Cancer-associated Mutations Activate the Nonreceptor Tyrosine Kinase Ack1
2010; Elsevier BV; Volume: 285; Issue: 14 Linguagem: Inglês
10.1074/jbc.m109.060459
ISSN1083-351X
AutoresVictoria Prieto-Echagüe, Azad L. Gucwa, Barbara P. Craddock, Deborah A. Brown, W. Todd Miller,
Tópico(s)Cancer-related gene regulation
ResumoAck1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802–990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe820) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity. Ack1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802–990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe820) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity. IntroductionAck1 is a nonreceptor tyrosine kinase (NRTK) 2The abbreviations used are: NRTKnonreceptor tyrosine kinaseSAMsterile α-motifCRIBCdc42-binding domainEGFepidermal growth factorEGFREGF receptorMHRMig6 homology regionSHSrc homologyE3ubiquitin-protein isopeptide ligasePMSFphenylmethylsulfonyl fluorideHAhemagglutininGFPgreen fluorescent proteinPBSphosphate-buffered salinePVDFpolyvinylidene difluorideWTwild typeGSTglutathione S-transferaseCasCrk-associated substrateWASPWiskott-Aldrich syndrome proteinEVempty vector. that was first isolated from a human hippocampal expression library because of its specific binding to GTP-bound Cdc42 (1.Manser E. Leung T. Salihuddin H. Tan L. Lim L. Nature. 1993; 363: 364-367Crossref PubMed Scopus (257) Google Scholar). Ack1 belongs to a family of nonreceptor tyrosine kinases that includes Ack1, Tnk, and homologous proteins in Drosophila and Caenorhabditis elegans (2.Hoehn G.T. Stokland T. Amin S. Ramírez M. Hawkins A.L. Griffin C.A. Small D. Civin C.I. Oncogene. 1996; 12: 903-913PubMed Google Scholar, 3.Hopper N.A. Lee J. Sternberg P.W. Mol. Cell. 2000; 6: 65-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4.Sem K.P. Zahedi B. Tan I. Deak M. Lim L. Harden N. Mol. Cell Biol. 2002; 22: 3685-3697Crossref PubMed Scopus (18) Google Scholar). Ack1 is a 120-kDa protein with an N-terminal sterile α-motif (SAM) domain (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar), a kinase domain, an SH3 domain, and a Cdc42-binding domain (CRIB) (see Fig. 1A). The large C-terminal portion of Ack1 contains several proline-rich sequences as well as a clathrin-binding motif (6.Teo M. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 18392-18398Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), a ubiquitin-binding domain (7.Shen F. Lin Q. Gu Y. Childress C. Yang W. Mol. Biol. Cell. 2007; 18: 732-742Crossref PubMed Scopus (75) Google Scholar), and a region homologous to Mig6 (8.Zhang X. Pickin K.A. Bose R. Jura N. Cole P.A. Kuriyan J. Nature. 2007; 450: 741-744Crossref PubMed Scopus (273) Google Scholar). The domain architecture of Ack1 is unique among NRTKs; it is the only NRTK with a CRIB domain, and the position of the SH3 domain (C-terminal to the kinase domain) differs from all other families of NRTKs.Recent data implicate Ack1 in different stages of cancer. Amplification of the Ack1 gene correlates with metastasis and poor prognosis in lung and prostate cancer, and overexpression increases invasiveness (9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar). In v-Ras-transformed mammalian cells, Ack1 is required for the maintenance of the transformed phenotype (10.Nur-E-Kamal A. Zhang A. Keenan S.M. Wang X.I. Seraj J. Satoh T. Meiners S. Welsh W.J. Mol. Cancer Res. 2005; 3: 297-305Crossref PubMed Scopus (19) Google Scholar). Ack1 contributes to prostate tumorigenesis by phosphorylating the tumor suppressor protein Wwox, leading to its degradation (11.Mahajan N.P. Whang Y.E. Mohler J.L. Earp H.S. Cancer Res. 2005; 65: 10514-10523Crossref PubMed Scopus (161) Google Scholar), and by phosphorylating androgen receptor (12.Mahajan N.P. Liu Y. Majumder S. Warren M.R. Parker C.E. Mohler J.L. Earp H.S. Whang Y.E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8438-8443Crossref PubMed Scopus (194) Google Scholar).Ack1 is ubiquitously expressed in mammals, with the highest expression in spleen, thymus, and brain (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar). Ack1 is phosphorylated and activated in response to a number of stimuli, including EGF, platelet-derived growth factor, bradykinin, agonists of the M3 muscarinic receptor, and integrin-mediated cell adhesion (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar, 9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar, 13.Yang W. Lin Q. Guan J.L. Cerione R.A. J. Biol. Chem. 1999; 274: 8524-8530Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14.Linseman D.A. Heidenreich K.A. Fisher S.K. J. Biol. Chem. 2001; 276: 5622-5628Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15.Yang W. Cerione R.A. J. Biol. Chem. 1997; 272: 24819-24824Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Upon EGF stimulation, Ack1 phosphorylates and activates the guanine exchange factor Dbl (16.Kato-Stankiewicz J. Ueda S. Kataoka T. Kaziro Y. Satoh T. Biochem. Biophys. Res. Commun. 