Title: 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 (222) Google Scholar, 21Mayer B.J. Jackson P.K. Van Etten R.A. Baltimore D. Mol. Cell. Biol. 1992; 12: 609-618Crossref PubMed Scopus (238) Google Scholar). However, deletion of the SH3 domain (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus (222) Google Scholar, 22Franz W.M. Berger P. Wang J.Y.J. EMBO J. 1989; 8: 137-147Crossref PubMed Scopus (161) Google Scholar) and SH3 point mutations that block PXXP ligand binding (23Van Etten R.A. Debnath J. Zhou H. Casasnovas J.M. Oncogene. 1995; 10: 1977-1988PubMed Google Scholar) does stimulate Abl kinase activity in vivo as does the mutation of a proline residue (Pro-242) in the Abl SH2-CD linker region that is homologous to Src Pro-250 (24Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar). Whereas the SH2-CD linker mutation implies an intramolecular role for the SH3 domain in Abl regulation, immunoprecipitated c-Abl and SH3-mutated Abl have similar high levels of kinase activity in vitro (22Franz W.M. Berger P. Wang J.Y.J. EMBO J. 1989; 8: 137-147Crossref PubMed Scopus (161) Google Scholar, 23Van Etten R.A. Debnath J. Zhou H. Casasnovas J.M. Oncogene. 1995; 10: 1977-1988PubMed Google Scholar, 25Mayer B.J. Baltimore D. Mol. Cell. Biol. 1994; 14: 2883-2894Crossref PubMed Scopus (158) Google Scholar), suggesting that the regulatory function of the Abl SH3 domain is only apparent within the cell. An alternative model is that SH3 regulates Abl kinase activity through the binding of a cellular inhibitor (26Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar). The expression of c-Abl in cells at up to 10-fold over endogenous levels does not result in Abl autophosphorylation (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus (222) Google Scholar, 27Van Etten R.A. Jackson P. Baltimore D. Cell. 1989; 58: 669-678Abstract Full Text PDF PubMed Scopus (338) Google Scholar), but the expression at higher levels (20–50-fold) results in tyrosine phosphorylation of Abl and other cellular proteins (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar, 26Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar). Furthermore, c-Abl kinase activity is suppressed when expressed in Saccharomyces cerevisiae 2B. B. Brasher and R. A. Van Etten, unpublished observations. andXenopus oocytes (28Dorey K. Barila D. Gavin A.C. Nebreda A.R. Superti-Furga G. Biol. Chem. Hoppe-Seyler. 1999; 380: 223-230Crossref PubMed Google Scholar) but not in Schizosaccharomyces pombe (29Walkenhorst J. Goga A. Witte O.N. Superti-Furga G. Oncogene. 1996; 12: 1513-1520PubMed Google Scholar). These observations suggest that wild-type and SH3-mutated c-Abl have similar intrinsic kinase activity, but the SH3-mutated c-Abl can no longer associate with a cellular inhibitor. By inference, the putative inhibitor is abundant but can be titrated upon the overexpression of Abl, does not efficiently immunoprecipitate with Abl, and is absent in fission yeast. Several Abl SH3-binding proteins have been identified as candidates for such an inhibitor, including Pag/Msp23 (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar), AAP1 (30Zhu J. Shore S.K. Mol. Cell. Biol. 1996; 16: 7054-7062Crossref PubMed Scopus (37) Google Scholar), Abi-1 (31Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar), and Abi-2 (32Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar). Of these inhibitors, Pag/Msp23 has been shown to inhibit c-Abl kinase activity upon co-expression in vivo (7Wen S.-T. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar). To better understand the regulation of c-Abl tyrosine kinase activity, we purified c-Abl in a form that should correspond to its inactive state by using the specific Abl kinase inhibitor STI 571 to prevent activation and autophosphorylation upon overexpression in vivo. We found that unphosphorylated c-Abl had substantial intrinsic catalytic activity relative to inactive c-Src, and this basal activity was further stimulated by autophosphorylation at two distinct regulatory tyrosine residues. Surprisingly, we found that the mutation of the SH3 domain significantly increased the basal activity of c-Abl, supporting an intramolecular regulatory role for SH3. Together, these results suggest a model where c-Abl is activated in vivo by dissociation of an inhibitor followed by phosphorylation at tyrosine residues within the catalytic domain and the SH2-CD linker region. The murine type IV c-abl cDNA in the vector pcDNA3 (Invitrogen) was modified to include six histidine codons at the 3′ end. The alteration changed the C-terminal amino acid sequence from …DIVRR to …DIVRRMYPRGNGGGHHHHHH. Abl mutants were generated by inverse polymerase chain reaction and confirmed by DNA sequencing. Abl proteins were expressed by transient transfection of 293T cells as described previously (33DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (918) Google Scholar), except that medium was supplemented with 50 μm STI 571 (Novartis) where indicated. 