Interaction of Hematopoietic Progenitor Kinase 1 and c-Abl Tyrosine Kinase in Response to Genotoxic Stress
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m007294200
ISSN1083-351X
AutoresYasumasa Ito, Pramod S. Pandey, Pradeep Sathyanarayana, Pin Ling, Ajay Rana, Ralph R. Weichselbaum, Tse‐Hua Tan, Donald Küfe, Surender Kharbanda,
Tópico(s)Quinazolinone synthesis and applications
ResumoThe c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The hematopoietic progenitor kinase 1 (HPK1) has also been shown to act upstream to the SAPK/JNK signaling pathway. We report here that exposure of hematopoietic Jurkat T cells to genotoxic agents is associated with activation of HPK1. The results demonstrate that exposure of Jurkat cells to DNA-damaging agents is associated with translocation of active c-Abl from nuclei to cytoplasm and binding of c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks c-Abl-mediated induction of SAPK/JNK. These findings indicate that activation of HPK1 and formation of HPK1/c-Abl complexes are functionally important in the stress response of hematopoietic cells to genotoxic agents. The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The hematopoietic progenitor kinase 1 (HPK1) has also been shown to act upstream to the SAPK/JNK signaling pathway. We report here that exposure of hematopoietic Jurkat T cells to genotoxic agents is associated with activation of HPK1. The results demonstrate that exposure of Jurkat cells to DNA-damaging agents is associated with translocation of active c-Abl from nuclei to cytoplasm and binding of c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks c-Abl-mediated induction of SAPK/JNK. These findings indicate that activation of HPK1 and formation of HPK1/c-Abl complexes are functionally important in the stress response of hematopoietic cells to genotoxic agents. ionizing radiation stress-activated protein kinase/c-Jun N-terminal kinase hematopoietic progenitor kinase mitogen-activated protein kinase kinase MAPKK/extracellular signal-regulated kinase kinase kinase mixed lineage kinase 1-β-d-arabinofuranosylcytosine polyacrylamide gel electrophoresis glutathione S-transferase proline-rich sequence myelin basic protein amino acid(s) SAP kinase/extracellular signal-regulated kinase kinase1 hemagglutinin The cellular response to ionizing radiation (IR)1 and other genotoxic agents includes cell cycle arrest, activation of DNA repair, and apoptosis or programmed cell death. However, the intracellular signals that control these events are largely unclear. The available evidence supports a role for the c-Abl protein tyrosine kinase in the induction of apoptosis (1Kharbanda S. Yuan Z. Weichselbaum R. Kufe D. Oncogene. 1998; 17: 3309-3318Crossref PubMed Scopus (153) Google Scholar, 2Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar). Transient transfection studies with wild-type but not kinase-inactive c-Abl have demonstrated induction of an apoptotic response (2Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar). Also, cells that stably express the dominant negative c-Abl(K-R) mutant exhibit resistance to induction of apoptosis by IR and other DNA-damaging agents (2Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar, 3Huang Y. Yuan Z.M. Ishiko T. Nakada S. Utsugisawa T. Kato T. Kharbanda S. Kufe D.W. Oncogene. 1997; 15: 1947-1952Crossref PubMed Scopus (40) Google Scholar). Similar results have been obtained in Abl−/− fibroblasts (2Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar, 3Huang Y. Yuan Z.M. Ishiko T. Nakada S. Utsugisawa T. Kato T. Kharbanda S. Kufe D.W. Oncogene. 1997; 15: 1947-1952Crossref PubMed Scopus (40) Google Scholar). The apoptosis-resistant phenotype is more pronounced in cells expressing c-Abl(K-R) than in c-Abl null cells. In addition, a proapoptotic role for c-Abl is supported by c-Abl-dependent induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the response to genotoxic stress (4Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar, 5Kharbanda S. Pandey P. Ren R. Feller S. