Cloning and Characterization of shk2, a Gene Encoding a Novel p21-activated Protein Kinase from Fission Yeast*

Peirong Yang, Sanjay Kansra, Ruth A. Pimental, Mary GilbrethDagger , and Stevan Marcus§

From the Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 yeast shk2 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (1-3). Homologs of cdc42 have been cloned from the evolutionarily distant yeasts Saccharomyces cerevisiae (4) and Schizosaccharomyces pombe (5), the nematode Caenorhabditis elegans (6), insects (7), and mammals (8). 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)1 cascades (1-3). 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 (9). 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 (alpha -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 (10). PAKs induce c-Jun N-terminal kinase/stress-activated protein kinase activation in vitro (11, 12), 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 alpha -Pak/Pak1 induce dissolution of actin stress fibers and reorganization of focal complexes (13). 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 (14, 15). WASP binds to Cdc42, but not to Rac or Rho GTPases (14). WASP is highly enriched in polymerized actin (14), and T lymphocytes from patients with Wiskott-Aldrich syndrome, an immunodeficiency disease, exhibit highly aberrant cytoskeletal organization (15). 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 yeast S. 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 (1, 16). Cdc42 is also required for induction of the filamentous growth phase of S. cerevisiae, a process that involves some, but not all, of the components of the pheromone signaling pathway, as well as Ras protein function (17, 18). Two PAK homologs, Ste20 (19-22) and Cla4 (23), and perhaps a third, Skm1 (24), are probable effectors for Cdc42 in S. cerevisiae. Ste20 and Cla4 are partially redundant in function. S. cerevisiae mutants deleted of the cla4 gene are morphologically aberrant (23) but mating-competent, while ste20 null mutants are sterile and defective in filamentous growth induction (19, 20). Mutants deleted of both STE20 and CLA4 are inviable and cannot undergo cytokinesis (23). Thus, Ste20 and Cla4 share at least one essential cellular function. While skm1 null mutants are viable and exhibit no obvious phenotypic defects, overexpression of skm1 leads to aberrant cell morphology, suggesting a role for the Skm1 protein in morphological regulation (24).

The fission yeast S. pombe possesses a single known cdc42 gene, which is essential for cell viability (5). Wild type fission yeast cells are rod-shaped, whereas cdc42 null cells are spheroidal in morphology and exhibit mislocalization of actin (5). This phenotype suggests a role for Cdc42 in cytoskeletal regulation. Chang et al. (25) 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 (1). Previously, we provided evidence that a Ste20/PAK homolog, Shk1 (also known as Pak1 (26)) is a critical effector for Cdc42 in S. pombe (27). Cdc42 and Shk1 interact physically, as determined by both two-hybrid assays (26, 27) and coprecipitation experiments (26). shk1, like cdc42, is an essential gene, and the terminal phenotypes of cdc42 and shk1 null mutants are similar (26, 27). Furthermore, overexpression of shk1 partially suppresses the mating defect of S. pombe mutants expressing a dominant negative mutant allele of cdc42 (27). 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 (26, 27). 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 ras1 null mutants (28). 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 of shk2, 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Strains, Manipulation, and Genetic Analysis-- S. pombe strains used in this study were SP870 (h90 ade6-210 leu1-32 ura4-D18) (from D. Beach), SP870D (h90 ade6-210 leu1-32 ura4-D18/h90 ade6-210 leu1-32 ura4-D18) (from V. Jung), CHP428 (h+ ade6-210 his7-366 leu1-32 ura4-D18) (from E. Chang), SP66 (leu2-32 ade6-216) (from D. Beach), SP42N17 (h90 ade6-216 leu1-32 ura4::adh1-cdc42N17) (27), SPGLD (h90 ade6-210 leu1-32 ura4-D18 gpa1::LEU2/h90 ade6-210 leu1-32 ura4-D18 gpa1::LEU2) (29), SPRN1 (h90 ade6-210 leu1-32 ura4-D18 ras1Delta ) (30), SPRN1D (h90 ade6-210 leu1-32 ura4-D18 ras1Delta /h90 ade6-210 leu1-32 ura4-D18 ras1Delta ) (28), SP206U (h90 ade6-210 leu1-32 ura4-D18/h90 ade6-210 leu1-32 ura4-D18 shk1::ura4) (27), and SPSHK2U (h90 ade6-210 leu1-32 ura4-D18 shk2::ura4) (see below). S. cerevisiae strains used were L40 (MATa ade2 his3 leu2 trp1 LYS2::lexA-HIS3 URA3::lexA-lacZ) (31), HF7c (MATa ade2-101 his3-200 leu2-3, 112 lys2-801 trp1-901 ura3-52 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL417mers(x3)-CYC1TATA-lacZ) (32), SFY526 (MATa ade2-201 his3-200 leu2-3, 112 lys2-801 trp1-901 ura3-52 canR gla4-542 gal80-538, URA3::GAL1UAS-GAL1TATA-lacZ) (CLONTECH), FY40 (MATa, HMLa, HMRa, ho-Bgal, ura3, HIS4, ade2-1, canI-100, met, his3, leu2-3, 112, trp1-1, bar1::HisG, ste20::del, cla4::LEU2, Ycp TRP1 cla4-75(c2816)), and MJY8 (MATa, cla4::LEU2 his3-100 ura3-1 leu2-3, 112 can1-100) (from F. Cvrckova, Institute of Molecular Pathology, Wien, Austria). Standard yeast culture media and genetic methods were used (33, 34). S. pombe cultures were grown on either 0.5% yeast extract, 3% dextrose, and 75 mg/liter adenine (YEA) or Edinburgh minimal medium (EMM) with appropriate auxotrophic supplements (34). S. cerevisiae cultures were grown on either 1% yeast extract, 2% peptone, and 2% dextrose (YPD) or drop-out medium with appropriate auxotrophic supplements (33). Yeasts were transformed by the lithium acetate procedure (34). The shk2::ura4 strain, SPSHK2U, was constructed by transforming SP870D with a 3.2-kb HpaI-Ecl136II shk2::ura4 fragment from the plasmid pBSIIshk2::ura4. Diploid transformants carrying a single disrupted and a single wide-type copy of shk2 were identified by Southern blot analysis, and shk2::ura4 transformants were isolated by tetrad dissection.

