CA2213680A1 - Nucleotide sequences and protein sequences - Google Patents

Abstract

A nucleotide sequence is described. The nucleotide sequence or the expression product of the nucleotide sequence has the capability of not substantially affecting the interaction of G.beta. with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof.

Description

- CA 02213680 1997-10-1~

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NUCLEOTIDE SEQUENCES AND PROTEIN SEQUENCES

The present invention relates to nucleotide sequences and protein sequences. In particular, the present invention relates to nucleotide sequences and protein sequences 5 that affect interactions of cellular components.

According to Cerione and Zheng (The Dbl family of oncogenes Current Opinion In Cell Biology 8, 216-222 (1996)), genetic screening and biochemical studies during the past years have led to the discovery of a certain family of cell growth regulatory proteins and o oncogene products for which the Dbl oncoprotein is the prototype. Another review on Dbl is presented by Machesky and Hall (1996 Trends In Cell Biology 6 pp 3-4-310).

Cerione and Zheng (ibid) say that proto-Dbl is a 115 kDa cytoskeleton-associated protein that is found in tissues such as brain, ovary, testis and adrenal glands. Oncogenic 5 activation of Dbl occurs as a result of an amino-terminal truncation of proto-Dbl which leaves residues 498-925 fused with the product of an as yet unidentified gene which is localised on chromosome 3.

Cerione and Zheng also say that a region located between residues 498 and 674 of proto-20 Dbl - which is retained by oncogenic Dbl - has significant similarities with the S.
cerevisiae cell division cycle molecule Cdc24 and the breakpoint cluster gene product Bcr (see also Hart et al 1991 Nature 354 311-314; Miyamoto et al 1991 Biochem Biophys Res Commun 181 604-610; Ron et al 1991 New Biol 3 372-379). This region - which is referred to as being the DH domain - was later shown to be responsible for the GEF (i.e.
25 guanine nucleotide exchange factor) activity of the Dbl oncoprotein and to be critical for its transforming function (see also Hart et al J Biol Chem 269 62-65).

Cerione and Zheng also report that since the initial identification of Dbl as a GEF for Rho-type GTP binding proteins, a number of oncogene products and growth regulatory 30 molecules have been shown to contain a DH domain in tandem with another region designated PH (i.e. a pleckstrin homology domain which is found between residues 703-812 in of proto-Dbl). Many of these products and molecules - such as Bcr, Cdc24, Sos, CA 02213680 1997- lO- l~

' ' 2 Vav, ect-2, 0st, Tim, Lbc, Lfc and Dbc - have been implicated in cell growth regulation.
Cerione and Zheng provide details on each of these products and molecules. In addition, these and other products and molecules are discussed below.

5 Cerione and Zheng (ibid:) end their Abstract by saying:

"Despite the increasing interest in the Dbl family of proteins, there is still a good deal to learn regarding the biochemical mechanisms that underlie their diverse biological functions. "

As mentioned above, it is known that proto-Dbl has significant similarities with the S.
cerevisiae cell division cycle molecule Cdc24 (see again Hart et al 1991 Nature ~ 311-314; Miyamoto et al 1991 Biochem Biophys Res Commun 181 604-610; Ron et al 1991 New Biol 3 372-379). However, whilst it is known that the Rho-family GTPases and15 their regulators are essential for cytoskeletal reorganisation and transcriptional activation in response to extracellular signalsl 2, little is known about what lirlks these molecules to membrane receptors. For example, in the budding yeast Saccharomyces cerevisiae, haploid cells respond to mating pheromone through a G-protein coupled receptor via G~
resulting in cell cycle arrest, transcriptional activation, and polarised growth towards a 20 mating partner45. Recently, the Rho-family GTPase Cdc42p and its exchange factor Cdc24p have been implicated in the mating process6 7 but their specific role is unknown.

However, in our studies (which are presented below) on Saccharomyces cerevisiae we have been able to identify hitherto unrecognised regions that play a key role in the 25 interaction of cellular components. This finding has broad implications - not only for the design of anti-fungal drugs (such as those that could be directed against the yeast Candida) but also in the screening and design of agents that can affect oncogenes such as Dbl, in particular proto-Dbl.

Moreover, in our studies (which are presented beolw), we have identified novel cdc24 30 alleles which do not affect vegetative growth but drastically reduce the ability of yeast cells to mate. When exposed to mating pheromone these mutants arrest growth, activate transcription, and undergo characteristic morphological and actin cytoskeleton polarisation. However, the mllt~nt~ are unable to orient towards a pheromone gradient and instead position their mating projection adjacent to their previous bud site.
Strikingly, these mut~nt.c are specifically defective in the binding of Cdc24p to G~r. This 5 work demonstrates that the association of an exchange factor and the ~r-subunit of a hetero-trimeric G-protein links receptor-mediated activation to oriented cell growth.

Aspects of the present invention are set out in the claims.

0 It is to be noted that the nucleotide sequence presented as SEQ ID No. l is quite different to the DH domain and the PH domain discussed by Cerione and Zheng (ibi~). It is also to be noted that the nucleotide sequence presented as SEQ ID No.l is in a region quite different to the DH domain and the PH domain.

5 One important aspect of the present invention is that we have found it is possible to affect the interation of Cdc24 with a ~ subunit or even a ~r subunit of a hetero-trimeric G-protein (hereinafter collectively referred to as "G~"). If the interaction is detrimentally affected (such as lost) then this may in turn prevent (or at least reduce) sign~lling (possibly GEF activity) being passed to Rho-family GTPase. Hence, the present 20 invention also covers the use of any one or more of the aforementioned aspects of the present invention to have an effect on a signal being passed to Rho-family GTPases.

The term "derivative or homologue" in relation to the nucleotide sequence of the present invention includes any substitution of, modification of, replacement of, deletion of or 25 addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence or the expression product thereof has the capability of not substantially affecting the interaction of G~ with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof. Inparticular, the term "homologue" covers homology with respect to function. With 30 respect to sequence homology (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. l in the attached sequence listings. More preferably there is at least 95 %, CA 02213680 1997-10-1~

such as at least 98%, homology to the sequence shown as SEQ ID No. 1 in the attached sequence listings.

The term "derivative or homologue" in relation to the protein sequence of the present 5 invention includes any substitution of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has the capability of not substantially affecting the interaction of G,B
with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof. In particular, the term "homologue" covers o homology with respect to function. With respect to sequence homology (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No.2 in the attached sequence listings.
More preferably there is at least 95%, such as at least 98%, homology to the sequence shown as SEQ ID No. 2 in the attached sequence listings.

Examples of homologues of Cdc24p include any one or more of the homologues listed above or below, such as Dbl or proto-Dbl.

The term "mutant" in relation to the nucleotide sequence of the present invention 20 means a variant of SEQ ID No. 1 but wherein that variant or the expression product thereof has the capability of substantially affecting the interaction of G~3 with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof.

2s Preferred mut~ntc of the nucleotide sequence of the present invention include any one or more of the nucleotide sequences presented as SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7.

The term "mutant" in relation to the protein sequence of the present invention means a 30 variant of SEQ ID No. 2 but wherein that variant has the capability of substantially affecting the interaction of G,B with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof.

Preferred mutants of the protein sequence of the present invention include any one or more of the protein sequences presented as SEQ ID No. 4, SEQ ID No. 6 or SEQ ID
No. 8.
s The term "growth behaviour" includes growth per se (but not vegetative growth ofyeast), growth control and growth orientation of cells. In some aspects, it includes at least growth orientation of cells. The term may also include the mating pattern (e.g.
mating per se or mating behaviour) of cells.

