Linkage of Caspase-mediated Degradation of Paxillin to Apoptosis in Ba/F3 Murine Pro-B Lymphocytes
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m111639200
ISSN1083-351X
AutoresKee-Oh Chay, Sung Sup Park, J. Frederic Mushinski,
Tópico(s)Cell death mechanisms and regulation
ResumoWe have cloned the complete cDNA from mouse paxillin, a 68-kDa adapter protein found in focal adhesions. We found that paxillin was degraded by caspases in Ba/F3 cell apoptosis induced by withdrawal of interleukin-3 (IL-3), a survival factor for this cell, and by ionizing radiation. Also, paxillin was degraded in vitro by incubation with recombinant caspase-3. Western blot analyses of degradation products of overexpressed green fluorescence protein-tagged paxillin and site-specific mutants demonstrated that Asp-102 and Asp-301 were early caspase cleavage sites, and Asp-5, Asp-146, Asp-165, and Asp-222 were late cleavage sites. Overexpression of paxillin delayed apoptosis of Ba/F3 after IL-3 withdrawal. Furthermore, this anti-apoptotic effect of paxillin was augmented by a triple mutation in aspartic acids at caspase cleavage sites. These results suggest that paxillin plays a critical role in cell survival signaling and that the cleavage of paxillin by caspases might be an important event for focal adhesion disassembly during cell apoptosis, contributing to detachment, rounding, and death. We have cloned the complete cDNA from mouse paxillin, a 68-kDa adapter protein found in focal adhesions. We found that paxillin was degraded by caspases in Ba/F3 cell apoptosis induced by withdrawal of interleukin-3 (IL-3), a survival factor for this cell, and by ionizing radiation. Also, paxillin was degraded in vitro by incubation with recombinant caspase-3. Western blot analyses of degradation products of overexpressed green fluorescence protein-tagged paxillin and site-specific mutants demonstrated that Asp-102 and Asp-301 were early caspase cleavage sites, and Asp-5, Asp-146, Asp-165, and Asp-222 were late cleavage sites. Overexpression of paxillin delayed apoptosis of Ba/F3 after IL-3 withdrawal. Furthermore, this anti-apoptotic effect of paxillin was augmented by a triple mutation in aspartic acids at caspase cleavage sites. These results suggest that paxillin plays a critical role in cell survival signaling and that the cleavage of paxillin by caspases might be an important event for focal adhesion disassembly during cell apoptosis, contributing to detachment, rounding, and death. When cells adhere to the extracellular matrix, integrin receptors initiate signals to cluster more integrins together and to recruit cytoskeleton proteins (such as talin, tensin, vinculin, zyxin, and α-actinin), adapters (such as paxillin, Crk-associate substrate (p130CAS) 1The abbreviations used are: p130CASCrk-associated substrateCrkchicken tumor virus no. 10 (CT10) regulator of kinaseIL-3interleukin-3FAKfocal adhesion kinaseLDconsensus sequence of LDXLLXXLLIMzinc finger motifs originally described in homeo-box-containing proteins such as Lin-II, Isl-I, and Mec-3ARF-GAPADP-ribosylation factor-GTPase-activating proteinCAKcell adhesion kinasez-VAD-fmkbenzoxycarbonyl-Val-Ala-Asp-fluoromethoxy ketonez-DEVD-fmkbenzoxycarbonyl-Asp-Glu-Val-Asp-fluoromethoxy ketoneALLNacetyl-leucyl-leucyl-norleucinalALLMN-acetyl-Leu-Leu-methioninalAEBSF4-(2-aminoethyl)benzenesulfonyl fluorideGFPgreen fluorescence proteinPARPpoly-ADP-ribosyl polymeraseAc-DEVD-pNAN-acetyl-Asp-Glu-Val-Asp-p-nitroanilineCHAPS3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonatePBSphosphate-buffered salinePIpropidium iodideDTTdithiothreitol and Crk), and kinases (such as focal adhesion kinase (FAK), and Src) to their cytoplasmic tails, forming a “focal adhesion complex” (1.Gilmore A.P. Burridge K. Structure. 1996; 4: 647-651Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2.Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (497) Google Scholar, 3.Miyamoto S. Katz B.Z. Lafrenie R.M. Yamada K.M. Ann. N. Y. Acad. Sci. 1998; 857: 119-129Crossref PubMed Scopus (200) Google Scholar). Focal adhesions provide not only mechanical support to cells through the connection with the actin cytoskeleton but also signals necessary for anchorage-dependent cellular responses such as proliferation, migration, and inhibition of anoikis, a type of apoptosis induced by cell detachment (4.Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3821) Google Scholar, 5.Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2816) Google Scholar, 6.Craig S.W. Johnson R.P. Curr. Opin. Cell Biol. 1996; 8: 74-85Crossref PubMed Scopus (248) Google Scholar). Crk-associated substrate chicken tumor virus no. 10 (CT10) regulator of kinase interleukin-3 focal adhesion kinase consensus sequence of LDXLLXXL zinc finger motifs originally described in homeo-box-containing proteins such as Lin-II, Isl-I, and Mec-3 ADP-ribosylation factor-GTPase-activating protein cell adhesion kinase benzoxycarbonyl-Val-Ala-Asp-fluoromethoxy ketone benzoxycarbonyl-Asp-Glu-Val-Asp-fluoromethoxy ketone acetyl-leucyl-leucyl-norleucinal N-acetyl-Leu-Leu-methioninal 4-(2-aminoethyl)benzenesulfonyl fluoride green fluorescence protein poly-ADP-ribosyl polymerase N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate phosphate-buffered saline propidium iodide dithiothreitol Paxillin is a 68-kDa adapter protein discovered in focal adhesions as a substrate of Src kinase in Src-transformed cells (reviewed in Refs.7.Schaller M.D. Oncogene. 2001; 20: 6459-6472Crossref PubMed Scopus (439) Google Scholar, 8.Turner C.E. Nat. 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J. Biol. Chem. 1997; 272: 7437-7444Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) of paxillin are known in the human and two (α and β) (15.Mazaki Y. Uchida H. Hino O. Hashimoto S. Sabe H. J. Biol. Chem. 1998; 273: 22435-22441Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) in the mouse. A number of paxillin-like homologs have been reported, e.g.the 48–50-kDa proteins, Hic-5 (16.Fujita H. Kamiguchi K. Cho D. Shibanuma M. Morimoto C. Tachibana K. J. Biol. Chem. 1998; 273: 26516-26521Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and leupaxin (17.Lipsky B.P. Beals C.R. Staunton D.E. J. Biol. Chem. 1998; 273: 11709-11713Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In structure, paxillin can be divided into two halves. The N-terminal half contains five LD motifs, whereas the C-terminal half contains four LIM domains (see model in Fig. 8 B below). LD motifs (LDXLLXXL) (18.Brown M.C. Curtis M.S. Turner C.E. Nat. Struct. Biol. 1998; 5: 677-678Crossref PubMed Scopus (99) Google Scholar) and LIM domains (intertwined zinc finger motifs originally described in homeo-box-containing proteins such as Lin-11,Isl-1, and Mec-3) (19.Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (482) Google Scholar, 20.Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar) are protein-protein interaction motifs and conserved in all known paxillin homologs. LD motifs are essential for binding with a variety of proteins such as FAK (21.Tachibana K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar, 22.Hildebrand J.D. Schaller M.D. Parsons J.T. Mol. Biol. Cell. 1995; 6: 637-647Crossref PubMed Scopus (310) Google Scholar, 23.Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar), vinculin (23.Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar, 24.Wood C.K. Turner C.E. Jackson P. Critchley D.R. J. Cell Sci. 1994; 107: 709-717Crossref PubMed Google Scholar), paxillin kinase linker (an ARF-GAP) (25.Turner C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S.N. McDonald A.R. Bagrodia S. Thomas S. Leventhal P.S. J. Cell Biol. 1999; 145: 851-863Crossref PubMed Scopus (401) Google Scholar), actopaxin (26.Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1447Crossref PubMed Scopus (170) Google Scholar), integrin-linked kinase (27.Nikolopoulos S.N. Turner C.E. J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and bovine papilloma virus E6 protein (28.Tong X. Howley P.M. Proc. Natl. Acad. Sci. 1997; 94: 4412-4417Crossref PubMed Scopus (222) Google Scholar, 29.Tong X. Salgia R. Li J.L. Griffin J.D. Howley P.M. J. Biol. Chem. 1997; 272: 33373-33376Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The LIM2 and LIM3 domains are important for localization of paxillin to focal adhesions by a phosphorylation-dependent mechanism (30.Brown M.C. Perrotta J.A. Turner C.E. J. Cell Biol. 1996; 135: 1109-1123Crossref PubMed Scopus (300) Google Scholar, 31.Brown M.C. Perrotta J.A. Turner C.E. Mol. Biol. Cell. 1998; 9: 1803-1816Crossref PubMed Scopus (124) Google Scholar). Paxillin also binds directly to integrins α4 (32.Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar) and α9 (33.Liu S. Slepak M. Ginsberg M.H. J. Biol. Chem. 2001; 276: 37086-37092Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Paxillin has two main sites of tyrosine phosphorylation (Tyr-31 and Tyr-118), which are phosphorylated probably by FAK (34.Bellis S.L. Miller J.T. Turner C.E. J. Biol. Chem. 1995; 270: 17437-17441Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 35.Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (502) Google Scholar), CAKβ (36.Li X. Earp H.S. J. Biol. Chem. 1997; 272: 14341-14348Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 37.Salgia R. Avraham S. Pisick E. Li J. Raja S. Greenfield A.E. Sattler M. Avraham H. Griffin J.D. J. Biol. Chem. 1996; 271: 31222-31226Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), or Src (38.Thomas S.M. Soriano P. Imamoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (304) Google Scholar, 39.KlinghoÄer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar). These phosphotyrosine motifs provide binding sites for a SH2 domain of Crk (35.Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (502) Google Scholar, 40.Birge R.B. Fajardo J.E. Reichman C. Shoelson S.E. Songyang Z. Cantley L.C. Hanafusa H. Mol. Cell. Biol. 1993; 13: 4648-4656Crossref PubMed Scopus (248) Google Scholar). Accumulating evidence suggests that paxillin plays a pivotal role in integrin-mediated signal pathways for adhesion, migration, and anchorage-dependent survival of cells. Apoptosis, programmed cell death, is accompanied by a succession of characteristic changes in cellular morphology such as detachment from substratum, rounding, cytoplasm shrinkage, membrane blebbing, chromatin condensation, nuclear shrinkage and fragmentation, and DNA fragmentation (41.Zornig M. Hueber A. Baum W. Evan G. Biochim. Biophys. Acta. 2001; 1551: F1-F37PubMed Google Scholar). It is clear that all these processes are dependent on proteolytic cleavages by caspases, a family of proteases that are activated in cascade and degrade many key cellular proteins during apoptosis. However, the basic mechanisms responsible for these phenomena and how they are integrated or interdependent have not been explored in detail. Recent studies have shown that focal adhesions are disassembled and some of their constituents, such as FAK (42.Wen L.P. Fahrni J.A. Troie S. Guan J.L. Orth K. Rosen G.D. J. Biol. Chem. 1997; 272: 26056-26061Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 43.Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (227) Google Scholar) and p130CAS (44.Kook S. Shim S.R. Choi S.J. Ahnn J. Kim J.I. Eom S.H. Jung Y.K. Paik S.G. Song W.K. Mol. Biol. Cell. 2000; 11: 929-939Crossref PubMed Scopus (68) Google Scholar), are cleaved by caspases during apoptosis. However, cleavage of these particular components does not appear to be critical for focal adhesion disassembly during apoptosis, because release of FAK from focal adhesion precedes cleavage of FAK by caspase (45.van de Water B. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1999; 274: 13328-13337Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). It is more likely that caspase cleavages of components upstream of FAK are crucial for focal adhesion disassembly. One plausible candidate for the caspase target that is critical to focal adhesion disassembly is paxillin, because binding to LD motifs and tyrosine phosphorylation sites at the N-terminal half of paxillin is essential for recruiting other critical focal adhesion proteins, such as FAK (21.Tachibana K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar, 22.Hildebrand J.D. Schaller M.D. Parsons J.T. Mol. Biol. Cell. 1995; 6: 637-647Crossref PubMed Scopus (310) Google Scholar, 23.Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar), vinculin (23.Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar, 24.Wood C.K. Turner C.E. Jackson P. Critchley D.R. J. Cell Sci. 1994; 107: 709-717Crossref PubMed Google Scholar), Crk (35.Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (502) Google Scholar, 40.Birge R.B. Fajardo J.E. Reichman C. Shoelson S.E. Songyang Z. Cantley L.C. Hanafusa H. Mol. Cell. Biol. 1993; 13: 4648-4656Crossref PubMed Scopus (248) Google Scholar), p130CAS (46.Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (388) Google Scholar), and actopaxin (26.Nikolopoulos S.N. Turner C.E. J. Cell Biol. 2000; 151: 1435-1447Crossref PubMed Scopus (170) Google Scholar), to the focal adhesion complex. IL-3 is a survival and proliferation factor for hematopoietic cells (47.Rodriguez-Tarduchy G. Collins M. Lopez-Rivas A. EMBO J. 1990; 9: 2997-3002Crossref PubMed Scopus (207) Google Scholar). Ba/F3, a murine pro-B cell line, is dependent on IL-3 for not only survival and growth but also for its polarized and elongated shape (48.Romanova L.Y. Alexandrov I.A. Blagosklonny M.V. Nordan R.P. Garfield S. Acs P. Nguyen P. Trepel J. Blumberg P.M. Mushinski J.F. J. Cell. Physiol. 1999; 179: 157-169Crossref PubMed Scopus (20) Google Scholar). Ba/F3 cells express a considerable amount of paxillin, and cell adhesion and migration are essential for lymphocyte functions (37.Salgia R. Avraham S. Pisick E. Li J. Raja S. Greenfield A.E. Sattler M. Avraham H. Griffin J.D. J. Biol. Chem. 1996; 271: 31222-31226Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In this report, we show that paxillin is degraded by caspase during IL-3 withdrawal and radiation-induced Ba/F3 apoptosis. We studied the kinetics of caspase-mediated cleavage of paxillin and its cleavage sites in detail, and we demonstrate that paxillin is anti-apoptotic in IL-3 withdrawal-induced apoptosis of Ba/F3 cells. Active recombinant caspase-3 was purchased from Upstate Biotechnology (Lake Placid, NY). Cell-permeable caspase inhibitors (z-VAD-fmk and z-DEVD-fmk), colorimetric caspase-3 substrate (Ac-DEVD-pNA),N-acetyl-leucyl-leucyl-norleucinal (ALLN), andN-acetyl-Leu-Leu-methioninal (ALLM), proteasome inhibitor (clasto-Lactacystin β-lactone), aprotinin, leupeptin, and 4-(2-aminoethyl)benzenesulfonyl fluoride (ABESF) were purchased fromCalbiochem (La Jolla, CA), and CHAPS was obtained from Sigma Chemical Co. (St. Louis, MO). Alexa 647-conjugated annexin V was from Molecular Probes (Eugene, OR). Antibodies for paxillin (clone 349), green fluorescence protein (GFP) (monoclonal and polyclonal), actin (clone AC-40), and poly(A)DP-rybosyl polymerase (PARP) (polyclonal) were from Transduction Laboratories (San Diego, CA), CLONTECH (San Francisco, CA), Sigma, and Santa Cruz Biotechnologies (Santa Cruz, CA), respectively. Secondary antibodies, horseradish peroxidase-conjugated anti-rabbit and -mouse IgG, were from Amersham Biosciences, Inc. (Piscataway, NJ). Enhanced chemiluminescence substrate kits, Supersignal West Pico and Supersignal West Dura, and the BCA protein assay kit were from Pierce (Rockford, IL). Protein G-Sepharose 4 Fast Flow was from Amersham Biosciences, Inc. A QuikChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). pEGFP-C2 and pEGFP-N1 plasmids were from CLONTECH. The λ phagemid cDNA library of mouse testis was kindly provided by Dr. Konrad Huppi, NCI (National Institutes of Health). The IL-3-dependent mouse pro-B cell line Ba/F3 was maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum and 10% WEHI-3-conditioned media as a source of murine