Plasmids-- The two-hybrid plasmids pGADGH (for expression of GAD fusions), pHP5, and pGBT9 (for expression of GBD fusions) and pBTM116 and pVJL11 (for expression of LBD fusions) have been described previously (25, 31, 35, 36). The plasmids pGADCdc42, pGADCdc42(T17N), pGADCdc42(G12V), pGADShk1, pGADSTE20, pGADSkb1, pGADScd1, pGADScd2, pGADByr2, pGADByr1, pGADRas1, pGADSpk1, pGBDSkb1, pGBDScd1, pGBDScd2, pGBDByr1, pLBDCdc42, pLBDShk1, pLBDRaf, pLBDRas1, pLBDRas1G17V, pLBDGpa1, pLBDByr2, pLBDScd1, pLBDlamin, pSP206, pREP1Shk1, pAAUCMSkb1, pAAAU, and pAAAUByr2 have also been described (25, 27, 28, 36). pLBDRac and pLBDRhoG were provided by J. Camonis (Faculte de Medecine Lariboisiere, Paris). pRSETCdc42-Hs(G12V) and pRSETHa-Ras(G12V) were provided by J. Frost and M. Cobb (University of Texas Southwestern Medical Center, Dallas). The S. pombe-Escherichia coli shuttle vector pAAUCM was used for high level expression of coding sequences from the S. pombe adh1 promoter (27). pREP1 (37) was used for expressing coding sequences from the S. pombe nmt1 promoter. pREP1Shk2(K343R) was provided by J. Chernoff (Fox Chase Cancer Center, Philadelphia). FD44, a TRP1-based plasmid carrying the CLA4 gene, was provided by F. Cvrckova. pREP1Byr2 was made by cloning a SalI-SacI fragment of the byr2 coding sequence obtained from pAIS1 (38) into the corresponding sites of pREP1 and allows for overexpression of byr2 from the nmt1 promoter. A 2.1-kb fragment of the shk2 gene was amplified by polymerase chain reaction (PCR) using a plasmid harboring the shk2 gene (pSP204) as template and the primer pair 5'-TGCATCGTGTCGACAATGCTTTTAAGTGTAAGT and 5'-AGGCAGGTCGACAGTTAACTAACG. The PCR-amplified shk2 fragment was then digested by SalI and BclI and cloned into SalI and BamHI sites of pREP1, generating pREP1Shk2. pBSIIShk2 was constructed by cloning a SalI-Ecl136II fragment of shk2 from pREP1Shk2 into the SalI -EcoRV sites of pBluescript II SK (pBSII). pHP5Shk2 was obtained by cloning a SalI-Ecl136II fragment of shk2 from pBSIIShk2 into the SalI-NaeI sites of pHP5. A SalI-SacI fragment of shk2 was released from pREP1Shk2 and cloned into pAAUCM, pAUD6, and pAD5, generating pAAUCMShk2, pAUD6Shk2, and pAD5Shk2, respectively. pTrcHisBShk2 was constructed by cloning a SalI-PstI fragment from pBSIIshk2 into the XhoI-PstI sites of pTrcHisB (Invitrogen). pGADGHShk2 was constructed by two steps. First, an EcoRI fragment of Shk2 was released form pTrcHisBShk2 and cloned into the EcoRI sites of pGADGH producing pGADGHShk2-3'. A shk2 5'-fragment was released by BamHI from pHP5Shk2 and cloned into the 5'-end of pGADGHShk2-3' to generated pGADGHShk2. pHP5Cdc42 was made by cloning a BamHI-SalI fragment of cdc42 from pGADHGHCdc42 into the corresponding sites of pHP5. Shk2P1/P2 was obtained by PCR using pSP204 as template and the primer pair 5'-CCTAAAGAGCTCTCAGATATATAA and 5'-AGGCAGGTCGACAGTTAACTAACG. The resulting fragment was cut by SacI and SalI and cloned into the SacI-XhoI sites of pBSII to produce pBSIIshk2P1/P2. To construct pBSIIshk2::ura4, a 0.9-kb HindIII fragment of pBSIIShk2P1/P2 (see Fig. 2A) was replaced by a 1.8-kb HindIII fragment of the ura4 gene released from pBSIIura4.

beta -Galactosidase Assays-- The filter assay for testing two-hybrid interactions was performed as described previously (35). LexA two-hybrid experiments were conducted using LexA DNA binding domain (LBD) and Gal4 activating domain (GAD) pairs of fusion proteins. The liquid assay for beta -galactosidase activity was performed as described (33). beta -galactosidase activity was calculated using the following formula: (A420 × 1.7)/(0.0045 × protein concentration × extract volume × time). Protein concentration is expressed as mg/ml, extract volume in ml, and time as min.

