SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Oda, A. Articles by Fujita, H. Search for Related Content PubMed PubMed Citation Articles by Oda, A. Articles by Fujita, H. Related Collections Signal Transduction Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 March 2002, Vol. 99, No. 5, pp. 1850-1852 BRIEF REPORT Calpain is a signal transducer and activator of transcription (STAT) 3 and STAT5 protease Atsushi Oda, Hiroshi Wakao, and Hiroyoshi Fujita From the Laboratory of Environmental Biology, Department of Preventive Medicine, Hokkaido University School of Medicine, Sapporo, and the Helix Research Institute, Chiba, Japan.     Abstract Top Abstract Introduction Study design Results and discussion References Truncation of signal transducer and activator of transcription (STAT) 5 at the carboxy-terminal domain, either by genetic engineering or by proteolytic cleavage, results in generation of dominant-negative forms. A nuclear serine protease expressed in the myeloid precursor cells is known to mediate this cleavage, but other proteases responsible for this reaction were unknown. We found that calpain, a ubiquitously expressed cysteine protease, also trims STAT5 in vivo and in vitro, within the carboxy-terminal domain. Nuclear element is not necessary for calpain-mediated STAT5 cleavage, since this process occurs in platelets. We also found that STAT3 is a substrate for calpain in vivo and in vitro, indicating that calpain-mediated cleavage is a common feature of STAT3 and STAT5. Thus, our study reveals a novel pathway for posttranslational modification of STAT3 and STAT5. (Blood. 2002;99:1850-1852) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Many cytokines induce tyrosine phosphorylation and subsequent activation of signal transducers and activators of transcription (STATs) 5A and STAT5B, the products of 2 highly related STAT5 genes.1,2 Mice deficient in both genes were found to have defects in response to growth hormone and prolactin, and the functions of peripheral T lymphocytes were compromised.2 Loss of STAT5 also led to severe anemia in fetal mice.3 Recently, much attention has been focused on the dominant-negative forms of STAT5A and STAT5B, which lack carboxy-terminal domains.4-9 A serine protease was reported to cleave both isoforms, and there is evidence for a role of the truncated STAT5s in myeloid differentiation.6-9 We previously found that STAT3 and STAT5 are present in platelets and that tyrosine phosphorylation is induced on platelet stimulation by thrombopoietin.10-12 In addition, by using platelets, we and others identified numerous substrates for calpain, including p60src, PTP1B, and focal adhesion kinase.13-17 Therefore, in this study, we used platelets to find a novel nuclear-free cleavage pathway of STATs. We found that both STAT5 and STAT3 were substrates for calpain in vivo and in vitro.     Study design Top Abstract Introduction Study design Results and discussion References Calpeptin and µ-calpain were from Calbiochem18 (San Diego, CA). An anti-pan STAT5 monoclonal antibody (MoAb) and an anti-STAT3 MoAb against the amino-terminal domain of STAT3 were from Transduction Laboratories (Lexington, KY). The anti-STAT5A and anti-STAT5B polyclonal antibodies against the carboxyl-terminal domains of each STAT were from R&D Systems (Minneapolis, MN). A polyclonal antibody against the carboxy-terminal domain of STAT5 that reacts with both isoforms and a polyclonal antibody against the carboxy-terminal domain of STAT3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Dibucaine and human thrombin were from Sigma (St Louis, MO). Platelet preparation Washed platelets (3 × 108 cells/mL) were prepared as described previously10,11 and suspended for the experiments in modified HEPES-Tyrode buffer supplemented with apyrase (0.6 U/mL) and Arg-Gly-Asp-Ser peptide (200 µg/mL). Platelet stimulation and protein analysis Aliquots of platelets (0.5 mL; 3 × 108 cells/mL) were incubated in modified HEPES-Tyrode buffer supplemented with apyrase (0.6 U/mL) and Arg-Gly-Asp-Ser peptide (200 µg/mL).10,11 Preparation of platelet lysates and immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were done as described previously.10,11 In vitro cleavage of STATs by calpain Platelets were lysed and immunoprecipitation done as described previously.10,11 The precipitated proteins were cleaved in vitro by calpain as described previously.15     Results and discussion Top Abstract Introduction Study design Results and discussion References On gradient SDS-PAGE gels, the STAT5 bands often resolved into 2 closely comigrating bands (Figure 1A, left panel). The upper