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W2171396586 abstract "Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with γ or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death. Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with γ or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death. Proteins modified by the proteases and kinases that are activated during apoptosis are often involved in both the execution phase of cell death and in the development of autoantibodies in patients with systemic lupus erythematosus and mixed connective tissue disease (reviewed in Ref. 1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar). For example, at least 17 proteins that are known to be cleaved by caspases during apoptosis are autoantigens, including the 70-kDa component of the U1-small nuclear ribonuclear protein complex (U1–70 kDa) (2Casciola-Rosen L.A. Miller D.K. Anhalt G.J. Rosen A. J. Biol. Chem. 1994; 269: 30757-30760Abstract Full Text PDF PubMed Google Scholar), poly(A) ribose polymerase (3Lazebnik Y. Kaufmann S. Desnoyers S. Poirier G. Earnshaw W. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2339) Google Scholar), DNA-dependent protein kinase (DNA-PK) (4Casciola-Rosen L.A. Anhalt G.J. Rosen A. J. Exp. Med. 1995; 182: 1625-1634Crossref PubMed Scopus (399) Google Scholar), hnRNP C1 and C2 (5Waterhouse N. Kumar S. Song Q. Strike P. Sparrow L. Dreyfuss G. Alnemri E.S. Litwack G. Lavin M. Watters D. J. Biol. Chem. 1996; 271: 29335-29341Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), lamins A, B, and C (6Lazebnik Y. Takahashi A. Moir R. Goldman R. Poirier G. Kaufmann S. Earnshaw W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9042-9046Crossref PubMed Scopus (482) Google Scholar), the nuclear mitotic apparatus protein (NuMA) (7Weaver V. Carson C. Walker P. Chaly N. Lach B. Raymond Y. Brown D. Sikorska M. J. Cell Sci. 1996; 109: 45-56Crossref PubMed Google Scholar, 8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), topoisomerases 1 and 2 (8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), the nucleolar protein UBF/NOR-90 (8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), and α-fodrin (9Marin S. O'Brien G. Nishioka W. McGahon A. Mahboubi A. Saido T. Green D. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 10Haneji N. Nakamura T. Takio K. Yanagi K. Higashiyama H. Saito I. Noji S. Sugino H. Hayashi Y. Science. 1997; 276: 604-607Crossref PubMed Scopus (384) Google Scholar) (reviewed in Ref. 1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar). In addition, phosphorylated serine/arginine splicing factors have recently been shown to specifically associate with the U1-small nuclear RNP autoantigen complex during apoptosis (11Utz P.J. Hottelet M. van Venrooij W. Anderson P. J. Exp. Med. 1998; 187: 547-560Crossref PubMed Scopus (81) Google Scholar, 12Utz P.J Hottelet M. Schur P. Anderson P. J. Exp. Med. 1997; 185: 843-854Crossref PubMed Scopus (202) Google Scholar). These observations have led to the hypothesis that proteins modified during apoptosis can be presented to the immune system in a way that bypasses tolerance to self proteins. Although the molecular mechanisms by which this occurs are not known, the data suggests that patient-derived autoantisera may be useful in the identification of proteins that contribute to the execution phase of apoptosis.While screening a panel of human autoantisera for their ability to precipitate new phosphoproteins from apoptotic Jurkat cell lysates, we serendipitously identified several sera that precipitated phosphoproteins from extracts prepared from untreated Jurkat cells that were no longer observed when extracts were prepared from apoptotic Jurkat cells. One of these phosphorylated autoantigens has been identified as the 72-kDa component of the signal recognition particle (SRP). 1The abbreviations used are: SRP, signal recognition particle; ER, endoplasmic reticulum; HI-FCS, heat-inactivated fetal calf serum; PAGE, polyacrylamide gel electrophoresis; SAP, shrimp alkaline phosphatase. 1The abbreviations used are: SRP, signal recognition particle; ER, endoplasmic reticulum; HI-FCS, heat-inactivated fetal calf serum; PAGE, polyacrylamide gel electrophoresis; SAP, shrimp alkaline phosphatase.SRP is a ribonucleoprotein complex comprising the 7 S RNA in association with six distinct polypeptides. SRP functions to recognize the signal peptide of nascent transcripts, attach the translating ribosome to the endoplasmic reticulum (ER), and facilitate translocation into the ER lumen. The 72-kDa SRP protein is essential for protein translocation. In this report we demonstrate that SRP 72 is constitutively phosphorylated on serine residues. In Jurkat cells subject to apoptotic stimuli, SRP 72 is cleaved by caspases to liberate a 6-kDa carboxyl-terminal phosphopeptide. Our results suggest that phosphorylation and caspase cleavage might regulate translocation of secretory proteins into the ER lumen during apoptosis.DISCUSSIONSRP, a highly conserved cytoplasmic complex composed of a 7 S structural RNA molecule and 6 polypeptides, mediates the targeting of secretory proteins to the endoplasmic reticulum (26Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar, 27Walter P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7112-7116Crossref