2001; 284: 470-477Crossref PubMed Scopus (40) Google Scholar). In response to Cdc42 activation, Ack1 mediates phosphorylation of p130Cas to promote cell migration (17.Eisenmann K.M. McCarthy J.B. Simpson M.A. Keely P.J. Guan J.L. Tachibana K. Lim L. Manser E. Furcht L.T. Iida J. Nat. Cell Biol. 1999; 1: 507-513Crossref PubMed Scopus (168) Google Scholar, 18.Modzelewska K. Newman L.P. Desai R. Keely P.J. J. Biol. Chem. 2006; 281: 37527-37535Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Other identified substrates of Ack1 include WASP (Wiskott-Aldrich syndrome protein) (19.Yokoyama N. Lougheed J. Miller W.T. J. Biol. Chem. 2005; 280: 42219-42226Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and the sorting nexin SH3PX1 (20.Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar). A number of SH3-containing proteins have been identified as ligands for the proline-rich region of Ack1, including the SH3 domains of Hck (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), Grb2 (22.Satoh T. Kato J. Nishida K. Kaziro Y. FEBS Lett. 1996; 386: 230-234Crossref PubMed Scopus (49) Google Scholar), and SNX9 (20.Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar, 22.Satoh T. Kato J. Nishida K. Kaziro Y. FEBS Lett. 1996; 386: 230-234Crossref PubMed Scopus (49) Google Scholar) and the WW domain of the E3 ubiquitin ligase Nedd4–2 (23.Chan W. Tian R. Lee Y.F. Sit S.T. Lim L. Manser E. J. Biol. Chem. 2009; 284: 8185-8194Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Although the physiological functions of Ack1 are not completely understood, several studies have focused on the role of Ack1 in EGF receptor trafficking and dynamics. Ack1 is recruited to EGFR following EGF stimulation, and overexpression of Ack1 interferes with the proper trafficking of EGFR (24.Gr⊘vdal L.M. Johannessen L.E. R⊘dland M.S. Madshus I.H. Stang E. Exp. Cell Res. 2008; 314: 1292-1300Crossref PubMed Scopus (35) Google Scholar). EGFR stability is regulated by interactions between Ack1 and multiple partner proteins including ubiquitin (7.Shen F. Lin Q. Gu Y. Childress C. Yang W. Mol. Biol. Cell. 2007; 18: 732-742Crossref PubMed Scopus (75) Google Scholar), clathrin heavy chain (6.Teo M. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 18392-18398Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 25.Yang W. Lo C.G. Dispenza T. Cerione R.A. J. Biol. Chem. 2001; 276: 17468-17473Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and SH3PX1 (26.Childress C. Lin Q. Yang W. Biochem. J. 2006; 394: 693-698Crossref PubMed Scopus (17) Google Scholar).The activities of other NRTKs are controlled through intramolecular interactions and by conformational changes in the kinase activation loops (27.Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar). In Src family kinases, the inactive conformation is stabilized by an interaction between the SH3 domain and a polyproline type II helix and an interaction between the SH2 domain and the C-terminal phosphotyrosine (28.Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 29.Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). Activating signals (such as SH3 or SH2 ligands) disrupt these interactions and promote the phosphorylation of Tyr416 in the activation loop of Src (30.Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar, 31.Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 32.Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 33.Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). In c-Abl, the SH2 and SH3 domains also participate in autoinhibitory interactions. These interactions are stabilized by an N-terminal cap region and by an interaction between the N-terminal myristoyl group and the base of the kinase catalytic domain (34.Pluk H. Dorey K. Superti-Furga G. Cell. 2002; 108: 247-259Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Based on the distinctive domain organization of Ack1, the regulatory mechanisms are likely to be divergent from the Src and Abl family paradigm.Using purified Ack1, we previously showed that the major autophosphorylation site is Tyr284 in the activation loop (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The crystal structure of the isolated kinase domain of Ack1 has been solved in both the phosphorylated (Tyr(P)284) and unphosphorylated forms (35.Lougheed J.C. Chen R.H. Mak P. Stout T.J. J. Biol. Chem. 2004; 279: 44039-44045Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In both of these structures, the conformation of the activation loop resembles the conformation seen in other active kinases. Phosphorylation of Tyr284 in Ack1 stimulates kinase activity (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), but not as strongly as in Src or Abl NRTKs. Thus, the regulatory importance of Ack1 autophosphorylation appears to be intermediate between Src/Abl (strongly phosphorylation-dependent) (35.Lougheed J.C. Chen R.H. Mak P. Stout T.J. J. Biol. Chem. 2004; 279: 44039-44045Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and kinases such as EGFR or Cdks (in which formation of the activated state is phosphorylation-independent) (36.Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar, 37.Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (874) Google Scholar, 38.Nolen B. Taylor S. Ghosh G. Mol. Cell. 2004; 15: 661-675Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar). Ligands for the SH3 or the CRIB domains of Ack1 do not activate the purified enzyme (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar); thus, the regulatory mechanism for Ack1 is poorly understood at present.Recently, the genes encoding 518 protein kinases were sequenced in a large collection of human cancers to identify somatic mutations (39.Greenman C. Stephens P. Smith R. Dalgliesh G.L. Hunter C. Bignell G. Davies H. Teague J. Butler A. Stevens C. Edkins S. O'Meara S. Vastrik I. Schmidt E.E. Avis T. Barthorpe S. Bhamra G. Buck G. Choudhury B. Clements J. Cole J. Dicks E. Forbes S. Gray K. Halliday K. Harrison R. Hills K. Hinton J. Jenkinson A. Jones D. Menzies A. Mironenko T. Perry J. Raine K. Richardson D. Shepherd R. Small A. Tofts C. Varian J. Webb T. West S. Widaa S. Yates A. Cahill D.P. Louis D.N. Goldstraw P. Nicholson A.G. Brasseur F. Looijenga L. Weber B.L. Chiew Y.E. DeFazio A. Greaves M.F. Green A.R. Campbell P. Birney E. Easton D.F. Chenevix-Trench G. Tan M.H. Khoo S.K. Teh B.T. Yuen S.T. Leung S.Y. Wooster R. Futreal P.A. Stratton M.R. Nature. 2007; 446: 153-158Crossref PubMed Scopus (2355) Google Scholar). Four missense mutations were identified in Ack1: two mutations in the N terminus (R34L and R99Q) were identified in lung adenocarcinoma and ovarian carcinoma, respectively; a mutation in the kinase catalytic domain (E346K) was identified in ovarian endometroid carcinoma; and a mutation in the SH3 domain (M409I) was found in gastric adenocarcinoma. The probability of each mutation being a driver (i.e. a mutation that confers an advantage to the cancer cell and that is therefore subject to positive selection pressure) was estimated by comparing the rate of nonsynonymous and synonymous somatic mutations in each gene. The genes were ranked according to their probability of carrying at least one driver mutation, and Ack1 ranked in the top 5%. The effect of these mutations on Ack1 activity and downstream signaling is unknown.The aims of this study were: 1) to test the effects of the cancer-associated mutations on Ack1 function and activity and 2) to use the mutations to gain insight into the regulation of Ack1. We report that cancer-associated mutations stimulate Ack1 activity without affecting its subcellular localization, suggesting that point mutations represent a new mechanism for the oncogenic activation of Ack1. Moreover, we propose that an autoinhibitory interaction exists between the kinase domain and the C-terminal Mig6 homology region and that mutations that disrupt this interaction (such as the cancer-associated mutation E346K) activate Ack1.DISCUSSIONAck1 has previously been implicated in tumorigenesis (11.Mahajan N.P. Whang Y.E. Mohler J.L. Earp H.S. Cancer Res. 2005; 65: 10514-10523Crossref PubMed Scopus (161) Google Scholar, 12.Mahajan N.P. Liu Y. Majumder S. Warren M.R. Parker C.E. Mohler J.L. Earp H.S. Whang Y.E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8438-8443Crossref PubMed Scopus (194) Google Scholar), cell survival (10.Nur-E-Kamal A. Zhang A. Keenan S.M. Wang X.I. Seraj J. Satoh T. Meiners S. Welsh W.J. Mol. Cancer Res. 2005; 3: 297-305Crossref PubMed Scopus (19) Google Scholar), and metastasis (9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar). The Ack1 gene is amplified and overexpressed in a number of tumors, and the copy number change correlates with later stage, more aggressive tumors (9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar). Prior to this study, it was not clear whether mutations represent an additional mechanism for the oncogenic activation of Ack1. The four nonsynonymous mutations in Ack1 studied in this paper were identified in a study in which 210 cancer tissue samples were screened for somatic mutations in protein kinase genes (39.Greenman C. Stephens