Quantitative S. pombe Mating Assays-- Mating assays were performed as described previously (39). Briefly, transformants were grown on EMM agar for 4 days to induce sexual activity. Zygotes, asci, and unmated cells within individual clones were then quantitated by microscopy.

Preparation of Yeast Cell Lysates, Immunoprecipitations, Immunoblotting, and Myelin Basic Protein (MBP) Kinase Assays-- Yeast cultures were grown to about 107 cells/ml in either drop-out medium (for S. cerevisiae strains) or EMM (for S. pombe strains), harvested by centrifugation, resuspended with yeast lysis buffer (20 mM HEPES (pH 7.6), 200 mM KCl, 2 mM EGTA, 2 mM EDTA, 10 mM sodium molybdate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium vanadate, 0.1% Nonidet P-40, 10% glycerol, 10 µM E64, 100 µM leupeptin, 1 µM pepstatin, 1 mM phenylmethanesulfonyl fluoride, 2 µg/ml aprotinin), and ground with glass beads. Crude lysates were centrifuged at 16,000 × g for 15 min, and the supernatant and particulate fractions were aliquoted and quick frozen in liquid nitrogen prior to storing at -80 °C.

Immunoprecipitations of c-Myc epitope-tagged proteins were performed by incubating yeast lysate (1 mg of protein) with 5 µl of anti-c-Myc monoclonal antibody 9E10 (40) ascites on an orbital rotator for 2 h at 4 °C. Immune complexes were washed three times with yeast lysis buffer and then resuspended in SDS-PAGE sample buffer and boiled for 3 min prior to SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes, and c-Myc-tagged proteins were detected by immunoblotting using 9E10 ascites (1:2500 dilution).

For detection of Cdc42 proteins, particulate fractions (50 µg of protein) of SP42N17 cell lysates were boiled in SDS-PAGE sample buffer prior to SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes, and Cdc42 proteins were detected using anti-Cdc42-Hs polyclonal antibody sc-87 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

MBP kinase assays were performed by resuspending immune complexes in 25 µl of MBP kinase buffer (50 mM Tris, pH 7.4, 0.1 M NaCl, 10 mM MgCl2, 1 mM MnCl2, 0.1 mg/ml MBP, 10 mM ATP, 0.4 µCi/µl [gamma -32P]ATP (6000 Ci/mmol)) and incubating for 20 min at 30 °C. Reactions were stopped by adding SDS-PAGE sample buffer and boiling for 3 min prior to SDS-PAGE. After electrophoresis, gels were fixed by exchanging six times with 5% trichloroacetic acid, 3% sodium pyrophosphate, dried, and exposed to film.

Filter Binding Assay for Cdc42-- The filter binding assay for detection of Cdc42-Shk interactions was performed as described (41). Briefly, His6-tagged Shk1, Shk2, Cdc42-Hs(G12V), Ha-Ras(G12V), and TrcHis peptide (THP) were purified from bacterial cell lysates using nickel-agarose following the manufacturer's instructions (Invitrogen). GST protein was purified from bacterial lysates using glutathione-agarose and the manufacturer's instructions (Amersham Pharmacia Biotech). GST and His6-tagged Shk1, Shk2, and THP were immobilized on nitrocellulose membranes using a vacuum dot blotter. The membranes were blocked for 2 h at room temperature in 5% dried milk. 10 µg of Hiss-tagged Cdc42-Hs(G12V) and Ha-Ras(G12V) were each incubated with 10 µCi of [gamma -32P]GTP (6000 Ci/mmol) for 10 min at 30 °C in 30 µl of 50 mM Tris, pH 7.5, 5 mM EDTA, and 0.5 mg/ml bovine serum albumin. Nucleotide exchange was stopped on ice by adding MgCl2 to 10 mM. The nitrocellulose filter was washed twice with buffer A (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol) and incubated in 10 ml of buffer A containing 5% skim milk and [gamma -32P]GTP-bound Cdc42-Hs(G12V) or Ha-Ras(G12V). After incubation for 5 min at 4 °C, the membrane was washed three times with cold buffer A containing 5% dried milk and subjected to autoradiography to visualize bound Cdc42-Hs(G12V) or Ha-Ras(G12V) protein.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequence Analysis of the shk2 Gene-- In a previous study, we reported on the cloning and characterization of shk1, a S. pombe gene encoding a homolog of the S. cerevisiae PAK Ste20 (27). shk1 was cloned independently by Ottilie et al. (26), who named the gene pak1. The original shk1 fragment was amplified from S. pombe genomic DNA using the PCR and degenerate oligonucleotide primers based on peptide sequences in the catalytic domain of the S. cerevisiae Ste20 protein kinase (27). The product resulting from this PCR contained the partial shk1 gene fragment as well as a partial fragment of a related sequence that we named shk2, for Ste20 homologous kinase 2. Analysis of several recombinant plasmids generated from the PCR for STE20-related S. pombe sequences indicated that shk1 and shk2 were represented in roughly equal proportions, with no other sequences being identified. The full-length shk2 gene was isolated by using the PCR-derived shk2 fragment as a probe to screen a S. pombe genomic DNA library. The nucleotide sequence of the full-length shk2 gene (GenBankTM accession number U45981) revealed an intronless open reading frame of 1770 base pairs encoding a predicted protein 589 amino acids in length (Fig. 1A).


