Caspase Activation of Mammalian Sterile 20-like Kinase 3 (Mst3)
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m202468200
ISSN1083-351X
AutoresChi‐Ying F. Huang, Yi-Mi Wu, Chiung‐Yueh Hsu, Wan-Shu Lee, Ming-Derg Lai, Te‐Jung Lu, Chia-Lin Huang, Tzeng‐Horng Leu, Hsiu-Ming Shih, Hsin-I Fang, Dan R. Robinson, Hsing‐Jien Kung, Chiun-Jye Yuan,
Tópico(s)Cell death mechanisms and regulation
ResumoMammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH2 terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD313, which is at the junction of the NH2-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis. Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH2 terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD313, which is at the junction of the NH2-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis. mitogen-activated protein kinase kinase mitogen-activated protein kinase/extracellular signal-regulated kinase germinal center kinase green fluorescent protein hemagglutinin 3-[ (3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid nuclear localization signal mammalian Sterile 20-like kinase human embryonic kidney Apoptosis (or programmed cell death) is a naturally occurring process that evolved early, and one that has been conserved throughout all the animal kingdoms (1Glucksmann A. Arch. Biol. 1965; 76: 419-437PubMed Google Scholar, 2Truman J.W. Annu. Rev. Neurosci. 1984; 7: 171-188Crossref PubMed Scopus (72) Google Scholar). The dysregulation of apoptotic cell death may be involved in the pathogenesis of a variety of human diseases, including cancers, autoimmune diseases, neurodegenerative disorders, and viral infection (3Sarraf C.E. Bowen I.D. Cell Tissue Kinet. 1988; 21: 45-49PubMed Google Scholar, 4Williams G.T. Cell. 1991; 65: 1097-1098Abstract Full Text PDF PubMed Scopus (831) Google Scholar, 5Raff M.C. Barres B.A. Burne J.F. Coles H.S. Ishizaki Y. Jacobson M.D. Science. 1993; 262: 695-700Crossref PubMed Scopus (1344) Google Scholar, 6Vaux D.L. Haecker G. Strasser A. Cell. 1994; 76: 777-779Abstract Full Text PDF PubMed Scopus (688) Google Scholar). Growing evidence suggests that although apoptotic stimuli vary from cell to cell, there seems to be a basic biochemical mechanism underlying this process, which involves the activation of a family ofCys-dependent, Asp-specific proteases known as the caspases (7Ellis R.E. Yuan J.Y. Horvitz H.R. Annu. Rev. 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Science. 1997; 276: 1571-1574Crossref PubMed Scopus (601) Google Scholar), protein kinase C isoforms δ (19Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 20Pongracz J. Webb P. Wang K. Deacon E. Lunn O.J. Lord J.M. J. Biol. Chem. 1999; 274: 37329-37334Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 21Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) and θ (22Datta R. Kojima H. Yoshida K. Kufe D. J. Biol. Chem. 1997; 272: 20317-20320Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), PKN (23Takahashi M. Mukai H. Toshimori M. Miyamoto M. Ono Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11566-11571Crossref PubMed Scopus (119) Google Scholar), Etk (24Wu Y.M. Huang C.L. Kung H.J. Huang C.Y. J. Biol. Chem. 2001; 276: 17672-17678Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), Mst1 (25Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar, 26Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar, 28Reszka A.A. Halasy-Nagy J.M. Masarachia P.J. Rodan G.A. J. Biol. Chem. 1999; 274: 34967-34973Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), Mst2 (26Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar), ROCK1 (29Coleman M.L. Sahai E.A. Yeo M. Bosch M. Dewar A. Olson M.F. Nat. Cell Biol. 2001; 3: 339-345Crossref PubMed Scopus (953) Google Scholar, 30Sebbagh M. Renvoize C. Hamelin J. Riche N. Bertoglio J. Breard J. Nat. Cell Biol. 2001; 3: 346-352Crossref PubMed Scopus (685) Google Scholar), SLK (31Sabourin L.A. Tamai K. Seale P. Wagner J. Rudnicki M.A. Mol. Cell. Biol. 2000; 20: 684-696Crossref PubMed Scopus (91) Google Scholar), and HPK1 (32Chen Y.R. Meyer C.F. Ahmed B. Yao Z. Tan T.H. Oncogene. 