P. Smith R. Dalgliesh G.L. Hunter C. Bignell G. Davies H. Teague J. Butler A. Stevens C. Edkins S. O'Meara S. Vastrik I. Schmidt E.E. Avis T. Barthorpe S. Bhamra G. Buck G. Choudhury B. Clements J. Cole J. Dicks E. Forbes S. Gray K. Halliday K. Harrison R. Hills K. Hinton J. Jenkinson A. Jones D. Menzies A. Mironenko T. Perry J. Raine K. Richardson D. Shepherd R. Small A. Tofts C. Varian J. Webb T. West S. Widaa S. Yates A. Cahill D.P. Louis D.N. Goldstraw P. Nicholson A.G. Brasseur F. Looijenga L. Weber B.L. Chiew Y.E. DeFazio A. Greaves M.F. Green A.R. Campbell P. Birney E. Easton D.F. Chenevix-Trench G. Tan M.H. Khoo S.K. Teh B.T. Yuen S.T. Leung S.Y. Wooster R. Futreal P.A. Stratton M.R. Nature. 2007; 446: 153-158Crossref PubMed Scopus (2355) Google Scholar). Using this approach, Ack1 emerged as a likely candidate to carry driver mutations. We show here that point mutations activate Ack1 and that cells expressing the cancer-associated mutants exhibit aspects of the transformed phenotype. Mutations located in the N terminus (R34L and R99Q), in the C-lobe of the kinase domain (E346K), and in the SH3 domain (M409I) activated Ack1 in functional and biochemical assays. Our data reinforce the previous characterization of Ack1 as an oncogene and suggest that the somatic mutations may contribute to cancer development.Consistent with published data (9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar), we found that the overexpression of wild type Ack1 promotes the transformed phenotype. Cells overexpressing WT Ack1 showed higher levels of migration than control cells (Fig. 5). The expression of WT Ack1 also caused a loss of contact inhibition in cells growing attached to a surface and allowed the cells to survive in nonanchored conditions (Fig. 4). Our studies were carried out in NIH3T3 cells, and they are similar to the results obtained in human mammary epithelial cells and in 4T1 mouse mammary tumor cells, suggesting that Ack1 promotes cell growth and migration independently of cell context.The two cancer-associated mutations located in the N terminus of Ack1 increased the level of Ack1 autophosphorylation in cells (Fig. 1B). This was due to enhanced Ack1 kinase activity, because the mutants also phosphorylated a synthetic peptide at a higher rate (Fig. 1C). Neither of the N-terminal mutations displayed any significant enhancement of anchored or nonanchored growth over the level observed with WT Ack1. On the other hand, the R34L mutation did increase the effect of Ack1 on cell migration. The N terminus of Ack1 contains a region that was identified as a SAM domain and as a membrane localization motif (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar). The observed activation of Ack1 by these mutations could potentially have been explained by the mislocalization of Ack1 caused by the disruption of the membrane localization domain. However, the intracellular localization of Ack1 was not affected by the cancer-associated mutations located in the N terminus (Fig. 2). Thus, our data indicate that the R34L and R99Q mutations increase Ack1 autophosphorylation and activity. Although the mechanism is not clear, one possibility is that the N-terminal mutations disrupt an inhibitory interaction. This putative interaction could be either intramolecular or intermolecular (in the latter case, involving other molecules or the formation of an Ack1 homodimer). SAM domains share an overall secondary structure rich in α-helices and have been found in more than 1000 proteins, including the Eph family of tyrosine kinase receptors (46.Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Crossref PubMed Scopus (211) Google Scholar, 47.Thanos C.D. Goodwill K.E. Bowie J.U. Science. 1999; 283: 833-836Crossref PubMed Scopus (199) Google Scholar), diacylglycerol kinase δ (48.Harada B.T. Knight M.J. Imai S. Qiao F. Ramachander R. Sawaya M.R. Gingery M. Sakane F. Bowie J.U. Structure. 2008; 16: 380-387Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and the transcriptional repressor TEL (49.Kim C.A. Phillips M.L. Kim W. Gingery M. Tran H.H. Robinson M.A. Faham S. Bowie J.U. EMBO J. 2001; 20: 4173-4182Crossref PubMed Scopus (194) Google Scholar, 50.Kim C.A. Gingery M. Pilpa R.M. Bowie J.U. Nat. Struct. Biol. 2002; 9: 453-457PubMed Google Scholar). The three-dimensional structures of several SAM domains have been solved, and they form dimeric and polymeric associations. Although the biological roles of these interactions are not yet clear for all of the SAM-containing proteins, diacylglycerol kinase δ was reported to be regulated by SAM-mediated polymerization (48.Harada B.T. Knight M.J. Imai S. Qiao F. Ramachander R. Sawaya M.R. Gingery M. Sakane F. Bowie J.U. Structure. 