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Fig. 1.   Shk2 sequence analysis and alignment with representative yeast and mammalian PAKs. A, deduced amino acid sequence of the shk2 gene product. The shk2 nucleic acid sequence was submitted to GenBankTM (accession number U45981). B, predicted structural organization of the Shk2 protein. A predicted protein kinase catalytic domain composes residues ~295-589. The protein kinase subdomain I signature sequence GXGXXG comprises residues 316-321 (GQGASG). The sequence VAIK, containing the invariant subdomain II lysine, lies at positions 340-343. The predicted regulatory domain (amino residues 1 to ~294) can be subdivided into three subdomains. A highly conserved CRIB motif is designated as R2 and occupies residues 129-184. The sequence N-terminal to the R2/CRIB subdomain is designated as R1, and the sequence between the R2/CRIB and catalytic domains is designated as R3. C, amino acid sequence alignments of rat alpha -Pak/Pak1 (Pak1), S. pombe Shk2 and Shk1, and S. cerevisiae Cla4. Identical amino acid residues are indicated by black boxes. Cdc42/Rac1-binding domains (R2/CRIB) are highlighted by the outlined shaded box.

The structural organization of the predicted Shk2 protein is similar to that of previously described PAKs (Fig. 1B). The N-terminal half of Shk2 (amino acid residues 1 to ~294) comprises a presumptive regulatory domain containing a potential Cdc42/Rac interactive binding (CRIB) (41) sequence (amino acid residues 129-184), while the C-terminal half of Shk2 (residues ~295-589) contains a predicted protein kinase catalytic domain. We have designated the CRIB sequence as regulatory subdomain 2, or R2; the regulatory sequence N-terminal to the CRIB domain (residues 1-128) as R1; and the domain between the R2/CRIB and catalytic domains (residues 185-294) as R3 (Fig. 1B). Shk2 is most closely related in structure to the S. cerevisiae PAKs Cla4 (45% identity) and Skm1 (44% identity); it exhibits a lesser degree of homology to Shk1 (38% identity), Ste20 (39% identity), and mammalian alpha -Pak/Pak1 (41% identity). An alignment of Shk2 with S. pombe Shk1, S. cerevisiae Cla4, and mammalian alpha -Pak/Pak1 is shown in Fig. 1C. The catalytic domain of Shk2 is highly homologous to other yeast and mammalian PAKs (52-60% identity). The R1 subdomain of Shk2 exhibits greatest homology to the corresponding domains of Shk1 (28% identity), Skm1 (28% identity), and Cla4 (24% homology). The subdomains of Cla4 and Skm1 corresponding to the Shk2 R1 domain were shown by others to exhibit homology with the pleckstrin homology domain consensus sequence (24). Shk2 exhibits a degree of homology with the pleckstrin homology domain consensus sequence similar to that of Skm1 and Cla4, although the extent of this homology is greatly dependent on the parameters used to generate the alignments (data not shown). By contrast, the Shk1 R1 subdomain lacks any discernible homology to either the corresponding domains of Cla4 and Skm1 or to the pleckstrin homology consensus sequence, despite the fact that it exhibits marked homology with the Shk2 R1 domain. The R2/CRIB and catalytic domains of Shk2 exhibit greatest similarity to the corresponding domains of Skm1 and Cla4 (56 and 52% identity, respectively). The R3 subdomain of Shk2 exhibits little homology to the corresponding domains of any other previously described PAK.

shk2 Is a Nonessential Gene-- To examine the function of Shk2 in S. pombe, a disruption of the shk2 gene was made by replacing the majority of the shk2 protein coding sequence with the ura4 gene (see Fig. 2A and "Experimental Procedures"). The shk2::ura4 DNA fragment was used to transform the wild type S. pombe diploid strain SP870D. Two independent shk2+/shk2::ura4 diploids were sporulated, and asci containing four spores were dissected for tetrad analysis. Most asci produced two viable Ura+ spores and two viable Ura- spores. Southern blotting was used to confirm that cells derived from Ura+ spores contained a disrupted copy of shk2 and lacked the wild type shk2 gene (data not shown). These results demonstrated that shk2 is not an essential gene. shk2-deleted cells exhibited no obvious growth defects at either 30 or 36 °C (Fig. 2B), and microscopic analysis revealed that they are indistinguishable from wild type S. pombe cells in morphology (Fig. 2, C and D). By contrast, S. pombe shk1 null mutants are inviable and spheroidal in shape (26, 27). We also determined that shk2 null cells mated with about the same efficiency as wild type cells (data not shown), indicating that Shk2 is dispensable for mating in S. pombe.