For a preferred aspect of the present invention, any one or more of the nucleotide sequence of the present invention or the expression product thereof, or the mutant nucleotide sequence of the present invention or the expression product thereof, or the protein of the present invention, or the mutant protein of the present invention may be 5 within a transgenic organism or cell (such as being an integral part thereof) - that is an organism or cell that is not a naturally occurring organism or cell and wherein the organism or cell has been prepared by use of recombinant DNA techniques. The transgenic cell may be part of or contained within tissue.

20 Preferably, the transgenic organism or cell is a yeast, an animal (such as a m~mm:~l) or an animal cell (such as a m:lmm~ n cell).

In preferred embodiments, the transgenic organism is a transgenic yeast or a transgenic mouse.
Transgenic yeast may be prepared by appropriately adapting the teachings of Ito et al Journal of Bacteriology 153 163-168; Rose et al 1991 Methods in yeast genetics: a laboratory course manual Cold Spring Harbor, N.Y.: Cold Spring Harbor Press) .

30 Transgenic m~mm~l.s or m~mm~ n cells may be prepared by appropriately adapting the teachings of Ausubel et al 1992 Short Protocols in Molecular Biology 2nd Ed. New York: John Wiley and Sons) .

The transgenic organism or transgenic cell of the present invention therefore provides a simple assay system that can be used to determine whether one or more agents (e.g.
compounds or compositions) have one or more beneficial properties. By way of 5 example, the assay system of the present invention may only utilise a mating phenotype and/or the assay system may be a two-hybrid interaction assay.

By way of example, if the transgenic organism is a transgenic yeast which comprises the nucleotide sequence presented as SEQ ID No. l or the expression product thereof o (namely the protein sequence presented as SEQ ID No. 2) then the yeast could be used to screen for agents that bind to this nucleotide sequence or the expression product thereof and in doing so affect the growth behaviour of the yeast. If an agent produces such a detrimental affect (such as drastically reducing the ability of the yeast to mate), then that agent may also affect the interaction of G~ with that or another Cdc24p entity 5 that is usually capable of being associated therewith. This aspect of the present invention could allow workers to screen for anti-fungal agents, such as agents that could be used to treat or combat Candida.

By way of further example, if the transgenic organism is a transgenic yeast which 20 comprises the nucleotide sequence presented as SEQ ID No. 1 or the expressionproduct thereof then the yeast could be used to screen for agents that bind to this nucleotide sequence or expression product thereof and in doing so affect the growth behaviour of the yeast. If an agent produces a detrimental affect (such as drastically reducing the ability of the yeast to mate), then that agent is likely to detrimentally 25 affect the interaction of G~ with a homologue of Cdc24p with which it is usually capable of being associated. This could allow workers to screen for compounds orcompositions that could for example influence the in vivo expression or behaviour of effect of oncogenes and the like - such as Dbl.

By way of further example, if the transgenic organism is a transgenic yeast which comprises a mutant of the nucleotide sequence in accordance with the present invention then the yeast could be used to screen for agents that affect the growth behaviour of the yeast. If an agent produces a marked affect - such as restoration to a normal growth 5 behaviour or a further detrimental growth behaviour - then workers could screen for compounds or compositions that could for example influence the in vivo expression or behaviour or effect or activity of Cdc24 homologue oncogenes - such as Dbl or Vav.

By way of further example, if the transgenic organism is a transgenic yeast which o comprises a homologue of the nucleotide sequence shown as SEQ ID No. 1 (e.g. Dbl) or an expression product thereof then workers could see if that homologue or theexpression product thereof had an affect on the growth behaviour of yeast, and thus also to see if it had an affect on the interaction of G~3 with a homologue of Cdc24p. In addition, workers could use those transgenic yeast to screen for agents that modified 5 the effect - such as enhance the growth behaviour or detrimentally affect the growth behaviour. In this aspect, agents that affect the growth behaviour may also influence the activity of oncogenes (or even parts thereof) and therefore have potential as therapeutic agents.

20 The assays of the present invention may also be used to screen for agents that affect the interaction of Cdc24 or a Cdc24 homologue with G~ to determine whether that effect has a downstream effect on on Rho-family GTPase.

For example, with the present invention - such as by use of the assays of the present 25 invention - it is possible to devise and/or to screen for peptide inhibitors which block GEF/G~3 interaction. In this regard, peptides and peptidyl derivatives based region encompassing m1lt~nt.~ may be used to block and/or antagonise GEF (such as the proto-oncogenes Dbl or Vav) G,~ interaction. Derivatives of these peptides (including peptide CA 02213680 1997-10-1~

mimics) which bind with higher affinity may also be used. The perturbation of these interactions may be of therapeutic value for example in treatment of cancers.

In addition, by use of the present invention it is possible to devise simple yeast based s assay systems (utilising mating function and interaction reporters). These assay systems will be extremely useful for high through-put screening to identify molecules perturbing the GEF/G~ interaction.

In addition, it is possible to devise and/or screen for agents that can interact with and 0 affect the Cdc24/G,B interactions. Hence, it would be possible to devise and/or to screen for anti-fungal agents directed at the pathogenic yeast Candida.

If the assay of the present invention utilises a transgenic organism according to the present invention then transgenic organim may comprise nucleotide sequences etc. that 15 are additional to the nucleotide sequences of the present invention in order to m~int~in the viability of the transgenic organism.

In the assays of the present invention, the agent can be any suitable compound, compostion as well as being (or even including) a nucleotide sequence of interest or the 20 expression product thereof. Hence, if any one of the nucleotide sequences of the present invention are contained within a transgenic organism - such as a transgenic yeast - then that transgenic organism may also contain that nucleotide sequence of interest. If the agent is a nucleotide sequence, then the agent may be, for example, nucleotide sequences from org~ni~m~ (e.g. higher org~ni~m~ - such as eukaryotes) that restore or increase the 25 growth behaviour. Agents which affect the growth behaviour may also influence the activity of homologous oncogenes and may therefore be potential therapeutic agents.

The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited 30 (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 lRY on 3 October 1997:

E.coli CMK603 PRS414CDC24 (WT) - Deposit Number NCIMB 40898 E.coli CMK603 PRS414CDC24 (Ml) - Deposit Number NCIMB 40899 E.coli CMK603 PRS414CDC24 (M2) - Deposit Number NCIMB 40900 E.coli CMK603 PRS414CDC24 (M3) - Deposit Number NCIMB 40901 Deposit NCIMB 40898 is in respect of Cdc24 (wt); Deposit NCIMB 40899 is in respect of cdc24-ml; Deposit NCIMB 40900 is in respect of cdc24m2; Deposit NCIMB 40901 is in respect of cdc24-m3.

In accordance with a pl~fell~d aspect of the present invention, the nucleotide sequence is s obtainable from, or the protein is expressable from the nucleotide sequence contained within, the respective deposit. By way of example, the respective nucleotide sequence may be isolated from the respective deposit by use of aL,plopliate restriction enzymes or by use of PCR techniques.

The present invention will now be described only by way of example, in which reference is made to the following Figures:

Figure 1 which presents some photographs and a graph;

Figure 2 which presents some images and graphs;

Figure 3 which presents some photographs, a sequence, and a pictorial representation of Cdc24 and DBD Cdc24; and Figure 4 which presents a pictorial representation of a cellular interaction.

The Figures are discussed in more detail later on.

CA 02213680 1997-10-1~

Materials and Methods General techniques Strains were constructed using standard techniques2l. All constructs were verif1ed by DNA dye terminator cycle sequencing (ABI377 sequencer).