2008; 16: 380-387Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The deletion of the N-terminal portion of Ack1 reduces the ability of full-length Ack1 to undergo autophosphorylation (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar). We speculate that the N-terminal SAM domain may be involved in protein-protein interactions that play a role in Ack1 regulation and that cancer-associated mutations in this region disrupt the normal regulation mechanism, rendering the enzyme more active. Future studies will be focused on the role of the N-terminal region in Ack1 regulation.The cancer-associated mutation M409I, located in the SH3 domain of Ack1, activated cellular autophosphorylation of Ack1 and promoted increased cell migration as compared with WT Ack1. On the other hand, the effect of this mutation on cell proliferation did not significantly differ from the effect of WT Ack1. The reason for this discrepancy is not clear; however, the functional assays that we used measure different biological processes. Ack1 may activate several downstream effectors and pathways through its different domains. Therefore, it is possible that the SH3 domain is involved in the regulation of motility-related rather than cell proliferation pathways. The methionine residue mutated in Ack1 is not conserved in other closely related SH3 domains, and it is not predicted to lie in the binding site for polyproline-containing ligands. Thus, the molecular basis for Ack1 activation by M409I is not understood presently.The cancer-associated mutation E346K is located in the C-lobe of the kinase domain. We found that the expression of E346K Ack1 potentiated the effects of the expression of WT Ack1 on migration and nonanchored growth. Thus, our functional data suggested that the C-terminal portion of Ack1 plays a role in its regulation. Next, we focused on the study of this region to gain insight into a regulatory mechanism that involves the kinase domain and the C-terminal Mig6 homology region.Relatively little information was previously available concerning Ack1 regulation. Ack1 autophosphorylation produced modest activation in a construct containing the N terminus, the catalytic domain, the SH3 domain, and the CRIB domain of Ack1 (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Also, the addition of Cdc42 (ligand for CRIB domain) or poly-proline peptides (ligands for SH3 domain) did not activate this purified construct in vitro. However, in cells expressing full-length Ack1, the co-expression of Cdc42 did activate Ack1 (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The introduction of point mutations into the CRIB and SH3 domains in full-length Ack1 expressed in cells also produced increased autophosphorylation, suggesting that the different domains of Ack1 participate in regulatory interactions (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar).The structure of the kinase domain of EGFR in a complex with the inhibitor protein Mig6 (8.Zhang X. Pickin K.A. Bose R. Jura N. Cole P.A. Kuriyan J. Nature. 2007; 450: 741-744Crossref PubMed Scopus (273) Google Scholar) suggested that the C-lobe of the kinase domain could be important for an intramolecular interaction with the Mig6 homology region located in the C terminus of Ack1 (Fig. 7B). Based on previous studies on EGFR, we introduced point mutations designed to disrupt this putative interaction. We found that point mutations located either in the C-lobe of the kinase domain (V365R) or in the Mig6 homology region (F820A) resulted in the activation of full-length Ack1 (Fig. 8, B and C). Although F820A is not a naturally occurring mutation, it was able to recapitulate the functional effects that we observed for the cancer-associated mutations. Likewise, it produced a 50% increase in cell migration and nonanchored growth (Fig. 9). The ability of the F820A mutation to produce a large (∼80-fold) increase in kinase activity suggests that previous work on Ack1, which was carried out with a construct lacking the Mig6 homology region (MHR), underestimated the dynamic range of kinase activation.To further test the interaction predicted by the model and suggested by the cell transfection experiments, we conducted binding experiments using purified GST-tagged MHR and purified His6-tagged Ack1 kinase domain. The purified kinase domain binds to the purified WT MHR (Fig. 10, A and B). This interaction is prevented by the F820A mutation in the MHR (Fig. 10A) and by the E346K mutation in the kinase domain (Fig. 10B). The interaction between these two minimal segments provides additional evidence for a direct interaction between the kinase domain and the MHR in the context of full-length Ack1. Based on the data