IL-3. To withdraw IL-3, cells were washed four times by centrifugation and resuspension in PBS at room temperature (pH 7.4), and 2 × 106 cells were plated into 100-mm dishes containing 10 ml of pre-warmed IL-3-free medium and incubated in a CO2incubator for various times with or without exposure to 20 Gy of γ-irradiation from a 137Cs source. A cDNA that included the entire open reading frame of mouse paxillin α was constructed using DNA from a λ phagemid library of mouse testis as a template for a series of PCR reactions. The 5′ and 3′ portions of paxillin were amplified separately using vector sequences and mouse paxillin α-specific sequences from a central part of the transcript (15.Mazaki Y. Uchida H. Hino O. Hashimoto S. Sabe H. J. Biol. Chem. 1998; 273: 22435-22441Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). λgt10 forward primer (5′-GCAAGTTCAGCCTGGTTAAGTCCAAG-3′) was paired with paxillin α-specific reverse (5′-TGGGCCATGAACTTGAAATCTGACAG-3′) primer, and λgt10 reverse primer (5′-GGTGGCTTATGAGTATTTCTTCCAGGGT-3′) was paired with paxillin α-specific forward primer (5′-CTGTCAGATTTCAAGTTCATGGCCCA-3′). The two internal paxillin primers were complementary to one another, and they crossed the splice site where paxillin β 2A complete cDNA clone for mouse paxillin β cDNA was also obtained from this library and will be reported elsewhere. is sometimes inserted by variant splicing (15.Mazaki Y. Uchida H. Hino O. Hashimoto S. Sabe H. J. Biol. Chem. 1998; 273: 22435-22441Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) to prevent cloning paxillin β. The 5′ and 3′ halves of paxillin α were PCR amplified three or four times and sequenced. Consensus sequences (eliminating the occasional Taq polymerase mistake) were recognized by comparing the DNA sequences obtained from three or four independent PCR amplifications. A cDNA encoding the entire open reading frame was obtained by PCR amplification using a pair of primers composed of the outermost sequences in the initial clones for the two halves of paxillin and restriction sites for BglII andEcoRI, respectively. This cDNA was then cloned into pEGFP-C2 and pEGFP-N1 to create GFP-paxillin and paxillin-GFP fusion proteins, respectively. All clones were verified by DNA sequencing. A QuikChange site-directed mutagenesis kit (Stratagene) was used forin vitro mutagenesis of potential caspase-targeting aspartic acids (see below) according to the manufacturer's instructions. cDNAs encoding GFP fusion proteins with paxillin and their mutants were transfected into BaF/3 cells by electroporation using Electro Square Porator ECM830 (BTX Division of Genetronics, Inc., San Diego, CA). In detail, Ba/F3 cells (4 × 106) were washed once with 5 ml of ice-cold 10 mm sodium phosphate buffer (pH 7.4) containing 250 mm sucrose and 1 mm MgCl2, resuspended in 0.4 ml of the same buffer, and put into 2-mm gap electroporation cuvettes that contained 20 μg of DNA. After gentle mixing and incubation on ice for 10 min, two electric pulses (375 V, 99-μs duration) were given with a 1-s interval, and cells were immediately added into 10 ml of pre-warmed medium in a 100-mm tissue culture dish. Cells were cultured for 2 days and then cloned into 96-well plates by limiting dilution under selection with 400 μg/ml G418. Clones that showed green fluorescence under the fluorescence microscope were selected for expansion, and the expression level of paxillin and GFP-paxillin fusion proteins was assayed by Western blot analysis using anti-paxillin or anti-GFP antibodies. Cells in 100-mm tissue culture dishes were washed twice by centrifugation at 4 °C and resuspension in cold PBS (pH 7.4). Appropriate amounts of ice-cold lysis buffer containing 0.5% Triton X-100, 0.5% Nonidet P-40, 0.5 mm EDTA, 0.5 mm EGTA, 150 mm NaCl, 10 mm Tris-HCl (pH 7.2), 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm ABESF, and 25 μm each of calpain inhibitors I and II, ALLN and ALLM, were added to the cell pellets. After brief sonication on ice and a 