Strains pRS414CDC24 contains the CDC24 ORF including 258 bp upstream of ATG.

Oligonucleotide-directed mutagenesis was used to introduce silent base changes that resulted in the following ten new restriction sites in CDC24: NheI (bp -12), KasI (bp 283), AatII (bp 681), PstI (bp 1207), RsrII (bp 1369), Bst~T~ (bp 1426), XhoI ~bp 1758), MluI (bp 1963), SaM (bp 2061), BamHI (bp 2485). RAY410 (MATa, leu2, CDC24::LEU2, ade2, lys2, his3, trpl, ura3, pEG(KT)CDC24) was derived from the diploid YOC38022 which was transformed with pEG(KT)CDC2423 and sporulated.
RAY950 is isogenic to RAY410 but has pRS416GalHis6CDC24 as a rescuing plasmid.
RAY928 (MATa, leu2-3, 112, ura3-52, his3-D200, trpl-D901, lys2-801, suc2-D9, CDC24::HIS5 pEG[KT]CDC24) and RAY931 (same as RAY928 but MATa, ade2, LYS2) were made by transformation of SEY6210 and 6211 with pEG(KT)CDC24 followed by PCR-based gene disruption of CDC24. The CDC24 ORF was replaced with S. pombe HIS524, flanked by LoxP sites. Replacement of CDC24 in SEY6211 with a PCR-generated integration cassette consisting of TRPI fused to 343 bp of CDC24 promoter followed by 1704 bp of CDC24 or cdc24-ml ORF was used to construct RAY1034 or RAY1035, respectively.

IDENTIFICATION OF cdc24 MUTANTS WITH SPECIFIC DEFECTS IN CELL
MATING:

A) Construction of a library of cdc24 random mutants Error-prone PCR was used to generate a library of cdc24 mllt~nt.s in a plasmid vector suitable for phenotypic screening in yeast.

5 1) Plasmid:

pRS414 CDC24 with upstream region and new restriction sites (referred to as pRS414CDC24).

o 2) Mutagenic PCRs:

Conditions from Fromant, M., Blanquet, S. & Plateau, P. Direct random mutagenesis of gene-sized DNA fragments using polymerise chain-reaction. Analytical Biochemistry 224, 347-353 (1995) Dirrelelll PCR-conditions were tested and the error-rate was determined by DNA
sequencing. The following conditions were used for constructing the library used in the screen.

Composition of PCR-reactions (25 ~11 each):

DNA pRS414CDC24 600pM

dATP 0.23 mM
dCTP 0.20 mM
dTTP 2.9 mM
dGTP 0.42 mM

o Buffer PCR Buffer supplied with Taq-polymerase MgCI2 4 rnM
MnC12 O.S mM
Taq (Ampli-Taq) 2 U per reaction Primer: ~ 0.5 rnM

PCR-cycles:

step 1 94 ~C 5 min step 2 91 ~C 1 min step 3 51 ~C 1 min step 4 72 ~C 3 min step 5 72 ~C S min step 6 4 ~C pause 16 cycles (steps 2-4) - CA 02213680 1997-10-1~

3) Library construction:

The PCR products were digested with AatII and NheI (680 bp corresponding to amino acid 1 - 227) were mutagenised and the resulting fragment ligated into pRS414CDC24 s (cut with the same enzymes). Ligations were transformed into E. coli by electroporation and > 50,000 transformants pooled for plasmid isolation.

B) Phenotypic screening for cell-mating specific cdc24 alleles Rationale:

To identify mutant cdc24 alleles which cause defects in cell mating but allow vegetative growth. Yeast strain RAY950, in which expression of CDC24 is repressed in glucose medium, was used.
1) Library plasmids were transformed into RAY950 and transformants selected on SC -trp plates which contained 2% glucose. As RAY950 does not grow on glucose plates this procedure elimin~ted all non-functional cdc24 mutants.

20 2) Transformants were replica-plated onto a lawn of WT (screen 1) or ~fusl~J~us2 (screen 2) tester cells, incubated at 30~C for 3 hrs and replica-plated onto plates selecting for diploids or RAY950 derived haploids. Mating defective ~ were identified by c~Jlllpalillg the pattern of colonies on the two sets of plates and candidate Ill~ were picked from the original transformation plates for retesting.
2s 3) Plasmids from mllt~nt~ were isolated by transformation into E. coli. Isolated plasmids were retransformed into RAY950, RAY928 and RAY931 for independent confirm~tion of phenotype and retested for defects in cell mating.

30 4) Mutations of confirmed ~1llll;-lll.~ were identified by DNA sequencing. Multiple mutations were separated by subcloning and the mutation responsible for the phenotype identified by mating tests in RAY950.

CA 02213680 1997-10-1~

~ ' 14 5) A total of ~ 5,000 yeast transformants were tested in each screen.

- Screen 1 identified two mutants (cdc24-ml, cdc24-m2).
s - Screen 2 identified one mutant (cdc24m3).

Phenotypic analyses Qll~n~it~tive m~ting~l~, m:~ting.c in the presence of saturating pheromonel3, halo-assays26 0 using sstl::URA3 strains, and FusllacZ measurements with pSG231" were carried out as described. Halo assays showed MATa and MATa cdc24ml cells secreted oc-factor and a-factor, respectively. Actin was visualised with rhodamine phalloidin27 on a Biorad-MRC-600 confocal microscope and pictures are projections of 4-6 0.5 mm z-series steps.
For (x-factor treatment, cells were incubated with 5 mM oc-factor for 2 hr. RAY1034 and RAY1035 cells were used to determine bud scar positions on zygotesl4 visualised with Calcoflour28. Sirnilar results were observed with the position of the bud scar on shmoos.
Direct measurement of cell orientation in a pheromone gradient was carried out essentially as describedl2. A pheromone gradient was generated using a micropipet filled with 80 mM a-factor injected at 105 kPa into lml of YEPD media layered on top of cells embedded in 2% LMP agarose. Cells shape was recorded by video microscopy on a heated stage at 35~ for 4 - 7 hr and data analysis was from traced cell outlinesl4.
Mating projections were formed at the same pheromone concentrations and budding, that is non-responding cells were seen at similar distances from the micropipet in both strains.

Two-Hybrid methods STE4, BEMl (372 - 551 aa), CDC42[C178S], and CDC24 Icdc24ml (1-288, 1-160, and 170-245 aa) were cloned by PCR into pGAD424 (AD, GAL4 activation domain) or pASl(DBD, GAL4 DNA binding domain). Plasmids were transformed into HF7c. For determination of STE18 requirement, PCR-based gene disruption was carried out in PJ69-4A (MATa, trpl-901, leu2-3,112, ura3-52, his3-200, gal4D, gal80D, GAL2-ADE2, LYS2: :GAL1-HIS3, met2: :GAL7-lacZ)29, replacing the entire STE18 ORF with K

- CA 02213680 1997-10- l=7 Lactis URA330. For all two-hybrid experiments, equal amounts of transformants were spotted on SC-leu-trp and SC-leu-trp-his plates, identical results were obtained with at least four transformants, and for Dstel 8 two independent deletion strains. All strains for two-hybrid analyses expressed similar amounts of AD- and DBD- fusion proteins of the s expected sizes, as determined by SDS-PAGE and immuno-blotting. None of the DBDfusions showed any self-activation using two different non-interacting AD fusions.