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Fig. 2.   Analysis of the shk2 null mutant. A, map of the shk2 gene showing the fragment deleted by ura4 in construction of the shk2::ura4 mutant strain, SPSHK2U (see "Experimental Procedures"). The bottom bar corresponds to the sequence encoding the Shk2 protein (Shk2p), subdivided into regulatory subdomains R1, R2, and R3, and the catalytic domain. B, the shk2 null mutant grows normally at 30 and 36 °C. S. pombe wild type (SP66) cells (left side of panel) and shk2::ura4 (SPSHK2U) cells (right side of panel) were streaked onto YEA as described under "Experimental Procedures" and grown at either 30 °C (top of panel) or 36 °C (bottom of panel) for 3 days. C and D, photomicrographs of wild type (C) and shk2::ura4 (D) S. pombe cells. shk2::ura4 cells are indistinguishable from wild type cells in morphology.

Shk2 Overexpression Restores Viability and Normal Morphology to the shk1 Deletion Mutant-- Having observed that shk2-deleted cells exhibit no obvious phenotypic defects, we asked whether Shk1 and Shk2 might be partially overlapping in function by determining whether overexpression of shk2 could suppress the shk1 null mutation. To do this, we constructed a LEU2-based plasmid, pREP1Shk2, for overexpressing shk2 from the thiamine-repressible nmt1 promoter (see "Experimental Procedures"). A shk1+/shk1::ura4 diploid strain was transformed with pREP1Shk2, the control plasmid pREP1, pWH5Shk1, a LEU2-based plasmid carrying the genomic shk1 sequence, and pREP1Shk2(K343R), which expresses a mutant Shk2 protein in which the invariant protein kinase subdomain II lysine is substituted by arginine. Transformants were induced to sporulate, and then spores were scored for viability. Viable shk1::ura4 spores were recovered from pWH5Shk1- and pREP1Shk2-transformed cells, but not from pREP1 or pREP1Shk2(K343R)-transformed cells. This result suggested that overexpression of functional shk2 can restore viability to shk1-deleted cells. Microscopic analysis of pREP1Shk2-transformed shk1::ura4 cells indicated that they are similar to wild type cells in morphology (Fig. 3, top panels). When grown in thiamine-containing medium, pREP1Shk2-transformed shk1::ura4 cells became spheroidal in morphology and growth-inhibited, whereas pWH5Shk1-transformed shk1::ura4 cells remained elongated (Fig. 3, bottom panels). These results demonstrate that shk2 can act as a high dosage suppressor of the viability and morphology defects of the shk1 null mutant.


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Fig. 3.   Overexpression of shk2 restores viability and wild type morphology to the shk1 null mutant. The shk1+/shk1::ura4 diploid strain SP206U was transformed with either a plasmid harboring a genomic copy of the shk1 gene (pWH5Shk1), a plasmid for thiamine-repressible overexpression of shk2 (pREP1Shk2), or the control plasmid pREP1. Transformants were sporulated, and then random spore analysis was performed by micromanipulation. Viable Ura+ spores were recovered only from pWH5Shk1- and pREP1Shk2-transformed cells. pWH5Shk1- (left side of panel) and pREP1Shk2- (right side of panel) transformed shk1::ura4 cells were grown in the absence (top of panel) or presence (bottom of panel) of thiamine in EMM medium. pREP1Shk2 restored elongate morphology to the shk1::ura4 mutant (top, right). Incubation of the pREP1Shk2-transformed shk1::ura4 mutant in thiamine (15 µM) resulted in reversion to the spheroidal morphology characteristic of the shk1 null phenotype (bottom, right).

Genetic and Biochemical Evidence for Interaction between Shk2 and Cdc42-- Previously characterized PAKs have been shown to bind Cdc42 and Rac GTPases but not other small GTPases, such as Ras and Rho. The two-hybrid assay was used to determine whether Shk2, like other known PAKs, interacts with Cdc42 or Rac or with proteins besides Cdc42 that are involved in Cdc42-dependent signaling in S. pombe. A summary of the two-hybrid interactions tested by the beta -galactosidase filter assay are shown in Table I, and results of a smaller subset of quantitative liquid beta -galactosidase assays are shown in Table II. Shk2 was found to form detectable two-hybrid complexes both with Cdc42 and, to a much lesser degree, with Rac1, but not with Ras1 or RhoG (Tables I and II). Shk2 interacted in the two-hybrid assay with both wild type and activated (G12V) forms of Cdc42 but not with a dominant negative mutant of Cdc42, Cdc42(T17N) (Tables I and II). The T17N mutation is analogous to mutations identified at the corresponding positions of yeast and mammalian Ras proteins that result in defective guanine nucleotide exchange (42-44). The inability of Cdc42(T17N) and Shk2 to interact in the two-hybrid assay suggests that the Cdc42-Shk2 interaction is GTP-dependent.

                              
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Table I
Analysis of Shk2 two-hybrid interactions
Values represent the presence of transformed colonies that expressed detectable beta -galactosidase activity (+) or did not (-). Activating domain fusions were to the activating domain of S. cerevisiae Gal4 (GAD). GBD-GAD combinations were expressed in the Gal4 two-hybrid tester strains HF7c and/or SF7526. LBD-GAD combinations were tested in the LexA tester strain, L40. At least eight independent transformants were tested for each determination.