48–60 h posttransfection, cells were collected and washed twice with phosphate-buffered saline supplemented with 5 mm EDTA, washed once with phosphate-buffered saline only, and then solubilized (1 ml/60-mm plate) in lysis buffer (0.5% Triton X-100, 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5% glycerol, 5 mm 2-mercaptoethanol, 0.1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 mmbenzamide, 0.7 μg/ml pepstatin, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and placed on ice for 15 min. Lysates were cleared by centrifugation at 13,000 × g for 20 min at 4 °C and then added to cobalt nitrilotriacetic acid-agarose (Talon resin,CLONTECH) at a ratio of 2 ml of lysate to 200 μl of (packed volume) agarose. Binding reactions were allowed to proceed at 4 °C for approximately 30 min with constant gentle shaking, and then the mixture was transferred to 5-ml disposable chromatography columns. Each column was washed with 1 ml of lysis buffer followed by 0.5 ml of wash buffer I (20 mm Tris, 10 mm imidazole, pH 8.0, 150 mm NaCl, 0.05% Brij35, 0.1 mm EGTA, and protease inhibitors), 0.5 ml of wash buffer II (same as wash buffer I but with 20 mmimidazole), and eluted with 0.5 ml of elution buffer (same as wash buffer I but with 100 mm imidazole). Eluted products were adjusted to 2 mm EDTA and 1 mm dithiothreitol and then dialyzed overnight against 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1 mm EDTA, 0.01% Brij35, and 1 mm dithiothreitol. During dialysis, 15 μl of agarose-linked anti-phosphotyrosine antibodies (Oncogene Science, Inc.) was included to remove residual phosphotyrosine-containing proteins. Dialysates were cleared for 30 min at 13,000 × g and then stored on ice for up to 5 days. The concentration of purified Abl proteins was determined by SDS-polyacrylamide gel electrophoresis analysis and Coomassie Blue staining compared with purified bovine serum albumin standards (Pierce). These were quantitated by densitometry using a digital camera and NIH Image software. The typical yield from two transfected plates was 12–25 μg of total full-length Abl protein at 25–50 ng/μl. Abl phosphorylation reactions were carried out at various kinase concentrations at 30 °C in kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 2 mm dithiothreitol, 1 mmEGTA, and 0.01% Brij35) and in 500 μm ATP. For timed autophosphorylation reactions, all additions other than ATP were mixed and preheated for at least 5 min before the addition of ATP. Autophosphorylation reactions were terminated by the addition of Laemmli sample buffer and boiling. Proteins were then separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and detected with anti-phosphotyrosine (4G10, UBI), anti-Abl (3F12, a gift of R. Salgia, Dana-Farber Cancer Institute), monoclonal antibodies, and enhanced chemiluminescence (Amersham Pharmacia Biotech). Blots were digitized by scanning, and relative phosphorylation levels were determined using NIH Image and Origin 5.0 (Microcal) software. Specific activity of unphosphorylated Abl kinases was determined using a peptide substrate containing the preferred Abl substrate sequence (34Songyang Z. Carraway III, K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantely L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (848) Google Scholar) and modified with an amino-terminal biotin, biotin-GGEAIYAAPFKK-amide. Kinase assays were carried out at 30 °C in kinase buffer plus 50 μm ATP, [γ-32P]ATP at 5000–7000 cpm/pmol, and peptide substrate. Assays were done in triplicate for each substrate concentration and were allowed to proceed for 5 min before termination by the addition of guanidine hydrochloride to 2.5 m final concentration. After termination, portions of each reaction were spotted onto streptavidin-coated paper discs (SignaTECT,Promega) and then sequentially washed with 2 m NaCl followed by 2 m NaCl with 1% phosphoric acid as suggested by the manufacturer. Phosphate incorporation was determined by liquid scintillation counting of the discs. Background binding to the discs was determined by omitting peptide substrate in a series of assays and was usually less than 0.03% total input counts. Incorporated counts for each kinase/substrate combination were averaged, adjusted for background, and plotted on a double-reciprocal (1/V versus 1/[S]) graph using the Origin 5.0 program to calculate K m and V max values. In most assays, the concentration of kinase was 0.01 μm, and specific activity was calculated as picomoles of phosphate incorporated