Requirement for PAK4 in the Anchorage-independent Growth of Human Cancer Cell Lines

Abstract

p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth. p21-activated protein kinase (PAK) serine/threonine kinases are important effectors of Rho family GTPases and have been implicated in the regulation of cell morphology and motility, as well as in cell transformation. To further investigate the possible involvement of PAK kinases in tumorigenesis, we analyzed the expression of several family members in tumor cell lines. Here we demonstrate that PAK4 is frequently overexpressed in human tumor cell lines of various tissue origins. We also have identified serine (Ser-474) as the likely autophosphorylation site in the kinase domain of PAK4 in vivo. Mutation of this serine to glutamic acid (S474E) results in constitutive activation of the kinase. Phosphospecific antibodies directed against serine 474 detect activated PAK4 on the Golgi membrane when PAK4 is co-expressed with activated Cdc42. Furthermore, expression of the active PAK4 (S474E) mutant has transforming potential, leading to anchorage-independent growth of NIH3T3 cells. A kinase-inactive PAK4 (K350A,K351A), on the other hand, efficiently blocks transformation by activated Ras and inhibits anchorage-independent growth of HCT116 colon cancer cells. Taken together, our data strongly implicate PAK4 in oncogenic transformation and suggest that PAK4 activity is required for Ras-driven, anchorage-independent growth. p21-activated protein kinase p21 binding domain mitogen-activated protein mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase hemagglutinin glutathioneS-transferase amino acid(s) monoclonal antibody(ies) phosphate-buffered saline fluorescein isothiocyanate wild type At least 28 human STE20 group kinases have been identified that fall into ten distinct subfamilies based on phylogenic and structural comparisons of their regulatory and catalytic domains (1Dan I. Wanabe N. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). To date, six human PAK1 kinases, which fall into two subfamilies, have been identified. The first subfamily consists of PAK1 (PAKα), PAK2 (PAKγ, PAKΘ, hPAK65), and PAK3 (PAKβ), which exhibit 80–90% sequence identity within their catalytic domains. Members of the recently identified second PAK subfamily, PAK4, PAK5, and PAK6, are also highly related to each other but show only about 40–50% identity to the kinase domains of PAKs 1–3. All PAK family members are characterized by the presence of a p21 binding domain (PBD), which binds activated Rho family GTPases (2Manser E. Leung T. Salihuddin H. Zhao Z. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1305) Google Scholar, 3Bagrodia S. Taylor S. Creasy C. Chernoff J. Cerione R. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 4Knaus U. Morris S. Dong H. Chernoff J. Bokoch G. Science. 1995; 269: 221-223Crossref PubMed Scopus (358) Google Scholar, 5Martin G. Bollag G. McCormick F. Abo A. 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PAKs 1, 2, and 3 interact via their proline-rich domains with the SH3 domain of the PAK-interactive exchange factors PIX/Cool (11Manser E. Haung H. Loo T. Chen X. Dong J. Leung T Lim L. Mol. Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar,12Bagrodia S. Taylor S. Jordon K. Van Aelst C. Cerione R. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). In addition to the PIX (PAK-interacting exchange factor) family of DH-PH exchange factors, the PAKs can also bind to adaptors such as Nck and can be recruited directly to growth factor receptors (3Bagrodia S. Taylor S. Creasy C. Chernoff J. Cerione R. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 13Galisteo M. Chernoff J. Su Y. Skolnik E. Schlessinger J. J. Biol. Chem. 1996; 271: 20997-21000Abstract Full Text PDF PubMed Scopus (237) Google Scholar). Overall, both PAK subfamilies share the same organization and structure in their regulatory and catalytic domains, although PAKs 4, 5, and 6 do not have obvious PIX/Cool binding motifs. As effectors of Rho family GTPases, PAK kinases play an important role in the regulation of cell morphology and motility by regulating the actin cytoskeleton (14Sells M. Knaus U. Bagrodia S. Ambrose D. Bokoch G. Chernoff J. Biol. 7: Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. Chernoff J. J. Cell Biol. 2000; PubMed Scopus Google Scholar, R. J. D. J. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, Z. Manser E. Chen X. C. Leung T. Lim L. Mol. Cell. Biol. 1998; PubMed Google Scholar, D. L. Bokoch G. G. Nature Cell Biol. 1: PubMed Scopus Google Scholar, W. R. C. Bokoch G. M. J. Cell Biol. PubMed Scopus Google Scholar). PAK activity has been implicated in the the leading and the of cell motility (14Sells M. Knaus U. Bagrodia S. Ambrose D. Bokoch G. Chernoff J. 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We that PAK4 is frequently overexpressed in human tumor cell lines and that the PAK4 to an with human In we demonstrate that an activated of PAK4 has transforming and that an PAK4 mutant blocks anchorage-independent growth. results strongly implicate PAK4 in and suggest that PAK4 be important in cell transformation. The and J. J. 1998; 17: PubMed Scopus Google Scholar). by of human tumor human and two human on a and to with a of PAK4 and a of PAK4 the a and is to the sequence expression the to an to various of To and we a site of the a site within the and a site the by and by of the which into the expression The and and The with the The with the and the to site to of the The to the the of the kinase domain of PAK4 by which a and a site and as The site the expression A. T B. D. M. M. Nature. PubMed Scopus Google A. human S. S. K. B. F. Y. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). monoclonal antibodies against the of the the antibodies against the with The further on a protein A The of the protein A by against and As antibodies with the by the protein A the protein the protein with and in and and by and the with and protein for by several by with and by a with kinase kinase in kinase and with various in a of with for with a and by addition of and for analyzed by and NIH3T3 that been in the of T. M. and S. Cell lines in with and and in a and to as and in for kinase NIH3T3 for their of transformation as well as for their growth in in transformation by The with for with acid for in in and with and G. A. S. C. 1995; PubMed Scopus Google Scholar). transformation 3 on of in growth and with in growth with in growth in with a a of in in transformation and with various of PAK4 as in tissue and a of the The 6 and the to for with and with The a of and with antibodies in and of PAK4, protein directed against with and a of and and for by to to detect PAK4 and to detect The with and in the for with a with a with The and in and to a on a with a To overexpressed in human we an for several STE20 family kinases we the expression of PAK4 and in human and a of human tumor cell lines of various tissue origins. All with actin to of In human PAK4 In the related kinase although only be in and We the expression of PAK4 and in the tumor cell In of the tumor cell not of to the tissue only be in a of the cell lines. two of the colon cancer cell lines expression not be in colon In to PAK6, PAK4 in tumor cell lines of the tumor cell lines PAK4 to the tissue We our by PAK4 protein expression in human and tumor tissue PAK4 protein expression in and to and to of a can be the of expression of To the of PAK4 and protein an of the PAK4 we to the of PAK4 the for The PAK4 with a the the to within to by of the human the on the on of is in a of human and M. M. J. A. H. E. T. T. S. J. Google Scholar, and the to a tumor we to in a of human tumor cell lines. PAK4 about in and and about in not To PAK4 kinase we analyzed a of mutant PAK4 by in kinase this we a the PAK4 activation loop as a for this kinase. We a of activation loop on serine 474 A highly related the kinase kinase activation loop as a to PAK4 activation The kinase domain of PAK4 as a protein in and in kinase as that is a for PAK4 the the activation loop of kinase as as Furthermore, of serine 474 the of the by which a to the PAK4 kinase domain the we have of not the activation loop sequence of PAK4 is a in for the and be the autophosphorylation site in we do not the PAK4 for the with a in the motif is of The motif is in the activation loop of other STE20 group members and be a site for regulation of by other this we characterized of of PAK4 by serine 474 to glutamic To a kinase-inactive we the in the binding In addition to the amino acid in domain PAK4 has an both to In kinase on PAK4 and PAK4 expression in are in PAK4 kinase activity the PAK4 mutant activity the protein and data not The catalytic domain kinase activity in which be to the of an PAK4 catalytic activity in the that is likely a regulatory autophosphorylation site In addition to the autophosphorylation site PAK4 also to on within the the of this is not PAK4 that the to show autophosphorylation kinase the PAK4 that has the has autophosphorylation We this is to the that PAK4 is on when in tissue not and of cell expression of PAK4 (K350A,K351A), which