PubMed Scopus (302) Google Scholar). The intact particle has at least three separable activities: (i) binding to newly synthesized proteins bearing signal sequence as they emerge from the ribosome; (ii) elongation arrest during translation; and (iii) binding to the SRP receptor, leading to release from elongation arrest and translocation of the targeted protein into the lumen of the endoplasmic reticulum. Biochemical mutagenesis experiments have implicated individual domains of the SRP complex in each of these three functions (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 29Walter P. Blobel G. Cell. 1983; 34: 525-533Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Thus, the 54-kDa polypeptide is required for binding to the signal sequence, the 14- and 9-kDa polypeptides are involved in elongation arrest, and the 68- and 72-kDa proteins have been implicated in binding to the SRP receptor and promoting the directional translocation of newly translated proteins bearing a signal sequence into the lumen of the ER (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar). The role played by the 7 S RNA molecule is currently unknown.Deletion analysis of canine SRP 68 and SRP 72 has demonstrated that these proteins associate with each other through their carboxyl termini, forming a stable complex with the 7 S RNA (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). A 57-kDa fragment that includes the amino terminus of canine SRP 72 (i.e. a smaller fragment than that generated by caspase cleavage of SRP 72) generated by elastase digestion is still capable of interacting in vitro with SRP 68, while a 42-kDa fragment is not (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Interestingly, an elastase-generated carboxyl fragment of ∼4 kDa was observed in this analysis, suggesting that a portion of the carboxyl terminus of SRP 72 is exposed when associated with other components of the SRP particle (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Our results are consistent with those of Lütcke et al. (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar) since caspase-cleaved SRP 72 remains associated with the SRP complex in immunoprecipitates (Fig.1 B), and migrates at ∼11 S by sucrose gradient centrifugation when comparing complexes prepared from untreated and apoptotic Jurkat cells (data not shown). SRP has been observed to migrate in a larger complex of ∼40 S (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar), and it remains possible that cleavage of SRP 72 may disrupt the formation of the larger complex.Based on biological and chemical mutagenesis experiments (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar, 28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar), we predict that cleavage of SRP 72 during apoptosis may inhibit binding of the complex to the SRP receptor on the endoplasmic reticulum membrane. This would result in (i) globally preventing the localization of secretory proteins to the endoplasmic reticulum; and (ii) irreversible elongation arrest of newly translated proteins bearing a signal sequence. While this is unlikely to play an important role in the execution phase of most types of cell death, cleavage of SRP 72 may play a critical role when apoptosis is induced by enveloped viruses, since many viral proteins pass through the ER. This would be thwarted if SRP 72 was no longer capable of targeting nascent peptides to its receptor on the outer lumen of the ER. While the in vitroresults shown in Fig. 8 argue against this model, it remains possible that removal of the carboxyl terminus of SRP 72 during apoptosis may have a more subtle effect on ER transport in vivo, particularly since the cleaved carboxyl-terminal peptide is likely to harbor the serine phosphorylation site(s).Targeting of secretory proteins to the ER occurs by a strongly conserved, and presumably highly regulated, mechanism. However, little is known about how this process is governed in eukaryotic cells. Protein translation is regulated in response to exogenous stress at an early step (initiation) by several kinases, including: mitogen-activated protein kinases, which repress translation by phosphorylating the mRNA cap-binding protein eIF4E (30Sonenberg N. Gingras A. Curr. Opin. Cell Biol. 1998; 10: 268-275Crossref PubMed Scopus (503) Google Scholar); the interferon-inducible, double stranded RNA-regulated protein kinase, which inhibits translation by phosphorylating the eIF2 initiation factor (31de Haro C. Mendez R. Santoyo J. FASEB J. 1996; 10: 1378-1387Crossref PubMed Scopus (235) Google Scholar, 32Clemens M. Int. J. Biochem. Cell Biol. 1997; 29: 945-949Crossref PubMed Scopus (169) Google Scholar); the target of rapamycin which phosphorylates PHAS-1, a regulator of eIF4E; and the rapamycin-sensitive p70 S6 kinase, which modulates translation through phosphorylation of the S6 component of ribosomes (33Proud C. