AU4158100A

AU4158100A - Human protein kinase bgamma polypeptides and a method for disrupting protein kinase bgamma in a non-human

Info

Publication number: AU4158100A
Application number: AU41581/00A
Authority: AU
Inventors: Anneli Attersand
Original assignee: Pharmacia and Upjohn AB
Current assignee: Swedish Orphan Biovitrum AB
Priority date: 1999-03-25
Filing date: 2000-03-23
Publication date: 2000-10-16
Also published as: SE9901115D0; CA2368379A1; EP1163327A1; WO2000058446A1; JP2002539823A

Description

WO 00/58446 PCT/SEOO/00571 HUMAN PROTEIN KINASE By POLYPEPTIDES AND A METHOD FOR DISRUPTING PROTEIN KINASE By IN A NON-HUMAN TECHNICAL FIELD 5 The present invention relates to human protein kinase B polypeptides and to nucleic acid molecules coding for such polypeptides. The invention further relates to vectors and cells comprising said nucleic acid molecules, as well as to methods for screening test compounds which affect the insulin signaling pathway. 10 BACKGROUND ART Insulin plays a key role in regulating a wide range of cellular processes. The activation of class la phopshoinositide 3-kinase (PI 3-kinase) is necessary to elicit many of insulin's 15 effects on glucose and lipid metabolism (for reviews, see Shepherd, P.R. et al. (1998) Biochem. J. 333, 471-490; Alessi, D.R. & Downes, C.P. (1998) Biochim. Biophys. Acta 1436, 151-164). One of the major ways the lipid products of PI 3-kinase act in insulin signaling is by binding to pleckstrin homology (PH) domains of phosphoinositide dependent protein kinase (PDK1) and protein kinase b (PKB). This mechanism regulates 20 the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. Protein kinase B (for reviews, see e.g. Coffer, P.J. et al. (1998) Biochem. J. 335, 1-13: Alessi, D.R. & Cohen, P. (1998) Current Opinion in Genetics & Development 8, 55-62) 25 has also been called Akt or RAC (Related to protein kinase A and C). There are three mammalian isoforms of PKB, namely PKBa, PKBp and PKBy, but most studies of the regulation and specificity of this enzyme have so far been focused on the PKBa isoform. The activation of PKBca by insulin or insulin growth factor 1 (IGF1) results from its phosphorylation at two residues, Thr 308 and Ser 473 , which are phosphorylated by distinct 30 enzymes.