                              
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Table II
Quantitative beta -galactosidase assays for representative Shk2 two-hybrid tests
Representative yeast transformants tested by the beta -galactosidase filter assay (Table I) were also tested by quantitative liquid beta -galactosidase assays. Lamin, Cdc42, Cdc42(T17N), Rac1, Ras1, and RhoG were tested as LBD fusion proteins in the two-hybrid tester strain L40. Snf4 and Skb1 were tested as GBD fusion proteins in SFY526. The GBD-Skb1 fusion protein tested lacks the first 23 amino acids of the full-length Skb1 protein and was used because the full-length Skb1 GBD fusion protein weakly autoactivates in the two-hybrid assay.

The Shk2-Cdc42 interaction was confirmed biochemically by a filter binding assay (41) using bacterially expressed recombinant proteins. His6-tagged Shk1 and Shk2 proteins, as well as His6-tagged pTrcHis peptide (His6-THP) and GST protein, were immobilized on nitrocellulose membranes using a dot blot apparatus. After blocking, the membranes were incubated with His6-Cdc42-Hs(G12V)·[gamma -32P]GTP or His6-Ha-Ras(G12V)·[gamma -32P]GTP and then washed and exposed to film. As shown in Fig. 4, His6-Cdc42-Hs(G12V)·[gamma -32P]GTP bound to both His6-Shk1 and His6-Shk2, but not to His6-THP or GST. This result demonstrates that both Shk1 and Shk2 bind directly to Cdc42. Binding was not detected between His6-Ha-Ras(G12V)·[gamma -32P]GTP and either His6-Shk1 or His6-Shk2.


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Fig. 4.   Direct binding of Cdc42 to Shk1 and Shk2 proteins. Approximately 100 ng of His6-tagged Shk1, Shk2, and THP and approximately 10 µg of GST were each immobilized onto nitrocellulose filters using a vacuum dot blotter and incubated with [gamma -32P]GTP·Cdc42-Hs(G12V) (top) or [gamma -32P]GTP·Ha-Ras(G12V) (bottom) as described under "Experimental Procedures." After washing, bound GTPase was visualized by autoradiography.

Shk2 also formed a two-hybrid complex with Skb1 (Tables I and II), a protein that we previously identified by a two-hybrid screen for Shk1-interacting proteins and that we showed by genetic analyses to positively modulate Shk1 function (28). However, we were unable to recapitulate the Shk2-Skb1 interaction in vitro by coprecipitation experiments using recombinant bacterially expressed proteins.

Having determined that Cdc42 and Shk2 interact physically, we examined whether the two proteins interact functionally in S. pombe. We first examined whether cooverexpression of shk2 and cdc42 affects cell growth differently than overexpression of each gene separately. cdc42 was overexpressed from a plasmid containing the thiamine-repressible nmt1 promoter (pREP1Cdc42), while shk2 was overexpressed from a plasmid containing the constitutive adh1 promoter (pAAUCMShk2). The wild type S. pombe strain CHP428 was cotransformed with pREP1Cdc42, pAAUCMShk2, and/or control plasmids, and transformants were assayed for growth in the presence and absence of thiamine. As shown in Fig. 5A, cells transformed with pREP1Cdc42 or pAAUCMShk2 grew equally well on medium with or without thiamine. However, cells cotransformed with pREP1Cdc42 and pAAUCMShk2 produced normal size colonies only when grown on thiamine. When grown in the absence of thiamine, cells cotransformed with pREP1Cdc42 and pAAUCMShk2 produced only microcolonies, indicating that cooverexpression of cdc42 and shk2 is toxic. We also examined cells overexpressing cdc42 and/or shk2 microscopically. Cells overexpressing cdc42 (Fig. 5C) were virtually indistinguishable from wild type cells (Fig. 5B) in morphology, while a substantial percentage of cells overexpressing shk2 alone were somewhat distorted in shape (Fig. 5D). However, cells cooverexpressing both cdc42 and shk2 were drastically distorted in morphology and much larger than wild type cells or cells overexpressing cdc42 or shk2 alone (Fig. 5E). These results provide evidence for functional interaction between Cdc42 and Shk2 in regulating cell morphology in S. pombe.


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Fig. 5.   Evidence for functional interaction between Cdc42 and Shk2 in S. pombe. A, wild type (CHP428) S. pombe cells were transformed with pREP1Cdc42 (for overexpression of cdc42), pAAUCMShk2 (for overexpression of shk2), and/or the control plasmid pREP1 or pAAUCM and plated onto EMM medium containing 15 µM thiamine for repression of cdc42 expression. Transformants were then streaked onto EMM with (left) or without (right) thiamine (15 µM) and grown at 30 °C for 4 days. Overexpression of cdc42 or shk2 alone did not affect the growth rate of cells. However, cooverexpression of both genes was toxic, suggesting that the Cdc42 and Shk2 proteins functionally interact in vivo. B-E, the same transformants shown in A were grown in liquid EMM with thiamine, washed, and grown overnight in EMM without thiamine and observed microscopically. B, cells cotransformed with pREP1 and pAAUCM; C, cells cotransformed with pREP1Cdc42 and pAAUCM; D, cells cotransformed with pREP1 and pAAUCMShk2 (note aberrant, bulbous shape of cells indicated by arrows); E, cells cotransformed with pREP1Cdc42 and pAAUCMShk2 (note that most cells are highly aberrant in shape).