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Our observation that SRP 72 is phosphorylated on serine residue(s) in all cell types tested suggests that a serine kinase and/or phosphatase may regulate gene expression at a later step than the translation initiation checkpoint that is regulated by the above kinases.Our results are most consistent with caspase-mediated cleavage of the carboxyl terminus of SRP 72, generating a 6-kDa peptide containing the serine phosphorylation site(s). The fate of the 6-kDa fragment is unknown. We cannot, however, exclude the possibility that SRP 72 is a target for both a phosphatase and a caspase during apoptosis. It is also possible that a comigrating serine kinase (e.g. p70 S6 kinase) is coprecipitated with the SRP complex, and that the carboxyl terminus of SRP 72 is required for the interaction between the kinase and SRP. We have not consistently observed a serine kinase activity in these immunoprecipitates in in vitro kinase assays, however, arguing against the later possibility (data not shown).Comparison of the carboxyl terminus of SRP 72 from different organisms (Table I) shows striking conservation of eight serine residues (labeled with an asterisk, *) that are distal to the proposed caspase cleavage site (bold letters). Three of these serine residues are conserved from homo sapiens to Schistosoma mansoni (underlined). Although SRP 72 from Caenorhabditis elegans and Saccharomyces cerevisiae lacks these conserved serine residues, both proteins have carboxyl-terminal serines (i.e. amino acids 660, 663, and 693, C. elegans; aa 620 and 627, S. cerevisiae) that are potential phosphorylation sites. We have not yet determined whether SRP 72 derived from yeast, C. elegans, and S. mansoniare similarly phosphorylated in vivo.The results shown in Fig. 8 demonstrate cleavage of SRP 72 in vitro by caspase 3; however, the caspase(s) responsible for cleaving SRP 72 in vivo remain unidentified. Based on published data, it is most likely that SRP 72 is cleaved in vivo by a group III caspase such as caspase-6 (Mch 2), caspase-8 (MACH, FLICE, Mch 5), or caspase-9 (ICE-LAP6, Mch 6), reviewed in Ref.21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar. Group III caspases cleave non-DXXD motifs characteristically found in other caspases or in components of the nuclear or cytosolic skeleton (e.g. actin, Gas2, and nuclear lamins (21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar)). The SELD/A motif of SRP 72 most resembles the SELD/A cleavage site of SREBP-2, a transcription factor involved in regulation of cholesterol metabolism that is also associated with the endoplasmic reticulum (34Wang X. Pai J-T. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In support of the hypothesis that SRP 72 is also a substrate for group III caspases, we have observed cleavage of SRP 72 (generated in an in vitro translation reaction) by several group III caspases in vitro, including caspases 8 and 9. 4P. J. Utz, T. M. Le, S. J. Kim, M. E. Geiger, and P. Anderson, unpublished data. It will also be of great interest to determine if SRP 72 from C. elegans and S. cerevisiae (both of which lack a conserved caspase cleavage site), and SRP 72 from S. mansoni (which possesses such a site at665SEWD/A669) can be cleaved in vivoor in vitro.Autoantibodies directed against the 54-kDa polypeptide of SRP were first described independently in 3 patients with dermatomyositis (17Reeves W. Nigam S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9507-9511Crossref PubMed Scopus (139) Google Scholar,35Okada N. Mimori T. Mukai R. Kashiwagi H. Hardin J. J. Immunol. 1987; 138: 3219-3223PubMed Google Scholar). It is currently unknown whether phosphorylation or caspase-mediated cleavage of SRP 72 contributes to the production of autoantibodies reactive with components of the SRP complex. Several studies have identified autoantigens as constituents of membrane-bound blebs on the surface of apoptotic cells, where it has been proposed that they are ideally situated for presentation to the immune system (1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar, 36Casciola-Rosen L.A. Anhalt G. Rosen A. J. Exp. Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1527) Google Scholar, 37LeFeber W.P. Norris D.A. Ryan S.R. Huff J.C. Lee L.A. Kubo M. Boyce S.T. Kotzin B.L. Weston W.L. J. Clin. Invest. 1984; 74: 1545Crossref PubMed Scopus (289) Google Scholar, 38Golan T.D. Elkon K.B. Gharavi A.E. Krueger J.G. J. Clin. Invest. 1992; 90: 1067-1076Crossref PubMed Scopus (158) Google Scholar). Consistent with this hypothesis, we have observed SRP components as prominent constituents of apoptotic blebs by immunofluorescence.4 Like SRP 72, several proteins that are modified by caspases, or become associated with highly phosphorylated proteins during apoptosis are targets for autoantibody production in patients with autoimmune disease. The presence of autoantibodies reactive with components of the SRP is associated with a particularly aggressive form of dermatomyositis (39Targoff I. Johnson A. Miller F. Arthritis Rheum. 1990; 33: 1361-1370Crossref PubMed Scopus (226) Google Scholar). Whether this is a reflection of increased levels of apoptosis in affected tissues remains to be determined. Proteins modified by the proteases and kinases that are activated during apoptosis are often involved in both the execution phase of cell death and in the development of autoantibodies in patients with systemic lupus erythematosus and mixed connective tissue disease (reviewed in Ref. 1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar). For example, at least 17 proteins that are known to be cleaved by caspases during apoptosis are autoantigens, including the 70-kDa component of the U1-small nuclear ribonuclear protein complex (U1–70 kDa) (2Casciola-Rosen L.A. Miller D.K. Anhalt G.J. Rosen A. J. Biol. Chem. 1994; 269: 30757-30760Abstract Full Text PDF PubMed Google Scholar), poly(A) ribose polymerase (3Lazebnik Y. Kaufmann S. Desnoyers S. Poirier G. Earnshaw W. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2339) Google Scholar), DNA-dependent protein kinase (DNA-PK) (4Casciola-Rosen L.A. Anhalt G.J. Rosen A. J. Exp. Med. 1995; 182: 1625-1634Crossref PubMed Scopus (399) Google Scholar), hnRNP C1 and C2 (5Waterhouse N. Kumar S. Song Q. Strike P. Sparrow L. Dreyfuss G. Alnemri E.S. Litwack G. Lavin M. Watters D. J. Biol. Chem. 1996; 271: 29335-29341Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), lamins A, B, and C (6Lazebnik Y. Takahashi A. Moir R. Goldman R. Poirier G. Kaufmann S. Earnshaw W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9042-9046Crossref PubMed Scopus (482) Google Scholar), the nuclear mitotic apparatus protein (NuMA) (7Weaver V. Carson C. Walker P. Chaly N. Lach B. Raymond Y. Brown D. Sikorska M. J. Cell Sci. 1996; 109: 45-56Crossref PubMed Google Scholar, 8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), topoisomerases 1 and 2 (8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), the nucleolar protein UBF/NOR-90 (8Casiano C.A. Martin S.J. Green D.R. Tan E.M. J. Exp. Med. 1996; 184: 765-770Crossref PubMed Scopus (244) Google Scholar), and α-fodrin (9Marin S. O'Brien G. Nishioka W. McGahon A. Mahboubi A. Saido T. Green D. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 10Haneji N. Nakamura T. Takio K. Yanagi K. Higashiyama H. Saito I. Noji S. Sugino H. Hayashi Y. Science. 1997; 276: 604-607Crossref PubMed Scopus (384) Google Scholar) (reviewed in Ref. 1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar). In addition, phosphorylated serine/arginine splicing factors have recently been shown to specifically associate with the U1-small nuclear RNP autoantigen complex during apoptosis (11Utz P.J. Hottelet M. van Venrooij W. Anderson P. J. Exp. Med. 1998; 187: 547-560Crossref PubMed Scopus (81) Google Scholar, 12Utz P.J Hottelet M. Schur P. Anderson P. J. Exp. Med. 1997; 185: 843-854Crossref PubMed Scopus (202) Google Scholar). These observations have led to the hypothesis that proteins modified during apoptosis can be presented to the immune system in a way that bypasses tolerance to self proteins. Although the molecular mechanisms by which this occurs are not known, the data suggests that patient-derived autoantisera may be useful in the identification of proteins that contribute to the execution phase of apoptosis. While screening a panel of human autoantisera for their ability to precipitate new phosphoproteins from apoptotic Jurkat cell lysates, we serendipitously identified several sera that precipitated phosphoproteins from extracts prepared from untreated Jurkat cells that were no longer observed when extracts were prepared from apoptotic Jurkat cells. One of these phosphorylated autoantigens has been identified as the 72-kDa component of the signal recognition particle (SRP). 1The abbreviations used are: SRP, signal recognition particle; ER, endoplasmic reticulum; HI-FCS, heat-inactivated fetal calf serum; PAGE, polyacrylamide gel electrophoresis; SAP, shrimp alkaline phosphatase. 1The abbreviations used are: SRP, signal recognition particle; ER, endoplasmic reticulum; HI-FCS, heat-inactivated fetal calf serum; PAGE, polyacrylamide gel electrophoresis; SAP, shrimp alkaline phosphatase.SRP is a ribonucleoprotein complex comprising the 7 S RNA in association with six distinct polypeptides. SRP functions to recognize the signal peptide of nascent transcripts, attach the translating ribosome to the endoplasmic reticulum (ER), and facilitate translocation into the ER lumen. The 72-kDa SRP protein is essential for protein translocation. In this report we demonstrate that SRP 72 is constitutively phosphorylated on serine residues. In Jurkat cells subject to apoptotic stimuli, SRP 72 is cleaved by caspases to liberate a 6-kDa carboxyl-terminal phosphopeptide. Our results suggest that phosphorylation and caspase cleavage might regulate translocation of secretory proteins into the ER lumen during apoptosis. DISCUSSIONSRP, a highly conserved cytoplasmic complex composed of a 7 S structural RNA molecule and 6 polypeptides, mediates the targeting of secretory proteins to the endoplasmic reticulum (26Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar, 27Walter P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7112-7116Crossref PubMed Scopus (302) Google Scholar). The intact particle has at least three separable activities: (i) binding to newly synthesized proteins bearing signal sequence as they emerge from the ribosome; (ii) elongation arrest during translation; and (iii) binding to the SRP receptor, leading to release from elongation arrest and translocation of the targeted protein into the lumen of the endoplasmic reticulum. Biochemical mutagenesis experiments have implicated individual domains of the SRP complex in each of these three functions (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 29Walter P. Blobel G. Cell. 1983; 34: 525-533Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Thus, the 54-kDa polypeptide