [Schizosacc. +3 769 8.7e-115 2 gij 1870020 (X97657) serine/threonine kinase [Neurospor. +3 725 1.2e-102 2 gij 641979 (U14989) kinase [Colletotrichum trifolii] +3 733 4.5e-100 2 gi 11870019 (X97657) serine/threonine kinase [Neurospor. +3 725 1.3e-99 2 gij 2304746 (A52140) HUMAN NDR [unidentified] +3 647 1.5e-93 2 gi I 854170 (Z35102) Ndr protein kinase [Homo sapiens] +3 647 1.5e-93 2 gi j 853820 (Z35103) Ndr protein kinase [Drosophila mel . +3 629 2.7e-92 2 gij 2304742 (A52135) D. MELANOGASTER NDR [unidentified] +3 372 1.8e-89 3 gij 1086654 (U41016) coded for by C. elegans cDNA CEESC. +3 628 2.0e-85 2 gij 853791 (Z34989) Ndr protein kinase [Caenorhabditis. +3 618 l.le-80 1 gij 506534 (X71057) protein kinase [Nicotiana tabacum] +3 435 l.le-74 3 gij 3135270 (AC003058) putative protein kinase [Arabido. +3 533 5.8e-74 2 gi 1457709 (Z30330) protein kinase [Spinacia oleracea] +3 533 6.3e-74 2 gijl914507 (Z81594) T20F10.1 [Caenorhabditis elegans] +3 575 1.9e-72 1 gij 903942 (U29608) LATS [Drosophila melanogaster] +3 466 2.0e-71 3 gi I 755008 (L39837) tumor suppressor [Drosophila melan. +3 466 2.0e-71 3 gi 1457689 (Z30329) protein kinase [Mesembryanthemum c. +3 548 1.4e-69 1 gij 1200533 (M28500) protein kinase [Euplotes crassus] +3 469 5.0e-60 1 gi 11200509 (U47679) protein kinase [Euplotes crassus] +3 469 3.5e-58 1 gi 11276901 (U43195) Rho-associated, coiled-coil contai. +3 300 3.7e-54 2 gij 1514696 (U58512) Rho-associated, coiled-coil contai. +3 300 3.7e-54 2 gij 1438567 (U61266) Rho-associated kinase beta [Rattus. +3 300 3.8e-54 2 gi I 2736153 (AF021936) myotonic dystrophy kinase-relate. +3 280 1.6e-51 2 gi|l81605 (L08835) myotonic dystrophy kinase [Homo sa. +3 271 2.2e-51 2 gi|l81606 (L08835) myotonic dystrophy kinase [Homo sa., +3 271 2.2e-51 2 gij 976145 (L00727) myotonin-protein kinase, Form VIII.. +3 271 2.4Θ-51 2 gi 11061299 (Z67757) unknown [Schizosaccharomyces pombe] +3 422 2.6e-51 1 gi 1186756 (M94203) protein kinase [Homo sapiens] +3 271 3.0e-51 2 gij 976143 (L00727) myotonin-protein kinase, Form V [H.. +3 271 3.2e-51 2 gij 976146 (L00727) myotonin-protein kinase, Form VII .. +3 271 3.5e-51 2 gi 1307177 (L19268) protein kinase [Homo sapiens] +3 271 3.8e-51 2 gi|l81603 (L08835) myotonic dystrophy kinase [Homo sa .. +3 271 3.8e-51 2 gi|633865 (S72883) myotonin protein kinase, MtPK=thym.. +3 271 3.8e-51 2 gi|l81604 (L08835) myotonic dystrophy kinase [Homo sa.. +3 271 3.9e-51 2 gi 1976147 (L00727) myotonin-protein kinase, Form VI [.. +3 271 4.0e-51 2 giJ976144 (L00727) myotonin-protein kinase, Form I [H.. +3 271 4.1e-51 2 gij 1384133 (U38481) ROK-alpha [Rattus norvegicus] +3 285 5.7e-51 2 gi 11326078 (U36909) Rho-associated kinase [Bos taurus] +3 285 5.7e-51 2 gi 11514698 (U58513) Rho-associated, coiled-coil contai.. +3 285 5.7e-51 2 gij 556903 (Z21503) DM protein kinase [Mus musculus] +3 268 6.8e-51 2 gijl695873 (U59305) ser-thr protein kinase PK428 [Homo.. +3 266 8.2e-51 2 gi|563526 (Z38015) myotonic dystrophy protein kinase .. +3 268 1.0e-50 2 gi 1171380 (M62506) putative [Saccharomyces cerevisiae] +3 402 1.4e-50 2 gi 11938422 (U97001) Similar to serine/threonine-protei .. +3 253 2.6e-50 3 gi|2982220 (AF037073) Rho-associated kinase alpha [Xen.. +3 283 3.8e-50 2 gijl323137 (Z72877) ORF YGR092 [Saccharomyces cerevis.. +3 413 4.4e-50 1