15-min centrifugation at maximum speed in the microcentrifuge, supernatants (soluble fraction) were assayed for protein concentration, mixed with SDS-PAGE sample buffer, heated at 100 °C for 5 min, and loaded onto a 4–20% gradient Tris-glycine polyacrylamide gel. For the insoluble fraction, the pellet was resuspended and sonicated in an equal volume of lysis buffer and analyzed as above. Western blots of these gels were developed with antibodies and chemiluminescence. For immunoprecipitations, protein G-conjugated beads (10-μl bed volume) were added to cell extracts (0.5 mg of protein) and continuously inverted for 2 h at 4 °C. After a brief centrifugation, 1 μg of paxillin antibody and 10 μl of protein G-conjugated beads were added to the supernatant and continuously inverted overnight at 4 °C. Cell extracts (10 μg of protein) were incubated with 200 μm caspase-3 substrate (Ac-DEVD-pNA) in 100 μl of 25 mmHEPES buffer (pH 7.5) containing 1 mm EDTA, 2 mm dithiothreitol (DTT), 0.1% CHAPS, and 10% sucrose, for 1 h at 37 °C. Absorbance at 405 nm was measured by a microplate reader. To study in vitro cleavage of paxillin by caspase, paxillin was isolated by immunoprecipitation from Ba/F3 cell extracts as described above. Beads were washed four times with PBS and once by 1 ml of caspase reaction buffer containing 25 mm HEPES (pH 7.5), 1 mm EDTA, 2 mm DTT, 0.1% CHAPS, 10% sucrose, incubated with 50 μl of reaction buffer in the absence or presence of 100 ng of active recombinant caspase-3 with or without 10 μm z-DEVD-fmk inhibitor for 1 h at 37 °C, and analyzed by Western blot analysis of half of each reaction mixture. Cells (1 × 106) were washed once with PBS containing 1 g/liter sucrose and then fixed and permeabilized overnight in 1 ml of 70% ethanol. After centrifugation at 3000 × g for 10 min, cell pellets were resuspended in PBS containing 1 μg/ml PI and incubated at room temperature for 1 h. One drop of this suspension of stained cells was put on a slide glass under a coverslip. Confocal fluoromicroscopic images were taken with an LSM 510 confocal laser-scanning microscope (Zeiss, Thornwood, NY). Cells (1 × 106) were washed once with PBS containing 1 g/liter sucrose and resuspended in 100 μl of binding buffer containing 10 mm HEPES (pH 7.4), 140 mm NaCl, and 2.5 mm CaCl2 (50.Andree H.A. Reutelingsperger C.P. Hauptmann R. Hemker H.C. Hermens W.T. Willems G.M. J. Biol. Chem. 1990; 265: 4923-4928Abstract Full Text PDF PubMed Google Scholar). Cells were incubated at room temperature for 15 min after addition of 5 μl of Alexa 647 (excitation: 647 nm; emission: 665 nm)-conjugated annexin V stock solution and 1 μg/ml PI and analyzed with the FACSCalibur system (BD PharMingen immunocytometry system, San Jose, CA). A bicinchoninic acid (BCA) protein assay kit was used, and bovine serum albumin was used as protein standard (51.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar). Ba/F3 cells are dependent on IL-3 for their survival and proliferation in culture, and IL-3 withdrawal-induced apoptosis of Ba/F3 cells has been well studied (47.Rodriguez-Tarduchy G. Collins M. Lopez-Rivas A. EMBO J. 1990; 9: 2997-3002Crossref PubMed Scopus (207) Google Scholar,52.Leverrier Y. Thomas J. Perkins G.R. Mangeney M. Collins M.K. Marvel J. Oncogene. 1997; 14: 425-430Crossref PubMed Scopus (59) Google Scholar, 53.Mathieu A.L. Gonin S. Leverrier Y. Blanquier B. Thomas J. Dantin C. Martin G. Baverel G. Marvel J. J. Biol. Chem. 2001; 276: 10935-10942Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 54.Johnson D.E. Front. Biosci. 