is required for binding to the signal sequence, the 14- and 9-kDa polypeptides are involved in elongation arrest, and the 68- and 72-kDa proteins have been implicated in binding to the SRP receptor and promoting the directional translocation of newly translated proteins bearing a signal sequence into the lumen of the ER (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar). The role played by the 7 S RNA molecule is currently unknown.Deletion analysis of canine SRP 68 and SRP 72 has demonstrated that these proteins associate with each other through their carboxyl termini, forming a stable complex with the 7 S RNA (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). A 57-kDa fragment that includes the amino terminus of canine SRP 72 (i.e. a smaller fragment than that generated by caspase cleavage of SRP 72) generated by elastase digestion is still capable of interacting in vitro with SRP 68, while a 42-kDa fragment is not (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Interestingly, an elastase-generated carboxyl fragment of ∼4 kDa was observed in this analysis, suggesting that a portion of the carboxyl terminus of SRP 72 is exposed when associated with other components of the SRP particle (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Our results are consistent with those of Lütcke et al. (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar) since caspase-cleaved SRP 72 remains associated with the SRP complex in immunoprecipitates (Fig.1 B), and migrates at ∼11 S by sucrose gradient centrifugation when comparing complexes prepared from untreated and apoptotic Jurkat cells (data not shown). SRP has been observed to migrate in a larger complex of ∼40 S (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar), and it remains possible that cleavage of SRP 72 may disrupt the formation of the larger complex.Based on biological and chemical mutagenesis experiments (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar, 28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar), we predict that cleavage of SRP 72 during apoptosis may inhibit binding of the complex to the SRP receptor on the endoplasmic reticulum membrane. This would result in (i) globally preventing the localization of secretory proteins to the endoplasmic reticulum; and (ii) irreversible elongation arrest of newly translated proteins bearing a signal sequence. While this is unlikely to play an important role in the execution phase of most types of cell death, cleavage of SRP 72 may play a critical role when apoptosis is induced by enveloped viruses, since many viral proteins pass through the ER. This would be thwarted if SRP 72 was no longer capable of targeting nascent peptides to its receptor on the outer lumen of the ER. While the in vitroresults shown in Fig. 8 argue against this model, it remains possible that removal of the carboxyl terminus of SRP 72 during apoptosis may have a more subtle effect on ER transport in vivo, particularly since the cleaved carboxyl-terminal peptide is likely to harbor the serine phosphorylation site(s).Targeting of secretory proteins to the ER occurs by a strongly conserved, and presumably highly regulated, mechanism. However, little is known about how this process is governed in eukaryotic cells. Protein translation is regulated in response to exogenous stress at an early step (initiation) by several kinases, including: mitogen-activated protein kinases, which repress translation by phosphorylating the mRNA cap-binding protein eIF4E (30Sonenberg N. Gingras A. Curr. Opin. Cell Biol. 1998; 10: 268-275Crossref PubMed Scopus (503) Google Scholar); the interferon-inducible, double stranded RNA-regulated protein kinase, which inhibits translation by phosphorylating the eIF2 initiation factor (31de Haro C. Mendez R. Santoyo J. FASEB J. 1996; 10: 1378-1387Crossref PubMed Scopus (235) Google Scholar, 32Clemens M. Int. J. Biochem. Cell Biol. 1997; 29: 945-949Crossref PubMed Scopus (169) Google Scholar); the target of rapamycin which phosphorylates PHAS-1, a regulator of eIF4E; and the rapamycin-sensitive p70 S6 kinase, which modulates translation through phosphorylation of the S6 component of ribosomes (33Proud C. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Our observation that SRP 72 is phosphorylated on serine residue(s) in all cell types tested suggests that a serine kinase and/or phosphatase may regulate gene expression at a later step than the translation initiation checkpoint that is regulated by the above kinases.Our results are most consistent with caspase-mediated cleavage of the carboxyl terminus of SRP 72, generating a 6-kDa peptide containing the serine phosphorylation site(s). The fate of the 6-kDa fragment is unknown. We cannot, however, exclude the possibility that SRP 72 is a target for both a phosphatase and a caspase during apoptosis. It is also possible that a comigrating serine kinase (e.g. p70 S6 kinase) is coprecipitated with the SRP complex, and that the carboxyl terminus of SRP 72 is required for the interaction between the kinase and SRP. We have not consistently observed a serine kinase activity in these immunoprecipitates in in vitro kinase assays, however, arguing against the later possibility (data not shown).Comparison of the carboxyl terminus of SRP 72 from different organisms (Table I) shows striking conservation of eight serine residues (labeled with an asterisk, *) that are distal to the proposed caspase cleavage site (bold letters). Three of these serine residues are conserved from homo sapiens to Schistosoma mansoni (underlined). Although SRP 72 from Caenorhabditis elegans and Saccharomyces cerevisiae lacks these conserved serine residues, both proteins have carboxyl-terminal serines (i.e. amino acids 660, 663, and 693, C. elegans; aa 620 and 627, S. cerevisiae) that are potential phosphorylation sites. We have not yet determined whether SRP 72 derived from yeast, C. elegans, and S. mansoniare similarly phosphorylated in vivo.The results shown in Fig. 8 demonstrate cleavage of SRP 72 in vitro by caspase 3; however, the caspase(s) responsible for cleaving SRP 72 in vivo remain unidentified. Based on published data, it is most likely that SRP 72 is cleaved in vivo by a group III caspase such as caspase-6 (Mch 2), caspase-8 (MACH, FLICE, Mch 5), or caspase-9 (ICE-LAP6, Mch 6), reviewed in Ref.21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar. Group III caspases cleave non-DXXD motifs characteristically found in other caspases or in components of the nuclear or cytosolic skeleton (e.g. actin, Gas2, and nuclear lamins (21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar)). The SELD/A motif of SRP 72 most resembles the SELD/A cleavage site of SREBP-2, a transcription factor involved in regulation of cholesterol metabolism that is also associated with the endoplasmic reticulum (34Wang X. Pai J-T. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In support of the hypothesis that SRP 72 is also a substrate for group III caspases, we have observed cleavage of SRP 72 (generated in an in vitro translation reaction) by several group III caspases in vitro, including caspases 8 and 9. 4P. J. Utz, T. M. Le, S. J. Kim, M. E. Geiger, and P. Anderson, unpublished data. It will also be of great interest to determine if SRP 72 from C. elegans and S. cerevisiae (both of which lack a conserved caspase cleavage site), and SRP 72 from S. mansoni (which possesses such a site at665SEWD/A669) can be cleaved in vivoor in vitro.Autoantibodies directed against the 54-kDa polypeptide of SRP were first described independently in 3 patients with dermatomyositis (17Reeves W. Nigam S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9507-9511Crossref PubMed Scopus (139) Google Scholar,35Okada N. Mimori T. Mukai R. Kashiwagi H. Hardin J. J. Immunol. 1987; 138: 3219-3223PubMed Google Scholar). It is currently unknown whether phosphorylation or caspase-mediated cleavage of SRP 72 contributes to the production of autoantibodies reactive with components of the SRP complex. Several studies have identified autoantigens as constituents of membrane-bound blebs on the surface of apoptotic cells, where it has been proposed that they are ideally situated for presentation to the immune system (1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar, 36Casciola-Rosen L.A. Anhalt G. Rosen A. J. Exp. Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1527) Google Scholar, 37LeFeber W.P. Norris D.A. Ryan S.R. Huff J.C. Lee L.A. Kubo M. Boyce S.T. Kotzin B.L. Weston W.L. J. Clin. Invest. 1984; 74: 1545Crossref PubMed Scopus (289) Google Scholar, 38Golan T.D. Elkon K.B. Gharavi A.E. Krueger J.G. J. Clin. Invest. 1992; 90: 1067-1076Crossref PubMed Scopus (158) Google Scholar). Consistent with this hypothesis, we have observed SRP components as prominent constituents of apoptotic blebs by immunofluorescence.4 Like SRP 72, several proteins that are modified by caspases, or become associated with highly phosphorylated proteins during apoptosis are targets for autoantibody production in patients with autoimmune disease. The presence of autoantibodies reactive with components of the SRP is associated with a particularly aggressive form of dermatomyositis (39Targoff I. Johnson A. Miller F. Arthritis Rheum. 1990; 33: 1361-1370Crossref PubMed Scopus (226) Google Scholar). Whether this is a reflection of increased levels of apoptosis in affected tissues remains to be determined. SRP, a highly conserved cytoplasmic complex composed of a 7 S structural RNA molecule and 6 polypeptides, mediates the targeting of secretory proteins to the endoplasmic reticulum (26Walter P. Blobel G. Nature. 1982; 299: 691-698Crossref PubMed Scopus (460) Google Scholar, 27Walter P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7112-7116Crossref PubMed Scopus (302) Google Scholar). The intact particle has at least three separable activities: (i) binding to newly synthesized proteins bearing signal sequence as they emerge from the ribosome; (ii) elongation arrest during translation; and (iii) binding to the SRP receptor, leading to release from elongation arrest and translocation of the targeted protein into the lumen of the endoplasmic reticulum. Biochemical mutagenesis experiments have implicated individual domains of the SRP complex in each of these three functions (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 29Walter P. Blobel G. Cell. 1983; 34: 525-533Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Thus, the 54-kDa polypeptide is required for binding to the signal sequence, the 14- and 9-kDa polypeptides are involved in elongation