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (460) Google Scholar). The SAPK/JNK signaling cascade plays a critical role in the responses stimulated by DNA damage, heat shock, interleukin 1, tumor necrosis factor α, and Fas (4–15). SAPK is phosphorylated and activated by immediate upstream mitogen-activated protein kinase kinases (MAPKKs), MAPKK4 (MKK4)/SEK1 (8Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1411) Google Scholar, 16Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar) and MKK7 (17Tournier C. Hess P. Yang D. Xu J. Turner T. Nimmual A. Bar-Sagi D. Jones S. Flavell R. Davis R. Science. 2000; 288: 870-874Crossref PubMed Scopus (1541) Google Scholar). These MAPKKs are activated, in turn, by the upstream MAPKK kinases including MAPKK/extracellular signal-regulated kinase kinase kinase 1 (MEKK-1) (18Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar), mixed lineage kinase 3 (MLK-3) (19Teramoto H. Coso O. Miyata H. Igishi T. Miki T. Gutkind J. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), transforming growth factor β-activated kinase 1 (TAK1) (20Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1175) Google Scholar), tumor progression locus 2 (Tpl-2) (16Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar), mitogen-activated protein kinase upstream kinase (21Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar), and apoptosis signal-regulating kinase 1 (22Ichijo H. Nishida E. Irie K. ten Dijke P. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2022) Google Scholar). Furthermore, several Ste20-related protein kinases that activate SAPK through MAPKK kinases have been identified as MAPKK kinase kinases, including hematopoietic progenitor kinase 1 (HPK1) (23Hu M. Qiu W. Wang X. Meyer C. Tan T. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar, 24Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (199) Google Scholar), germinal center kinase (25Katz P. Whalen G. Kehrl J.H. J. Biol. Chem. 1994; 269: 16802-16809Abstract Full Text PDF PubMed Google Scholar, 26Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar), HPK1/germinal center kinase-like kinase/Nck-interacting kinase (27Su Y. Han J. Xu S. Cobb M. Skolnik E. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (218) Google Scholar,28Yao Z. Zhou G. Wang X. Brown A. Diener K. Gan H. Tan T. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), germinal center kinase-like kinase (29Diener K. Wang X. Chen C. Meyer C. Keesler G. Zukowski M. Tan T. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (117) Google Scholar), and kinase homologous to Ste20/Sps1/germinal center kinase-related kinase (30Shi C. Kehrl J. J. Biol. Chem. 1997; 272: 32102-32107Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 31Tung R. Blenis J. Oncogene. 1997; 14: 653-659Crossref PubMed Scopus (66) Google Scholar). HPK1, a 97-kDa serine/threonine kinase, is restricted to hematopoietic tissues in adults (23Hu M. Qiu W. Wang X. Meyer C. Tan T. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar, 24Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (199) Google Scholar). Studies have shown that HPK1 interacts with MEKK-1 (23Hu M. Qiu W. Wang X. Meyer C. Tan T. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar), MLK-3 (24Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (199) Google Scholar), and TAK1 (32Wang W. Zhou G. Hu M. Yao Z. Tan T. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), which, in turn, can activate MKK4/SEK1 and thereby result in activation of the SAPK signaling pathway. Previous studies have demonstrated that four proline-rich motifs in HPK1 are potential binding sites for SH3 domain-containing proteins. HPK1 interacts with the SH2/SH3 domain-containing adaptor proteins Crk and CrkL (33Ling P. Yao Z. Meyer C. Wang X. Oehri W. Feller S. Tan T. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar). Using yeast two-hybrid analysis, HPK1 has also been shown to associate with the c-Abl SH3 domain (24Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (199) Google Scholar). The demonstration that Abl−/− cells exhibit a defective SAPK response in response to certain DNA-damaging agents has provided support for c-Abl as an upstream effector in the SAPK pathway (5Kharbanda S. Pandey P. Ren R. Feller S. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (460) Google Scholar) and has raised the possibility of a functional interaction between c-Abl and HPK1. The present studies demonstrate that exposure of Jurkat cells to IR is associated with activation of HPK1. Similar results were obtained with another genotoxic agent, 1-β-d- arabinofuranosylcytosine (ara-C). The results also demonstrate that activated HPK1 forms a complex with cytoplasmic c-Abl in the cellular response to genotoxic agents. The functional significance of the c-Abl/HPK1 interaction is supported by the finding that HPK-1-induced activation of SAPK is inhibited by a dominant negative c-Abl and that kinase-inactive mutants of HPK1 block c-Abl-mediated induction of SAPK activity. Human Jurkat T cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum and antibiotics. MCF-7/neo and MCF-7/c-Abl(K-R) (34Yuan Z.M. Huang Y. Whang Y. Sawyers C. Weichselbaum R. Kharbanda S. Kufe D. Nature. 1996; 382: 272-274Crossref PubMed Scopus (210) Google Scholar) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, antibiotics, and 500 μg/ml Geneticin sulfate (Life Technologies, Inc.). Cells were seeded at a density of 1 × 106 cells/100-mm culture dish for 24 h before treatment with 20 Gy of IR or 10 μmara-C (Sigma). Irradiation was performed at room temperature with a γ-ray source (Cs173; Gamma Cell 1000; Atomic Energy of Canada, Ontario, Canada) at a fixed dose rate of 0.76 Gy/min. Cytosolic fractions were prepared as described previously (35Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (369) Google Scholar). Cells were washed twice with phosphate-buffered saline and then suspended in ice-cold buffer (20 mm HEPES, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin and aprotinin) containing 250 mm sucrose. The cells were disrupted by five strokes in a Dounce homogenizer. After centrifugation of the lysate at 10,000 × g for 5 min at 4 °C, the supernatant fraction was centrifuged at 105,000 × g for 30 min at 4 °C. The resulting supernatant was used as the soluble cytosolic fraction. Nuclear proteins were isolated as described previously (36Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar). In brief, cells were washed three times with phosphate-buffered saline and resuspended in 4 cell volumes of hypotonic lysis buffer (10 mm HEPES, pH 7.5, 2 mm MgCl2, 10 mm KCl, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). After incubation on ice for 30 min to allow swelling, the cells were disrupted in a Dounce homogenizer (15–20 strokes). The homogenate was layered on a cushion of 1m sucrose in hypotonic solution and subjected to centrifugation for 10 min. The nuclei were then suspended in lysis buffer containing 0.5% Nonidet P-40. After incubation at 4 °C for 30 min, the suspension was centrifuged at 12,000 × g,and the supernatant was used as the nuclear fraction. Total cell lysates were prepared as described in lysis buffer containing 1% Nonidet P-40 (37Kumar S. Avraham S. Bharti A. Goyal J. Pandey P. Kharbanda S. J. Biol. Chem. 1999; 274: 30657-30663Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Equal amounts of total, cytosolic, or nuclear proteins were subjected to immunoprecipitation with anti-c-Abl (K-12; Santa Cruz Biotechnology) or anti-HPK1 (32Wang W. Zhou G. Hu M. Yao Z. Tan T. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Immune complexes were recovered by incubation with protein A-Sepharose for 1 h at 4 °C, washed three times with lysis buffer, separated by SDS-PAGE, and then transferred to nitrocellulose filters. After blocking with 5% dried milk in phosphate-buffered saline-Tween, the filters were incubated with anti-HPK1, anti-c-Abl (Ab-3; Oncogene Research Products), anti-Flag M2 (Sigma), anti-P-Tyr (4G10; Upstate Biotechnology), anti-Lamin A (Santa Cruz Biotechnology), anti-β-actin (Santa Cruz Biotechnology), or anti-SAPK (Santa Cruz Biotechnology). The filters were analyzed by ECL (Amersham Pharmacia Biotech). The pSRα-c-Abl wild-type and pSRα-c-Abl(K-290R) have been described previously (6Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (460) Google Scholar, 34Yuan Z.M. Huang Y. Whang Y. Sawyers C. Weichselbaum R. Kharbanda S. Kufe D. Nature. 1996; 382: 272-274Crossref PubMed Scopus (210) Google Scholar). HA-c-Abl was provided by Dr. Jean Y. J. Wang (University of California, San Diego, CA); GST-Jun(1–102) as described (38Saleem A. Yuan Z.-M. Kufe D.W. Kharbanda S.M. J. Immunol. 1995; 154: 4150-4156PubMed Google Scholar); pEBG-SAPK, pEBG-SEK1 as described (6Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (460) Google Scholar); pCIneo-Flag-HPK1 wild-type, pCIneo-Flag-HPK1(M46), GST-HPK1KD, GST-HPK1CD as described (23Hu M. Qiu W. Wang X. Meyer C. Tan T. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar). The plasmid GST-Crk(120–225) was provided by Dr. Stephan Feller (Bavarian Julius-Maximilians University, Wurzburg Germany). The peptides PR1 (H2N-PELPPAIPRR-COOH), PR2 (H2N-PPPLPPKPK-COOH), PR3 (H2N-PPPNSPRPGPPP-COOH), and PR4 (H2N-KPPLLPPKKE-COOH) were prepared as described previously (33Ling P. Yao Z. Meyer C. Wang X. Oehri W. Feller S. Tan T. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar). GST and GST-Abl SH3 (39Ren R. Ye Z. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (290) Google Scholar) were purified by affinity chromatography using glutathione-Sepharose beads and equilibrated in lysis buffer. Cell lysates were incubated with 5 μg of immobilized GST or GST-c-Abl SH3 for 2 h at 4 °C. The resulting protein complexes were washed three times with lysis buffer and boiled for 5 min in SDS sample buffer. The complexes were then separated by SDS-PAGE and subjected to immunoblot analysis with anti-HPK1. GST-c-Abl SH3 fusion protein was incubated with PR2 (33Ling P. Yao Z. Meyer C. Wang X. Oehri W. Feller S. Tan T. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar), PR3, or PR4. The fusion protein-peptide mixtures were incubated separately with cell lysates for 30 min at room temperature. After washing, bound proteins were analyzed by immunoblotting. Cell lysates were subjected to immunoprecipitation with anti-HPK1 or anti-c-Abl as described previously (35Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (369) Google Scholar). The protein complexes were washed and incubated in kinase buffer (20 mm HEPES, pH 7.4, and 10 mmMgCl2) containing 2.5 μCi of [γ-32P]ATP and either GST-Crk(120–225) (40Feller S. Ren R. Hanafusa H. Baltimore D. Trends Biochem. Sci. 1994; 19: 453-458Abstract Full Text PDF PubMed Scopus (182) Google Scholar) or myelin basic protein (MBP; Sigma) as substrates for 15 min at 30 °C. The reaction products were analyzed by SDS-PAGE and autoradiography. 293T cells were transfected with pEBG-SAPK, pEBG-SEK-1, Flag-HPK1, Flag-HPK1(M46), and c-Abl or c-Abl(K-R). After 12 h of incubation at 37 °C, the medium was replaced, and the cells were incubated for another 24 h. Cell lysates were prepared as described, and 200–250 μg of soluble proteins were incubated with 5 μg of immobilized GST for 30 min at 4 °C. The protein complexes were washed with lysis buffer and then incubated in kinase buffer containing [γ-32P]ATP and GST-c-Jun(2–100) (38Saleem A. Yuan Z.-M. Kufe D.W. Kharbanda S.M. J. Immunol. 1995; 154: 4150-4156PubMed Google Scholar) for 15 min at 30 °C. Reactions were terminated by the addition of SDS-PAGE sample buffer and boiling. Phosphorylated proteins were resolved by SDS-PAGE and analyzed by autoradiography. Cell lysates were also subjected to immunoblotting with anti-GST (Santa Cruz Biotechnology). 293T cells were cotransfected by the calcium phosphate method with HA-c-Abl and Flag-HPK1. After incubation for 36 h, the cells were lysed in lysis buffer containing 1% Nonidet P-40 and then subjected to immunoprecipitation with anti-HA (Boehringer Mannheim), and the immunoprecipitates were analyzed by immunoblotting with anti-Flag. 293T cells were also transiently transfected by the calcium phosphate method with Flag-HPK1 or Flag-HPK1(M46) in the presence of c-Abl or c-Abl(K-R). After incubation for 36 h, the cells were lysed in lysis buffer containing 1% Nonidet P-40 and then subjected to HPK1 kinase assay or immunoblot analysis with anti-Flag. MCF-7/neo or MCF-7/c-Abl(K-R) cells were transiently transfected with Flag-HPK1 by LipofectAMINE (Life Technologies, Inc.). Total cell lysates were subjected to immunoprecipitation with anti-Flag and then subjected to immunoblot analysis with anti-P-Tyr. Autoradiograms were scanned by laser densitometry, and the intensity of the signals was quantitated with the ImageQuant program (Molecular Dynamics, Sunnyvale, CA). Recombinant c-Abl protein was incubated with GST-HPK1-KD or GST-HPK1-CD fusion proteins in the presence of [γ-32P]ATP for 30 min at 30 °C. Phosphorylation of the reaction products was assessed by SDS-PAGE and autoradiography. 