c-Abl Has High Intrinsic Tyrosine Kinase Activity That Is Stimulated by Mutation of the Src Homology 3 Domain and by Autophosphorylation at Two Distinct Regulatory Tyrosines

Abstract

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K m of 204 μm and V max of 33 pmol min−1. Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K m = 91 μm,V max = 112 pmol min−1). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245. Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K m of 204 μm and V max of 33 pmol min−1. Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K m = 91 μm,V max = 112 pmol min−1). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245. Src homology 2 Src homology glutathione S-transferase c-Abl is a non-receptor tyrosine kinase of which the precise functions are not known, but roles for Abl in growth factor and integrin signaling, cell cycle regulation, neurogenesis, and responses to DNA damage and oxidative stress have been suggested (1Van Etten R.A. Trends Cell Biol. 1999; 9: 179-186Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including ionizing radiation (2Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (463) Google Scholar), entry into S phase (3Welch P.J. Wang J.Y.J. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (368) Google Scholar), integrin activation (4Lewis J.M. Baskaran R. Taagepera S. Schwartz M.A. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15174-15179Crossref PubMed Scopus (269) Google Scholar), and platelet-derived growth factor stimulation (5Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (373) Google Scholar). The mechanism of regulation of Abl tyrosine kinase activity by these processes is not well understood. Ionizing radiation may activate Abl kinase activity through phosphorylation of the Abl catalytic domain at Ser-465 by the Atm kinase (6Baskaran R. Wood L.D. Whitaker L.L. Canman C.E. Morgan S.E. Xu Y. Barlow C. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y.J. Nature. 1997; 387: 516-519Crossref PubMed Scopus (485) Google Scholar), whereas platelet-derived growth factor stimulation is associated with tyrosine phosphorylation of c-Abl by c-Src (5Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (373) Google Scholar). In contrast, the activation of nuclear c-Abl in S phase is through the detachment of the inhibitor Rb protein (3Welch P.J. Wang J.Y.J. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (368) Google Scholar), whereas Abl may be activated by free radicals through dissociation of Pag/Msp23, an antioxidant protein that also inhibits Abl (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar). Abl kinase activity can also be stimulated by the binding of several activator proteins, including the transcription factors c-Jun (8Barila D. Mangano R. Gonfloni S. Kretzschmar J. Moro M. Bohmann D. Superti-Furga G. EMBO J. 2000; 19: 273-281Crossref PubMed Scopus (77) Google Scholar) and RFX1 (9Agami R. Shaul Y. Oncogene. 1998; 16: 1779-1788Crossref PubMed Scopus (25) Google Scholar), and the adapter protein Nck (10Smith J.M. Katz S. Mayer B.J. J. Biol. Chem. 1999; 274: 27956-27962Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These observations suggest complex regulation of c-Abl at multiple levels through binding or dissociation of activators and inhibitors and via direct phosphorylation. Although the NH2-terminal sequence of c-Abl is very similar to members of the Src family, biochemical and genetic studies suggest that the structural basis of regulation of c-Abl catalytic activity is significantly different from the catalytic activity of Src. When co-expressed with another kinase, such as Csk, that can phosphorylate the COOH-terminal regulatory tyrosine 527, c-Src (and the Src family member Hck) can be purified as an inactive monomer in which the phosphorylated Tyr-527 residue binds the SH21 domain in an intramolecular fashion. In this structure, the SH3 domain contacts the linker region between SH2 and the catalytic domain (the SH2-CD linker) in an atypical interaction involving a single proline (Pro-250) (11Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar,12Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). Mutation or deletion of Tyr-527 (13Piwinica-Worms H. Saunders K. Roberts T. Smith A. Cheng S. Cell. 1987; 49: 75-82Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 14Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (411) Google Scholar) or mutation of the SH2 or SH3 domains (15Hirai H. Varmus H.E. Mol. Cell. Biol. 1990; 10: 1307-1318Crossref PubMed Google Scholar, 16Seidel-Dugan C. Meyer B.E. Thomas S.M. Brugge J.S. Mol. Cell. Biol. 1992; 12: 1835-1845Crossref PubMed Scopus (150) Google Scholar) dysregulates and increases Src kinase activity both in vitro and in vivo. The precise mechanism of physiological activation of Src kinases is unknown, but in vitro studies demonstrate that activation may involve discrete steps that independently increase catalytic activity. In the presence of ATP and magnesium, Src or Hck that is monophosphorylated at the Tyr-527 homologue undergoes slow autophosphorylation at Tyr-412 that increases kinase activity about 10-fold (17Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar, 18Boerner R.J. Kassel D.B. Barker S.C. Ellis B. DeLacy P. Knight W.B. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar, 19Porter M. Schindler T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; 275: 2721-2726Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Dissociation of the SH2-Tyr-527 interaction by dephosphorylation or a competing SH2 ligand stimulates activity 2.5-fold, whereas the disruption of the SH3-linker interaction by an activating SH3 ligand, such as Nef (17Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar), induces a further 3-fold increase in catalytic activity to a maximally activated state. Unlike Src kinases, c-Abl lacks phosphotyrosine in its inactive state, and deletion of the C terminus or mutation of SH2 does not activate Abl (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus Google Scholar, B.J. Van Etten R.A. Baltimore D. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar). deletion of the SH3 domain (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus Google Scholar, P. Wang J.Y.J. EMBO J. 1989; 8: PubMed Scopus Google Scholar) and SH3 point that ligand binding Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar) does Abl kinase activity in vivo as does the mutation of a proline residue in the Abl SH2-CD linker region that is homologous to Src D. Superti-Furga G. 1998; PubMed Scopus Google Scholar). the SH2-CD linker mutation an intramolecular for the SH3 domain in Abl regulation, c-Abl and Abl have similar levels of kinase activity in vitro P. Wang J.Y.J. EMBO J. 1989; 8: PubMed Scopus Google Scholar, Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar, B.J. Baltimore D. Mol. Cell. Biol. PubMed Scopus Google Scholar), that the regulatory of the Abl SH3 domain is only the model is that SH3 Abl kinase activity through the binding of a inhibitor A.M. R. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of c-Abl in at to 10-fold levels does not in Abl autophosphorylation (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus Google Scholar, Etten R.A. P. Baltimore D. Cell. 1989; Full Text PDF PubMed Scopus Google Scholar), but the at higher levels results in tyrosine phosphorylation of Abl and (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar, A.M. R. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). c-Abl kinase activity is in B. and R. A. Van K. D. Superti-Furga G. Biol. Chem. 1999; PubMed Google Scholar) but not in J. A. Superti-Furga G. Oncogene. 1996; 12: Google Scholar). These observations suggest that wild-type and c-Abl have similar intrinsic kinase but the c-Abl can with a the inhibitor is but can be the of does not with and is in Abl have been as for such an including (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar), J. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar), Y. K. Genes Dev. 1995; 9: PubMed Scopus Google Scholar), and Pendergast A.M. Genes Dev. 1995; 9: PubMed Scopus Google Scholar). these been to c-Abl kinase activity in vivo (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar). the regulation of c-Abl tyrosine kinase we purified c-Abl in a that to its inactive by using the specific Abl kinase inhibitor STI to activation and autophosphorylation in vivo. found that unphosphorylated c-Abl had substantial intrinsic catalytic activity to inactive and this activity was further stimulated by autophosphorylation at regulatory tyrosine we found that the mutation of the SH3 domain significantly increased the activity of an intramolecular regulatory for these results suggest a model where c-Abl is activated in vivo by dissociation of an inhibitor by phosphorylation at tyrosine the catalytic domain and the SH2-CD linker The murine type IV in the was to at the The the sequence from to Abl by and by DNA Abl by of as P. Miller Mol. Cell. Biol. 1987; PubMed Scopus Google Scholar), that was with μm STI where and with with with and in and and on for by at for at and to at a of 2 of to of to at for with and the was to was with of by of and of as but with and with of as but with to 2 and and and of was to for at and on for to The concentration of purified Abl was by and with purified These by using a and The from was of Abl protein at Abl phosphorylation at kinase at in kinase 2 and and in μm autophosphorylation than ATP and for at the of Autophosphorylation by the of and by to and with a of R. and by and phosphorylation levels using and activity of unphosphorylated Abl kinases was using a peptide the Abl sequence Eck M.J. Harrison S.C. R.A. M. J. Smith C. M.J. Mayer B.J. Nature. 