arrest, and the 68- and 72-kDa proteins have been implicated in binding to the SRP receptor and promoting the directional translocation of newly translated proteins bearing a signal sequence into the lumen of the ER (28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar). The role played by the 7 S RNA molecule is currently unknown. Deletion analysis of canine SRP 68 and SRP 72 has demonstrated that these proteins associate with each other through their carboxyl termini, forming a stable complex with the 7 S RNA (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). A 57-kDa fragment that includes the amino terminus of canine SRP 72 (i.e. a smaller fragment than that generated by caspase cleavage of SRP 72) generated by elastase digestion is still capable of interacting in vitro with SRP 68, while a 42-kDa fragment is not (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Interestingly, an elastase-generated carboxyl fragment of ∼4 kDa was observed in this analysis, suggesting that a portion of the carboxyl terminus of SRP 72 is exposed when associated with other components of the SRP particle (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar). Our results are consistent with those of Lütcke et al. (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar) since caspase-cleaved SRP 72 remains associated with the SRP complex in immunoprecipitates (Fig.1 B), and migrates at ∼11 S by sucrose gradient centrifugation when comparing complexes prepared from untreated and apoptotic Jurkat cells (data not shown). SRP has been observed to migrate in a larger complex of ∼40 S (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar), and it remains possible that cleavage of SRP 72 may disrupt the formation of the larger complex. Based on biological and chemical mutagenesis experiments (14Lütcke H. Prehn S. Ashford A. Remus M. Rainer F. Dobberstein B. J. Cell Biol. 1993; 121: 977-985Crossref PubMed Scopus (40) Google Scholar, 28Siegel V. Walter P. Cell. 1988; 52: 39-49Abstract Full Text PDF PubMed Scopus (171) Google Scholar), we predict that cleavage of SRP 72 during apoptosis may inhibit binding of the complex to the SRP receptor on the endoplasmic reticulum membrane. This would result in (i) globally preventing the localization of secretory proteins to the endoplasmic reticulum; and (ii) irreversible elongation arrest of newly translated proteins bearing a signal sequence. While this is unlikely to play an important role in the execution phase of most types of cell death, cleavage of SRP 72 may play a critical role when apoptosis is induced by enveloped viruses, since many viral proteins pass through the ER. This would be thwarted if SRP 72 was no longer capable of targeting nascent peptides to its receptor on the outer lumen of the ER. While the in vitroresults shown in Fig. 8 argue against this model, it remains possible that removal of the carboxyl terminus of SRP 72 during apoptosis may have a more subtle effect on ER transport in vivo, particularly since the cleaved carboxyl-terminal peptide is likely to harbor the serine phosphorylation site(s). Targeting of secretory proteins to the ER occurs by a strongly conserved, and presumably highly regulated, mechanism. However, little is known about how this process is governed in eukaryotic cells. Protein translation is regulated in response to exogenous stress at an early step (initiation) by several kinases, including: mitogen-activated protein kinases, which repress translation by phosphorylating the mRNA cap-binding protein eIF4E (30Sonenberg N. Gingras A. Curr. Opin. Cell Biol. 1998; 10: 268-275Crossref PubMed Scopus (503) Google Scholar); the interferon-inducible, double stranded RNA-regulated protein kinase, which inhibits translation by phosphorylating the eIF2 initiation factor (31de Haro C. Mendez R. Santoyo J. FASEB J. 1996; 10: 1378-1387Crossref PubMed Scopus (235) Google Scholar, 32Clemens M. Int. J. Biochem. Cell Biol. 1997; 29: 945-949Crossref PubMed Scopus (169) Google Scholar); the target of rapamycin which phosphorylates PHAS-1, a regulator of eIF4E; and the rapamycin-sensitive p70 S6 kinase, which modulates translation through phosphorylation of the S6 component of ribosomes (33Proud C. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Our observation that SRP 72 is phosphorylated on serine residue(s) in all cell types tested suggests that a serine kinase and/or phosphatase may regulate gene expression at a later step than the translation initiation checkpoint that is regulated by the above kinases. Our results are most consistent with caspase-mediated cleavage of the carboxyl terminus of SRP 72, generating a 6-kDa peptide containing the serine phosphorylation site(s). The fate of the 6-kDa fragment is unknown. We cannot, however, exclude the possibility that SRP 72 is a target for both a phosphatase and a caspase during apoptosis. It is also possible that a comigrating serine kinase (e.g. p70 S6 kinase) is coprecipitated with the SRP complex, and that the carboxyl terminus of SRP 72 is required for the interaction between the kinase and SRP. We have not consistently observed a serine kinase activity in these immunoprecipitates in in vitro kinase assays, however, arguing against the later possibility (data not shown). Comparison of the carboxyl terminus of