293T cells were transiently transfected with pCIneo-Flag-HPK1. Cell lysates were subjected to immunoprecipitation with anti-Flag, and the precipitates were incubated with recombinant purified c-Abl or kinase-inactive c-Abl(K-R) in the presence of [γ-32P]ATP for 30 min at 30 °C. Phosphorylation of the reaction products was assessed by SDS-PAGE and autoradiography. To assess whether c-Abl and HPK1 associate in cells, lysates from human Jurkat T cells were subjected to immunoprecipitation with anti-HPK1, and the protein precipitates were analyzed by immunoblotting with anti-c-Abl. Immunoblot analysis of precipitates using a control antibody or preimmune rabbit serum demonstrated little, if any, detection of c-Abl (Fig.1 a; data not shown). However, a similar analysis of anti-HPK1 immunoprecipitates demonstrated the coprecipitation of HPK1 and c-Abl (Fig. 1 a). To assess interactions between c-Abl and HPK1 in response to genotoxic agents, Jurkat cells were treated with 10 μm ara-C and harvested at 3 h. Analysis of anti-HPK1 immunoprecipitates by immunoblotting with anti-c-Abl demonstrated induction of HPK1-c-Abl complexes (Fig.1 b). Whereas ara-C incorporates into DNA and inhibits DNA replication (41Major P.P. Egan E.M. Herrick D. Kufe D.W. Biochem. Pharmacol. 1982; 31: 2937-2941Crossref PubMed Scopus (129) Google Scholar), IR induces single- and double-strand DNA breaks. The finding that exposure of Jurkat cells to IR is also associated with increased binding of c-Abl and HPK1 indicated that this response is induced by diverse types of genotoxic stress (Fig. 1 b). To further determine the interaction of c-Abl with HPK1, we transiently overexpressed Flag-HPK1 with HA-c-Abl in 293T cells and analyzed anti-HA immunoprecipitates with anti-Flag. Reactivity of anti-Flag with a 100-kDa protein supported the coprecipitation of HPK1 with c-Abl (Fig. 2 a). In the reciprocal experiment, anti-Flag immunoprecipitates were subjected to immunoblot analysis with anti-HA. The results confirmed the detection of complexes containing HPK1 and c-Abl (data not shown). Lysates from transfected 293T cells were also subjected to immunoprecipitation with anti-HA. Analysis of protein precipitates by immunoblotting with anti-HA demonstrated equal levels of c-Abl (Fig. 2 a). Taken together, these findings indicate that c-Abl associates with HPK1 in cells. To assess whether interaction between c-Abl and HPK1 is induced by genotoxic stress under conditions overexpressing c-Abl and HPK1, 36 h after the transfection with Flag-HPK1 and HA-c-Abl, cells were treated with 10 μm ara-C for 3 h. Analysis of anti-HA immunoprecipitates by immunoblotting with anti-Flag demonstrated a significant induction of HPK1-c-Abl complex in response to ara-C (Fig. 2 b). Four proline-rich sequences (PR1–PR4) are present in the C-terminal region of HPK1 (33Ling P. Yao Z. Meyer C. Wang X. Oehri W. Feller S. Tan T. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar). One of these proline-rich sequences (SGPPPNSPRPGPPPS; aa 430–444) displays homology with motifs located in the C-terminal domains of 3BP1, 3BP2, and ST5 that bind c-Abl SH3. To determine whether the c-Abl SH3 domain binds to HPK1, lysates from irradiated Jurkat cells were incubated with GST or GST-c-Abl SH3, and the resulting precipitates were analyzed by immunoblotting with anti-HPK1. The results demonstrate that in contrast to GST, HPK1 was detectable in the adsorbates to GST-c-Abl SH3 (Fig.2 c). Because the HPK1 proline-rich motif PR3 (but not PR1, PR2, or PR4) matches the c-Abl SH3-binding consensus motif (PXXXXPXPP), we examined the ability of HPK1 proline-rich peptides to block the formation of c-Abl/HPK1 complexes. The results demonstrate that the PR3 proline-rich peptide efficiently blocks the interaction of HPK1 with c-Abl, whereas PR2 and PR4 had at best a marginal effect on c-Abl/HPK1 complex (Fig. 2 d). These findings collectively indicate that the interaction between HPK1 and c-Abl likely involves c-Abl-SH3 and HPK1-Pro. It is possible that the coprecipitation of HPK1 and c-Abl is due to interactions of each kinase with other molecules. HPK1 is localized primarily in the cytoplasm (23Hu M. Qiu W. Wang X. Meyer C. Tan T. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (194) Google Scholar). To define the subcellular localization of the interaction between c-Abl and HPK1, we subjected nuclear and cytoplasmic lysates from control and ara-C-treated cells to immunoprecipitation with anti-HPK1. The immunoprecipitates were then analyzed by immunoblotting with anti-c-Abl. Signal intensities from the anti-c-Abl immunoblotting experiments (n = 3) were analyzed by densitometric scanning. Immunoblot analysis of the immunoprecipitates from control and ara-C-treated cells demonstrated little if any reactivity with anti-c-Abl in the nuclear fraction (Fig.3 a). Formation of HPK1/c-Abl complexes was significantly increased in the cytoplasm but not in the nucleus of ara-C-treated cells (Fig. 3 a). Studies have shown that although c-Abl contains three nuclear localization signals, it is not localized exclusively to the nucleus (42Van Etten R.A. Jackson P.K. Baltimore D. Sanders M.C. Matsuddaira P.T. Janmey P.A. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar, 43Wen S.-T. Jackson P.K. Van Etten R.A. EMBO J. 1996; 15: 1583-1595Crossref PubMed Scopus (185) Google Scholar). c-Abl contains a functional nuclear export signal, and the subcellular localization of c-Abl is determined by a balance of nuclear import and export (44Taagepera S. McDonald D. Loeb J. Whitaker L. McElroy A. Wang J. Hope T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7457-7462Crossref PubMed Scopus (264) Google Scholar). To assess whether c-Abl translocates to the cytoplasm in response to genotoxic stress, Jurkat cells were treated with ara-C for different intervals of time. Nuclear and cytoplasmic fractions were isolated and analyzed by immunoblotting with anti-c-Abl. As controls, nuclear and cytoplasmic fractions were also analyzed by immunoblotting with anti-Lamin A and anti-β-actin, respectively. The results demonstrate that treatment of Jurkat cells with ara-C was associated with significant decreases in nuclear c-Abl levels (Fig. 3 b, left panel). Moreover, levels of cytoplasmic c-Abl were increased in response to ara-C (Fig. 3 b, right panel). Translocation of c-Abl from nucleus in the response to genotoxic stress may initiate formation of complexes with multiple molecules in cytoplasm. Indeed, densitometric scanning of autoradiograms and quantitative analysis demonstrate that the formation of c-Abl complexes with HPK1 in cytoplasm is significantly less than the translocation of c-Abl from the nucleus to the cytoplasm. These findings support a model in which c-Abl is activated in the nucleus in response to genotoxic stress, translocates to the cytoplasm, and thereby interacts with HPK1. To determine whether the kinase function of HPK1 is necessary for the interaction with c-Abl, we transiently cotransfected Flag-HPK1 or a kinase-inactive Flag-HPK1(M46) mutant with c-Abl in 293T cells. After treatment with ara-C, anti-c-Abl immunoprecipitates were analyzed by immunoblotting with anti-Flag. The results demonstrate that in contrast to HPK1(M46), overexpression of wild-type HPK1 is associated with an increase in binding with c-Abl (Fig. 3 c). Because c-Abl is also activated by ara-C and IR (11Kharbanda S. Saleem A. Shafman T. Emoto Y. Weichselbaum R. Woodgett J. Avruch J. Kyriakis J. Kufe D. J. Biol. Chem. 1995; 270: 18871-18874Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we asked whether the association of HPK1 with c-Abl is also dependent on the c-Abl kinase function. To address this issue, 293T cells were transiently transfected with Flag-HPK1 and c-Abl or dominant negative c-Abl(K-R) mutant and then treated with ara-C. Anti-c-Abl immunoprecipitates were analyzed by immunoblotting with anti-Flag. The results demonstrate that the interaction between HPK1 and c-Abl is significantly increased in cells overexpressing wild-type c-Abl (Fig. 3 d). Taken together, these findings suggest that the kinase function of c-Abl and HPK1 may be necessary for their interaction. However, our data do not rule out the possibility that the loss of interaction between c-Abl and HPK1 might also be due to improper folding of these mutants. Additional studies using purified recombinant c-Abl and HPK1 proteins are required to delineate this issue. To assess in part the functional significance of the interaction of c-Abl and HPK1, we incubated purified recombinant c-Abl with GST-HPK1-KD (HPK1 kinase domain; aa 1–291) or GST-HPK1-CD (C-terminal domain; aa 292–833) (Fig. 4 a) fusion proteins in the presence of [γ-32P]ATP. Analysis of the re
Referência(s)