Genbank search results using 384286E10. si .seq (search 1 and search 2)

Blast Results

WARNING: -hspmax 100 was exceeded with 1 of the database sequences, with as many as 139 HSPs being found at one time. Smallest Sum Reading High Probability

Sequences producing High-scoring Segment Pairs: Frame Score P(N) N gi I 295681 (M24929) protein kinase [Saccharomyces cere... +3 284 5.0e-32 1 gi|817862 (Z49702) Ypk2p [Saccharomyces cerevisiae] +3 284 5.0e-32 1 gij 172181 (M21307) protein kinase [Saccharomyces cere... +3 280 1.8e-31 1 giJ486213 (Z28126) ORF YKL126w [Saccharomyces cerevis... +3 280 1.8e-31 1 gi|2911462 (AF046923) serine/threonine protein kinase ... +3 194 1.7e-20 2 gi|458284 (U05811) serine/threonine protein kinase [T... +3 193 5.0e-20 2 gi|206181 (M18330) protein kinase C delta subspecies ... +3 175 2.6e-15 1 gi 1189510 (M60725) p70 ribosomal S6 kinase alpha-II [... +3 174 3.3e-15 1 gi|206840 (M57428) S6 kinase [Rattus norvegicus] +3 174 3.3e-15 1 gij 1562 (X54415) G3 serine/threonine kinase [Orycto... +3 174 3.4e-15 1 gij 189508 (M60724) p70 ribosomal S6 kinase alpha-I [H... +3 174 3.4e-15 1 gij 2222775 (Y12002) protein kinase C homologue [Neuros... +3 173 5.7e-15 1 giJ200381 (M69042) [Mouse protein kinase C delta mRNA... +3 172 7.2e-15 1 gi|53437 (X60304) protein kinase [Mus musculus] +3 172 7.2e-15 1 gi|501075 (U10016) protein kinase C [Trichoderma reesei] +3 172 8.0e-15 1 gi|2687849 (Y15839) protein kinase C [Cochliobolus het... +3 169 2.2e-14 1 gij 507900 (U10549) protein kinase C [Aspergillus niger] +3 168 3.1e-14 1 gi J206842 (M58340) S6 protein kinase [Rattus norvegicus] +3 165 7.3e-14 1 gij 1136302 (D38108) protein kinase [Schizosaccharomyce... +3 162 l.le-13 2 gij 165563 (M20014) protein kinase C [Oryctolagus cuni... +3 161 3.1e-13 1 giJ35495 (X65293) protein kinase C epsilon [Homo sap... +3 161 3.1e-13 1 gi|206183 (M18331) protein kinase C epsilon subspecie... +3 160 4.3e-13 1 gi J2605804 (AF028009) protein kinase C epsilon [Mus mu... +3 159 6.0e-13 1 gi I 832908 (X81142) protein kinase C [Candida albicans] +3 159 6.5e-13 1 gij 558100 (L07032) protein kinase C-theta [Homo sapiens] +3 156 1.6e-12 1 giJ558099 (L01087) protein kinase C-theta [Homo sapiens] +3 156 1.6e-12 1 gi J458923 (U00029) Sch9p: cAMP-dependent protein kina... +3 156 1.7e-12 1 giJ4426 (X12560) SCH9 protein (AA 1-824) [Saccharom... +3 156 1.7e-12 1 gij 5279 (X57629) Sch9 [Saccharomyces cerevisiae] +3 156 1.7e-12 1 gijl778590 (U82935) protein kinase C2 A isoform [Caeno... +3 150 2.5e-12 2 gijl000069 (U15210) rac-alpha serine/threonine kinase ... +3 151 2.6e-12 2 gi|214789 (M20188) S6 kinase II beta [Xenopus laevis] +3 154 3.1e-12 1 giJ3114960 (Y13104) Protein kinase C-related kinase (P... +3 149 3.4e-12 2 gijl73427 (L07637) protein kinase C [Schizosaccharomy ... +3 154 3.4e-12 1 gi|303941 (D14338) protein kinase [Schizosaccharomyce... +3 154 3.4e-12 1 gij 401772 (L07598) ribosomal protein S6 kinase 2 [Horn... +3 152 5.9e-12 1 gi|220527 (D90242) nPKC-eta [Mus musculus] +3 152 6.3e-12 1 gi|l033033 (X85106) ribosomal S6 kinase [Homo sapiens] +3 152 6.3e-12 1 gij 551556 (M28488) ribosomal protein S6 kinase [Gallu... +3 152 6.4e-12 1 gij 56916 (X68400) protein kinase [Rattus norvegicus] +3 151 8.7e-12 1