per min per pmol of kinase. To determine the effects of autophosphorylation on kinase specific activity, the two assays detailed above were combined. Autophosphorylation reactions were carried out as described and typically contained unlabeled ATP at 500 μm and kinase at 0.04 μm final concentrations. At indicated times, aliquots of autophosphorylation reactions were withdrawn and terminated for SDS-polyacrylamide gel electrophoresis-Western blot analysis or added to prewarmed mixtures of kinase buffer, [γ-32P]ATP, and peptide substrate. Peptide phosphorylation reactions were done in duplicate or triplicate for 5 min as described above using a single concentration of peptide substrate (100 μm final concentration) to measure activity. Dilution of Abl kinases in the transfer from autophosphorylation reactions to peptide kinase reactions resulted in a final kinase concentration of 0.01 μm in the peptide kinase reactions. Full-length myristoylated (type IV) murine c-Abl proteins containing a C-terminal hexahistidine tag were expressed in 293T cells and purified in a single step by affinity chromatography on Co2+-agarose. c-Abl normally lacks detectable levels of tyrosine phosphorylation in vivo in its inactive state (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus (222) Google Scholar), but high level expression of Abl in mammalian or insect cells results in significant levels of Abl tyrosine phosphorylation (26Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar). To isolate unphosphorylated c-Abl, transfected cells were grown in the presence of Novartis STI 571, an Abl-specific kinase inhibitor (35Buchdunger E. Zimmermann J. Mett H. Meyer T. Müller M. Druker B.J. Lydon N.B. Cancer Res. 1996; 56: 100-104PubMed Google Scholar), and any residual tyrosine-phosphorylated proteins were removed from purified kinase preparations with agarose-conjugated anti-phosphotyrosine antibodies. c-Abl was the predominant polypeptide in the final preparation by Coomassie Blue staining (Fig.1 A) and contained no detectable phosphotyrosine by immunoblotting with anti-phosphotyrosine antibody (Fig. 1 B). Peptide phosphorylation by the purified wild-type c-Abl was completely STI 571-inhibitable with an IC50 of 0.4 μm (Fig. 1 C), demonstrating that c-Abl was the only detectable tyrosine kinase present in the preparation. To study the in vitrocatalytic activity of purified c-Abl, we employed a sensitive kinase assay using a biotinylated peptide with a sequence preferred by Abl kinases (34Songyang Z. Carraway III, K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantely L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (848) Google Scholar). Unphosphorylated wild-type c-Abl demonstrated substantial activity toward the peptide substrate with an averageV max of 33 pmol of phosphate min−1and K m of 204 μm (Fig. 1 D). Deletions (20Jackson P. Baltimore D. EMBO J. 1989; 8: 449-456Crossref PubMed Scopus (222) Google Scholar, 22Franz W.M. Berger P. Wang J.Y.J. EMBO J. 1989; 8: 137-147Crossref PubMed Scopus (161) Google Scholar) and some point mutations (23Van Etten R.A. Debnath J. Zhou H. Casasnovas J.M. Oncogene. 1995; 10: 1977-1988PubMed Google Scholar) in the c-Abl SH3 domain dysregulate Abl kinase activity in vivo, inducing high levels of tyrosine phosphorylation of Abl and many other proteins and usually causing cellular transformation. However, the in vitro kinase activities of wild-type and SH3-mutated c-Abl are similar when measured after immunoprecipitation (22Franz W.M. Berger P. Wang J.Y.J. EMBO J. 1989; 8: 137-147Crossref PubMed Scopus (161) Google Scholar, 23Van Etten R.A. Debnath J. Zhou H. Casasnovas J.M. Oncogene. 1995; 10: 1977-1988PubMed Google Scholar). In contrast, we found that a transforming c-Abl protein containing a point mutation in the SH3 domain (P131L) that disrupts the binding of proline-rich ligands (23Van Etten R.A. Debnath J. Zhou H. Casasnovas J.M. Oncogene. 1995; 10: 1977-1988PubMed Google Scholar) exhibited significantly higher catalytic activity than did c-Abl when measured in solution with the peptide substrate. Unphosphorylated c-Abl P131L protein had aV max approximately 3.5 times higher (112 pmol min−1) and a reduced K m (91 μm) relative to wild-type c-Abl (Fig.1 D). A Co2+-agarose affinity-purified preparation from non-transfected cells had only background levels of activity, again confirming that Abl was the sole kinase activity measured in our assay (data not shown). Therefore, the mutation or deletion of the SH3 domain appears to significantly increase the intrinsic tyrosine kinase activity of unphosphorylated c-Abl. We have also used a physiological protein substrate of c-Abl, GST-Crk (36Feller S.M. Knudsen B. Hanafusa H. EMBO J. 1994; 13: 2341-2351Crossref