Overexpression of shk2 Restores Elongate Morphology to the ras1 Deletion Mutant-- Given the physical and functional interactions observed previously among Ras1, Cdc42, and Shk1 (25, 27, 28), we asked whether shk2 overexpression might suppress the morphological defect resulting from deletion of the ras1 gene. ras1 null mutants are spheroidal in shape (Fig. 6A). When transformed with a plasmid harboring the ras1 gene, elongate morphology is restored to the ras1 mutant (Fig. 6B). As shown in Fig. 6, C and D, overexpression of shk2 restores elongate morphology to the ras1 deletion mutant, providing further evidence that Shk2, like Shk1 and Cdc42, participates in the Ras1-dependent morphological control pathway in S. pombe.


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Fig. 6.   Overexpression of shk2 restores elongate morphology to the ras1 null mutant. A S. pombe ras1 deletion mutant (SPRN1) was transformed with the control plasmids pAAUCM and pREP1 (A); pAUR, which carries the wild type ras1 gene (B); or pREP1Shk2 for overexpression of Shk2 (C and D). Overexpression of shk2 restored elongate morphology to ras1-deleted cells. Transformants were patched onto EMM and grown overnight at 30 °C prior to photomicroscopy.

Overexpression of Byr2 Suppresses the Mating Defect of an S. pombe Mutant Partially Defective for Shk1 Function-- A role for Shk1 in mediating sexual responses in S. pombe was suggested from previous studies in which it was shown that overexpression of dominant negative mutants of shk1 impaired sexual responses (26, 27). To further explore this aspect of Shk1 function, we tested whether pREP1Shk2-transformed shk1::ura4 cells exhibit a defect in mating. As shown in Table III, the mating efficiency of pREP1Shk2-transformed shk1::ura4 cells was nearly 30-fold lower than that of shk1+ cells. Two conclusions can be drawn from this experiment: (i) the shk1 gene is required for normal mating in S. pombe, and (ii) overexpression of shk2, while capable of restoring both viability and normal morphology to the shk1 deletion mutant, does not fully restore mating functions to the shk1 deletion mutant.

                              
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Table III
shk1-deleted cells overexpressing shk2 exhibit a mating defect that is partially suppressed by overexpression of byr2

Other investigators have demonstrated by genetic analyses that the S. cerevisiae PAK Ste20 functions upstream of the protein kinase Ste11 (19), a MAPK kinase kinase homolog required for pheromone-induced signal transduction (46). Furthermore, it has been shown that Ste20 immune complexes can phosphorylate Ste11 protein in vitro, implicating Ste11 as a potential Ste20 substrate (47). The fission yeast protein kinase Byr2 is a structural and functional homolog of S. cerevisiae Ste11 (29). Because shk1 is an essential gene, it had been difficult to test for a genetic interaction between shk1 and byr2. However, the above described finding that pREP1Shk2-transformed shk1::ura4 cells exhibit a significant mating defect made this test possible. Indeed, we found that pREP1Shk2-transformed shk1::ura4 cells that overexpressed byr2 mated with about 16-fold higher efficiency than shk1Delta pREP1Shk2 cells transformed with a control plasmid and with only about 2-fold lower efficiency than shk1+ cells (Table III). Thus, byr2 is a high dosage suppressor of the mating defect resulting from partial loss of shk1. These results provide the first direct genetic evidence for interaction between PAKs and the MAPK module/cascade required for mating response in S. pombe.

Next, we examined the genetic interaction between cdc42 and shk2 with respect to mating. We showed previously that S. pombe cells overexpressing the dominant negative cdc42(T17N) mutant gene exhibit a marked mating defect, which can be partially suppressed by overexpression of Shk1 (27). As shown in Table IV, cells harboring an integrated copy of an adh1-cdc42(T17N) fusion gene and transformed with a high copy shk2 plasmid mated with about 4-fold greater efficiency than cdc42(TN17)-expressing cells transformed with a control plasmid. This result suggests that Shk2, like Shk1, participates in the Ras and Cdc42-dependent mating response pathway of S. pombe. To rule out the trivial explanation that differences in the level of expression of the cdc42(T17N) gene are responsible for the differences in mating efficiency, lysates were prepared from the various cdc42(T17N) strains tested and subjected to immunoblot analysis. As shown in Fig. 7, comparable levels of Cdc42 protein were detected in all strains, except the one transformed with a high copy plasmid for overexpression of wild type Cdc42, from which, as expected, a substantially greater amount of Cdc42 protein was detected.

                              
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Table IV
Overexpression of shk2 results in partial suppression of the mating defect caused by expression of the cdc42T17N mutant gene


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Fig. 7.   Level of Cdc42(T17N) in S. pombe transformants shown in Table IV. Cell lysates were prepared from S. pombe strains listed in Table IV as described under "Experimental Procedures." Particulate fractions of the cell lysates (50 µg of protein) were resolved by SDS-PAGE and transferred to nitrocellulose, and immunoblots were performed using alpha -Cdc42-Hs monoclonal antibody for detection of Cdc42. The wild type S. pombe strain SP66, which carries only the endogenous cdc42 gene, was used as a control to show that the Cdc42 protein detected in SP42N17 cultures was predominantly Cdc42(T17N), except for the pREP1Cdc42-transformed culture, which overexpresses both wild type and T17N forms of Cdc42.