Genbank search results using 384362E11. sl.seq (search 1 and search 2) Blast Results

Smallest Sum Reading High Probability

Sequences producing High-scoring Segment Pairs: Frame Score P(N) N gi 1486213 (Z28126) ORF YKL126w [Saccharomyces cerevis... +2 391 6, ,6e-65 3 gij 172181 (M21307) protein kinase [Saccharomyces cere... +2 387 2. ,4e-64 3 giJ295681 (M24929) protein kinase [Saccharomyces cere... +2 380 5, .8e-63 3 gi I 817862 (Z49702) Ypk2p [Saccharomyces cerevisiae] +2 380 5.8e-63 3 gi 12911462 (AF046923) serine/threonine protein kinase ... . +2 355 1.6e-58 3 gi|458284 (U05811) serine/threonine protein kinase [T... . +2 353 7.5e-58 3 gi 11000069 (U15210) rac-alpha serine/threonine kinase ... . +2 267 2.1e-52 3 gi 1558100 (L07032) protein kinase C-theta [Homo sapiens] 1 +2 274 3.1e-43 2 gij 558099 (L01087) protein kinase C-theta [Homo sapiens] 1 ++22 274 3.1e-43 2 gij 220574 (D11091) 'Protein Kinase¹ [Mus musculus] +2 271 l.le-42 2 gij 1834511 (Y10032) serine/threonine protein kinase [H.. . +2 270 1.3e-42 2 gi 12463201 (A 000512) serine/threonine protein kinase .. . +2 270 1.3e-42 2 gi|l673493 (Z81140) W10G6.2 [Caenorhabditis elegans] +2 263 1.6e-42 2 gij 294637 (L01624) serine/threonine protein kinase [R.. +2 269 1.7e-42 2 gijll36302 (D38108) protein kinase [Schizosaccharomyce.. +2 144 3.2e-42 3 giJ206191 (M15522) protein kinase [Rattus norvegicus] +2 237 1.5e-40 2 gi I 56920 (X04139) protein kinase C C-terminal region.. +2 237 1.5e-40 2 gi|2996092 (U63742) rac serine-threonine kinase homolo.. +2 189 1.9e-40 3 giJ210068 (M80675) gag:akt fusion protein [AKT8 retro., +2 222 2.5e-39 4 gij 3116064 (AJ223715) s-sgkl [Squalus acanthias] +2 264 l.le-38 2 gij 3116066 (AJ223716) s-sgk2 [Squalus acanthias] +2 264 2.8e-38 2 gij 942578 (U22445) serine/threonine kinase [Mus muscu.. +2 225 3.4e-38 3 gi 12586064 (AF027183) protein kinase C-related kinase .. +2 244 3.8e-38 2 gi 12652950 (Z50028) F46F6.2 [Caenorhabditis elegans] +2 256 4.3e-38 3 gij 1667370 (Y07611) protein kinase [Mus musculus] +2 250 6.0e-38 2 gi 11220289 (Z70043) unknown [Schizosaccharomyces pombe] +2 135 6.4e-38 3 gij 206181 (M18330) protein kinase C delta subspecies .. +2 235 7.1e-38 2 gi 1167718 (M59744) protein kinase 2 [Dictyostelium di .. +2 232 8.7e-38 3 gij 1778590 (U82935) protein kinase C2 A isoform [Caeno.. +2 247 1.0e-37 2 gij 1000125 (U33052) PRK2 [Homo sapiens] +2 245 l.le-37 2 gi|914100 (S75548) protein kinase PRK2 [human, DX3 B- .. , +2 245 l.le-37 2 gi|200642 (M94335) protein kinase [Mus musculus] +2 224 1.2e-37 3 gi J2062375 (U75358) myeloma protein kinase [Rattus nor.. , +2 245 1.5e-37 2 gi|l89510 (M60725) p70 ribosomal S6 kinase alpha-II [... +2 242 1.7e-37 2 gi|206840 (M57428) S6 kinase [Rattus norvegicus] +2 242 1.7e-37 2 gij 1562 (X54415) G3 serine/threonine kinase [Orycto... +2 242 2.0e-37 2 gij 189508 (M60724) p70 ribosomal S6 kinase alpha-I [H... +2 242 2.0e-37 2 giJ287807 (X65687) serine/threonine protein kinase [M... +2 222 2.3e-37 3 gi J485403 (D30040) RAC protein kinase alpha [Rattus