1998; 3: d313-d324Crossref PubMed Scopus (29) Google Scholar). Ba/F3 cells are sensitive to ionizing radiation. Exposure to 4 Gy of γ-radiation in the absence of IL-3 is enough to induce apoptosis in this cell line (53.Mathieu A.L. Gonin S. Leverrier Y. Blanquier B. Thomas J. Dantin C. Martin G. Baverel G. Marvel J. J. Biol. Chem. 2001; 276: 10935-10942Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), but IL-3 protects this cell from irradiation-induced apoptosis (55.Collins M.K. Marvel J. Malde P. Lopez-Rivas A. J. Exp. Med. 1992; 176: 1043-1051Crossref PubMed Scopus (216) Google Scholar, 56.Canman C.E. Gilmer T.M. Coutts S.B. Kastan M.B. Genes Dev. 1995; 9: 600-611Crossref PubMed Scopus (400) Google Scholar). Ba/F3 cells also depend on IL-3 for cell shape (48.Romanova L.Y. Alexandrov I.A. Blagosklonny M.V. Nordan R.P. Garfield S. Acs P. Nguyen P. Trepel J. Blumberg P.M. Mushinski J.F. J. Cell. Physiol. 1999; 179: 157-169Crossref PubMed Scopus (20) Google Scholar). They have an elongated shape under optimum growth conditions, although every culture contains a low percentage of round cells. The entire cell population becomes round immediately after removal of IL-3. During the study of regulation of Ba/F3 cell shape by IL-3, we noticed that paxillin protein levels decreased following IL-3 withdrawal. Western blot analysis of cell extracts with monoclonal anti-paxillin antibody revealed two protein bands, and the intensity of both bands decreased after IL-3 withdrawal (Fig. 1 A). The upper 68-kDa band is paxillin, and the lower 48- or 50-kDa band could be Hic-5 (16.Fujita H. Kamiguchi K. Cho D. Shibanuma M. Morimoto C. Tachibana K. J. Biol. Chem. 1998; 273: 26516-26521Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) or leupaxin (17.Lipsky B.P. Beals C.R. Staunton D.E. J. Biol. Chem. 1998; 273: 11709-11713Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), previously described paxillin-like proteins. The lower band was not detected by commercial antibody for Hic-5 (Transduction Laboratories, San Diego, CA, data not shown). Thus, we presume that this band is leupaxin, for which no commercial antibody is available. Paxillin was degraded more rapidly after exposure to 20 Gy of γ-radiation after withdrawal of IL-3 (Fig. 1 B). However, in neither case did we see the appearance of smaller breakdown products concurrent with disappearance of p68 paxillin or p48 presumed leupaxin (Fig. 1). Because the cell extracts used in this Western blot analysis included only the Triton X-100-soluble fraction, we also checked the insoluble fraction as described under “Experimental Procedures.” No significant band was detected in the insoluble fraction using the same paxillin antibody (data not shown). We assumed 1) that Ba/F3 cells underwent apoptosis after IL-3 withdrawal and γ-irradiation, 2) that paxillin was cleaved by proteases that were activated during the apoptosis, and 3) that this cleavage destroyed the epitope site for the monoclonal paxillin antibody or left too small epitope-bearing fragments to be detected in Western blot analyses. IL-3 withdrawal-induced apoptosis of Ba/F3 cells was analyzed with annexin V conjugation, to detect early apoptotic cells, and with parallel PI staining to detect late apoptotic cells (Fig. 2 A). Under normal conditions of culture, more than 99% of the Ba/F3 cell population was negative for both annexin V and PI staining (Fig. 2 A, 0 h). After 6 h without IL-3, a considerable proportion (19%) of the cells was converted to annexin V-positive, but the major population (80%) of the annexin V-positive cells remained negative for PI staining (early apoptotic cells). The percentage of annexin V- positive cells increased to 81% of total cells after 24-h incubation. At this time point, most of annexin V-positive cells (95%) were converted to positive for PI staining (late apoptotic cells). Apoptosis was much more rapid in cells exposed to radiation in addition to IL-3 withdrawal. Characteristic early apoptotic changes in nuclear morphology (Fig. 2 B) were revealed by PI staining of ethanol-fixed cells 8 h after IL-3 withdrawal and γ-irradiation. Virtually all the nuclei appeared apoptotic, namely, shrunken and fragmented, and the chromatin stained brighter and more homogenously (8 h), compared with nuclei in control cells (0 h). The percentage of apoptotic nuclei is plotted in Fig. 2 C, along with a
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