The Apoptosis Inhibitor ARC Undergoes Ubiquitin-Proteasomal-mediated Degradation in Response to Death Stimuli

Abstract

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death. Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death. Apoptosis is activated by two major pathways, one involving death receptors (extrinsic pathway) and the other involving the mitochondria/endoplasmic reticulum (intrinsic pathway) (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4059) Google Scholar). Each of these pathways is opposed by several inhibitory proteins, which characteristically act on only one or the other pathway and antagonize very circumscribed step(s) within that pathway. For example, FLIP (Fas-associated death domain protein-like interleukin-1β-converting enzyme inhibitory protein) inhibits the extrinsic pathway by interfering with the activation of procaspase-8 in the Death-inducing Signaling Complex (DISC) (2Peter M.E. Biochem. J. 2004; 382 (Pt. 2): e1-3Crossref PubMed Scopus (348) Google Scholar). In contrast, Bcl-2 (B cell leukemia/lymphoma-2 protein) and XIAP (X-linked inhibitor of apoptosis protein) are examples of intrinsic pathway inhibitors with Bcl-2 blocking permeabilization of the mitochondrial outer membrane via undetermined mechanisms (3Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar) and XIAP inhibiting already activated downstream caspases by blocking substrate access (4Liston P. Fong W.G. Korneluk R.G. Oncogene. 2003; 22: 8568-8580Crossref PubMed Scopus (390) Google Scholar). Studies in Drosophila and mammalian cells have shown that efficient cell killing requires neutralization of inhibitory proteins as well as activation of death-promoting proteins (5Salvesen G.S. Abrams J.M. Oncogene. 2004; 23: 2774-2784Crossref PubMed Scopus (213) Google Scholar, 6Potts P.R. Singh S. Knezek M. Thompson C.B. Deshmukh M. J. Cell Biol. 2003; 163: 789-799Crossref PubMed Scopus (121) Google Scholar, 7Potts M.B. Vaughn A.E. McDonough H. Patterson C. Deshmukh M. J. Cell Biol. 2005; 171: 925-930Crossref PubMed Scopus (92) Google Scholar). ARC 3The abbreviations used are: ARC, apoptosis repressor with caspase recruitment domain; HEK, human embryonic kidney; HA, hemagglutinin. (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that is expressed primarily in terminally differentiated cells, including cardiac and skeletal myocytes and neurons (8Geertman R. McMahon A. Sabban E.L. Biochim. Biophys. Acta. 1996; 1306: 147-152Crossref PubMed Scopus (40) Google Scholar, 9Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google Scholar). ARC is also induced in a variety of human cancer cell lines and primary human cancers (10Mercier I. Vuolo M. Madan R. Xue X. Levalley A.J. Ashton A.W. Jasmin J.F. Czaja M.T. Lin E.Y. Armstrong R.C. Pollard J.W. Kitsis R.N. Cell Death Differ. 2005; 12: 682-686Crossref PubMed Scopus (56) Google Scholar, 11Wang M. Qanungo S. Crow M.T. Watanabe M. Nieminen A.L. FEBS Lett. 2005; 579: 2411-2415Crossref PubMed Scopus (55) Google Scholar). In contrast to most endogenous inhibitors of apoptosis, ARC antagonizes both the extrinsic and intrinsic pathways (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Inhibition of the extrinsic pathway is mediated by non-homotypic death-fold interactions of ARC with Fas (CD95/Apo-1) and FADD (Fas-associated death domain protein) that preclude the conventional homotypic interactions required for death-inducing signaling complex assembly (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Inhibition of the intrinsic pathway involves interactions between ARC and the C-terminal regulatory domain of Bax (Bcl-2-associated X protein) that prevent the conformational activation of Bax and its translocation to the mitochondria in response to apoptotic stimuli (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 13Gustafsson A.B. Tsai J.G. Logue S.E. Crow M.T. Gottlieb R.A. J. Biol. Chem. 2004; 279: 21233-21238Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Overexpression of ARC blocks cell death in response to activators of both extrinsic and intrinsic pathways (9Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google Scholar, 12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 13Gustafsson A.B. Tsai J.G. Logue S.E. Crow M.T. Gottlieb R.A. J. Biol. Chem. 2004; 279: 21233-21238Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius F.C. II I Nunez G. Circ. Res. 1999; 85: e70-77Crossref PubMed Google Scholar, 15Neuss M. Monticone R. Lundberg M.S. Chesley A.T. Fleck E. Crow M.T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Gustafsson A.B. Sayen M.R. Williams S.D. Crow M.T. Gottlieb R.A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar, 17Chatterjee S. Bish L.T. Jayasankar V. Stewart A.S. Woo Y.J. Crow M.T. Gardner T.J. Sweeney H.L. J. Thorac. Cardiovasc. Surg. 2003; 125: 1461-1469Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Conversely, knock down of endogenous ARC promotes activation of both pathways (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Similarly, inactivation of ARC in the mouse increases cardiac myocyte apoptosis in models of myocardial ischemia-reperfusion and hemodynamic overload (18Donath S. Li P. Willenbockel C. Al-Saadi N. Gross V. Willnow T. Bader M. Martin U. Bauersachs J. Wollert K.C. Dietz R. von Harsdorf R. Circulation. 