n... +2 222 2.3e-37 3 giJ35481 (X61037) human protein kinase B [Homo sapiens] +2 222 2.8e-37 3 gi I 520587 (D10495) protein kinase C delta-type [Homo ... +2 238 4.1e-37 2 gi 13114960 (Y13104) Protein kinase C-related kinase (P... +2 236 4.5e-37 2 gi|200381 (M69042) [Mouse protein kinase C delta mRNA... +2 235 4.9e-37 2 gi|l89985 (L07861) protein kinase C-delta 13 [Homo sa... +2 238 4.9e-37 2 gijl89680 (L07860) protein kinase C-delta 13 [Homo sa... +2 238 4.9e-37 2 gij 173427 (L07637) protein kinase C [Schizosaccharomy... +2 252 5.4e-37 2 gi|303941 (D14338) protein kinase [Schizosaccharomyce... +2 252 5.4e-37 2 gi 1190828 (M63167) rac protein kinase-alpha [Homo sap... +2 222 6.1e-37 3 giJ485405 (D30041) RAC protein kinase beta [Rattus no... +2 215 8.4e-37 3 giJ414286 (U02967) protein kinase C [Lytechinus pictus] +2 246 8.7e-37 2 giJ53437 (X60304) protein kinase [Mus musculus] +2 233 9.2e-37 2 gi|206169 (K03486) protein kinase C type III [Rattus ... +2 237 1.2e-36 2 gij 825510 (D26180) PKN [Rattus norvegicus] +2 246 1.7e-36 2 gi 11778592 (U82936) protein kinase C2 B isoform [Caeno... +2 247 1.8e-36 2 gi 11213263 (Z69795) unknown [Schizosaccharomyces pombe] +2 244 2.2e-36 2 gi|631 (X61036) bovine protein kinase B [Bos taurus] +2 222 2.2Θ-36 3 gi|303939 (D14337) protein kinase [Schizosaccharomyce... +2 244 2.5e-36 2 gij 868172 (U29376) similar to protein kinase C [Caeno... +2 247 2.8e-36 2 gi|l58129 (J04848) protein kinase C [Drosophila melan... +2 238 2.9e-36 2 gij 1041183 (D43890) PKN [Xenopus sp.] +2 248 3.0e-36 2 gij 206842 (M58340) S6 protein kinase [Rattus norvegicus] +2 242 3.5e-36 2 gij 8353 (X05076) protein kinase C (AA 1-639) [Droso... +2 247 4.0e-36 2 gi 12822146 (AC002299) Protein kinase C beta (5' partia... +2 237 4.1e-36 2 gij 2065190 (Y12237) protein kinase C [Hydra vulgaris] +2 242 4.3e-36 2 gi |163530 (M13973) protein kinase C [Bos taurus] +2 242 4.5e-36 2 gi j 1673 (X04796) PKC-gamma (AA 1-672) [Oryctolagus ... +2 242 4 .5e-36 2 gi 2297395 (A41791) unnamed protein product [unidentif ... +2 242 4 .5e-36 2 gi 35483 (X52479) protein kinase C alpha (AA 1-672) ... +2 242 4 .5e-36 2 g 200363 (M25811) protein kinase C-alpha [Mus musculus] +2 242 4 .5e-36 2 gi 49939 (X52685) protein kinase C [Mus musculus] +2 242 4 .5e-36 2 gi 56914 (X07286) protein kinase C alpha (AA 1-672) ... +2 242 4 .5e-36 2 gi 55132 (X52684) protein kinase C [Mus musculus] +2 242 4 .5e-36 2 gi 3114956 (Y13099) Serine/Threonine protein kinase [S... +2 248 4 5e-36 2 gi 1487920 (Z75953) F57F5.5 [Caenorhabditis elegans] +2 237 5 3e-36 2 gi 1000127 (U33053) PRK1 [Homo sapiens] +2 250 6 le-36 2 gi 914098 (S75546) protein kinase PRK1 [human, fetal ... +2 250 6 le-36 2 gi 825505 (D26181) PKN [Homo sapiens] +2 250 6 le-36 2 gi 1671 (X04795) PKC-beta (AA 1-671) [Oryctolagus c... +2 237 8 5e-36 2 gi 35489 (X06318) PKC beta 1 (AA 1-671) [Homo sapiens] +2 237 8 5e-36 2 gi 206175 (M19007) protein kinase C beta-1 [Rattus no... +2 237 8 5e-36 2 gi 56959 (X04439) protein kinase C (AA 1-671) [Rattu... +2 237 8. 5e-36