To further PAK4 activity and we antibodies directed against serine 474 of with and the To demonstrate the of the we the against several to the PAK4 activation loop results demonstrate that the is highly for in which serine 474 is The two only in To demonstrate that of serine 474 with PAK4 kinase we wild type PAK4 and the PAK4 kinase domain which is active and with the both to only PAK4 with the with the data in 2, results demonstrate that of serine 474 with PAK4 kinase have that PAK4 to the Golgi membrane with activated A. Qu J. Cammarano M. Dan C. Fritsch A. Baud V. Belisle B. Minden A. EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (317) Google Scholar). is not interaction with is to the kinase activity of Golgi In to show activation of PAK4 by activated 7Abo A. Qu J. Cammarano M. Dan C. Fritsch A. Baud V. Belisle B. Minden A. EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (317) Google and data not To this we the to detect activated We wild type PAK4 with activated in NIH3T3 and analyzed PAK4 by with with data A. Qu J. Cammarano M. Dan C. Fritsch A. Baud V. Belisle B. Minden A. EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (317) Google expression of PAK4 results in in the presence of activated PAK4 to the Golgi membrane 4, A when co-expressed with activated the PAK4 is by the In PAK4 kinase PAK4 co-expressed with activated not with the 4, A and the of PAK4 expression in a of human tumor we in PAK4 in cell transformation. To this we co-expressed of PAK4 in NIH3T3 and with activated As a with the to 3 the and analyzed for of the kinase not kinase-inactive PAK4 strongly the of activated Ras to In we the of wild type and kinase PAK4 on cell transformation by other activated but not a not suggest that kinase PAK4 with NIH3T3 transformation by oncogenic Furthermore, this is not to as PAK4 also Ras transformation of the kinase PAK4 can with both and transformation of NIH3T3 is not to with transformation We activated of PAK4 anchorage-independent growth to NIH3T3 cells. We PAK4 in and for 3 As in wild type PAK4 and the not in the activated mutant PAK4 (S474E) to anchorage-independent growth to NIH3T3 expression of activated PAK4 only on the growth of NIH3T3 not Taken together, the of activated PAK4 to anchorage-independent growth and the of kinase PAK4 to with suggest that PAK4 a role in transformation. To this we to PAK4 the growth of a tumor this we lines of HCT116 human colon of HCT116 an of S. M. N. T. Science. PubMed Scopus Google Scholar). HCT116 with wild type PAK4, the kinase-inactive PAK4 mutant the analyzed for anchorage-independent growth in As in expression of kinase-inactive PAK4 of HCT116 in of is a of growth and is frequently by activation of by of various protein kinases, members of the protein kinase family such as and P. T. Nature. 2001; PubMed Scopus Google for are frequently with only a members of the serine/threonine kinases have been to have oncogenic and to be overexpressed in such serine/threonine kinases as and J. J. 1998; 17: PubMed Scopus Google Scholar, T. M. M. T. U. PubMed Scopus Google Scholar, S. J. J. PubMed Scopus Google Scholar). Here we demonstrate that the serine/threonine kinase PAK4 is overexpressed in a of human tumor cell lines. PAK4 expression in be the of expression We the by the of the PAK4 to a on that is in a of human to In we of PAK4 in two tumor cell lines. The a of expression is in colon and a and M. M. J. A. H. E. T. T. S. J. 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We do not of and PAK4 with the same members of the two PAK subfamilies distinct of Ras our that PAK4 not transformation by other such as that the is likely to of To date, the of PAK4 and effectors has not been have implicated PAK4 in the regulation of cell PAK4 are to and PAK4 can the in N. Qu J. Minden A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In tumor the is we to the possible of PAK4 on to the results by N. Qu J. Minden A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google we that is a for PAK4 in we also that of PAK4 in tissue can in an in of on not we are not PAK4 to is only and in be also to activation of the kinase We are this be of to the of PAK4 on cell transformation least in on of the In our data strongly implicate PAK4 in and suggest that PAK4 be important for Ras transformation. of the and the by which PAK4 and other PAK family members Ras further to our of cell transformation and the of human We and for cell expression and expression We members of the group for their and on this We also and for their and of this We for in the of the