WO 00/58446 PCT/SEOO/00571 -2 In Caenorhabditis elegans it has been shown that two Akt/PKB homologs, called akt- 1 and akt-2, transduce insulin receptor-like signals (Paradis, S. & Ruvkun, G. (1998) Genes & Development 12, 2488-2498). 5 The molecular cloning of human PKB . is disclosed by Jones, P. F. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4171-4175. Human PKB P is disclosed by Cheng et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9267-9271. RAC-protein kinase is disclosed in WO 97/18303. 10 The article by Konishi, H. et al. (1995) Biochem. Biophys. Res. Comm. 216, 526-534 discloses cloning of PKB y from a rat brain cDNA library. An isolated cDNA clone had an open reading frame encoding a sequence of 454 amino acids with the molecular weight of 52,845. The amino acid sequence showed approximately 83% and 77% sequence homology with rat PKB c and rat PKB P, respectively. 15 Like PKB a and PKB P, PKB y possess an N-terminal PH domain and a residue equivalent to Thr 30 8 (Thr 305 in PKB y). However, on the basis of the disclosure by Konishi et al., it has been generally believed that PKB y has a C-terminal truncation of 23 residues and thereby lacks a residue equivalent to Ser 473 (see e.g. Coffer et al., supra; Alessi & Downes, 20 supra; Alessi & Cohen, supra; Walker et al. (1998) Biochem. J. 331, 299-308). It has thus been believed that PKB y is activated solely via phosphorylation of Thr 30 5 . DISCLOSURE OF THE INVENTION 25 It has surprisingly been found that human protein kinase B y is a protein comprising 479 amino acids, including the 23 amino acid C-terminal region lacking in the enzyme cloned from rat brain (Konishi et al., supra). Human protein kinase B y comprises not only the phosphorylation residue Thr 305 but also a putative second phosphorylation residue Ser 472 . 30 WO 00/58446 PCT/SEOO/00571 -3 In a first aspect this invention provides an isolated nucleic acid molecule selected from: (a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 and which codes for human protein kinase B y; (b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing under 5 stringent hybridization conditions to a nucleotide sequence complementary the polypeptide coding region of a DNA molecule as defined in (a) and which codes for human protein kinase B y, or a functionally equivalent modified form thereof; and (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b) and which codes io for human protein kinase B y, or a functionally equivalent modified form thereof. The term "stringent hybridization conditions" is known in the art from standard protocols (e.g. Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A is laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989) and could be understood as e.g. hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65'C, and washing in 0.1xSSC / 0.1% SDS at +68*C. 20 In a preferred form of the invention, the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NO: 1 of the Sequence Listing. However, the nucleic acid molecule according to the invention is not to be limited strictly to the sequence shown as SEQ ID NO: 1. Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which 25 nevertheless encode proteins having substantially the biochemical activity of protein kinase B y. Included in the invention are consequently nucleic acid molecules, the nucleotide sequence of which is at least 85% homologous, preferably at least 90 or 95% homologous, with the nucleotide sequence shown as SEQ ID NO: I in the Sequence Listing. 30 Included in the invention is also a DNA molecule which nucleotide sequence is degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO: 1. A sequential grouping of three nucleotides, a "codon", codes for one amino acid. Since there WO 00/58446 PCT/SE00/00571 -4 are 64 possible codons, but only 20 natural amino acids, most amino acids are coded for by more than one codon. This natural "degeneracy", or "redundancy", of the genetic code is well known in the art. It will thus be appreciated that the DNA sequence shown in the Sequence Listing is only an example within a large but definite group of DNA sequences 5 which will encode the polypeptide as described above. In a further aspect the invention includes a nucleic acid molecule as described above wherein the said human protein kinase B y, or the functionally equivalent form thereof, comprises at least two regulation phosphorylation sites. By comparison with the previously 1o known PKB ax and PKB P amino acid sequences, regulation phosphorylation sites can be assigned to the residues Thr 305 and Ser 472 . In a preferred form of the invention, the nucleic acid molecule according to the invention is coding for human protein kinase B y, or a functionally equivalent form thereof, which is comprises at least 455 amino acids, or more preferably comprises essentially the amino acid sequence shown as positions 455 to 480 in SEQ ID NO: 2. In a further aspect this invention provides an isolated human protein kinase B y polypeptide, encoded by a nucleic acid molecule according to the invention. In a preferred 20 form, the said human protein kinase B y polypeptide has an amino acid sequence according to SEQ ID NO: 2 of the Sequence Listing. However, the polypeptide according to the invention is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or 25 inversions, which polypeptides nevertheless have substantially the biological activities of protein kinase B y. Included in the invention are consequently polypeptides, the amino acid sequence of which is at least 85% homologous, preferably at least 90% or 95% homologous, with the amino acid sequence shown as SEQ ID NO: 2 in the Sequence Listing. 30 Included in the invention are variants, derivatives, and fragments of a protein kinase B y polypeptide. The skilled person will readily be able to determine whether such a variant, WO 00/58446 PCT/SEO0/00571 -5 derivative, or fragment of a protein kinase B y polypeptide displays protein kinase B y activity by subjecting the variant, derivative, or fragment to a standard protein kinase assay. Such assays are well known in the art, see e.g. Konishi et al. (1995) Biochem. Biophys. Res. Comm. 216, 526-534. 5 The present invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as host cells transformed with such vectors. Any of the nucleic acid molecules of the invention may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. Because the invention also 10 provides protein kinase B y polypeptides expressed from the nucleic acid molecules described above, vectors for the expression of protein kinase B y are preferred. Such expression vectors include DNA encoding a protein kinase B y polypeptide, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include is transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding protein kinase B y. Thus, a promoter nucleotide sequence is operably linked to a PKB DNA sequence if the promoter nucleotide sequence directs the transcription of the PKB sequence. 20 The vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of 25 chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini chromosome or virus. Alternatively, the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Examples of suitable vectors are a bacterial expression vector and a yeast expression vector. The vector of the invention may 30 carry any of the DNA molecules of the invention as defined above.

