Cloning and Characterization of shk2, a Gene Encoding a Novel p21-activated Protein Kinase from Fission Yeast
1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês
10.1074/jbc.273.29.18481
ISSN1083-351X
AutoresPeirong Yang, Sanjay Kansra, Ruth A. Pimental, Mary Gilbreth, Stevan Marcus,
Tópico(s)Cancer-related Molecular Pathways
ResumoWe describe the characterization of a novel gene,shk2, encoding a second p21cdc42/rac-activated protein kinase (PAK) homolog in fission yeast. Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro. While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo. Fission yeastshk2 null mutants are viable and exhibit no obvious phenotypic defects. Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation. Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast. We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast. Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function. We describe the characterization of a novel gene,shk2, encoding a second p21cdc42/rac-activated protein kinase (PAK) homolog in fission yeast. Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro. While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo. Fission yeastshk2 null mutants are viable and exhibit no obvious phenotypic defects. Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation. Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast. We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast. Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function. The cdc42 gene has been highly conserved through evolution and encodes a small GTPase belonging to the Rho family of Ras-related guanine nucleotide-binding proteins (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 2Ridley A.J. Curr. Opin. Genet. Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 3Hotchin N.A. Hall A. Cancer Surveys. 1996; 27: 311-322PubMed Google Scholar). Homologs ofcdc42 have been cloned from the evolutionarily distant yeasts Saccharomyces cerevisiae (4Johnson D.I. Pringle J.R. J. Cell. Biol. 1990; 111: 143-152Crossref PubMed Scopus (403) Google Scholar) andSchizosaccharomyces pombe (5Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar), the nematodeCaenorhabditis elegans (6Chen W. Lim H.H. Lim L. J. Biol. Chem. 1993; 268: 13280-13285Abstract Full Text PDF PubMed Google Scholar), insects (7Luo L. Liao Y.J. Jan L.Y. Jan Y.N. Genes Dev. 1994; 8: 1787-1802Crossref PubMed Scopus (806) Google Scholar), and mammals (8Shinjo K. Koland J.G. Hart M.J. Narasimhan V. Johnson D.I. Evans T. Cerione R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9853-9857Crossref PubMed Scopus (172) Google Scholar). Until recently, the cellular functions of Cdc42 proteins were unknown. However, recent studies from a variety of model systems have provided substantial insights into Cdc42 function. In mammalian cells, Cdc42 and a related GTPase, Rac, have been shown to participate in regulation of the actin cytoskeleton, cell cycle control, and mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PAK, p21cdc42/rac-activated protein kinase; WASP, Wiskott-Aldrich syndrome protein; kb, kilobase pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; THP, TrcHis peptide; CRIB, Cdc42/Rac interactive binding; GST, glutathione S-transferase; LBD, LexA DNA binding domain; GAD, Gal4 activating domain; GBD, Gal4 DNA-binding domain. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PAK, p21cdc42/rac-activated protein kinase; WASP, Wiskott-Aldrich syndrome protein; kb, kilobase pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; THP, TrcHis peptide; CRIB, Cdc42/Rac interactive binding; GST, glutathione S-transferase; LBD, LexA DNA binding domain; GAD, Gal4 activating domain; GBD, Gal4 DNA-binding domain.cascades (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 2Ridley A.J. Curr. Opin. Genet. Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 3Hotchin N.A. Hall A. Cancer Surveys. 1996; 27: 311-322PubMed Google Scholar). At least two types of proteins have been implicated as effectors for Cdc42 in mammalian cells. The first are members of a recently elucidated family of protein kinases referred to as p21cdc42/rac-activated kinases, or PAKs (9Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (263) Google Scholar). PAKs, like Cdc42, are conserved from yeasts to mammals and are activated by Cdc42 and Rac GTPases but not by other small GTPases, such as Ras and Rho. In a recent study, evidence was provided that p65PAK(α-Pak/Pak1) is required for Cdc42-induced activation of the c-Jun N-terminal kinase/stress-activated protein kinase cascade, but not for Cdc42-induced cytoskeletal remodeling or DNA synthesis (10Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenström P. Bridges T. Chant J. Hall A. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). PAKs induce c-Jun N-terminal kinase/stress-activated protein kinase activation in vitro (11Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), so it would appear that they are likely mediators of Cdc42-induced c-Jun N-terminal kinase/stress-activated protein kinase activation in vivo. In another recent study, it was shown that dominant-activated mutants of α-Pak/Pak1 induce dissolution of actin stress fibers and reorganization of focal complexes (13Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). Thus, a role for PAKs in cytoskeletal regulation is likely, although the exact nature of this function is, at present, unclear. A second putative Cdc42 effector in mammalian cells is the Wiskott-Aldrich syndrome protein, or WASP (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar,15Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar). WASP binds to Cdc42, but not to Rac or Rho GTPases (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). WASP is highly enriched in polymerized actin (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), and T lymphocytes from patients with Wiskott-Aldrich syndrome, an immunodeficiency disease, exhibit highly aberrant cytoskeletal organization (15Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar). Thus, WASP is likely to mediate at least part of the cytoskeletal regulatory functions of Cdc42.Substantial insights into the function and regulation of Cdc42 GTPases have come from studies using yeast model systems. In the budding yeastS. cerevisiae, Cdc42 is required for activation of a mating pheromone-induced MAPK cascade and for proper bud site selection, a process involving reorganization of the actin cytoskeleton (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 16Simon M.N. De Virgilio C. Souza B. Pringle J.R. Abo A. Reed S.I. Nature. 1995; 376: 702-705Crossref PubMed Scopus (179) Google Scholar). Cdc42 is also required for induction of the filamentous growth phase ofS. cerevisiae, a process that involves some, but not all, of the components of the pheromone signaling pathway, as well as Ras protein function (17Roberts R.L. Fink G.R. Genes Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (522) Google Scholar, 18Mosch H.U. Roberts R.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5352-5356Crossref PubMed Scopus (297) Google Scholar). Two PAK homologs, Ste20 (19Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar, 20Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar, 21Peter M. Neiman A.M. Park H.O. Vanlohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (189) Google Scholar, 22Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 59-66Crossref PubMed Scopus (188) Google Scholar) and Cla4 (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar), and perhaps a third, Skm1 (24Martin H. Mendoza A. Rodriguezpachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar), are probable effectors for Cdc42 in S. cerevisiae. Ste20 and Cla4 are partially redundant in function. S. cerevisiae mutants deleted of thecla4 gene are morphologically aberrant (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar) but mating-competent, while ste20 null mutants are sterile and defective in filamentous growth induction (19Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar, 20Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar). Mutants deleted of both STE20 and CLA4 are inviable and cannot undergo cytokinesis (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar). Thus, Ste20 and Cla4 share at least one essential cellular function. While skm1 null mutants are viable and exhibit no obvious phenotypic defects, overexpression ofskm1 leads to aberrant cell morphology, suggesting a role for the Skm1 protein in morphological regulation (24Martin H. Mendoza A. Rodriguezpachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar).The fission yeast S. pombe possesses a single knowncdc42 gene, which is essential for cell viability (5Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar). Wild type fission yeast cells are rod-shaped, whereas cdc42 null cells are spheroidal in morphology and exhibit mislocalization of actin (5Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar). This phenotype suggests a role for Cdc42 in cytoskeletal regulation. Chang et al. (25Chang