In vitro binding studies 0 A fragment of CDC24 (1-472 aa) in pGEX-2T (Pharmacia) and His6Ste4p (pTrcSte4) were expressed in E. coli. Cells were resuspended in buffer A (PBS, 0.1% TX-100,PMSF, leupeptin, chymostatin, pepstatin, aprotinin) and lysed by snap freezing in liquid nitrogen followed by sonication. Insoluble material was removed by centrifugation (10,OOOg). Mixed supernatants (denoted cell extracts) cont~ining His6Ste4 and GSTCdc24 fusions were incubated with GSH-agarose (Sigma Chemical Co.) at 4~ for 1 hr. Resin was washed 3 times with buffer A. Resin samples (referred to as eluates) and extracts were analyzed by SDS-PAGE, immuno-blotting probed with Omni-probe anti-sera (Santa Cruz), and visualised with enhanced chemiluminescence (Amersham).
GSTCdc24p (1-127 aa), similar to GST, did not bind His6Ste4p. Similar results were observed in 5 independent experiments.

RESULTS

Table 1 Cdc24ml is defective incell mating Strain Tester % Mating effficiency CDC24 MATa MATa WT 100 (21) cdc24-ml MATa MATa WT 0.5 (0.2) CDC24 MATa MATa WT 100 (20) cdc24-ml MATa MATa WT 3.8 (1 6) 1S CDC24 MATa MATa ~fusl ~fus2 100(17) cdc24-ml MATa MATa ~fusl ~fus2 < 0.02 CDC24 MATa CDC24 MATa 100(18) cdc24-ml MATa cdc24-ml MATa < 0.0006 Mating effficiencies are the number of diploid cells divided by the total cells with CDC24 WT set to 100%. The values are means of 4 determinations with standard deviation ().
Absolute mating efficiency was 14-15% with MATa and MATa testers, 1.8% with ~fusl ~fus2 tester, and 3.4% with CDC24 tester.

Some of the results are also shown in the accompanying Figures. These Figures are now discussed in more detail.

30 FIG. 1 Cdc24-ml phenotypes. a, Actin cytoskeleton of cdc24-ml cells shows polarised distribution. Bar equals 5 mm. b, Pheromone-induced growth arrest is similar in cdc24-ml with WT cells. Sterile filter disks spotted with a-factor (1, 0.5, 0.2, 0.1, 0.05, and 35 0.012 mg) were placed onto cells in agarose. c, MAP-kinase pathway sign:~lling is unaffected in cdc24ml. LacZ activities are the average of 2 experiments (2-3 d~Lellllillations per experiment) with standard deviation. WT maximum (29.6 Miller Units) was set to 100%.

- CA 02213680 1997-10-1~

FIG. 2 Cdc24-ml cells are unable to orient in a pheromone gradient. a, Excess pheromone has a negligible effect on cdc24-ml mating. MATa cells were mated with a WT tester andmating efficiency for CDC24 (2.8%) was set to 100%. Values are means (n=2). b, Cdc24-ml cells are unable to orient in a pheromone gradient. A trace of cell shapes after 6-7 hr in a pheromone gradient is shown with arrowheads indicating orientation.
Qu~ntit~tion of cell projection angle relative to the micropipet (needle) from 4-7 separate experiments (n=112 CDC24 and 167 cdc24-ml cells). The average cosine of the angle of cell projection relative to the micropet was 0.52 for CDC24 and -0.02 for cdc24-ml cells (a cosine of 1 represents perfect orientation and 0, random orientation). c, Cdc24 ml cells position their shmoos adjacent their bud scar. The position of the bud scar on zygotes was d~L~lmilled for approximately 120 cells.

FIG. 3 Cdc24-m mutants are defective in mating and Ste4p binding. a, Location of Cdc24pmating mutations. Mating patches show diploids from mating with MATa WT tester.
Ste4 2-H patch growth on -leu-trp-his indicates an interaction of Cdc24p (1-288 aa) with Ste4p. Similar results were obtained using a LacZ reporter in strain Y187 (relative Miller Units 100 for Cdc24/Ste4 and 3 for Cdc24-ml/Ste4). b, Two hybrid interactions of Cdc24p. For interactions with Ste4p, a fragment of Cdc24p (1-288 aa) was used, however, full length Cdc24p also interacts with Ste4p. c, Region of Cdc24p necessary for Ste4p interaction. Numbers refer to Cdc24p aa fused to DBD. d, Cdc24p binds to Ste4p in the absence of other yeast proteins. Mixed bacterial cell extracts (1 eq) cont;~ining either His6Ste4p and GST or GSTCdc24p (1-472 aa), and GSH-agarose eluates (800 eq) were separated by SDS-PAGE, immllno-blotted and probed with anti-sera to His6Ste4p. Anti-GST sera showed similar amounts of GST and GSTCdc24p in eluates.Due to proteolysis, His6Ste4p migrates as a doublet.

FIG. 4 Model for signal transduction pathway required for cell orientation. For clarity we have omiKed components of MAP-kinase cascade. The role of Cdc42p (a Rho-family GTPase) 5 in cell orientation is speculative. Pheromone binds the pheromone receptor (Ste2p or Ste3p) resulting in the dissociation of Ga (Gpalp) from G~l~ (Ste4p/Stel8p). Direct binding of Cdc24p to G~ (in the vicinity of the receptor) activates or recruits Cdc24p which is n~ocess~ry for oriented growth towards a mating partner.

I o SF.OUENCF, ANAT ,YSIS

The DH and PH sequences were analysed by a Blast homology search. In addition, an analysis of the amino acid identity over the entire protein to S. cerevisiae Cdc24p was conducted. DH refers to the Dbl homology region (GEF region) - see Hart et al 1991 Nature ~ 311-314; Miyamoto et al 1991 Biochem Biophys Res Comrnun 181 604-610;
Ron et al 1991 New Biol 3 372-379. PH refers to the Pleckstrin homology region - see Musacchio et al Trends Biochem Sci 18 343-348.

The results are as follows.
A. Blast homology search using Cdc24 DH and PH region TBLASTN 1.4.9 MP

Query= yeast Cdc24p DH PH (392 aa):

2~ K I IK~ rATERKyvHDLEILDKyRQQLLDsNLITsEELyMLFpNLGDAIDFQRR ,FLI
SLEINALVEPSKQRIGALFMH~ KLYEPWSIGQNAAIEFLSSTLHKMRVDESQ
RFIINNKLELQSFLYKPVQRLCRYPLLVKELLAESSDDNNTKELEAALDISKNLARS
INENQRRTENHQVVKKLYGRVVNWKGYRISKFGELLYFDKVFISTTNSSSEPEREF
EVYL~ Kllll ,FSEVVTKKSASST ,TT .KKK-ssTsAsIsAsNITDNNGspHHsyHKRHsNs ,o SSSNNIHLSSSSAAAIIHSSTNSSDNNSNNSSSSSLFKLSANEPKLDLRGRIMIMNLN
QIIPQNNRSLNITWESIKEQGNFLLKFKNEETRDNWSSCLQQLIHDLKN
(SEQ ID NO: 9) - ' 19 Database: Non-re~lln-l~nt Genbank + EMBL + DDBJ + PDB sequences 349,525 sequences; 540,957,745 total letters Reference: Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (1990). Basic local alignment search tool. J. Mol.Biol. 215:403-410.