band comigrated with a band recognized by anti-STAT5A (Figure 1A, right panel), whereas the lower band comigrated with the lower band, reactive with anti-STAT5B. These 2 bands likely corresponded to STAT5A and STAT5B. When platelets were treated with dibucaine, a recognized activator of calpain,13-16 we observed a significant loss of immunoreactive STAT5 and the generation of bands (Figure 1A, left panel, arrowheads) of lower molecular weights, reactive with anti-STAT5 (Figure 1A, middle lane). Inhibition of calpain activation by calpeptin resulted in inhibition of cleavage of STAT5 (Figure 1A, right panel). None of these STAT5-like molecules reacted with antibodies against the carboxy-terminal domains of STAT5A or STAT5B (Figure 1A, middle and right panels), thereby indicating that the bands were STAT5 molecules, which lack the carboxy-terminal domains. Thus, truncation of the carboxy-terminal domains of STAT5 can occur independently of nuclear elements; and calpain, a cysteine protease, is probably responsible for truncation of STAT5. View larger version (27K): [in this window] [in a new window]   Figure 1. STAT5 is cleaved by calpain in vivo and in vitro. (A) Dibucaine treatment of platelets leads to cleavage of STAT5. The platelets were incubated with either calpeptin (20 µM) or dimethyl sulfoxide (DMSO; vehicle of calpeptin; final, 0.1%) for 5 minutes. The platelets were then treated with dibucaine (1 mM) for 15 minutes. Whole-platelet lysates (1.5 × 107 cells/lane) were analyzed by 7.5% to 15% gradient SDS-PAGE. The separated proteins were electrophoretically transferred from the gel to nitrocellulose membranes. STAT5 was detected by immunoblotting with a pan anti-STAT5 MoAb (left panel), anti-STAT5A (middle panel), or anti-STAT5B (right panel). Arrows indicate the relative position of intact STAT5; arrowheads show the positions of cleaved products of STAT5. R indicates control resting platelets; D, dibucaine-treated cells after incubation in DMSO; D + C, dibucaine plus calpeptin; and IB, immunoblotting. (B) In vitro cleavage of STAT5. STAT5 was precipitated by either anti-STAT5A or anti-STAT5B. The immunoprecipitates were untreated or incubated with purified µ-calpain in the presence or absence of calcium ion. After denaturing in SDS, STAT5 was detected by immunoblotting with a pan anti-STAT5 MoAb. (C) Truncated STAT5 molecules cleaved in vitro were not recognized by the anticarboxy-terminal domain of STAT5. IP indicates immunoprecipitation. Methods were the same as those as described in the legend for Figure 1B except that STAT5 was recognized by an antibody against the carboxy-terminal domain of STAT5. (D) Cleavage of STAT5 during platelet aggregation. Platelets were stimulated with thrombin (1 U/mL) for 30 minutes with or without stirring. After addition of ethyleneglycotetraacetic acid (5 mM) and EDTA (5 mM), platelets were lysed by boiling in SDS sample buffer. STAT5 was detected by immunoblotting as described in the legend for Figure 1A by using the pan anti-STAT5 MoAb. Lane 1 shows resting platelets; lane 2, results after thrombin stimulation of platelets for 30 minutes, with stirring, in the absence of Arg-Gly-Asp-Ser; and lane 3, results after thrombin stimulation for 30 minutes without stirring. The arrow indicates full-length STAT5 and the arrowhead a cleaved form of STAT5. We next investigated whether calpain cleaves STAT5 in vitro. STAT5A and STAT5B were immunoprecipitated, and the precipitates were incubated with µ-calpain in the presence or absence of ionized calcium. The data from the blot analysis shown in Figure 1B indicate that, after incubation with calpain, the apparent molecular weights of both STAT5A and STAT5B decreased in a fashion dependent on ionized calcium, resulting in the generation of multiple bands reactive with an anti-STAT5 MoAb. None of these generated bands were recognized by a polyclonal antibody against the carboxy-terminal domain of STAT5, thereby indicating that all of the bands were truncated STAT5 molecules devoid of the carboxy-terminal domain (Figure 1C). When platelets were activated with thrombin and simultaneously stirred to induce platelet aggregation, which activates calpain,19,20 a significant loss of STAT5 immunoreactivity was observed (Figure 1D), resulting in appearance of a major cleaved band (as in Figure 1A). The band was not recognized by anti-STAT5 antibodies reactive with the carboxy-terminal domain of STAT5 (data not shown). It is known that STAT3 variants (termed STAT3) can be generated through alternative splicing leading to a loss or major defect in the carboxy-terminal domain of STAT3.20 We also