E.C. Barr M. Wang Y. Jung V. Xu H.P. Wigler M.H. Cell. 1994; 79: 131-141Abstract Full Text PDF PubMed Scopus (242) Google Scholar) showed that Cdc42 participates in a Ras-mediated morphological control pathway in S. pombe. These investigators provided genetic and biochemical evidence that Cdc42 and Ras1, the single known S. pombe Ras homolog, are part of a complex of interacting proteins that includes the putative Cdc42 guanine nucleotide exchange factor Scd1 and Scd2, an SH3 domain-containing protein of unknown function. Scd1 and Scd2 are homologous to Cdc24 and Bem1, respectively, which have been shown to regulate Cdc42 function in S. cerevisiae (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar). Previously, we provided evidence that a Ste20/PAK homolog, Shk1 (also known as Pak1 (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar)) is a critical effector for Cdc42 in S. pombe (27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). Cdc42 and Shk1 interact physically, as determined by both two-hybrid assays (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar) and coprecipitation experiments (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar). shk1, like cdc42, is an essential gene, and the terminal phenotypes of cdc42 and shk1 null mutants are similar (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). Furthermore, overexpression of shk1partially suppresses the mating defect of S. pombe mutants expressing a dominant negative mutant allele of cdc42 (27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). Like Cdc42, Shk1 is also linked to Ras function in S. pombe. Overexpression of dominant negative forms of shk1 results in inhibition of Ras-dependent mating responses (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). In addition, cooverexpression of shk1 and skb1, a gene we recently described that encodes a second putative Shk1 regulator, restores elongate morphology to S. pombe ras1null mutants (28Gilbreth M. Yang P.R. Wang D. Frost J. Polverino A. Cobb M.H. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13802-13807Crossref PubMed Scopus (60) Google Scholar). These various data suggest that Shk1 is a key mediator of the Ras1/Cdc42 signaling complex in S. pombe.In this report, we describe the cloning and characterization ofshk2, a novel gene encoding a second Ste20/PAK-related protein kinase in S. pombe. Shk2 is more closely related in structure to the S. cerevisiae PAKs Cla4 and Skm1 than to other known yeast and metazoan PAKs. We provide evidence for physical and functional interaction between Shk2 and Cdc42 and for involvement of Shk2 in Ras1/Cdc42-mediated morphological control and mating response pathways. We show that Shk2 is not essential for viability, normal morphology, or mating in S. pombe and provide evidence that its functions substantially overlap with those of Shk1, with Shk1 being the dominant protein in function. We also provide genetic evidence corroborating a role for Shk1 in MAPK cascade-dependent mating response in S. pombe. Finally, we show that, despite the structural relatedness of the two proteins, Shk2 cannot substitute for Cla4 in budding yeast, suggesting that Shk2 and Cla4 are not functional homologs.DISCUSSIONIn this report, we have described the cloning and characterization of shk2, a gene encoding a novel PAK in the fission yeast,S. pombe. Our results suggest that Shk2, like the previously described fission yeast PAK Shk1 (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar, 28Gilbreth M. Yang P.R. Wang D. Frost J. Polverino A. Cobb M.H. Marcus S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13802-13807Crossref PubMed Scopus (60) Google Scholar), participates in Ras- and Cdc42-dependent morphological control and mating response pathways. While shk2 deletion mutants exhibit no obvious defects in growth, morphology, or mating, overexpression ofshk2 restores viability and elongate morphology to theS. pombe shk1 null mutant. Overexpression of shk2does not restore full mating competence to the shk1 null mutant. These results suggest that Shk1 and Shk2 may be partially redundant, with Shk1 being the dominant protein in function. We cannot rule out the possibility that an additional Shk2-related PAK exists inS. pombe. However, from our PCR-based cloning approach, which utilized degenerate oligonucleotide primers based on the S. cerevisiae Ste20 protein sequence, we identified only theshk1 and shk2 genes.Results of previous studies by us and others provided evidence for involvement of Shk1 in the S. pombe