Reading High Smallest Smallest Frame Score Sum Sum Probability Probability P(N) N
gblU125381SPU12538 S.~ .yces +3 171 1.0e-51 6 pombe scdl emb l X57298 l MMMCF2PO M.m~cc~ c Mcf2 proto- + 1 128 8.3e-10 3 oncogene (Mcf2 is Dbl) gblU16296lHSU16296 HumanT-lymphoma +3 88 2.3e-09 3 invasion and metastasis inducing TIAM1 gblU05245lMMU05245 MusmusculusBALB/c +3 88 5.5e-09 3 invasion inducing protein (Tiam-1) gblJ03639lHUMDBLTP HumanDBLoncogene +2 121 2.1e-07 3 encoding a ~ r~....;llg protein gblS76992lS76992 VAV2=VAV oncogene +3 125 2.6e-07 2 homolog human dbj l D86547 l D86547 Fruitfly still life type 1 +2 76 5.4e-07 5 gblU37017lMMU37017 Mus musculus Vav2 +1 126 6.4e-07 2 oncogene dbj ¦ D86546 ¦ D86546 Fruitfly still life type 2 + 1 76 l .Oe-06 5 gb¦U39476lRNU39476 Rattus norvegicus p95 Vav +3 116 6.3e-06 proto-oncogene gb l S76838 l S76838 Dbs (Dbl guanine nucleotide +3 112 4.4e-05 2 exchange factor homolog) murine dbjlAB0023601AB002360 HumanKIAA0362 +2 113 4.5e-05 2 emb l Z356541 RNOSTOG R.norvegicus 0st oncogene + 1 112 4.9e-05 2 emblX83931lHSVAVONCO H.sapiensVAVoncogene +1 109 5.5e-05 gblAF0031471CELCllD9 Ca~.. u.l.aldili~elegans +3 81 0.0070 3 CllD9 - ' 20 gblU966341MMU96634 Mus musculus p85SPR +2 62 0.016 3 emblY10159¦DDY10159 D.di~oid~ -l racGAP +1 71 0.025 3 gblU582031MMU58203 MusmusculusLsc +2 75 0.044 2 oncogene emblY091601HSSUB15 H.sapiens Subl 5 +1 80 0.063 2 gblAF0037401CELC41Dll Ca~.. u-llab.liLi~ elegans +2 81 0.064 4 gblU020811HSU02081 Human guanine nucleotide +1 77 0.12 2 regulatory protein (NET1) gb I U000551 CELR02F2 Ca~llulllab.liLi~ elegans + 1 85 0.13 gb I U641051 HSU64105 Human guanine nucleotide + 1 77 0.14 exchange factor pl 15-RhoGEF
gblU423901HSU42390 Homo sapiens Trio +1 74 0.33 3 gblM246031HUMBCRD Humanbcrproteinamino +1 58 0.91 3 end emblX025961HSBCRR Human bcr (breakpoint +3 58 0.996 3 cluster region) in Pl l; I ,'/1~ 1~ ,1,; A ~;111 Ul IIOSUI 11.~
gb I U116901 HSU11690 Human faciogenital +2 73 0.999 dysplasia (FGD1) gblU223251MMU22325 Musmusculusfaciogenital +3 73 0.9997 2 dysplasia (Fgdl) gblM150251HUMBCRABL HumanBCR/ABLproduct +3 58 0.99995 5 of the translocation of t(222ql1; 9q34) - CA 02213680 1997-10- l~

B. Amino acid identity over entire protein to S. cerevisiae Cdc24p Organism gene protein % identity size (aa) (aa) Schizosac~lldlulllyces pombe Scdl 834 21.9 Mouse Fgdl 960 16.7 Human Fgdl 961 16.5 Mouse Vav2 868 16.5 10 Mouse Ect2 768 16.2 Human Vav2 878 15.8 Worm Q18479 860 15.4 Mouse Vav 844 14.6 Rat Vav 843 14.5 15 Human Vav 846 14.4 Mouse Dbs 1150 14.3 Human Tim 519 14.0 Human proto-Dbl 925 13.4 Human pllSRhoGEF 912 13.4 20 Mouse Lfc 572 13.4 Rat 0st 872 12.9 Worm Q22354 862 12.9 Mouse Lsc 919 12.5 Human Lbc 424 12.4 25 Human Netl 460 12.3 Human BCR 1271 11.9 Mouse Tiaml 1591 11.2 Human Tiaml 1591 10.9 Mouse proto-Dbl 320 (partial) 9.7 30 Drosophila Still Life 1 2064 9.0 Drosophila Still Life 2 2044 8.4 Protein name key:
Scdl: Srl~ s,~r~ l UIlly~,e pombe Cdc24p~~l .
35 Fgdl Faciogenital Dysplasia Protein. FGD also known as Aarskog-Scott syndrome, is an X-linked developmental disorder'02.
Vav/Vav2 A oncogene derived from hellldLupo;~lic cellsl03.
Q18479 (similar to Vav) Q22354 (similar to Vav) 40 Ect2 Oncogene expressed in epithelial cells and possessing Lld.~rollllillg potential~04.
Tim Mammary epithelial oncogene~05.
Dbl/Dbs Diffuse b-cell lymphoma (dbl) oncogenel06~ ~07.
pl l5RhoGEF Regulates cell proliferation, induces the transformation of cells~03.
Lfc Hematopoietic oncogene~09.
45 0st Osteosarcoma derived proto-oncogene. Truncation is oncogenic and highly tumorigenic in micel 10.
Lsc Oncoproteinl 1 l .
Lbc Oncogene involved in chronic myeloid leukemias~2.
Netl Neuroepithelioma lldl~OIlllillg oncogene~3.
50 BCR bcr (breakpoint cluster region), an oncogene which is the translocation breakpoint in chronic myeloid le-lkrmi~c (CML)I14 ~5.
Tiaml Human invasion- and mrt~ct~ci~-inducirlg tiaml gene and is expressed in tumor-cell lines of different tissue originl'6.
Still Life 1/2 A synaptic terminal proteinll7.

DISCUSSION

CDC42 and its GDP/GTP exchange factor (GEF) CDC24 are required for vegetative growth8 9 and cell mating6 7 l0. The precise function of these proteins in cell mating has 5 been difficult to study because they are essential for viability. In accordance with the present invention, we reasoned that if CDC24 has a specific function in the mating pathway, cdc24 alleles should exist which affect cell mating but not vegetative growth.
To identify such alleles, a collection of CDC24 random mutants was screened and three recessive mating mut~nt~, cdc24-ml-3 were isolated (Figure 3A). This screen required o isolated cdc24 mutants to be able to support vegetative growth. Further characterisation of cdc24-m cells revealed normal growth between 18~ and 37~ and cell morphology, bud site selection, and actin distribution were similar to WT cells (see below and Figure lA).
The specificity of the cdc24-m phenotype is in contrast to that of all other described cdc24 ml]t~nt~ which have strong defects in vegetative growth8~l0.

To elucidate the role of CDC24 in mating, we ex~min~l cdc24ml cells for defects in the mating pathway. The mating efficiency of cdc24-ml cells with a WT partner was reduced approximately 100-fold compared to WT (Table I), and this effect was essentially independent of mating type. When cdc24ml or an enfeebled mater defective 20 in cell fusion were used as mating partners, significantly stronger defects were observed.
Such bilateral mating defects suggest impairment in a process such as shmoo (mating projection) formation, orientation, or fusion in which a WT mating partner can partially compensate for the mutant strain.

