Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts

Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.

[1]  G. Bokoch,et al.  Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells , 1997, Current Biology.

[2]  C. Der,et al.  Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways , 1997, Molecular and cellular biology.

[3]  Wange Lu,et al.  Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck , 1997, Current Biology.

[4]  C. Der,et al.  p120 GAP Modulates Ras Activation of Jun Kinases and Transformation* , 1997, The Journal of Biological Chemistry.

[5]  C. Hall,et al.  Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. , 1996, European journal of biochemistry.

[6]  D. Bar-Sagi,et al.  RAC Regulation of Actin Polymerization and Proliferation by a Pathway Distinct from Jun Kinase , 1996, Science.

[7]  J. Chant,et al.  Rac and Cdc42 Induce Actin Polymerization and G1 Cell Cycle Progression Independently of p65PAK and the JNK/SAPK MAP Kinase Cascade , 1996, Cell.

[8]  J. Schlessinger,et al.  The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1 , 1996, The Journal of Biological Chemistry.

[9]  S. Oldham,et al.  Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Frost,et al.  Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members , 1996, Molecular and cellular biology.

[11]  M. Wigler,et al.  Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation , 1996, Molecular and cellular biology.

[12]  M. Wigler,et al.  Stimulation of Membrane Ruffling and MAP Kinase Activation by Distinct Effectors of RAS , 1996, Science.

[13]  M. White,et al.  Ras Interaction with Two Distinct Binding Domains in Raf-1 5 Be Required for Ras Transformation (*) , 1996, The Journal of Biological Chemistry.

[14]  S. Yokoyama,et al.  Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras (*) , 1995, The Journal of Biological Chemistry.

[15]  A. Hall,et al.  A Conserved Binding Motif Defines Numerous Candidate Target Proteins for Both Cdc42 and Rac GTPases (*) , 1995, The Journal of Biological Chemistry.

[16]  S. Forsburg,et al.  Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating. , 1995, The EMBO journal.

[17]  B. Dérijard,et al.  Cdc42 and PAK-mediated Signaling Leads to Jun Kinase and p38 Mitogen-activated Protein Kinase Activation (*) , 1995, The Journal of Biological Chemistry.

[18]  K. Clark,et al.  Pheromone Response in Yeast: Association of Bem1p with Proteins of the MAP Kinase Cascade and Actin , 1995, Science.

[19]  J. Frost,et al.  Activation of Mitogen-activated Protein Kinase Cascades by p21-activated Protein Kinases in Cell-free Extracts of Xenopus Oocytes (*) , 1995, Journal of Biological Chemistry.

[20]  A. Abo,et al.  Rac GTPase interacts with GAPs and target proteins through multiple effector sites. , 1995, The EMBO journal.

[21]  Jiahuai Han,et al.  Rho Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1 (*) , 1995, The Journal of Biological Chemistry.

[22]  M. Marshall,et al.  Ras target proteins in eukaryotic cells , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  J. Chernoff,et al.  Identification of a Mouse p21Cdc42/Rac Activated Kinase (*) , 1995, The Journal of Biological Chemistry.

[24]  M. Karin,et al.  Selective activation of the JNK signaling cascadeand c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs , 1995, Cell.

[25]  P. Crespo,et al.  The small GTP-binding proteins Rac1 and Cdc42regulate the activity of the JNK/SAPK signaling pathway , 1995, Cell.

[26]  M. Wigler,et al.  Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  G. Prendergast,et al.  Critical role of Rho in cell transformation by oncogenic Ras. , 1995, Oncogene.

[28]  C. Der,et al.  Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[29]  C. Der,et al.  Two Distinct Raf Domains Mediate Interaction with Ras (*) , 1995, The Journal of Biological Chemistry.

[30]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[31]  F. McCormick,et al.  An essential role for Rac in Ras transformation , 1995, Nature.

[32]  J. Colicelli,et al.  A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1 , 1995, Molecular and cellular biology.

[33]  N. Ahn,et al.  Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade , 1995, Molecular and cellular biology.

[34]  Richard Treisman,et al.  Transcriptional Regulation by Extracellular signals: Mechanisms and Specificity , 1995, Cell.

[35]  C. Marshall,et al.  Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation , 1995, Cell.

[36]  L. Zon,et al.  Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1 , 1994, Nature.

[37]  G L Johnson,et al.  Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. , 1994, Science.

[38]  S. Fields,et al.  Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Michael J. Fry,et al.  Phosphatidylinositol-3-OH kinase direct target of Ras , 1994, Nature.

[40]  M. Greenberg,et al.  Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB , 1994, Cell.

[41]  L. Lim,et al.  A brain serine/threonine protein kinase activated by Cdc42 and Rac1 , 1994, Nature.

[42]  Mark S. Boguski,et al.  Proteins regulating Ras and its relatives , 1993, Nature.

[43]  M. Karin,et al.  Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. , 1993, Genes & development.

[44]  R. Weinberg,et al.  The pathway to signal achievement , 1993, Nature.

[45]  S. Elledge,et al.  Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1 , 1993, Nature.

[46]  M. Wigler,et al.  Complex formation between RAS and RAF and other protein kinases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[47]  R L Smith,et al.  Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. , 1993, Science.

[48]  R. W. Davis,et al.  A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[49]  M. Whiteway,et al.  The protein kinase homologue Ste20p is required to link the yeast pheromone response G‐protein beta gamma subunits to downstream signalling components. , 1992, The EMBO journal.

[50]  R. Weinberg,et al.  Molecular cloning of cDNAs encoding the GAP-associated protein p190: Implications for a signaling pathway from ras to the nucleus , 1992, Cell.

[51]  G. Tennekoon,et al.  Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters. , 1989, Experimental cell research.

[52]  D. Bar-Sagi,et al.  Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. , 1986, Science.

[53]  C. Der,et al.  Biological assays for cellular transformation. , 1994, Methods in enzymology.

[54]  A. Hall,et al.  Small GTP-binding proteins and the regulation of the actin cytoskeleton. , 1994, Annual review of cell biology.

[55]  D. Lowy,et al.  Function and regulation of ras. , 1993, Annual review of biochemistry.

[56]  J. Nickoloff,et al.  Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. , 1992, Analytical biochemistry.

[57]  M. Barbacid.  ras genes. , 1987, Annual review of biochemistry.