WO 00/58446 PCT/SEOO/00571 -6- A suitable host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism. The host cell can thus e.g. be a bacterial cell such as an E. coli cell; a cell from a yeast such as Saccharomyces cervisiae or Pichia pastoris, or a mammalian cell. The methods employed to effect introduction of the vector into the host 5 cell are standard methods well-known to a person familiar with recombinant DNA methods. Included in the invention is a process for production of a human protein kinase B y polypeptide, which comprises culturing a host cell as described above under conditions 10 whereby said polypeptide is produced, and optionally recovering said polypeptide. A further aspect of the invention is a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with at least a part of the nucleic acid molecule according to the invention, said part having a sequence shown as positions 1363 to 1446 in 15 SEQ ID NO: 1. The invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to at least a part of the nucleic acid molecule according to the invention, said part having a sequence shown as positions 1363 to 1446 in SEQ ID NO: 1. 20 Fragments of the nucleic acid molecules described herein, as well as polynucleotides capable of hybridizing to such nucleic acid molecules may be used as a probe or as primers in a polymerase chain reaction (PCR). Such probes may be used, e.g., to detect the presence of nucleic acids coding for PKB in in vitro assays, as well as in Southern and Northern blots. Cell types expressing PKB may also be identified by the use of such probes. Such procedures 25 are well known, and the skilled artisan will be able to choose a probe of a length suitable to the particular application. For PCR, 5' and 3' primers corresponding to the termini of a desired PKB nucleic acid molecule are employed to isolate and amplify that sequence using conventional techniques. 30 The polypeptides of the present invention may also be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting PKB polypeptide expression. Such antibodies may be prepared by conventional techniques. See, for example, WO 00/58446 PCT/SEOO/00571 -7-_ Antibodies: A Laboratoiv Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980). 5 Included in the invention is a method for screening for compounds which affect the insulin signaling pathway, comprising (a) providing a human protein kinase B y polypeptide; (b) contacting a test compound to the said human protein kinase B y polypeptide; and (c) determining the effect of said test compound on said human protein kinase B y 10 polypeptide. The invention also provides a method for disrupting the protein kinase B y gene in a non human embryonic stem cell, said method comprising (a) providing an oligonucleotide capable of disrupting said protein kinase By gene; and is (b) introducing said oligonucleotide into the non-human embryonic stem-cell under conditions such that the said oligonucleotide is homologously recombined into at least one of the naturally occurring protein kinase B y genes in the genome of said cell to produce a cell containing at least one disrupted protein kinase B y allele. The invention further provides a non-human transgenic animal expressing reduced levels of protein kinase B y, 20 wherein the protein kinase B y gene has been disrupted by this method. The invention also provides a non-human transgenic animal whose genome comprises an antisense nucleic acid molecule which hybridizes to mRNA encoding human protein kinase B y, thereby reducing its translation. A transgenic non-human mammal can e.g. be 25 mammalian animal, such a mouse, or an animal of the species Caenorhabditis elegans. The invention also provides a method for screening compounds for affecting the insulin signaling pathway comprising (a) providing a non-human animal as described above; 30 (b) providing a composition comprising a test compound in a form suitable for administration to the said non-human animal; WO 00/58446 PCT/SEOO/00571 -8 (c) administering said test compound to said non-human animal; and (d) determining the effect of the test compound on the insulin-signaling pathway. 