25 Pheromone activation results in a number of responses including cell cycle arrest, MAP-kinase cascade mediated induction of mating $pecific genes, and changes in cell morphology 4'5. Pheromone-in(lllcecl growth arrest determined by halo-assays showed both cdc24-ml and WT cells responded similarly (Figure lB). Furthermore, overexpression of the ~-subunit of the yeast hetero-trimeric G-protein, Ste4p, from an 30 inducible promoter arrested growth of both cdc24-ml and WT cells (data not shown).
Microscopic ex~min~tion revealed identical numbers of WT and cdc24-ml cells (78%, n= 1600) formed shmoos after 4 hr exposure to 10 mM pheromone. The actin - CA 02213680 1997-10-1~

distribution of cdc24ml budding and shmooing cells was also similar to that of WT cells (Figure lA), demonstrating that the mating defect was not due to an inability to polarise the actin cytoskeleton. The level of pheromone induced FUSl-lacZ expression, a reporter used to measure induction of mating specific genesll, was similar in cdc24-ml and WT cells (Figure lC). However, e~min~tion of mating mixtures of cdc24ml and WT tester cells showed a greater than ten-fold decrease in the number of zygotes, indicating that the cdc24ml defect occurs prior to cell fusion. Thus cdc24-m cells appear normal for cell cycle arrest, shmoo formation, actin cytoskeleton polarisation, and MAP-kinase sign~lling, yet are defective at a step prior to cell fusion.

During mating, polarised growth towards a mating partner requires a pheromone gradientl2 and saturation with pheromone during mating results in random orientation of growth and mating partner selection, and hence a decrease in mating efficiencyl3 l4. WT
cells showed a 16-fold decrease in mating efficiency in the presence of saturating pheromone (20 mM), whereas only 10% reduction was observed with cdc24ml cells (Figure 2A), suggesting that this mutant is unable to orient towards a pheromone gradient during mating. Similar results were observed with cdc24-m2 and cdc24m3 cells. Totest directly whether cdc24-ml cells are defective in mating projection orientation their response to an artificial pheromone gradient created by a micropipet was examined.
While CDC24 cells oriented growth towards the pheromone source (greater than 70% of cells oriented within 60~ angle of micropipet), cdc24ml cells did not show a pL~r~ d orientation (Figure 2B). No difference in the sensitivity of WT or mutant cells to pheromone was observed.

Although cdc24-ml cells oriented randomly in a pheromone gradient, the choice ofshmoo site could be dictated by an internal cue, such as the previous bud site. To examine this possibility, the location of the bud scar (in cells with a single bud scar) relative to the neck of the zygote was determined. While WT cells showed a random position of their bud scar on the zygotes, 86% of cdc24-ml zygotes had formed a shmoo adjacent to their previous bud site (Figure 2C). Together these results establish a specific role for Cdc24p in orientation towards a mating partner.

CA 022l3680 l997- lO- l~

- ' 24 Sequencing of cdc24m alleles revealed mutations that changed one of two adjacentamino acid residues (Figure 3A). Cdc24-ml and cdc24-m3 both have a single amino acid change from Ser 189 to either a Phe or Pro. Cdc24-m2 had two amino acid substitutions and subcloning demonstrated that the mutation responsible for the mating 5 defect is Asp to Gly at residue 190. The grouping of these mutations suggests that this region of Cdc24p is important for an interaction required for oriented growth.

Previous two-hybrid studies have suggested that the amino-terminus of Cdc24p might interact with Ste4p7, however, the in vivo significance of this association was unclear.
o We determined whether Cdc24p mating mutants could interact with Ste4p (Figure 3A).
In contrast to the wild-type Cdc24p, the mutants did not show a detectable interaction with Ste4p. In agreement with the clustering of the cdc24-m mutations, amino acid residues 170 to 245 of Cdc24p were sufficient for the Ste4p interaction (Figure 3C), while an amino-terminal fragment consisting of the first 160 amino acid residues, 15 although expressed, failed to interact. Consistent with a functional significance of the Cdc24p Ste4p interaction, we have isolated mutants in STE4, using a two-hybrid screen, which are unable to interact with Cdc24p and are phenotypically similar to cdc24m mnt~nt~.

20 To assess the specificity of the defect in the interaction between Ste4p and Cdc24-mlp, interactions with Cdc42p and Bemlp, two proteins known to bind to Cdc24pls l6 were investigated. Bemlp is an SH3 domain protein involved in bud formation and matingl7.
Cdc24-mlp was able to interact with both Cdc42p and Bemlp (Figure 3B) consistentwith the absence of an effect of cdc24-ml on vegetative growth.

While the cdc24-ml phenotype along with the two-hybrid results indicates that the interaction between Cdc24p and G,B is central to cell orientation, these results do not address whether this interaction is direct or indirect. G~ typically functions as a complex with the third subunit of a hetero-trimeric G-protein, G~. We therefore determined 30 whether the yeast Gy, Stel8p, was required for the Cdc24p Ste4p interaction. Deletion of 51~18 abolished the Cdc24p Ste4p two-hybrid interaction (data not shown), suggesting that Cdc24p interacts with the G~y-complex. To determine if Cdc24p could directly bind . - CA 02213680 1997-10-1 - ' 25 Ste4p, these proteins were expressed in bacteria. Hexahistidine-tagged Ste4p specifically bound to GSTCdc24p (Figure 3D). These results demonstrate that Cdc24p can directly bind G~ in the absence of any other yeast proteins. We attribute the requirement for G~
in the two-hybrid assays to its stabilisation of G~18.

Pheromone receptor activation results in dissociation of G~3r from Ga at the receptor.
Our results indicate that the orientation defect in cdc24-m cells is due to a specific defect in the Cdc24p G~ interaction. This suggests a model in which direct binding of Cdc24p to G~ results in recruitment (to the vicinity of the receptor) or activation of Cdc24p and o that this local concentration of activated Cdc24p is responsible for oriented growth towards a pheromone gradient (Figure 4). In the absence of this recruitment or activation a site adjacent to the previous bud site appears to function as a default site for shmoo formation. Our results together with previous studies implicating Cdc24p in bud site selection8, suggest that Cdc24p acts as a crucial component required both for bud and 15 shmoo site selection, perhaps functioning as a kind of molecular selector switch between internal signals for bud site selection and external signals for shmoo site selection. It is likely that local activation of Cdc24p recruits and activates the Rho GTPase Cdc42p, which could then interact with downstream targets required for orientation of the cytoskeleton. Cdc42p interactions with the protein kinase Ste20pl92~ are not nPcess:~ry 20 for cell orientation20, suggesting that novel targets of Cdc42p are required for oriented growth towards a mating partner.

Cdc24p belongs to a diverse family of GEFs which include many mamm~ n proto-oncogenes2. This group of proteins shares a conserved region consisting of a Dbl-domain 25 (named after the human proto-oncogene Dbl) followed by a plecktstrin-homology domain (PH). Sequence comparison revealed similarity between a small stretch of amino acids flankin~ the cdc24 mating mutations and Dbl (Figure 3A). Our results indicate that an association between Cdc24p and G~ links pheromone receptor activation to shmoo orientation. We propose that other GEFs, such as the proto-oncogene Dbl, provide a 30 similar connection between G-protein coupled receptor activation and polarised cell growth.

Hence, in accordance with the present invention there are provided the following uses and utilities of Cdc24/Ste4 interaction and cdc24-m mutants 1) Peptide inhibitors which block GEF/G~ interaction. Peptides and peptidyl derivatives 5 based region encompassing mut:~nt~ will be used to block and/or antagonise GEF (such as the proto-oncogenes Dbl or Vav) G,B interaction. Derivatives of these peptides (including peptide mimics) which bind with higher affinity will also be used. The perturbation of these interactions will be of therapeutic value for example in treatment of cancers.

o 2) Simple yeast based assays systems (utilising mating function and interaction reporters) will be extremely useful for high through-put screening to identify molecules perturbing this GEF/G~ interaction.