1995; PubMed Scopus Google Scholar) and with an at in kinase μm at and peptide in for concentration and to for by the of to m of and with 2 m by 2 m with as suggested by the was by of the binding to the was by peptide in a of and was than for for and on a using the to K m and V max In assays, the concentration of kinase was and specific activity was as of pmol of the of autophosphorylation on kinase specific the Autophosphorylation as and ATP at μm and kinase at μm of autophosphorylation and for or to of kinase and peptide phosphorylation in or for as using a single concentration of peptide μm to activity. of Abl kinases in the from autophosphorylation to peptide kinase in a kinase concentration of μm in the peptide kinase murine c-Abl a in and purified in a single by on c-Abl lacks levels of tyrosine phosphorylation in vivo in its inactive (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus Google Scholar), but of Abl in or results in levels of Abl tyrosine phosphorylation A.M. R. F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). unphosphorylated in the presence of STI 571, an kinase inhibitor J. H. Meyer T. M. B.J. 1996; Google Scholar), and from purified kinase with c-Abl was the in the by and phosphotyrosine by with phosphorylation by the purified wild-type c-Abl was STI with an of μm that c-Abl was the only tyrosine kinase in the the in activity of purified we a kinase using a peptide with a sequence by Abl kinases Eck M.J. Harrison S.C. R.A. M. J. Smith C. M.J. Mayer B.J. Nature. 1995; PubMed Scopus Google Scholar). Unphosphorylated wild-type c-Abl substantial activity the peptide with an max of 33 pmol of K m of 204 μm (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus Google Scholar, P. Wang J.Y.J. EMBO J. 1989; 8: PubMed Scopus Google Scholar) and point Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar) in the c-Abl SH3 domain Abl kinase activity in levels of tyrosine phosphorylation of Abl and and the in vitro kinase of wild-type and c-Abl are similar measured P. Wang J.Y.J. EMBO J. 1989; 8: PubMed Scopus Google Scholar, Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar). In contrast, we found that a transforming c-Abl protein a point mutation in the SH3 domain (P131L) that the binding of Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar) exhibited significantly higher catalytic activity than c-Abl measured in solution with the peptide Unphosphorylated c-Abl P131L protein had max higher pmol and a reduced K m to wild-type c-Abl A from had only levels of that Abl was the kinase activity measured in not the mutation or deletion of the SH3 domain to significantly increase the intrinsic tyrosine kinase activity of unphosphorylated have also a physiological protein of S.M. B. H. EMBO J. 13: PubMed Scopus Google Scholar), as a in this and similar results not and of Abl can to c-Abl and may Abl kinase activity. this the peptide was in this Autophosphorylation to increased catalytic activity for kinases including c-Src A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and we the was for c-Abl and the P131L mutant to and intrinsic kinase activity was measured with the peptide assay. c-Abl and c-Abl in the presence of and ATP Autophosphorylation of c-Src and Hck is an intermolecular mechanism (17Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S.C. Kassel D.B. D. M.A. Knight W.B. Biochemistry. 1995; PubMed Scopus Google Scholar). When the Abl concentration was increased in the autophosphorylation the to Abl phosphotyrosine and the rate of phosphotyrosine increased 2 These results demonstrate that the c-Abl autophosphorylation rate is dependent on kinase concentration and suggest that autophosphorylation by c-Abl is also an intermolecular autophosphorylation an increased of c-Abl to phosphorylate the peptide was Although was in autophosphorylation-induced activation of c-Abl peptide kinase activity among different Abl increased peptide kinase activity the increase in Abl phosphotyrosine in c-Abl catalytic activity the peptide to increase to as as the of autophosphorylation 2 The kinase activity of the c-Abl was also stimulated by autophosphorylation 2 that the SH3 mutation and autophosphorylation independently to