SRP 72 from different organisms (Table I) shows striking conservation of eight serine residues (labeled with an asterisk, *) that are distal to the proposed caspase cleavage site (bold letters). Three of these serine residues are conserved from homo sapiens to Schistosoma mansoni (underlined). Although SRP 72 from Caenorhabditis elegans and Saccharomyces cerevisiae lacks these conserved serine residues, both proteins have carboxyl-terminal serines (i.e. amino acids 660, 663, and 693, C. elegans; aa 620 and 627, S. cerevisiae) that are potential phosphorylation sites. We have not yet determined whether SRP 72 derived from yeast, C. elegans, and S. mansoniare similarly phosphorylated in vivo. The results shown in Fig. 8 demonstrate cleavage of SRP 72 in vitro by caspase 3; however, the caspase(s) responsible for cleaving SRP 72 in vivo remain unidentified. Based on published data, it is most likely that SRP 72 is cleaved in vivo by a group III caspase such as caspase-6 (Mch 2), caspase-8 (MACH, FLICE, Mch 5), or caspase-9 (ICE-LAP6, Mch 6), reviewed in Ref.21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar. Group III caspases cleave non-DXXD motifs characteristically found in other caspases or in components of the nuclear or cytosolic skeleton (e.g. actin, Gas2, and nuclear lamins (21Nicholson D. Thornberry N. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar)). The SELD/A motif of SRP 72 most resembles the SELD/A cleavage site of SREBP-2, a transcription factor involved in regulation of cholesterol metabolism that is also associated with the endoplasmic reticulum (34Wang X. Pai J-T. Wiedenfeld E.A. Medina J.C. Slaughter C.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 18044-18050Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In support of the hypothesis that SRP 72 is also a substrate for group III caspases, we have observed cleavage of SRP 72 (generated in an in vitro translation reaction) by several group III caspases in vitro, including caspases 8 and 9. 4P. J. Utz, T. M. Le, S. J. Kim, M. E. Geiger, and P. Anderson, unpublished data. It will also be of great interest to determine if SRP 72 from C. elegans and S. cerevisiae (both of which lack a conserved caspase cleavage site), and SRP 72 from S. mansoni (which possesses such a site at665SEWD/A669) can be cleaved in vivoor in vitro. Autoantibodies directed against the 54-kDa polypeptide of SRP were first described independently in 3 patients with dermatomyositis (17Reeves W. Nigam S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9507-9511Crossref PubMed Scopus (139) Google Scholar,35Okada N. Mimori T. Mukai R. Kashiwagi H. Hardin J. J. Immunol. 1987; 138: 3219-3223PubMed Google Scholar). It is currently unknown whether phosphorylation or caspase-mediated cleavage of SRP 72 contributes to the production of autoantibodies reactive with components of the SRP complex. Several studies have identified autoantigens as constituents of membrane-bound blebs on the surface of apoptotic cells, where it has been proposed that they are ideally situated for presentation to the immune system (1Utz P.J. Anderson P. Arthritis Rheum. 1998; 41: 1152-1160Crossref PubMed Scopus (181) Google Scholar, 36Casciola-Rosen L.A. Anhalt G. Rosen A. J. Exp. Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1527) Google Scholar, 37LeFeber W.P. Norris D.A. Ryan S.R. Huff J.C. Lee L.A. Kubo M. Boyce S.T. Kotzin B.L. Weston W.L. J. Clin. Invest. 1984; 74: 1545Crossref PubMed Scopus (289) Google Scholar, 38Golan T.D. Elkon K.B. Gharavi A.E. Krueger J.G. J. Clin. Invest. 1992; 90: 1067-1076Crossref PubMed Scopus (158) Google Scholar). Consistent with this hypothesis, we have observed SRP components as prominent constituents of apoptotic blebs by immunofluorescence.4 Like SRP 72, several proteins that are modified by caspases, or become associated with highly phosphorylated proteins during apoptosis are targets for autoantibody production in patients with autoimmune disease. The presence of autoantibodies reactive with components of the SRP is associated with a particularly aggressive form of dermatomyositis (39Targoff I. Johnson A. Miller F. Arthritis Rheum. 1990; 33: 1361-1370Crossref PubMed Scopus (226) Google Scholar). Whether this is a reflection of increased levels of apoptosis in affected tissues remains to be determined. We thank members of the Anderson laboratory and H. Li for insights and helpful comments; I. Miller and T. Gensler for critical review of the manuscript; the Brigham & Women's Hospital Clinical Immunology Laboratory, J. Craft, M. Kuwana, T. Medsger, N. Fertig, H. Lütcke, B. Dobberstein, N. Kedersha, and M. Robertson for providing antibodies used in this study; N. Kedersha for assistance with immunofluorescence; V. Hsu, M. Gupta, N. Kedersha, K. Hiramatsu, and D. Joyal for providing cell lines used in this study; H. Li and J. Yuan for providing cDNAs encoding recombinant caspases and substrates and for recombinant caspase 1; V. Shifrin for providing the p35 cDNA; and J. Reed for the gift of the bcl-2- and neo-overexpressing Jurkat cells." @default.
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W2171396586 title "The 72-kDa Component of Signal Recognition Particle Is Cleaved during Apoptosis" @default.
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