2006; 113: 1203-1212Crossref PubMed Scopus (101) Google Scholar). These observations indicate that ARC is an important inhibitor of apoptosis in vivo. Despite its inhibition of both central apoptosis pathways, even cells that express high base-line levels of ARC can be induced to undergo apoptosis. The susceptibility of ARC-expressing cells to apoptosis has been hypothesized to be due to the fact that ARC protein levels decrease markedly during apoptosis in some systems. These include hypoxia and oxidative stress in the H9c2 muscle cell line and in primary neonatal rat cardiac myocytes, ischemia-reperfusion in isolated, perfused rat hearts, and human heart failure (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 14Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius F.C. II I Nunez G. Circ. Res. 1999; 85: e70-77Crossref PubMed Google Scholar, 15Neuss M. Monticone R. Lundberg M.S. Chesley A.T. Fleck E. Crow M.T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18Donath S. Li P. Willenbockel C. Al-Saadi N. Gross V. Willnow T. Bader M. Martin U. Bauersachs J. Wollert K.C. Dietz R. von Harsdorf R. Circulation. 2006; 113: 1203-1212Crossref PubMed Scopus (101) Google Scholar, 19Yaniv G. Shilkrut M. Lotan R. Berke G. Larisch S. Binah O. Cardiovasc. Res. 2002; 54: 611-623Crossref PubMed Scopus (53) Google Scholar, 20Yaniv G. Shilkrut M. Larisch S. Binah O. Biochem. Biophys. Res. Commun. 2005; 336: 740-746Crossref PubMed Scopus (15) Google Scholar). The mechanisms responsible for reductions in ARC protein levels during apoptosis and whether these decreases are causally related to the subsequent cell death are not known. In this study, we show that decreases in ARC protein abundance in response to death stimuli are caused by destabilization of ARC protein. This process is mediated by the ubiquitin-proteasomal pathway. Moreover, mutation of ubiquitin acceptor residues renders ARC resistant to degradation, maintains its steady state levels, and enhances cytoprotection. Plasmids and Cloning—Mammalian expression vectors for ARC were generated by subcloning the open reading frame of human ARC into pcDNA3.1/pcDNA3.1-HA/pcDNA3.1-Myc-His B/pcDNA3.1-V5-His (Invitrogen). Mammalian expression vectors for procaspase-8 and ubiquitin were obtained from Drs. Gabriel Nunez and Wei Gu, respectively. Mutants derived from pcDNA3.1-ARC-HA were generated using the QuikChange II XL mutagenesis kit (Stratagene) according to the manufacturer’s protocol. Cell Culture and Transfection— H9c2, MCF-7, HeLa, and HEK293 cell lines (ATCC) were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Primary cultures of neonatal rat cardiac myocytes were prepared as described (21Bialik S. Cryns V.L. Drincic A. Miyata S. Wollowick A.L. Srinivasan A. Kitsis R.N. Circ. Res. 1999; 85: 403-414Crossref PubMed Scopus (240) Google Scholar). Cells were transfected with the indicated plasmids using Effectene (Qiagen). H9c2 or HEK293 ARC stable transfectants were generated using Hygromycin (Invitrogen)-resistant vectors. Antibodies, Immunoblotting, Immunostaining, and Immunoprecipitations—Polyclonal ARC antisera from Cayman or as generated by Dr. M. Crow (14Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius F.C. II I Nunez G. Circ. Res. 1999; 85: e70-77Crossref PubMed Google Scholar) were used for immunoblotting. The Cayman antibody was also used for immunoprecipitations. A polyclonal ARC antibody (Neomarkers) was used for immunostaining. Additionally, monoclonal antibodies against ubiquitin, HA, Myc (all from Santa Cruz Biotechnology), and α-tubulin (Sigma) were used. Immunostaining was performed on mouse heart sections as previously described (10Mercier I. Vuolo M. Madan R. Xue X. Levalley A.J. Ashton A.W. Jasmin J.F. Czaja M.T. Lin E.Y. Armstrong R.C. Pollard J.W. Kitsis R.N. Cell Death Differ. 