2..

Genbank sseeaarrcchh iresults using 396076E03. s2. seq (search and search

4)

Blast Results

Smallest Sum Reading High Probability

Sequences producing High-scoring Segment Pairs: Frame Score P (N) N

. I 663254 (Z48149) probable protein kinase [Saccharom.. . -3 417 8.2e-102 2

|1419952 (Z74842) ORF YOLlOOw [Saccharomyces cerevis.. . -3 417 8.2e-102 2 j 927745 (U33050) Ydr490cp; CAI: 0.11 [Saccharomyces.. . -3 409 2.1e-99 2

.|1431588 (X99280) protein kinase [Schizosaccharomyce.. . -3 409 2.0e-92 2

.J2832892 (AL021730) hypothetical protein kinase phos.. . -3 405 9.9e-81 2

,|2407613 (AF017995) 3-phosphoinositide dependent pro.. . -3 280 l.le-79 3

.12505936 (Y15056) PkB kinase [Homo sapiens] -3 280 l.le-79 3 j 2665356 (Y15748) PkB kinase [Rattus norvegicus] -3 278 2.3e-79 3

.j 927730 (U33050) Ydr466wp; CAI: 0.11 [Saccharomyces.. . -3 277 5.3e-74 4

.|2980851 (Y07908) serine/threonine protein kinase [D.. . -3 307 4.7e-63 2

.j 2435556 (AF026207) Similar to protein kinase [Caeno.. . -3 259 5.6e-49 2

11000069 (U15210) rac-alpha serine/threonine kinase .. . -3 197 3.9e-48 3

.j 3043598 (AB011109) KIAA0537 protein [Homo sapiens] -3 233 4.4e-46 5

.11401040 (D49836) RAC-PK gamma [Rattus norvegicus] -3 198 2.2e-45 4

.135479 (X07767) protein kinase catalytic subunit t.. . -3 188 6.2e-45 3

, j 191175 (M63311) cAMP-dependent protein kinase alph.. . -3 188 6.2e-45 3

J200367 (M12303) cAMP-dependent protein kinase cata.. . -3 190 1.2e-44 3

. I 387513 (M19960) cAMP-dependent protein kinase alph.. . -3 190 1.2e-44 3

.J1890142 (D83380) catalytic subunit of cAMP-dependen.. . -3 185 1.3e-44 3

.J2253148 (Z82096) ZK909.2a [Caenorhabditis elegans] -3 185 1.9e-44 3

J2299249 (A44422) unnamed protein product [Eimeria m.. . -3 148 2.3e-44 5

. I 56912 (X57986) cAMP-dependent protein kinase subu.. -3 188 2.4e-44 3

.|288120 (X53261) cAMP-dependent protein kinase cata.. -3 184 2.5e-44 3

J2253149 (Z82096) ZK909.2b [Caenorhabditis elegans] -3 185 3.4Θ-44 3

. j 633 (X67154) protein kinase [Bos taurus] -3 188 4.8Θ-44 3

.J2760821 (L06196) serine/threonine protein kinase [E.. -3 200 5.3e-44 3

. j 1673493 (Z81140) W10G6.2 [Caenorhabditis elegans] -3 269 l.le-43 2

. jl911 (X05998) C-beta subunit (338 AA) [Sus scrofa] -3 187 1.4e-43 3

. I 2632252 (Y12464) serine/threonine kinase [Sorghum b.. -3 185 1.6Θ-43 4

.|773642 (U23730) vinclozolin resistance protein [Us.. -3 191 2.6e-43 4 gi |200642 (M94335) protein kinase [Mus musculus] -3 132 2.8e-43 5 gi J2213667 (U83459) Riml5p [Saccharomyces cerevisiae] -3 248 2.8e-43 4 gi | 836721 (D50617) YFL033C [Saccharomyces cerevisiae] -3 248 2.8e-43 4 gi j 603542 (X83510) RAC protein kinase DRAC-PK66 [Dros .. -3 187 3.7e-43 4 gi J398924 (Z26242) Daktl serine-threonine protein kin.. -3 187 3.7e-43 4 gi | 166600 (M93023) SNFl-related protein kinase [Arabi.. -3 193 4.0e-43 4 gi J1742969 (X94757) ser/thr protein kinase [Arabidopsi .. -3 193 4.0e-43 4 gi j 1279425 (Z71757) calmodulin-domain protein kinase [.. -3 145 4.4e-43 5 gi J178326 (M95936) protein serine/threonine kinase [H.. -3 191 6.5e-43 4 gi j 903942 (U29608) LATS [Drosophila melanogaster] -3 210 7.3e-43 4 gi j 755008 (L39837) tumor suppressor [Drosophila melan.. -3 210 7.3e-43 4 gi J1335643 (X83510) RAC protein kinase DRAC-PK85 [Dros.. -3 187 9.3e-43 4 gi J337491 (M77198) rac protein kinase-beta [Homo sapi.. -3 191 1.2e-42 4 gi J287807 (X65687) serine/threonine protein kinase [M.. -3 127 1.4e-42 5 gi |485403 (D30040) RAC protein kinase alpha [Rattus n.. -3 127 1.4e-42 5 gi |496385 (D26602) protein kinase [Nicotiana tabacum] -3 189 1.4e-42 4 gi J1743009 (Y10036) SNFl-related protein kinase [Cucum.. , -3 192 1.8e-42 4 gi j 1935916 (U83797) StubSNFl protein [Solanum tuberosum] -3 196 7.0e-42 4 g j 191177 (M63312) cAMP-dependent protein kinase beta.. , -3 184 1.8e-41 3 gi| |200387 (J02626) cAMP-dependent protein kinase beta... -3 184 1.8e-41 3 gil [220704 (D10770) cAMP-dependent protein kinase cata... -3 184 1.8e-41 3 ^gil 163532 (J02647) protein kinase beta-catalytic subu... -3 183 1.8e-41 3 ^gil 189983 (M34181) cAMP-dependent protein kinase cata... -3 183 1.8e-41 3 ^gil 942578 (U22445) serine/threonine kinase [Mus muscu... -3 188 3.0e-41 4 gil 210068 (M80675) gag.-akt fusion protein [AKT8 retro... -3 127 5.5e-41 5 ^gil 172175 (J02665) protein kinase [Saccharomyces cere... -3 186 5.5e-41 3 gil 173009 (M17072) cAMP-dependent protein kinase subu... -3 186 5.5e-41 3 gil 1008352 (Z49439) ORF YJL164C [Saccharomyces cerevis... -3 186 5.5e-41 3 ^gil 162787 (M60482) cAMP-dependent protein kinase Il-b... -3 183 5.5e-41 3 ^gil 407516 (Z26878) unknown [Saccharomyces cerevisiae] -3 187 5.6e-41 3 gil 486291 (Z28166) ORF YKL166c [Saccharomyces cerevis... -3 187 5.6e-41 3 gil 156233 (M37120) cAMP-dependent protein kinase cata... -3 178 7.4e-41 3 gil 173011 (M17073) cAMP-dependent protein kinase subu... -3 188 8.3e-41

3..

Claims

1. A method of identifying a compound which modulates the activity to different extents of (a) a host yeast cell protein kinase or kinases and (b) an protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, wherein a compound is exposed to

(1) the host yeast cell is a pathogenic yeast and the source other than the host yeast cell is any source other than the host yeast cell or

2. A method of identifying a compound which modulates the activity to different extents of (a) a protein kinase derivable from a first source and (b) a protein kinase derivable from a second source, both said protein kinases being equivalent to the same host yeast cell protein kinase or kinases, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the first source and

2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the second source and the effect of the compound on the phenotype of the said yeast cells is measured.