conducted experiments to assess the possibility that STAT3 is a substrate for calpain in platelets. The results of the blot analysis shown in Figure 2 clearly indicate that STAT3 is a substrate for calpain in vivo and in vitro. The anti-STAT3 MoAb used for the blotting analysis (Figure 2A, left panel, and Figure 2B) recognizes the amino-terminal domain of STAT3. Because the cleaved STAT3 molecules were not recognized by the anticarboxy-terminal domain of STAT3 (Figure 2A, right panel), these truncated STAT3 molecules must lack the carboxy-terminal domain while retaining a relatively intact amino-terminal domain. In addition, we found that cleavage of STAT3 also occurred during platelet aggregation (Figure 2C). Although these data do not directly indicate that calpain mediates cleavage of STATs on platelet aggregation, they are consistent with such a hypothesis. View larger version (26K): [in this window] [in a new window]   Figure 2. STAT3 is cleaved by calpain in vivo and in vitro. (A,B) Methods were the same as those described in the legends for Figures 1A and 1B, respectively, except that the blots were probed by using anti-STAT3 antibodies. (C) Cleavage of STAT3 during platelet aggregation. Methods were the same as those described in the legend for Figure 1C except that STAT3 was recognized by the anti-STAT3 MoAb as in Figure 2A. To our knowledge, this is the first report of calpain-mediated cleavage of STAT3 and STAT5 in vivo and in vitro. Although we found that µ-calpain cleaves STATs, µ-calpain may also be involved in truncation of STATs in platelets, since A23187-induced or thrombin-induced cleavage of several substrates for calpain was observed in platelets from µ-calpain null mice.17 Because truncation of STATs occurred in platelets, the nuclear element is not necessary for calpain-mediated trimming of STATs. Although truncation of the carboxy-terminal domain of STAT5 by a serine protease resulted in generation of a dominant-negative form,6-9 additional trimming of the carboxy-terminal domain of STAT5A due to alternative splicing has also been reported.21 Overexpression of truncated STAT5A (devoid of transactivation and Src homology 2 domains) in interleukin 3 (IL-3)-dependent FDCP-1 cells increased DNA-binding activity of endogenous (wild-type) STAT5, enhanced proliferation, and prevented apoptosis after deprivation of IL-3.21 Our finding that cleavage of both STATs by calpain led to generation of multiple bands, some of which had molecular weights similar to those in earlier reports, indicates that the effects of truncation of STATs by calpain are complex.     Footnotes Submitted May 25, 2001; accepted August 15, 2001. Supported in part by grants-in-aid from the Ministry of Education, Science and Technology of Japan (HF), Human Frontier Science Program (AO), Kaiun Mishima Memorial Foundation (AO), Mochida Memorial Foundation (AO), Mitsui Insurance Welfare Foundation (AO), Daiwa Health Foundation (AO), Ichiro Kanehara Foundation (AO), and Welfide Medicinal Research Foundation (AO). The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Atsushi Oda, Laboratory of Environmental Biology, Department of Preventive Medicine, Hokkaido University School of Medicine, N15W7, Kita-ku, Sapporo, 060-8638, Japan.     References Top Abstract Introduction Study design Results and discussion References 1. Wakao H, Gouilleux F, Groner B. Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J. 1994;13:2182-2191[Medline] [Order article via Infotrieve]. 2. Teglund S, McKay C, Schuetz E, et al. Stat5a and Stat5b proteins have essential roles and nonessential, or redundant, roles in cytokine responses. Cell. 1998;93:841-850[CrossRef][Medline] [Order article via Infotrieve]. 3. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF. Fetal anemia and apoptosis of red cell progenitors in Stat5a/5b/ mice: a direct role for Stat5 in Bcl-XL induction. Cell. 1999;98:181-191[CrossRef][Medline] [Order article via Infotrieve]. 4. Mui AL-F, Wakao H, Kinoshita T, Kitamura T, Miyajima A. Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation. EMBO J. 1996;15:2425-2433[Medline] [Order article via Infotrieve]. 5. Moriggl R, Gouilleux-Gruart V, Jaehne R, et al. Deletion of the carboxy-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol Cell Biol. 1996;16:5691-5700[Abstract]. 6. Azam M, Lee C, Strehlow I, Schindler C. Functionally distinct isoforms of Stat5 are generated by protein processing. Immunity. 1997;6:691-701[CrossRef][Medline] [Order article via Infotrieve]. 7. Meyer J, Juker M, Ostertag W, Stocking C. Carboxy-truncated Stat5 is generated by a nucleus-associated serine protease in early hematopoietic progenitors. Blood. 