2005; 12: 682-686Crossref PubMed Scopus (56) Google Scholar). Immunoblots and immunoprecipitations were performed as described (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Assessment of ARC mRNA Levels—ARC mRNA levels were assessed using quantitative real-time reverse transcriptase PCR. Total RNA was extracted from cell lysates with TRIzol (Invitrogen). First strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). Quantitative real-time reverse transcriptase PCR was performed using the DNA Master Plus SYBR Green I kit (Roche Applied Science) with a LightCycler PCR machine (Roche Applied Science) according to the manufacturer’s directions. ARC primers were forward 5′-TGTGCCCAGCAAACAGTG-3′, reverse 5′-CTGGGCATGAAGGGTCATAG-3′. Glyceraldehyde-3-phosphate dehydrogenase primers were forward 5′-TGCCACTCAGAAGACTGTGG-3′ and reverse 5′-GGATGCAGGGATGATGTTCT-3′. The abundance of ARC mRNA was normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA. Assessment of ARC Protein Stability—Cells were washed with phosphate-buffered saline and incubated with methionine/cysteine-free Dulbecco’s modified Eagle’s medium (ATCC) for 2 h, following which cells were metabolically labeled with 500 μCi of [35S]cysteine (ICN) for 10 h. After labeling, cells were washed with phosphate-buffered saline and chased with complete Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum for the indicated times. Lysates were immunoprecipitated for ARC, resolved by SDS-PAGE, and analyzed by autoradiography. Cell Death Assay—Cell death was assessed by nuclear condensation as described (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Mouse Ischemia-Reperfusion Model—Male, 8–10-week-old C57Bl6 mice were subjected to sham operation or ischemia-reperfusion in vivo and mouse heart sections and tissue homogenates prepared as previously described (22Tarzami S.T. Miao W. Mani K. Lopez L. Factor S.M. Berman J.W. Kitsis R.N. Circulation. 2003; 108: 2387-2392Crossref PubMed Scopus (77) Google Scholar). Steady State ARC Protein Levels Decrease in Response to Death Stimuli in Various Systems—Previous work in muscle cells has shown that ARC protein levels decrease in response to oxidative stress and hypoxia (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 14Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius F.C. II I Nunez G. Circ. Res. 1999; 85: e70-77Crossref PubMed Google Scholar, 15Neuss M. Monticone R. Lundberg M.S. Chesley A.T. Fleck E. Crow M.T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Yaniv G. Shilkrut M. Lotan R. Berke G. Larisch S. Binah O. Cardiovasc. Res. 2002; 54: 611-623Crossref PubMed Scopus (53) Google Scholar, 20Yaniv G. Shilkrut M. Larisch S. Binah O. Biochem. Biophys. Res. Commun. 2005; 336: 740-746Crossref PubMed Scopus (15) Google Scholar). To test the generality of this observation, we studied a variety of cell types (including muscle cells and cancer cells that express endogenous ARC) and apoptotic inducers (including oxidative stress, simulated ischemia, doxorubicin, and etoposide). Oxidative stress (hydrogen peroxide) stimulated a brisk and marked decrease in endogenous ARC protein levels in H9c2 cells (skeletal myocyte-like cell line derived from embryonic rat heart), MCF-7 cells (human breast cancer cell line), and HeLa cells (human cervical cancer cell line) (Fig. 1A). These decreases in ARC protein abundance were specific as steady state levels of α-tubulin in the same cells remained constant or decreased only modestly. Endogenous ARC protein levels in H9c2 cells also decreased in response to simulated ischemia (serum/glucose/oxygen deprivation) (Fig. 1A). Doxorubicin decreased endogenous ARC protein levels in H9c2 cells (Fig. 1A), but not in MCF-7 cells (not shown). Etoposide did not affect ARC protein levels in any of these cell types (not shown). Thus, although ARC protein levels are unaffected in some contexts, they decrease in response to oxidative stress in a variety of cell types. Because oxidative stress is an integral component of myocardial ischemia-reperfusion (23Becker L.B. Cardiovasc. Res. 2004; 61: 461-470Crossref PubMed Scopus (589) Google Scholar), we postulated that endogenous ARC protein levels would decrease in the hearts of mice subjected to transient left coronary artery ligation. operation or of ischemia that cardiac myocyte apoptosis R.A. R.A. J. PubMed Scopus Google Scholar, S. R. J.W. S.M. Kitsis R.N. J. PubMed Scopus (348) Google Scholar), on ARC protein levels (Fig. and In contrast, ischemia by which cardiac myocyte apoptosis R.A. R.A. J. PubMed Scopus Google Scholar, P. M. Factor S.M. K. Kitsis R.N. J. 2003; PubMed Scopus Google Scholar), induced marked decreases in the abundance of ARC protein (Fig. and were obtained by (Fig. These data demonstrate that endogenous ARC levels in the decrease dramatically in response to ischemia-reperfusion in vivo. ARC Protein during the by which apoptotic stimuli decrease ARC protein levels, we assessed whether ARC mRNA abundance decreases during apoptosis. ARC protein levels decrease most markedly during the 2 of apoptosis in H9c2 cells (Fig. 1A). Despite ARC mRNA levels remained 2 and decreased only h, as assessed by quantitative real-time reverse transcriptase PCR (Fig. in ARC mRNA levels some in ARC protein levels, these data indicate the and of the decreases in ARC protein