Shk2 Cannot Substitute for Cla4 in S. cerevisiae-- As already noted, Shk2 is most similar in structure to the S. cerevisiae PAK Cla4. This prompted us to examine whether Shk2 and Cla4 are functionally related. S. cerevisiae cla4 deletion mutants are morphologically aberrant but competent for mating (23). S. cerevisiae ste20 mutants are sterile but normal in morphology (19, 20). Deletion of both ste20 and cla4 is lethal (23). To determine whether shk2 can substitute for cla4 or ste20, we constructed the plasmid pAUD6Shk2 for overexpressing shk2 from the strong S. cerevisiae ADH1 promoter. Two different cla4 mutants, one a deletion mutant and the other a temperature-sensitive mutant, were transformed with either pAUD6Shk2, a plasmid containing the cla4 gene, or a control plasmid. pAUD6Shk2 failed to restore normal morphology to either the deletion (Fig. 8C) or temperature-sensitive (data not shown) mutant of cla4, nor did it restore viability to a cla4 ste20 double mutant (data not shown). pAUD6Shk2 also failed to restore mating ability to a ste20 deletion mutant (data not shown). pAUD6Shk2 expresses a c-Myc epitope-tagged Shk2 protein (CMShk2). To confirm that CMShk2 was expressed in S. cerevisiae, lysates were prepared from the cla4Delta mutant transformed with either pAUD6 or pAUD6Shk2 and c-Myc-tagged proteins precipitated using monoclonal antibody 9E10. Immunoprecipitates were then subjected to Western blotting or assayed for MBP kinase activity. As shown in Fig. 8D, an approximately 68-kDa c-Myc-tagged protein (the predicted size for CMShk2) was detected in c-Myc immune complexes from lysates of cells expressing pAUD6Shk2 but not pAUD6, indicating that CMShk2 protein is expressed from the pAUD6Shk2 plasmid. Furthermore, MBP kinase activity was detected in pAUD6Shk2, but not pAUD6, immunoprecipitates (Fig. 8D), indicating that CMShk2 is catalytically active. We conclude that the Shk2 and Cla4, despite their similarity in structure, are not functionally interchangeable.


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Fig. 8.   Shk2 cannot substitute for Cla4 in S. cerevisiae. A cla4Delta mutant, MJY8, was transformed with FD44 (carries the cla4 gene) (A), pAUD6 (B), or pAUD6Shk2, for expression of c-Myc epitope-tagged Shk2 from the strong ADH1 promoter (C). Note that Shk2 expression could not restore normal morphology to the cla4Delta mutant. D, pAUD6- and pAUD6Shk2-transformed cla4Delta cells were lysed, and CM-tagged proteins were immunoprecipitated using alpha -c-Myc monoclonal antibody 9E10. Immune complexes were either subjected to immunoblot analysis using 9E10 antibody for detection of c-Myc-tagged proteins (left side of panel) or assayed for MBP kinase activity (right side of panel). MBP kinase activity was detected only in c-Myc immune complexes isolated from pAUD6Shk2-transformed cells.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In 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 (26-28), 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 of shk2 restores viability and elongate morphology to the S. pombe shk1 null mutant. Overexpression of shk2 does 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 in S. pombe. However, from our PCR-based cloning approach, which utilized degenerate oligonucleotide primers based on the S. cerevisiae Ste20 protein sequence, we identified only the shk1 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 (26). 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 (27). Finally, overexpression of shk1 partially bypassed the mating defect of S. pombe cells expressing the dominant inhibitory cdc42(T17N) allele (27). In this report, we have shown that S. pombe cells deleted of shk1 but overexpressing shk2 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 inhibitory cdc42(T17N) allele. Our results corroborate a role for Shk1 in the S. pombe mating response pathway, which was suggested in previous studies (26, 27). 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 (19, 47).

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 the shk2 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 (23). The third, Skm1, is completely dispensable (24). Deletion of STE20 results in sterility (19, 20), while deletion of CLA4 results in aberrant morphology (23). However, deletion of both CLA4 and STE20 genes is lethal (23). PAK wiring is clearly different in S. pombe, in which a single PAK, Shk1, has essential functions not shared by other PAKs (26, 27). These differences are not surprising, given that fact that S. cerevisiae and S. pombe are a half billion years diverged in evolution (45). It remains to be determined whether the essential functions of PAKs in S. pombe and S. cerevisiae are conserved in higher organisms or, for that matter, between the two distantly related yeasts.

    ACKNOWLEDGEMENTS

We thank F. Cvrckova for plasmids and strains; Jonathan Chernoff for plasmids and for communicating unpublished results; Anjana Kundu and Erin Mooney for technical assistance; and Jenny Henkel and Anthony Polverino for comments on the manuscript. We especially thank Michael Wigler (Cold Spring Harbor Laboratory), in whose laboratory this project was initiated.

    Note Added in Proof

The 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 gene pak2. Their results are consistent with and complement the results presented in this paper.

    FOOTNOTES

* This work was supported by National Institutes of Health (National Institutes of Health) Grant R01GM53239 (to S. M.). Part of the DNA sequencing was performed by the University of Texas M. D. Anderson Cancer Center Core DNA Sequencing Facility, which is supported by National Institutes of Health Grant P30CA16672.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Predoctoral Training Grant T32CA09299.

§ To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-745-2032; Fax: 713-794-4394; E-mail: smarcus{at}mdacc.tmc.edu.

1 The 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.

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Abstract
Introduction
Procedures
Results
Discussion
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