3) Similar Cdc24/G~ interactions will be ideal targets for anti-fungal drugs directed at 15 the pathogenic yeast Candida.

SUMMARY

1) We have identified an important interaction between two general cellular 20 components. This work has been exemplified by work done with yeast genes/proteills, however, both cellular components involved have homologs in humans.

2) We show the physiological consequence of this interaction and from these dataextrapolate to the general role of this interaction in human cells.

3) In addition, we have identified sequences required for this interaction.
Specifically, we have identified a short stretch of one protein (Cdc24p) encompassing 75 aa sufficient for this interaction and three amino acid changes (within this stretch) which block the interaction and have physiological consequences. These amino acid changes 30 fall within a 19 amino acid piece with similarity to the human proto-oncogene Dbl.
Indeed, removal of this region from proto-Dbl (when the amino terminus is removed) results in oncogenicity in tissue culture cells.

4) There is a wealth of information on human G~'s, Cdc24p homologs (GEF's _DP/GTP _xchange Factors), and the GTP-binding-protein the exchange factors workon (rho like Cdc42p). Most human GEF's are oncogenes such as Dbl, Vav, and Ect and s are involved in some way in growth control. Furtherrnore G~'s are involved in linking signals from receptors to intracellular responses. Although unproven, it is likely that interactions between human GEF's and G~'s are crucial in growth control and chemotaxis.

o 5) We propose the interaction we have identified will have broad cellular ramifications and manipulation of these interactions (such as peptidic inhibitors and peptides mimicking activated species) will be of therapeutic value.

6) In addition, simple yeast based assays systems could be extremely useful for high through-put screening to identify molecules perturbing this interaction.

7) We also believe similar interactions will be ideal targets for anti-fungal drugs directed at the pathogenic yeast Candida.

20 Modifications to the present invention will be apparent to those skilled in the art.

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CA 022l3680 l998-ll-30 SEQUENCE LISTING

APPLICANT: MEDICAL RESEARCH COUNCIL
TITLE OF INVENTION: NUCLEOTIDE SEQUENCES AND PROTEIN SEQUENCES
NUMBER OF SEQUENCES: 9 CORRESPONDENCE ADDRESS:
Gowling, Strathy & Henderson 160 Elgin St. Suite 2600 Ottawa, Ontario KlP lC3 COMPUTER-READABLE FORM
COMPUTER: PC IBM Compatable OPERATING SYSTEM: PC/DOS - MC/DOS
SOFTWARE: PatentIn Ver. 2.0 CURRENT APPLICATION DATA
APPLICATION N~MBER: 2,213,680 FILING DATE: October 15, 1997 PATENT AGENT INFORMATION:
NAME: Gowling, Strathy & Henderson REFERENCE NUMBER: 08-877468CA
INFORMATION FOR SEQ ID NO.:
SEQUENCE CHARACTERISTICS
LENGTH: 228 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: nucleic acid SEQUENCE DESCRIPTION: SEQ ID NO.:
CCCCTCTGTA TA~llll~AA CTCTGTGAAG CCGCAATTTA AATTACCGGT AATAGCATCT 60 GACGATTTGA AAGTCTGTAA AAAATCCATT TATGACTTTA TATTGGGCTG CAAGA~ACAC 120 TTTGCATTTA ACGATGAGGA G~llllCACT ATATCCGACG TTTTTGCCAA CTCGACGTCC 180 INFORMATION FOR SEQ ID NO.: 2 SEQUENCE CHARACTERISTICS
LENGTH: 76 TYPE: PRT
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: amino acid CA 022l3680 l998-ll-30 35a SEQUENCE DESCRIPTION: SEQ ID NO.: 2 Pro Leu Cys Ile Leu Phe Asn Ser Val Lys Pro Gln Phe Lys Leu Pro Val Ile Ala Ser Asp Asp Leu Lys Val Cys Lys Lys Ser Ile Tyr Asp Phe Ile Leu Gly Cys Lys Lys His Phe Ala Phe Asn Asp Glu Glu Leu Phe Thr Ile Ser Asp Val Phe Ala Asn Ser Thr Ser Gln Leu Val Lys Val Leu Glu Val Val Glu Thr Leu Met Asn Ser Ser INFORMATION FOR SEQ ID NO.: 3 SEQUENCE CHARACTERISTICS
LENGTH: 228 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: nucleic acid SEQUENCE DESCRIPTION: SEQ ID NO.: 3 CCCW'~'l'~'l'A TA~llll~AA CTCTGTGAAG CCGCAATTTA AATTACCGGT AATAGCATTT 60 GACGATTTGA AAGTCTGTAA AAAATccATT TATGACTTTA TATTGGGCTG CAAGA~ACAC 120 INFORMATION FOR SEQ ID NO.: 4 SEQUENCE CHARACTERISTICS
LENGTH: 76 TYPE: PRT
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: amino acid SEQUENCE DESCRIPTION: SEQ ID NO.: 4 Pro Leu Cys Ile Leu Phe Asn Ser Val Lys Pro Gln Phe Lys Leu Pro Val Ile Ala Phe Asp Asp Leu Lys Val Cys Lys Lys Ser Ile Tyr Asp Phe Ile Leu Gly Cys Lys Lys His Phe Ala Phe Asn Asp Glu Glu Leu Phe Thr Ile Ser Asp Val Phe Ala Asn Ser Thr Ser Gln Leu Val Lys 35b Val Leu Glu Val Val Glu Thr Leu Met Asn Ser Ser SEQUENCE INFORMATION FOR SEQ ID NO.: 5 SEQUENCE CHARACTERISTICS
LENGTH: 228 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: nucleic acid SEQUENCE DESCRIPTION: SEQ ID NO.: 5 TTTGCATTTA ACGATGAGGA G~ ACT ATATCCGACG TTTTTGCCAA CTCGACGTCC 180 SEQUENCE INFORMATION FOR SEQ ID NO.: 6 SEQUENCE CHARACTERISTICS
LENGTH: 76 TYPE: PRT
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: amino acid SEQUENCE DESCRIPTION: SEQ ID NO.: 6 Pro Leu Cys Ile Leu Phe Asn Ser Val Lys Pro Gln Phe Lys Leu Pro Val Ile Ala Ser Gly Asp Leu Lys Val Cys Lys Lys Ser Ile Tyr Asp Phe Ile Leu Gly Cys Lys Lys His Phe Ala Phe Asn Asp Glu Glu Leu Phe Thr Ile Ser Asp Val Phe Ala Asn Ser Thr Ser Gln Leu Val Lys Val Leu Glu Val Val Glu Thr Leu Met Asn Ser Ser INFORMATION FOR SEQ ID NO.: 7 SEQUENCE CHARACTERISTICS
LENGTH: 228 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: nucleic acid CA 022l3680 l998-ll-30 SEQUENCE DESCRIPTION: SEQ ID NO.: 7 CCCCTCTGTA TA~llll~AA ~l~l~lGAAG CCGCAATTTA AATTACCGGT AATAGCACCT 60 GACGATTTGA AAGTCTGTAA AAAATCCATT TATGACTTTA TATTGGGCTG CAAGA~ACAC 120 TTTGCATTTA ACGATGAGGA G~llll~ACT ATATCCGACG TTTTTGCCAA CTCGACGTCC 180 INFORMATION FOR SEQ ID NO.: 8 SEQUENCE CHARACTERISTICS
LENGTH: 76 TYPE: PRT
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: amino acid SEQUENCE DESCRIPTION: SEQ ID NO.: 8 Pro Leu Cys Ile Leu Phe Asn Ser Val Lys Pro Gln Phe Lys Leu Pro Val Ile Ala Pro Asp Asp Leu Lys Val Cys Lys Lys Ser Ile Tyr Asp Phe Ile Leu Gly Cys Lys Lys His Phe Ala Phe Asn Asp Glu Glu Leu Phe Thr Ile Ser Asp Val Phe Ala Asn Ser Thr Ser Gln Leu Val Lys Val Leu Glu Val Val Glu Thr Leu Met Asn Ser Ser INFORMATION OF SEQ ID NO.: 9 SEQUENCE CHARACTERISTICS:
LENGTH: 392 TYPE: PRT
ORGANISM: Artificial Sequence FEATURE
OTHER INFORMATION: Description of Artificial Sequence: amino acid SEQUENCE DESCRIPTION: SEQ ID NO.: 9 Lys Ile Ile Lys Glu Phe Val Ala Thr Glu Arg Lys Tyr Val His Asp Leu Glu Ile Leu Asp Lys Tyr Arg Gln Gln Leu Leu Asp Ser Asn Leu Ile Thr Ser Glu Glu Leu Tyr Met Leu Phe Pro Asn Leu Gly Asp Ala Ile Asp Phe Gln Arg Arg Phe Leu Ile Ser Leu Glu Ile Asn Ala Leu CA 022l3680 l998-ll-30 35d Val Glu Pro Ser Lys Gln Arg Ile Gly Ala Leu Phe Met His Ser Lys ~is Phe Phe Lys Leu Tyr Glu Pro Trp Ser Ile Gly Gln Asn Ala Ala ~le Glu Phe Leu Ser Ser Thr Leu His Lys Met Arg Val Asp Glu Ser Gln Arg Phe Ile Ile Asn Asn Lys Leu Glu Leu Gln Ser Phe Leu Tyr Lys Pro Val Gln Arg Leu Cys Arg Tyr Pro Leu Leu Val Lys Glu Leu Leu Ala Glu Ser Ser Asp Asp Asn Asn Thr Lys Glu Leu Glu Ala Ala Leu Asp Ile Ser Lys Asn Ile Ala Arg Ser Ile Asn Glu Asn Gln Arg ~rg Thr Glu Asn His Gln Val Val Lys Lys Leu Tyr Gly Arg Val Val Asn Trp Lys Gly Tyr Arg Ile Ser Lys Phe Gly Glu Leu Leu Tyr Phe Asp Lys Val Phe Ile Ser Thr Thr Asn Ser Ser Ser Glu Pro Glu Arg Glu Phe Glu Val Tyr Leu Phe Glu Lys Ile Ile Ile Leu Phe Ser Glu Val Val Thr Lys Lys Ser Ala Ser Ser Leu Ile Leu Lys Lys Lys Ser ~er Thr Ser Ala Ser Ile Ser Ala Ser Asn Ile Thr Asp Asn Asn Gly Ser Pro His His Ser Tyr His Lys Arg His Ser Asn Ser Ser Ser Ser Asn Asn Ile His Leu Ser Ser Ser Ser Ala Ala Ala Ile Ile His Ser Ser Thr Asn Ser Ser Asp Asn Asn Ser Asn Asn Ser Ser Ser Ser Ser Leu Phe Lys Leu Ser Ala Asn Glu Pro Lys Leu Asp Leu Arg Gly Arg 325 . 330 335 Ile Met Ile Met Asn Leu Asn Gln Ile Ile Pro Gln Asn Asn Arg Ser -35e Leu Asn Ile Thr Trp Glu Ser Ile Lys Glu Gln Gly Asn Phe Leu Leu Lys Phe Lys Asn Glu Glu Thr Arg Asp Asn Trp Ser Ser Cys Leu Gln Gln Leu Ile His Asp Leu Lys Asn