levels during apoptosis be for by alterations in ARC mRNA We whether the of ARC protein decreases during apoptosis, an in of the with which ARC protein levels by the of endogenous ARC protein was in MCF-7 cells (Fig. A was for transfected human ARC in H9c2 cells (Fig. and HEK293 cells (Fig. In contrast, of cells with the of ARC protein to in MCF-7 cells (Fig. and H9c2 cells (Fig. Similarly, activation of apoptosis using procaspase-8 ARC to in HEK293 cells (Fig. We conclude that ARC protein decreases markedly in response to apoptotic stimuli and that increases in ARC protein degradation an important in the decreases in steady state ARC protein levels during apoptosis. ARC in Response to test whether the ubiquitin-proteasomal pathway is in ARC degradation, we whether ARC be a substrate for which proteins for degradation in the The of ubiquitin and ARC in MCF-7 cells in of ARC (Fig. the abundance of which was markedly increased by the inhibitor (Fig. These data indicate that ARC can undergo We whether ARC undergo in response to apoptotic stimuli. of procaspase-8 in HEK293 cells induced of Moreover, of MCF-7 cells or primary cultures of neonatal rat cardiac myocytes stimulated of endogenous Thus, ARC protein in cell types in response to death stimuli. The for Decreases in Steady State ARC Protein Levels during whether the ubiquitin-proteasomal pathway is responsible for the decreased abundance of ARC protein during apoptosis, we an ARC mutant that is resistant to are to the of residues in the degradation substrate Biochem. 2001; PubMed Scopus Google Scholar). ARC and mutation of of these to of ARC in response to an apoptotic (Fig. that the mutant markedly increased protein both (Fig. and apoptotic steady state levels of the mutant protein were even induction of apoptosis, which ARC protein levels were (Fig. In contrast to the of or of these residues did not steady state protein levels during apoptosis (not shown). These data demonstrate that decreases in ARC levels during apoptosis are mediated by destabilization of the ARC protein via the ubiquitin-proteasomal pathway. ARC the ARC mutant protein is resistant to degradation during apoptosis, we whether exhibits improved cytoprotection. apoptosis was induced in HEK293 cell lines or mutant steady state levels of ARC decreased dramatically during apoptosis, levels of mutant ARC were (Fig. cell death was decreased by ARC as the mutant (Fig. of was decreased by the mutant by ARC (Fig. These data show that inhibition of ARC degradation in cytoprotection. This the contexts in which ARC protein levels are to decrease during apoptosis. These decreases are mediated by destabilization of ARC protein via the ubiquitin-proteasomal pathway. of its ubiquitin acceptor residues renders ARC resistant to degradation during apoptosis, maintains its steady state levels, and enhances its cytoprotection. Quantitative that the marked decrease in steady state ARC protein levels during apoptosis is not explained by in ARC mRNA These data that in ARC and mRNA not major in ARC protein levels in this These are with in human hearts (18Donath S. Li P. Willenbockel C. Al-Saadi N. Gross V. Willnow T. Bader M. Martin U. Bauersachs J. Wollert K.C. Dietz R. von Harsdorf R. Circulation. 2006; 113: 1203-1212Crossref PubMed Scopus (101) Google Scholar). In contrast, pulse-chase experiments in several cell types marked decreases in ARC protein in response to apoptotic stimuli. The of these that increases in ARC protein degradation a in decreases in steady state ARC protein levels during apoptosis. To the of ARC destabilization during apoptosis, we the of the ubiquitin-proteasomal pathway. We that ARC during apoptosis. Moreover, mutation of ubiquitin acceptor an ARC mutant during apoptosis, to be was resistant to degradation, and steady state These data that the ubiquitin-proteasomal pathway decreases in steady state levels of ARC protein during apoptosis. A related the of the ubiquitin for in the L. Kitsis R.N. M.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), is one stable transfectants with of ARC, we have previously shown even in the of ARC protein levels during apoptosis, levels cell death (12Nam Y.J. Mani K. Ashton A.W. Peng C.F. Krishnamurthy B. Hayakawa Y. Lee P. Korsmeyer S.J. Kitsis R.N. Mol. Cell. 2004; 15: 901-912Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). These data that ARC protein abundance levels is for cell of a degradation-resistant ARC mutant is required to test this The ARC mutant ARC protein levels in the of an apoptotic which in ARC, levels decrease during apoptosis. These data that decreases in ARC protein abundance are an in apoptosis. this be important to the pathways that death stimuli with ARC these pathways with which to ARC in heart and and in We Drs. Wei Gu, and Gabriel Nunez for and for with