3. A method according to claim 1 or 2 wherein the host yeast cell other than the host yeast cell that is a pathogenic yeast is from any one of the genera Saccharomyces, including Saccharomyces cerevisiae, Candida, including Candida albicans, Pichia, Kluyveromyces, Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces,

Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, Aspergillus, including Aspergillus fumigatus, Cryptococcus, including Cryptococcus neoformans, and Histoplasma, including Histoplasma capsulatum.

4. A method according to any one of claims 1 to 3 wherein a yeast host cell which is not capable of expressing the said host yeast cell protein kinase or kinases is substantially not capable of growing unless the said yeast host cell is capable of expressing the said protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases.

5. A method according to any one of claims 1 to 4 wherein at least one protein kinase from a source other than the host yeast cell is a human protein kinase.

6. A method according to any one of claims 1 to 5 wherein the said host yeast cell protein kinase or kinases is Pkhl and/or Pkh2, wherein Pkhl is the polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae and Pkh2 is the polypeptide encoded by open reading frame YOLlOOw of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae.

1. A method according to any one of claims 1 to 6 wherein the said protein kinase equivalent to the said host yeast cell protein kinase or kinases is PDKl.

8. A method according to any one of claims 1 to 5 wherein the said host yeast cell protein kinase or kinases is Ypkl and/or Yrk2.

9. A method according to any one of claims 1 to 5 and 8 wherein the said protein kinase equivalent to the said host yeast cell protein kinase or kinases is serum and glucocorticoid induced protein kinase (SGK) or protein kinase B (PKB).

10. The method of any one of claims 2 to 9 wherein the first source is a human and the second source is a pathogenic yeast from any one of the genera Candida spp, Blastomyces spp, for example B. dermatitidis, Coccidioides spp, for example C. immitis, Histoplasma spp, for example H. capsulatum, Sporothrix spp, for example S. schenckii, Aspergillus spp, for example A. fumigatus, A. flavus, A. niger, Phialophora compacta (Fonsecaea compacta), P. pedrosoi (F. pedrosi), P. verrucosa, Cladosporium carrionii, Rhinocladiella aquaspersa, Cryptococcus spp, for example C. neoformans, Cephalosporium spp, Fusarium spp, Histoplasma spp, for example H. capsulatum, Pneumocystis carinii, Rhizopus spp, Rhizomucor spp, Madurella spp, for example M. mycetomatis, M. grisea, Pseudallescheria boydii, Paracoccidioides spp, for example P. brasiliensis, Prototheca spp, for example P. wickerhamii, Epidermophyton spp, Microsporum spp, Trichophyton spp, Malassezia spp, for example M. furfur (Pityrosporum orbiculare)

11. A method of identifying a compound that modulates (inhibits) the activity of PDKl derivable from a first source, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of human PDKl (Pkhl and Pkh2) and (b) is capable of expressing PDKl derivable from the said first source and optionally

2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide that is a functional equivalent of human PDKl (Pkhl and/or Pkh2) and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified.

12. A method of identifying a compound that modulates (inhibits) the activity of a functional equivalent of Ypkl and/or Ykr2 derivable from a first source, wherein a compound is exposed to

1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of Ypkl and/or Yrk2 and (b) is capable of expressing a functional equivalent of Ypkl and/or Ykr2 (for example SGK) derivable from the said first source and optionally 2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide (for example, an endogenous polypeptide) that is a functional equivalent of Ypkl and/or Yrk2 and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified.

13. A yeast cell that is not capable of expressing Pkhl and Pkh2 or any functional equivalent thereof.

14. A yeast cell that is not capable of expressing endogenous Pkhl and/or Pkh2.

15. A yeast cell according to claim 14 that is capable of expressing a functional equivalent of Pkhl and/or Pkh2 that is not endogenous Pkhl or Pkh2.

16. The yeast cell of claim 15 wherein the said functional equivalent is human PDKl or a variant, fusion or derivative thereof.

17. A yeast cell according to any one of claims 13 to 15 wherein the open reading frame encoding Pkhl or Pkh2 is disrupted by insertion of a selectable marker.