1998;91:1901-1908[Abstract/Free Full Text]. 8. Lee C, Piazza F, Brutsaert S, et al. Characterization of the Stat5 protease. J Biol Chem. 1999;274:26767-26775[Abstract/Free Full Text]. 9. Bovolenta C, Testolin L, Benussi L, Lievens PM, Liboi E. Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative Stat5. J Biol Chem. 1998;273:20779-20784[Abstract/Free Full Text]. 10. Miyakawa Y, Oda A, Druker BJ, et al. Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets. Blood. 1996;87:439-446[Abstract/Free Full Text]. 11. Ozaki K, Oda A, Wakao H, et al. Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. Blood. 1998;92:4652-4662[Abstract/Free Full Text]. 12. Oda A, Wakao H, Fujihara M, et al. Thrombopoietin and interleukin-2 induce association of CRK with STAT5. Biochem Biophys Res Commun. 2000;278:299-305[CrossRef][Medline] [Order article via Infotrieve]. 13. Oda A, Druker BJ, Ariyoshi H, Smith M, Salzman EW. pp60src is an endogenous substrate for calpain in human blood platelets. J Biol Chem. 1993;268:12603-12608[Abstract/Free Full Text]. 14. Frangioni JV, Oda A, Smith M, Salzman EW, Neel BG. Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. EMBO J. 1993;12:4843-4856[Medline] [Order article via Infotrieve]. 15. Cooray P, Yuan Y, Schoenwaelder SM, Mitchell CA, Salem HH, Jackson SP. Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain. Biochem J. 1996;318:41-47. 16. Fox JE. On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb Haemost. 1999;82:385-391[Medline] [Order article via Infotrieve]. 17. Azam M, Andrabi SS, Sahr KE, Kamath L, Kuliopulos A, Chishti AH. Disruption of the mouse µ-calpain gene reveals an essential role in platelet function. Mol Cell Biol. 2001;21:2213-2220[Abstract/Free Full Text]. 18. Tsujinaka T, Kajiwara Y, Kambayashi J, et al. Synthesis of a new cell penetrating calpain inhibitor (calpeptin). Biochem Biophys Res Commun. 1988;153:1201-1208[CrossRef][Medline] [Order article via Infotrieve]. 19. Wencel-Drake JD, Okita JR, Annis DS, Kunicki TJ. Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation. Arterioscler Thromb. 1991;11:882-891[Abstract/Free Full Text]. 20. Schaefer TS, Sanders LK, Nathans D. Cooperative transcriptional activity of Jun and Stat3, a short form of Stat3. Proc Natl Acad Sci U S A. 1995;92:9097-9101[Abstract/Free Full Text]. 21. Bittorf T, Sasse T, Wright M, et al. cDNA cloning and functional analysis of a truncated STAT5a protein from autonomously growing FDCP-1 cells. Cell Signal. 2000;12:721-730[CrossRef][Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: I. J. Smith, S. H. Lecker, and P.-O. Hasselgren Calpain activity and muscle wasting in sepsis Am J Physiol Endocrinol Metab, October 1, 2008; 295(4): E762 - E771. [Abstract] [Full Text] [PDF] B. Benayoun, S. Baghdiguian, A. Lajmanovich, M. Bartoli, N. Daniele, E. Gicquel, N. Bourg, F. Raynaud, M.-A. Pasquier, L. Suel, et al. NF-{kappa}B-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A FASEB J, May 1, 2008; 22(5): 1521 - 1529. [Abstract] [Full Text] [PDF] H. M. Lockyer, E. Tran, and B. H. Nelson STAT5 Is Essential for Akt/p70S6 Kinase Activity during IL-2-Induced Lymphocyte Proliferation J. Immunol., October 15, 2007; 179(8): 5301 - 5308. [Abstract] [Full Text] [PDF] J. W. Darnowski, F. A. Goulette, Y.-j. Guan, D. Chatterjee, Z.-F. Yang, L. P. Cousens, and Y. E. Chin Stat3 Cleavage by Caspases: IMPACT ON FULL-LENGTH Stat3 EXPRESSION, FRAGMENT FORMATION, AND TRANSCRIPTIONAL ACTIVITY J. Biol. Chem., June 30, 2006; 281(26): 17707 - 17717. [Abstract] [Full Text] [PDF] J. Zamorano, M. D. Rivas, F. Setien, and M. Perez-G Proteolytic Regulation of Activated STAT6 by Calpains J. Immunol., March 1, 2005; 174(5): 2843 - 2848. [Abstract] [Full Text] [PDF] D. W. Sternberg and D. G. Gilliland The Role of Signal Transducer and Activator of Transcription Factors in Leukemogenesis J. Clin. Oncol., January 15, 2004; 22(2): 361 - 371. [Abstract] [Full Text] [PDF] T. J. Mitchell, S. J. Whittaker, and S. John Dysregulated Expression of COOH-Terminally Truncated Stat5 and Loss of IL2-Inducible Stat5-Dependent Gene Expression in Sezary Syndrome Cancer Res., December 15, 2003; 63(24): 9048 - 9054. [Abstract] [Full Text] [PDF] J. G Tidball and M. J Spencer Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse J. Physiol., December 15, 2002; 545(3): 819 - 828. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Oda, A. Articles by Fujita, H. Search for Related Content PubMed PubMed Citation Articles by Oda, A. Articles by Fujita, H. Related Collections Signal Transduction Brief Reports Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020