mating response pathway. First, overexpression of a catalytically defective mutant of Shk1 inhibited mating of S. pombe cells (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar). Second, overexpression of the N-terminal regulatory domain of Shk1 attenuated the hypersexual response of S. pombe cells expressing the dominant activated ras1(G17V) mutant (27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). Finally, overexpression of shk1 partially bypassed the mating defect of S. pombe cells expressing the dominant inhibitorycdc42(T17N) allele (27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). In this report, we have shown thatS. pombe cells deleted of shk1 but overexpressingshk2 exhibit a significant mating defect. Furthermore, we have shown that this defect can be largely suppressed by overexpression of the MAPK kinase kinase Byr2 and, additionally, that overexpression of shk2 partially bypasses the mating defect of S. pombe cells expressing the dominant inhibitorycdc42(T17N) allele. Our results corroborate a role for Shk1 in the S. pombe mating response pathway, which was suggested in previous studies (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). In addition, our results suggest that, with regard to mating responses, Byr2 acts downstream from the Shk kinases. Our results provide the first direct genetic evidence linking PAKs to regulation of a MAPK module S. pombe. A homolog of Byr2, Ste11, has been similarly implicated as a downstream target for the Shk1 homolog Ste20 in S. cerevisiae (19Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar, 47Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).Although our results suggest that Shk1 function is dominant over that of Shk2, it is possible that Shk2 is required for cellular functions for which we have not assayed. Interestingly, the R3 subdomains of Shk1 and Shk2 lack any discernible structural homology. It is possible that these domains might specify unique molecular functions for each kinase. Further insights into Shk2 function and perhaps PAK functions in general may be gained by conducting genetic screens for S. pombe mutants that are synthetically lethal with theshk2 null mutation.In both S. pombe and S. cerevisiae, PAKs are required not only for mating responses but also for essential cellular functions unrelated to mating. The specific nature of these essential functions has yet to be defined in either yeast. S. cerevisiae possesses three PAK-encoding genes. Two of these,STE20 and CLA4, are partially overlapping in function (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar). The third, Skm1, is completely dispensable (24Martin H. Mendoza A. Rodriguezpachon J.M. Molina M. Nombela C. Mol. Microbiol. 1997; 23: 431-444Crossref PubMed Scopus (47) Google Scholar). Deletion of STE20 results in sterility (19Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (344) Google Scholar, 20Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar), while deletion of CLA4 results in aberrant morphology (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar). However, deletion of both CLA4 and STE20 genes is lethal (23Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar). PAK wiring is clearly different in S. pombe, in which a single PAK, Shk1, has essential functions not shared by other PAKs (26Ottilie S. Miller P.J. Johnson D.I. Creasy C.L. Sells M.A. Bagrodia S. Forsburg S.L. Chernoff J. EMBO J. 1995; 14: 5908-5919Crossref PubMed Scopus (127) Google Scholar, 27Marcus S. Polverino A. Chang E. Robbins D. Cobb M.H. Wigler M.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6180-6184Crossref PubMed Scopus (135) Google Scholar). These differences are not surprising, given that fact that S. cerevisiae and S. pombe are a half billion years diverged in evolution (45Sipiczki M. Nasim A. Young P. Johnson B.F. Molecular Biology of the Fission Yeast. Academic Press, Inc., San Diego, CA1989: 431-452Crossref Google Scholar). It remains to be determined whether the essential functions of PAKs in S. pombe andS. cerevisiae are conserved in higher organisms or, for that matter, between the two distantly related yeasts.Note Added in ProofThe shk2 gene has been cloned independently by Sells et al. (Sells, M. A., Barratt, J. T., Caviston, J., Ottilie, S., Lebever, E., and Chernoff, J. (1998)J. Biol. Chem. 273,18490–18498), who named the genepak2.Their results are consistent with and complement the results presented in this paper. The cdc42 gene has been highly conserved through evolution and encodes a small GTPase belonging to the Rho family of Ras-related guanine nucleotide-binding proteins (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 2Ridley A.J. Curr. Opin. Genet. Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 3Hotchin N.A. Hall A. Cancer Surveys. 1996; 27: 311-322PubMed Google Scholar). Homologs ofcdc42 have been cloned from the evolutionarily distant yeasts Saccharomyces cerevisiae (4Johnson D.I. Pringle J.R. J. Cell. Biol. 1990; 111: 143-152Crossref PubMed Scopus (403) Google Scholar) andSchizosaccharomyces pombe (5Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar), the nematodeCaenorhabditis elegans (6Chen W. Lim H.H. Lim L. J. Biol. Chem. 1993; 268: 13280-13285Abstract Full Text PDF PubMed Google Scholar), insects (7Luo L. Liao Y.J. Jan L.Y. Jan Y.N. Genes Dev. 1994; 8: 1787-1802Crossref PubMed Scopus (806) Google Scholar), and mammals (8Shinjo K. Koland J.G. Hart M.J. Narasimhan V. Johnson D.I. Evans T. Cerione R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9853-9857Crossref PubMed Scopus (172) Google Scholar). Until recently, the cellular functions of Cdc42 proteins were unknown. However, recent studies from a variety of model systems have provided substantial insights into Cdc42 function. In mammalian cells, Cdc42 and a related GTPase, Rac, have been shown to participate in regulation of the actin cytoskeleton, cell cycle control, and mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PAK, p21cdc42/rac-activated protein kinase; WASP, Wiskott-Aldrich syndrome protein; kb, kilobase pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; THP, TrcHis peptide; CRIB, Cdc42/Rac interactive binding; GST, glutathione S-transferase; LBD, LexA DNA binding domain; GAD, Gal4 activating domain; GBD, Gal4 DNA-binding domain. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PAK, p21cdc42/rac-activated protein kinase; WASP, Wiskott-Aldrich syndrome protein; kb, kilobase pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; THP, TrcHis peptide; CRIB, Cdc42/Rac interactive binding; GST, glutathione S-transferase; LBD, LexA DNA binding domain; GAD, Gal4 activating domain; GBD, Gal4 DNA-binding domain.cascades (1Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 2Ridley A.J. Curr. Opin. Genet. Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 3Hotchin N.A. Hall A. Cancer Surveys. 1996; 27: 311-322PubMed Google Scholar). At least two types of proteins have been implicated as effectors for Cdc42 in mammalian cells. The first are members of a recently elucidated family of protein kinases referred to as p21cdc42/rac-activated kinases, or PAKs (9Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (263) Google Scholar). PAKs, like Cdc42, are conserved from yeasts to mammals and are activated by Cdc42 and Rac GTPases but not by other small GTPases, such as Ras and Rho. In a recent study, evidence was provided that p65PAK(α-Pak/Pak1) is required for Cdc42-induced activation of the c-Jun N-terminal kinase/stress-activated protein kinase cascade, but not for Cdc42-induced cytoskeletal remodeling or DNA synthesis (10Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenström P. Bridges T. Chant J. Hall A. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). PAKs induce c-Jun N-terminal kinase/stress-activated protein kinase activation in vitro (11Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), so it would appear that they are likely mediators of Cdc42-induced c-Jun N-terminal kinase/stress-activated protein kinase activation in vivo. In another recent study, it was shown that dominant-activated mutants of α-Pak/Pak1 induce dissolution of actin stress fibers and reorganization of focal complexes (13Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). Thus, a role for PAKs in cytoskeletal regulation is likely, although the exact nature of this function is, at present, unclear. A second putative Cdc42 effector in mammalian cells is the Wiskott-Aldrich syndrome protein, or WASP (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar,15Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar). WASP binds to Cdc42, but not to Rac or Rho GTPases (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). WASP is highly enriched in polymerized actin (14Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), and T lymphocytes from patients with Wiskott-Aldrich syndrome, an immunodeficiency disease, exhibit highly aberrant cytoskeletal organization (15Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar). Thu
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