18. A yeast cell wherein one or more genes encoding a functional equivalent of human PDKl is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human PDKl .

19. A yeast cell according to claim 18 wherein each such gene encoding a functional equivalent of human PDKl is mutated such that the yeast cell is not capable of expressing a functional equivalent of human PDKl .

20. A method of identifying a compound that modulates (inhibits) the activity of PDKl wherein a yeast cell according to any one of claims 13 to 19 is used.

21. Use of a yeast cell according to any one of claims 13 to 19 in a method of identifying a compound that modulates (inhibits) the activity of PDKl.

22. The method of claim 20 or use of claim 21 wherein the PDKl is mammalian PDKl .

23. The method of claim 20 or use of claim 21 wherein the PDKl is a yeast PDKl, for example Candida PDKl .

24. A protein kinase derivable from yeast capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa-Arg-Xaa-Xaa- (Ser/Thr)-Hyd.

25. A protein kinase derivable from yeast capable of being phosphorylated by Pkhl or Pkh2 or PDKl.

26. A protein kinase according to claim 24 or 25 wherein the said protein kinase is Ypkl from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp or Ykr2 from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp.

27. A variant, derivative, fragment or fusion or a fusion of a variant, derivative or fragment of a protein kinase as defined in claim 26 that is capable of being phosphorylated by Pkhl or Pkh2 or mammalian, preferably human, PDKl and/or capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa- Arg-Xaa-Xaa-Ser/Thr-Hyd.

28. A yeast, for example S. cerevisiae or Candida, cell wherein one or more endogenous genes encoding a functional equivalent of human SGK is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human SGK.

29. A yeast cell according to claim 28 wherein the said gene is Ypkl or Yrk2.

30. A yeast cell according to claim 27 or 29 wherein each such endogenous gene encoding a functional equivalent of human SGK or Ypkl or Yrk2 is mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or Ypkl or Yrk2.

32. A method of identifying a compound which blocks the activation of a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 and is not SGK, PKBα or p70S6 kinase by an interacting polypeptide, for example Pkhl, Pkh2 or PDKl, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBα or p70S6 kinase or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide that is a functional equivalent of Ypkl and/or Ykr2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof.

33. The use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDKl to phosphorylate and/or activate a polypeptide that is Ypkl and/or Ykr2 or SGK or PKBα or a functional equivalent thereof or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion.

34. The use of PDKl or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof to phosphorylate and/or activate a polypeptide that is Ypkl and/or Ykr2 or SGK or a functional equivalent thereof or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion that is not PKBα or p70S6 kinase.

35. A kit of parts comprising means useful for carrying out the method as defined in any one of Claims 1 to 10.

36. A kit of parts according to claim 35 comprising a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a first source and

2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from an source other than the first source.

37. Any novel protein kinase as herein disclosed.

38. A compound identifiable by the method of any one of claims 1 to 12, 20, 22, 23 or 32.

39. A compound according to claim 38 that is capable of inhibiting mammalian PDKl or SGK.

40. A compound according to claim 38 that is capable of inhibiting a fungal functional equivalent of PDKl (which may be Pkhl or Pkh2) or SGK (which may be Ypkl or Yrk2).

41. A compound according to any one of claims 38 to 40 for use in medicine.

42. Use of a compound according to claim 40 in the manufacmre of a medicament for the treatment of a fungal, for example a Candida infection, for example thrush.

43. Use of a compound according to claim 39 in the manufacmre of a medicament for the treatment of cancer.

44. A substantially pure polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative or a substantially pure polypeptide encoded by open reading frame YOLlOOw of

S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae. or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative wherein the polypeptide does not comprise the amino acid sequence of human PDKl or Drosophila PDKl (DSTPK61).

45. A recombinant polynucleotide suitable for expressing a polypeptide as defined in claim 44.

46. A host cell comprising a recombinant polynucleotide as defined in claim 45.

47. A method of making a polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative the method comprising culturing a host cell as defined in Claim 46 which expresses said polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative and isolating said polypeptide or a variant, fragment, derivative or fusion thereof or fusion of a said variant, or fragment or derivative.

48. A polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative obtainable by the method of Claim 47.

49. An antibody reactive towards a polypeptide as defined in claim 44 or 48.