increase the catalytic activity of The activation of c-Abl P131L than wild-type a the increase in activity of c-Abl P131L was only about 2 The activity of wild-type and c-Abl was very similar 2 and to or than the specific activity of c-Abl that was purified from with STI not These results suggest that c-Abl is of activation these and that autophosphorylation of c-Abl the intrinsic of the SH3 the catalytic of the c-Abl kinase domain is homologous to c-Src tyrosine and autophosphorylation at tyrosine been to the kinase activity of Src kinases M. Schindler T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; 275: 2721-2726Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is to be a site of tyrosine phosphorylation in transforming Abl S. J. PubMed Google Scholar). A c-Abl mutant phosphotyrosine in in the of STI that Tyr-412 is a in vivo tyrosine phosphorylation site of mutation of the homologue in Hck to kinase activity M. Schindler T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; 275: 2721-2726Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), unphosphorylated c-Abl similar peptide kinase as wild-type c-Abl that mutation of tyrosine to phenylalanine not significantly the catalytic activity of c-Abl was to with and ATP 3 but the of phosphotyrosine was reduced in with wild-type and phosphorylation was by Autophosphorylation of c-Abl was by increased peptide kinase but the increase was very at a of activation the of ATP 3 kinase activity and for the very similar to the of autophosphorylation These results demonstrate that Tyr-412 is for of the of autophosphorylation on Abl catalytic activity and suggest as for phosphorylation of this activation tyrosine increases c-Abl kinase activity. the that c-Abl can and increased catalytic activity that autophosphorylation may was that tyrosine is not for the of by c-Abl M. Baltimore D. Oncogene. 1993; 8: Google Scholar). the of Tyr-412 autophosphorylation to the catalytic activity of we also purified the c-Abl double mutant in the unphosphorylated and its autophosphorylation and kinase activity to c-Abl The c-Abl mutant to about the as c-Abl P131L 3 the catalytic activity of the double mutant was stimulated to only about catalytic activity of c-Abl P131L 3 These results demonstrate that phosphorylation at tyrosine also stimulates the activity of but the of Tyr-412 phosphorylation to Abl catalytic activity is in the presence of SH3 these also suggest the presence of Abl that can in vitro kinase activity studies of tyrosine phosphorylation in the transforming protein S. J. PubMed Google Scholar). Although specific not of that site was and the site was COOH-terminal to in c-Abl tyrosine is from a site and of these phosphorylation not at proline are for the site in and The site is not phosphorylated c-Abl is in J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the site is of is in the SH2-CD linker region to of which the mutation to D. Superti-Furga G. 1998; PubMed Scopus Google Scholar) or B. and R. A. Van stimulates c-Abl kinase activity in vivo and induces cell the that may be a site of regulatory tyrosine phosphorylation in we tyrosine to phenylalanine and purified c-Abl in the unphosphorylated state. The c-Abl mutant also significantly phosphotyrosine than wild-type Abl that is phosphorylated in vivo these unphosphorylated c-Abl very similar to wild-type that this mutation does not significantly the intrinsic activity of c-Abl 3 c-Abl exhibited stimulation of kinase activity in to autophosphorylation. The catalytic activity of c-Abl was stimulated about in the presence of and ATP but increased only minimally the a activity of only about that of wild-type c-Abl The kinases similar levels of phosphotyrosine 3 but the of phosphotyrosine to Abl protein was higher for wild-type at also a c-Abl Y245F/Y412F double mutant and autophosphorylation and stimulation of catalytic activity with autophosphorylation 3 These results suggest that autophosphorylation of phosphorylation at Tyr-412 but an on c-Abl kinase and phosphorylation at Tyr-412 and for the of the regulatory of autophosphorylation on c-Abl catalytic activity. to the activation of the c-Abl we the c-Abl mutant kinase for the unphosphorylated protein very similar to the peptide kinase of c-Abl 3 (and not that the mutation does not have an on intrinsic activity of unphosphorylated Abl The kinase had only a in the of tyrosine phosphorylation to c-Abl P131L and the c-Abl double mutant 3 that Abl can phosphorylate in the of The peptide kinase activity of the mutant increased with and the rate and of activation only reduced with c-Abl These results suggest that phosphorylation at on the intrinsic