Claims (24)

1. A nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof, wherein the expression product of the nucleotide sequence has the capability of not substantially affecting the interaction of G.beta. with Cdc24p or a homologue thereof that is usually capable of being associated therewith.

2. A mutant of the nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof, wherein the expression product of the mutant nucleotide sequence has the capability of substantially affecting the interaction of G.beta. with Cdc24p or a homologue thereof that is usually capable of being associated therewith.

3. A nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof for use in medicine.

4. A mutant of the nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof for use in medicine.

5. Use of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in the manufacture of a medicament to affect the growth behaviour of cells.

6. Use of a mutant of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in the manufacture of a medicament to affect the growth behaviour of cells.

7. Use of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in a screen to identify one or more agents that are capable of affecting the interaction of Cdc24p or a homologue thereof with a G.beta. or an associated Rho-family GTPase.

8. Use of a mutant of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in a screen to identify one or more agents that are capable of affecting the interaction of Cdc24p or a homologue thereof with a G.beta. or an associated Rho-family GTPase.

9. An assay comprising contacting an agent with a nucleotide sequence shown as SEQ
I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in the presence of a G.beta. capable of being associated with Cdc24p or a homologue thereof;
and determining whether the agent is capable of affecting the interaction of thenucleotide sequence or the expression product with the G.beta..

10. An assay comprising contacting an agent with a mutant of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof in the presence of a G.beta. capable of being associated with Cdc24p or a homologue thereof; and determining whether the agent is capable of affecting theinteraction of the mutant nucleotide sequence or the expression product with the G.beta..

11. A kit comprising a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof; and a G.beta. capable of being associated with Cdc24p or a homologue thereof.

12. A kit comprising a mutant of a nucleotide sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof or the expression product thereof; and a G.beta. capable of being associated with Cdc24p or a homologue thereof.

13. A protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof, wherein the protein has the capability of not substantially affecting the interaction of G.beta.
with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof.

14. A mutant of the protein sequence shown as SEQ I.D. No. 1 or a derivative or homologue thereof, wherein the mutant protein has the capability of substantially affecting the interaction of G.beta. with Cdc24p or a homologue thereof that is usually capable of being associated with the Cdc24p or the homologue thereof.

15. A protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof for use in medicine.

16. A mutant of the protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof for use in medicine.

17. Use of a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof in the manufacture of a medicament to affect the growth behaviour of cells.

18. Use of a mutant of a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof in the manufacture of a medicament to affect the growth behaviour of cells.

19. Use of a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof in a screen to identify one or more agents that are capable of affecting the interaction of Cdc24p or a homologue thereof thereof with a G.beta. or an associated Rho-family GTPase.

20. Use of a mutant of a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof in a screen to identify one or more agents that are capable ofaffecting the interaction of Cdc24p or a homologue thereof with a G.beta. or an associated Rho-family GTPase.

21. An assay comprising contacting an agent with a protein sequence shown as SEQI.D. No. 1 or a derivative or homologue thereof in the presence of a G.beta. capable of being associated with Cdc24p or a homologue thereof; and determining whether theagent is capable of affecting the interaction of the protein sequence with the G.beta. or the Rho-family GTPase.

22. An assay comprising contacting an agent with a mutant of a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof in the presence of G.beta.
capable of being associated with Cdc24p or a homologue thereof; and determining whether the agent is capable of affecting the interaction of the mutant protein sequence with the G.beta. or the Rho-family GTPase.

23. A kit comprising a protein sequence shown as SEQ I.D. No. 2 or a derivative or homologue thereof; and a G.beta. capable of being associated with Cdc24p or a homologue thereof.

24. A kit comprising a mutant of a protein sequence shown as SEQ I.D. No. 2 or aderivative or homologue thereof; and a G.beta. capable of being associated with Cdc24p or a homologue thereof.