presented in this paper, we propose a model for Ack1 regulation in which the MHR interacts with C-lobe of the kinase domain to stabilize a down-regulated structure (Fig. 10C). The Mig6 region might interact directly with the Ack1 active site as pictured in Fig. 10C or alternatively act indirectly to position an inhibitory segment in the active site. In preliminary in vitro experiments, the GST-MHR did not inhibit the purified Ack1 kinase domain, 3V. Prieto-Echagüe and W. T. Miller, unpublished observations. suggesting that additional regions of Ack1 are needed for direct or indirect autoinhibition.The C-terminal region of Ack1 is also involved in interactions with EGFR and other upstream and downstream signaling molecules. Thus, as observed for other NRTKs such as Src (51.Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar), Abl (52.Nagar B. Hantschel O. Young M.A. Scheffzek K. Veach D. Bornmann W. Clarkson B. Superti-Furga G. Kuriyan J. Cell. 2003; 112: 859-871Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar), and focal adhesion kinase (53.Lietha D. Cai X. Ceccarelli D.F. Li Y. Schaller M.D. Eck M.J. Cell. 2007; 129: 1177-1187Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), enzymatic activation of Ack can be coupled to interactions between the noncatalytic domains and allosteric regulators, effectors, and potential substrates. IntroductionAck1 is a nonreceptor tyrosine kinase (NRTK) 2The abbreviations used are: NRTKnonreceptor tyrosine kinaseSAMsterile α-motifCRIBCdc42-binding domainEGFepidermal growth factorEGFREGF receptorMHRMig6 homology regionSHSrc homologyE3ubiquitin-protein isopeptide ligasePMSFphenylmethylsulfonyl fluorideHAhemagglutininGFPgreen fluorescent proteinPBSphosphate-buffered salinePVDFpolyvinylidene difluorideWTwild typeGSTglutathione S-transferaseCasCrk-associated substrateWASPWiskott-Aldrich syndrome proteinEVempty vector. that was first isolated from a human hippocampal expression library because of its specific binding to GTP-bound Cdc42 (1.Manser E. Leung T. Salihuddin H. Tan L. Lim L. Nature. 1993; 363: 364-367Crossref PubMed Scopus (257) Google Scholar). Ack1 belongs to a family of nonreceptor tyrosine kinases that includes Ack1, Tnk, and homologous proteins in Drosophila and Caenorhabditis elegans (2.Hoehn G.T. Stokland T. Amin S. Ramírez M. Hawkins A.L. Griffin C.A. Small D. Civin C.I. Oncogene. 1996; 12: 903-913PubMed Google Scholar, 3.Hopper N.A. Lee J. Sternberg P.W. Mol. Cell. 2000; 6: 65-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4.Sem K.P. Zahedi B. Tan I. Deak M. Lim L. Harden N. Mol. Cell Biol. 2002; 22: 3685-3697Crossref PubMed Scopus (18) Google Scholar). Ack1 is a 120-kDa protein with an N-terminal sterile α-motif (SAM) domain (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar), a kinase domain, an SH3 domain, and a Cdc42-binding domain (CRIB) (see Fig. 1A). The large C-terminal portion of Ack1 contains several proline-rich sequences as well as a clathrin-binding motif (6.Teo M. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 18392-18398Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), a ubiquitin-binding domain (7.Shen F. Lin Q. Gu Y. Childress C. Yang W. Mol. Biol. Cell. 2007; 18: 732-742Crossref PubMed Scopus (75) Google Scholar), and a region homologous to Mig6 (8.Zhang X. Pickin K.A. Bose R. Jura N. Cole P.A. Kuriyan J. Nature. 2007; 450: 741-744Crossref PubMed Scopus (273) Google Scholar). The domain architecture of Ack1 is unique among NRTKs; it is the only NRTK with a CRIB domain, and the position of the SH3 domain (C-terminal to the kinase domain) differs from all other families of NRTKs.Recent data implicate Ack1 in different stages of cancer. Amplification of the Ack1 gene correlates with metastasis and poor prognosis in lung and prostate cancer, and overexpression increases invasiveness (9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar). In v-Ras-transformed mammalian cells, Ack1 is required for the maintenance of the transformed phenotype (10.Nur-E-Kamal A. Zhang A. Keenan S.M. Wang X.I. Seraj J. Satoh T. Meiners S. Welsh W.J. Mol. Cancer Res. 2005; 3: 297-305Crossref PubMed Scopus (19) Google Scholar). Ack1 contributes to prostate tumorigenesis by phosphorylating the tumor suppressor protein Wwox, leading to its degradation (11.Mahajan N.P. Whang Y.E. Mohler J.L. Earp H.S. Cancer Res. 2005; 65: 10514-10523Crossref PubMed Scopus (161) Google Scholar), and by phosphorylating androgen receptor (12.Mahajan N.P. Liu Y. Majumder S. Warren M.R. Parker C.E. Mohler J.L. Earp H.S. Whang Y.E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8438-8443Crossref PubMed Scopus (194) Google Scholar).Ack1 is ubiquitously expressed in mammals, with the highest