5 EXAMPLES EXAMPLE 1: Molecular Cloning and Sequence Analysis Marathon-Ready T M heart cDNA from Clontech was used as a template for cloning. 10 PCR primers shown as SEQ ID NOS: 3 to 6 were used. The PCR reactions were performed using Advantage® cDNA polymerase mix from Clontech. The 3'-RACE PCR reaction was performed as described in Marathon-Ready T M cDNA users manual (PT l156-1) using primers shown as SEQ ID NOS: 3 and 5 and with the following PCR program: +94'C for 5 seconds, +65*C for 30 seconds, +68*C for 3 min, during 30 (+ 5 cycles), with an initial is step of +94*C for 30 seconds and a last step of +72'C for 7 min. A Perkin Elmer 2400 machine was used. A smear of bands was seen on agarose gel and a second PCR reaction with a nested primer XKOW #43 was performed to see if any of the bands could be amplified further. A band of 20 approximately 2400 bp was distinguished from the others and was excised from agarose gel. After further amplification and purification, the DNA was cleaved with restriction enzymes (RcaI and PstI) to verify a similarity with rat PKB y (Konishi et al., supra). To clone the PCR fragment in a plasmid vector, the least amplified PCR material was used. 25 This material was purified from agarose gel by using the Wizard PCRpreps DNA purification system and cloned into the plasmid pCR ®2. 1-TOPO (Invitrogen) by using the TOPO TA cloning kit (Invitrogen). Plasmid DNA from three clones were obtained by using the QlAprep spin plasmid kit (QIAGEN) and the subsequent sequencing was performed by the dideoxy chain-termination method using ABI PrismTM 377 DNA 30 sequencer XL (Applied Biosystems) WO 00/58446 PCT/SEOO/00571 -9 EXAMPLE 2: Northern Blotting A probe for northern blot analysis was obtained from the cloned PKB y by a PCR reaction 5 with primers shown as SEQ ID NOS: 7 and 8 which gave a fragment of 362 bp in the PH domain of the gene. The PCR reactions was performed using Advantage@ cDNA polymerase mix from Clontech with the following PCR program: +94'C for 30 seconds, +54'C for 30 seconds, +72'C for 1 min for 30 cycles, with an initial step of +94'C for 3 min and a last step of +72'C for 7 min. A Perkin Elmer 9700 machine was used. The band 1a of interest was excised from agarose gel and purified by using QlAquick Gel Extraction Kit (QIAGEN). The probe was radioactively labeled according to the rediprime TM 11 kit (Amersham Pharmacia Biotech). Human Multiple Tissue Northern (MTN T M ) Blots (Clontech) was used for hybridization of the cDNA probe as described in the MTN Blots protocol. 15 EXAMPLE 3: Use of antisense oligonucleotides for producing knock-out animals Knowledge of the cDNA sequence of protein kinase B y will enable its use as a tool in 20 sense (Yuossoufian et al. (1993) Mol. Cell. Biol. 13, 98-104) or antisense (Eguchi et al. (1991) Ann. Rev. Biochem 60, 631-652) technologies for the investigation of gene function. Oligonucleotides, from genomic or cDNA's, comprising either the sense or antisense strand of the cDNA sequence can be used in vitro or in vivo to inhibit expression. Such technology is well known in the art, and oligonucleotides or other fragments can be 25 designed from various locations along the sequences. The gene of interest can be turned off in the short term by transfecting a cell or tissue with expression vectors which will flood the cell with sense or antisense sequences until all copies of the vector are disabled by endogenous nucleases.