catalytic activity of These studies the of c-Abl in solution and that the structural basis of regulation of Abl kinase activity significantly from that of Src family found that unphosphorylated c-Abl had substantial intrinsic catalytic to a peptide at to 33 pmol of contrast, purified Hck that was monophosphorylated at tyrosine phosphorylated peptide in a similar at about pmol of Hck (17Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar). Although we the that the c-Abl we have purified been activated by in vivo through The of purified c-Abl with not the kinase activity not that the Abl was not vivo through phosphorylation. the of of the Abl the of of an activator The substantial intrinsic catalytic activity of purified c-Abl be a of from a In support of this we have that protein can the peptide kinase activity of purified c-Abl autophosphorylation. B. B. and R. A. Van previous studies found the in vitro tyrosine kinase activity of wild-type and c-Abl to be similar in immune complex kinase P. Wang J.Y.J. EMBO J. 1989; 8: PubMed Scopus Google Scholar, Etten R.A. J. H. J.M. Oncogene. 1995; 10: Google Scholar) or as in solution B.J. Baltimore D. Mol. Cell. Biol. PubMed Scopus Google Scholar). In contrast, we found that an SH3 point mutation to a and increase in the intrinsic catalytic activity of unphosphorylated c-Abl that was to wild-type The this was not in studies is not autophosphorylation to the catalytic activity of wild-type and the increased concentration of Abl in an immune complex autophosphorylation of Abl and similar kinase activity measured at than autophosphorylation in vivo may for the similar catalytic activity of wild-type and c-Abl purified as B.J. Baltimore D. Mol. Cell. Biol. PubMed Scopus Google Scholar). A previous that the mutation of c-Abl in the SH2-CD linker or the of a between the SH3 and catalytic domains activated Abl kinase activity in D. Superti-Furga G. 1998; PubMed Scopus Google Scholar). suggested that the Abl SH3 domain with the SH2-CD linker region and the catalytic domain to Abl kinase similar to the mechanism for Src family results also support a model where the regulatory of the Abl SH3 domain is through an intramolecular SH3 is to in purified unphosphorylated be the of an Abl In Src proteins, the binding of phosphorylated Tyr-527 by SH2 the atypical SH3-linker whereas a in the SH2-CD linker S. F. M. Superti-Furga G. Biol. 1999; PubMed Scopus Google Scholar) of the of the catalytic domain by the SH3-linker Unphosphorylated c-Abl lacks and Abl a at the to Src a c-Src mutant increased catalytic activity of the SH2-CD linker by SH3 S. F. M. Superti-Furga G. Biol. 1999; PubMed Scopus Google Scholar). that purified Abl may several structural of Src that are for of catalytic activity. c-Abl may for this by binding an inhibitor in vivo to a inactive state. demonstrate that autophosphorylation of c-Abl is intermolecular and stimulates Abl catalytic activity. of Tyr-412 increases c-Abl kinase activity about an that is similar to the stimulation phosphorylation of the homologous tyrosine in Src kinases and in the The of the c-Abl catalytic domain with STI been T. W. P. Miller W.T. B. Kuriyan J. 2000; PubMed Scopus Google Scholar). In this structure, which the inactive Tyr-412 is as in a but a that is for is The of this activation tyrosine in Abl is to the of the L. Ellis L. Nature. PubMed Scopus Google Scholar) but from that found in the inactive Src kinases (11Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar, F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). is that phosphorylation of Tyr-412 the of the activation of that to an of active protein In Abl be activated by autophosphorylation or by phosphorylation of Tyr-412 by another tyrosine kinase, such as c-Src (5Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (373) Google Scholar). also found that phosphorylation of in c-Abl stimulates c-Abl kinase activity about the of phosphorylation of and Tyr-412 are not of activation is inhibited in the c-Abl mutant but only about in c-Abl a model where c-Abl be activated in vivo in a with phosphorylation at Tyr-412 by is that is from which is to the Abl SH3 is found in this in murine and c-Abl and but not in Src family Src a at this whereas Hck a and the (11Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar, F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar) of of SH3 2 by this is that phosphorylation of this tyrosine in Abl induces a or that the interaction of SH3 and the of SH3 mutation on the catalytic activity of c-Abl The Abl and peptide kinase be for of the regulation of c-Abl and its of for the of STI and Mayer of for and for the