PubMed Scopus (326) Google Scholar), as a substrate in this assay and observed similar results (data not shown). However, Crk and many other polypeptide substrates of Abl can stably bind to c-Abl and may perturb Abl kinase activity. For this reason, the peptide substrate was employed exclusively in this report. Autophosphorylation leads to increased catalytic activity for many kinases including c-Src (37Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (137) Google Scholar), and we tested whether the same was true for c-Abl. Wild-type c-Abl and the P131L mutant were allowed to autophosphorylate, and intrinsic kinase activity was measured with the peptide assay. Both c-Abl and SH3-mutated c-Abl were rapidly tyrosine-phosphorylated when incubated in the presence of magnesium ion and ATP (Fig.2 A). Autophosphorylation of c-Src and Hck is concentration-dependent, suggesting an intermolecular reaction 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, 37Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (137) Google Scholar, 38Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (159) Google Scholar). When the Abl concentration was increased in the autophosphorylation reactions, the minimum time required to detect Abl phosphotyrosine decreased, and the rate of phosphotyrosine accumulation after initial detection increased (Fig. 2 B). 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 event. As autophosphorylation progressed, an increased ability of c-Abl to phosphorylate the peptide substrate was observed (Fig.2 C). Although there was some variability in autophosphorylation-induced activation of c-Abl peptide kinase activity among different Abl preparations, increased peptide kinase activity closely matched the increase in Abl phosphotyrosine content in each experiment. c-Abl catalytic activity toward the peptide substrate continued to increase to as high as 22-fold over the basal level after 60 min of autophosphorylation (Fig. 2 D). The kinase activity of the SH3-mutated c-Abl was also stimulated by autophosphorylation (Fig. 2 C), demonstrating that the SH3 mutation and autophosphorylation act independently to increase the catalytic activity of c-Abl. The activation of c-Abl P131L proceeded somewhat more rapidly than wild-type c-Abl, reaching a maximum after 10 min; however, the increase in activity of c-Abl P131L was only about 3.6-fold (Fig. 2 D). The final activity of tyrosine-phosphorylated wild-type and SH3-mutated c-Abl was very similar (Fig. 2 C) and equal to or greater than the specific activity of c-Abl that was purified from cells without treatment with STI 571 (data not shown). These results suggest that c-Abl is capable of maximal activation under these conditions and that autophosphorylation of c-Abl ultimately overcomes the intrinsic inhibitory effect of the SH3 domain. Tyrosine 412 within the catalytic lobe of the c-Abl kinase domain is homologous to c-Src tyrosine 416, and autophosphorylation at tyrosine 416 has been shown to stimulate the kinase activity of Src kinases (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, 37Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (137) Google Scholar). Tyrosine 412 is known to be a major site of tyrosine phosphorylation in transforming Abl proteins (39Reynolds F.H. Oroszlan S. Stephenson J.R. J. Virol. 1982; 44: 1097-1101Crossref PubMed Google Scholar). A c-Abl Y412F mutant accumulated little phosphotyrosine upon high level expression in 293T cells in the absence of STI 571 (Fig.3 A), confirming that Tyr-412 is a major in vivo tyrosine phosphorylation site of c-Abl. Whereas mutation of the Tyr-416 homologue in Hck to alanine partially activates kinase activity (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), unphosphorylated c-Abl Y412F displayed similar peptide kinase kinetics as wild-type c-Abl (Fig.3 B), demonstrating that mutation of tyrosine 412 to phenylalanine did not significantly alter the basal catalytic activity of Abl. c-Abl Y412F was able to autophosphorylate upon incubation with magnesium and ATP (Fig. 3 C), but the amount of phosphotyrosine was reduced in comparison with wild-type c-Abl, and phosphorylation was maximal by 20 min. Autophosphorylation of c-Abl Y412F was accompanied by increased peptide kinase activity, but the increase was very modest, peaking at a maximum of 4-fold activation 5 min after the addition of ATP (Fig. 3 D). Peptide kinase activity then dropped slightly and remained steady for the next 60 min, very similar to the kinetics of autophosphorylation (Fig.3 C). These results demonstrate that Tyr-412 is required for most of the stimulatory effect of autophosphorylation on Abl catalytic activity and suggest that, as for c-Src, phosphorylation of this activation