expression in spleen, thymus, and brain (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar). Ack1 is phosphorylated and activated in response to a number of stimuli, including EGF, platelet-derived growth factor, bradykinin, agonists of the M3 muscarinic receptor, and integrin-mediated cell adhesion (5.Galisteo M.L. Yang Y. Ureña J. Schlessinger J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9796-9801Crossref PubMed Scopus (68) Google Scholar, 9.van der Horst E.H. Degenhardt Y.Y. Strelow A. Slavin A. Chinn L. Orf J. Rong M. Li S. See L.H. Nguyen K.Q. Hoey T. Wesche H. Powers S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15901-15906Crossref PubMed Scopus (112) Google Scholar, 13.Yang W. Lin Q. Guan J.L. Cerione R.A. J. Biol. Chem. 1999; 274: 8524-8530Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14.Linseman D.A. Heidenreich K.A. Fisher S.K. J. Biol. Chem. 2001; 276: 5622-5628Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15.Yang W. Cerione R.A. J. Biol. Chem. 1997; 272: 24819-24824Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Upon EGF stimulation, Ack1 phosphorylates and activates the guanine exchange factor Dbl (16.Kato-Stankiewicz J. Ueda S. Kataoka T. Kaziro Y. Satoh T. Biochem. Biophys. Res. Commun. 2001; 284: 470-477Crossref PubMed Scopus (40) Google Scholar). In response to Cdc42 activation, Ack1 mediates phosphorylation of p130Cas to promote cell migration (17.Eisenmann K.M. McCarthy J.B. Simpson M.A. Keely P.J. Guan J.L. Tachibana K. Lim L. Manser E. Furcht L.T. Iida J. Nat. Cell Biol. 1999; 1: 507-513Crossref PubMed Scopus (168) Google Scholar, 18.Modzelewska K. Newman L.P. Desai R. Keely P.J. J. Biol. Chem. 2006; 281: 37527-37535Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Other identified substrates of Ack1 include WASP (Wiskott-Aldrich syndrome protein) (19.Yokoyama N. Lougheed J. Miller W.T. J. Biol. Chem. 2005; 280: 42219-42226Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and the sorting nexin SH3PX1 (20.Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar). A number of SH3-containing proteins have been identified as ligands for the proline-rich region of Ack1, including the SH3 domains of Hck (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), Grb2 (22.Satoh T. Kato J. Nishida K. Kaziro Y. FEBS Lett. 1996; 386: 230-234Crossref PubMed Scopus (49) Google Scholar), and SNX9 (20.Yeow-Fong L. Lim L. Manser E. FEBS Lett. 2005; 579: 5040-5048Crossref PubMed Scopus (42) Google Scholar, 22.Satoh T. Kato J. Nishida K. Kaziro Y. FEBS Lett. 1996; 386: 230-234Crossref PubMed Scopus (49) Google Scholar) and the WW domain of the E3 ubiquitin ligase Nedd4–2 (23.Chan W. Tian R. Lee Y.F. Sit S.T. Lim L. Manser E. J. Biol. Chem. 2009; 284: 8185-8194Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Although the physiological functions of Ack1 are not completely understood, several studies have focused on the role of Ack1 in EGF receptor trafficking and dynamics. Ack1 is recruited to EGFR following EGF stimulation, and overexpression of Ack1 interferes with the proper trafficking of EGFR (24.Gr⊘vdal L.M. Johannessen L.E. R⊘dland M.S. Madshus I.H. Stang E. Exp. Cell Res. 2008; 314: 1292-1300Crossref PubMed Scopus (35) Google Scholar). EGFR stability is regulated by interactions between Ack1 and multiple partner proteins including ubiquitin (7.Shen F. Lin Q. Gu Y. Childress C. Yang W. Mol. Biol. Cell. 2007; 18: 732-742Crossref PubMed Scopus (75) Google Scholar), clathrin heavy chain (6.Teo M. Tan L. Lim L. Manser E. J. Biol. Chem. 2001; 276: 18392-18398Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 25.Yang W. Lo C.G. Dispenza T. Cerione R.A. J. Biol. Chem. 2001; 276: 17468-17473Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and SH3PX1 (26.Childress C. Lin Q. Yang W. Biochem. J. 2006; 394: 693-698Crossref PubMed Scopus (17) Google Scholar).The activities of other NRTKs are controlled through intramolecular interactions and by conformational changes in the kinase activation loops (27.Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar). In Src family kinases, the inactive conformation is stabilized by an interaction between the SH3 domain and a polyproline type II helix and an interaction between the SH2 domain and the C-terminal phosphotyrosine (28.Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1041) Google Scholar, 29.Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1242) Google Scholar). Activating signals (such as SH3 or SH2 ligands) disrupt these interactions and promote the phosphorylation of Tyr416 in the activation loop of Src (30.Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar, 31.Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 32.Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 33.Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (536) Google Scholar). In c-Abl, the SH2 and SH3 domains also participate in autoinhibitory interactions. These interactions are stabilized by an N-terminal cap region and by an interaction between the N-terminal myristoyl group and the base of the kinase catalytic domain (34.Pluk H. Dorey K. Superti-Furga G. Cell. 2002; 108: 247-259Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Based on the distinctive domain organization of Ack1, the regulatory mechanisms are likely to be divergent from the Src and Abl family paradigm.Using purified Ack1, we previously showed that the major autophosphorylation site is Tyr284 in the activation loop (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The crystal structure of the isolated kinase domain of Ack1 has been solved in both the phosphorylated (Tyr(P)284) and unphosphorylated forms (35.Lougheed J.C. Chen R.H. Mak P. Stout T.J. J. Biol. Chem. 2004; 279: 44039-44045Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In both of these structures, the conformation of the activation loop resembles the conformation seen in other active kinases. Phosphorylation of Tyr284 in Ack1 stimulates kinase activity (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), but not as strongly as in Src or Abl NRTKs. Thus, the regulatory importance of Ack1 autophosphorylation appears to be intermediate between Src/Abl (strongly phosphorylation-dependent) (35.Lougheed J.C. Chen R.H. Mak P. Stout T.J. J. Biol. Chem. 2004; 279: 44039-44045Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and kinases such as EGFR or Cdks (in which formation of the activated state is phosphorylation-independent) (36.Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar, 37.Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (874) Google Scholar, 38.Nolen B. Taylor S. Ghosh G. Mol. Cell. 2004; 15: 661-675Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar). Ligands for the SH3 or the CRIB domains of Ack1 do not activate the purified enzyme (21.Yokoyama N. Miller W.T. J. Biol. Chem. 2003; 278: 47713-47723Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar); thus, the regulatory mechanism for Ack1 is poorly understood at present.Recently, the genes encoding 518 protein kinases were sequenced in a large collection of human cancers to identify somatic mutations (39.Greenman C. Stephens P. Smith R. Dalgliesh G.L. Hunter C. Bignell G. Davies H. Teague J. Butler A. Stevens C. Edkins S. O'Meara S. Vastrik I. Schmidt E.E. Avis T. Barthorpe S. Bhamra G. Buck G. Choudhury B. Clements J. Cole J. Dicks E. Forbes S. Gray K. Halliday K. Harrison R. Hills K. Hinton J. Jenkinson A. Jones D. Menzies A. Mironenko T. Perry J. Raine K. Richardson D. Shepherd R. Small A. Tofts C. Varian J. Webb T. West S. Widaa S. Yates A. Cahill D.P. Louis D.N. Goldstraw P. Nicholson A.G. Brasseur F. Looijenga L. Weber B.L. Chiew Y.E. DeFazio A. Greaves M.F. Green A.R. Campbell P. Birney E. Easton D.F. Chenevix-Trench G. Tan M.H. Khoo S.K. Teh B.T. Yuen S.T. Leung S.Y. Wooster R. Futreal P.A. Stratton M.R. Nature. 2007; 446: 153-158Crossref PubMed Scopus (2355) Google Scholar). Four missense mutations were identified in Ack1: two mutations in the N terminus (R34L and R99Q) were identified in lung adenocarcinoma and ovarian carcinoma, respectively; a mutation in the kinase catalytic domain (E346K) was identified in ovarian endometroid carcinoma; and a mutation in the SH3 domain (M409I) was found in gastric adenocarcinoma. The probability of each mutation being a driver (i.e. a mutation that confers an advantage to the cancer cell and that is therefore subject to positive selection pressure) was estimated by comparing the rate of nonsynonymous and synonymous somatic mutations in each gene. The genes were ranked according to their probability of carrying at least one driver mutation, and Ack1 ranked in the top 5%. The effect of these mutations on Ack1 activity and downstream signaling is unknown.The aims of this study were: 1) to test the effects of the cancer-associated mutations on Ack1 function and activity and 2) to use the mutations to gain insight into the regulation of Ack1. We report that cancer-associated mutations stimulate Ack1 activity without affecting its subcellular localization, suggesting that point mutations represent a new mechanism for the oncogenic activation of Ack1. Moreover, we propose that an autoinhibitory interaction exists between the kinase domain and the C-terminal Mig6 homology region and that mutations that disrupt this interaction (such as the cancer-associated mutation E346K) activate Ack1.
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