Claims

1. An isolated nucleic acid molecule selected from: (a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID 5 NO: 1 and which codes for human protein kinase B y; (b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing under stringent hybridization conditions to a nucleotide sequence complementary the polypeptide coding region of a DNA molecule as defined in (a) and which codes for human protein kinase B y, or a functionally equivalent modified form thereof; and 10 (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b) and which codes for human protein kinase B y, or a functionally equivalent modified form thereof. 15

2. The nucleic acid molecule according to claim 1 which has a sequence homology of above 85% with the nucleic acid molecule having a nucleotide sequence shown as SEQ ID NO: 1.

3. The nucleic acid molecule according to claim 1 or 2 wherein the said human protein 20 kinase B y, or the functionally equivalent form thereof, comprises at least 455 amino acids.

4. The nucleic acid molecule according to claim 3 wherein the said human protein kinase B y, or the functionally equivalent form thereof, comprises essentially the 25 amino acid sequence shown as positions 455 to 479 in SEQ ID NO: 2.

5. The nucleic acid molecule according to claim 3 or 4 wherein the said human protein kinase B y, or the functionally equivalent form thereof, comprises at least two regulation phosphorylation sites. 30 WO 00/58446 PCT/SEOO/00571 - 11

6. The nucleic acid molecule according to claim 5 wherein the said human protein kinase B y, or the functionally equivalent form thereof, comprises regulation phosphorylation sites at least at positions 305 and 472 in SEQ ID NO: 2. 5

7. An isolated human protein kinase B y polypeptide, encoded by a nucleic acid molecule according to any one of claims I to 6.

8. An isolated human protein kinase B y polypeptide according to claim 7, having essentially an amino acid sequence shown as SEQ ID NO: 2 in the Sequence Listing. 10

9. A recombinant vector comprising a nucleic acid molecule according to any one of claims 1 to 6.

10. The vector according to claim 9 wherein the said nucleic acid molecule is operably 15 linked to the regulatory elements necessary for the expression of human protein kinase B y.

11. A cultured host cell harboring a vector according to claim 9 or 10. 20

12. A process for production of a human protein kinase B y polypeptide which comprises culturing a host cell according to claim 11 under conditions whereby said polypeptide is produced, and optionally recovering said polypeptide.

13. A nucleic acid probe comprising at least 15 nucleotides, which probe specifically 25 hybridizes with at least a part of the nucleic acid molecule according to claim 4, said part having a sequence shown as positions 1363 to 1446 in SEQ ID NO: 1.

14. An antisense oligonucleotide having a sequence capable of specifically hybridizing to at least a part of the nucleic acid molecule according to claim 4, said part having a 30 sequence shown as positions 1363 to 1446 in SEQ ID NO: 1. WO 00/58446 PCT/SE00/00571 - 12

15. An isolated antibody binding specifically to the human protein kinase B y polypeptide according to claim 7 or 8.

16. A method for screening for compounds which affect the insulin signaling pathway, 5 comprising (a) providing a human protein kinase B y polypeptide according to claim 7 or 8; (b) contacting a test compound to the said human protein kinase B y polypeptide; and (c) determining the effect of said test compound on said human protein kinase B y polypeptide. 10

17. A method for disrupting the protein kinase B y gene in a non-human embryonic stem cell, said method comprising (a) providing an oligonucleotide capable of disrupting said protein kinase B y gene; and 15 (b) introducing said oligonucleotide into the non-human embryonic stem-cell under conditions such that the said oligonucleotide is homologously recombined into at least one of the naturally occurring protein kinase B y genes in the genome of said cell to produce a cell containing at least one disrupted protein kinase B y allele. 20

18. A non-human transgenic animal expressing reduced levels of protein kinase B y, wherein the protein kinase B y gene has been disrupted by the method according to claim 17.

19. A non-human transgenic animal whose genome comprises an antisense nucleic acid 25 molecule which hybridizes to mRNA encoding human protein kinase B y, thereby reducing its translation.

20. The non-human transgenic animal according to claim 18 or 19 which is a mouse. 30

21. The non-human transgenic animal according to claim 18 or 19 which is an animal of the species Caenorhabditis elegans. WO 00/58446 PCT/SEOO/00571 - 13

22. A method for screening compounds for affecting the insulin signaling pathway comprising (a) providing a non-human transgenic animal according to claim 18 or 19; 5 (b) providing a composition comprising a test compound in a form suitable for administration to the said non-human animal; (c) administering said test compound to said non-human animal; and (d) determining the effect of the test compound on the insulin-signaling pathway.