1999; 18: 7370-7377Crossref PubMed Scopus (66) Google Scholar). Several of the aforementioned kinases, including Mst1, Mst2, SLK, and HPK1, belong to the Sterile 20 family of serine/threonine kinases. Together, these observations imply that protein phosphorylation and/or dephosphorylation may play an essential role in apoptotic signal transduction. Sterile 20 was originally discovered as a component of the pheromone-response pathway in budding yeast (Ref. 33Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar; reviewed in Ref.34Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). The mammalian Sterile 20-related kinases represent a rapidly growing kinase family, with 28 members identified in humans at the time of writing. Some of these kinases activate mitogen-activated protein kinase cascades, and serve as cellular sensors that respond to numerous stimuli (reviewed in Refs. 34Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar and 35Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5262Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Furthermore, they are traditionally divided into the p21-activated kinase and germinal center kinase (GCK) families, which are distinguished by the relative location of their kinase domains. The p21-activated kinase domains were located at their COOH termini, whereas the GCK homologs are at their NH2 termini. Four Mst family kinases are already known and can be divided into two subgroups, GCK-II (Mst1 and Mst2) and GCK-III (Mst3 and Mst4), based on their sequence homology within and outside their kinase domains, and additional information obtained from the Drosophila and/or Caenorhabditis elegans orthologs (34Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). While sharing considerable homology in the kinase domain, the Mst kinases have different biological activities. For instance, Fas-mediated cleavage of Mst1/Mst2 is required for full activation of these kinases during apoptosis (26Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar). Overexpression of either full-length wild-type Mst1 or its truncated form in BJAB cells induces morphological changes characteristic of apoptosis (25Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar), suggesting that Mst1 is involved in this process. On the other hand, it has been demonstrated that Mst4, a newly identified Sterile 20-related kinase that shares 88% sequence similarity to Mst3 and SOK1 (36Qian Z. Lin C. Espinosa R. LeBeau M. Rosner M.R. J. Biol. Chem. 2001; 276: 22439-22445Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), mediates the growth and transformation of the tumor cell lines, HeLa and Phoenix, via the MEK/ERK pathway (37Lin J.L. Chen H.C. Fang H.I. Robinson D. Kung H.J. Shih H.M. Oncogene. 2001; 20: 6559-6569Crossref PubMed Scopus (81) Google Scholar); whereas Mst1- mediated apoptosis occurs via the c-Jun NH2-terminal kinase pathway (38Ura S. Masuyama N. Graves J.D. Gotoh Y. Genes Cells. 2001; 6: 519-530Crossref PubMed Scopus (100) Google Scholar). These findings indicate that the Mst family members may recognize distinct downstream targets, and hence, act through distinct signaling pathways. Analysis of the data raises interesting questions with respect to identification of the structural features that make Mst4 act differently to Mst1, and whether Mst3 might have a role in apoptosis (like Mst1), or cell transformation (like Mst4). In this study, we report that similar to Mst1 and Mst2 but not Mst4, Mst3 can be cleaved by caspases during apoptosis. Mst4 is resistant to cleavage, which may be the reason for cell transformation. The cleavage of Mst3 generates two fragments, 35 and 15 kDa, respectively. Unlike the conserved caspase-cleavage sites in Mst1 and Mst2, the caspase-cleavage site in Mst3 is mapped to AETD313G. Finally, ectopic expression of the truncated Mst3 results in kinase activation, nuclear translocation, and the induction of apoptosis. Taken together, these observations implicate the involvement of Mst3 in the process of apoptosis. All restriction enzymes were purchased from New England BioLabs. Fetal bovine serum, Dulbecco's modified Eagle's medium, penicillin, streptomycin, and LipofectAMINETM were purchased from Invitrogen. The [γ-32P]ATP was also from New England BioLabs. The Ac-DEVD-CHO was purchased fromCalbiochem. Monoclonal antibody against GFP (CLONTECH) was used to detect the presence of the enhanced green fluorescent protein (EGFP)-tagged proteins. The bacterially expressed carboxyl terminus of Mst3 (amino acids 310–432) was prepared from pET21-Mst3. Recombinant Mst3 protein was purified, and rabbit antiserum against the Mst3 carboxyl terminus was generated. All cell lines were purchased from ATCC and maintained in a humidified incubator at 37 °C in the presence of 5% CO2. HeLa, COS-1, and 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's modified Eagle's medium containing 10% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Transfection of cells was performed with LipofectAMINETM according to the manufacturer's instructions. The cDNAs, encoding full-length (amino acids 1–431) and truncated (amino acids 1–313) forms of Mst3, were amplified by reverse transcriptase-PCR using HeLa cell messenger RNA as the template. These constructs are referred to as Mst3WT and Mst3WTΔ314, respectively. The Mst3 cDNA fragments were inserted in-frame into the HA-tagged (at the COOH terminus) expression vector, pcDNA 3.0 (Invitrogen), and the EGFP-tagged expression vector, pEGFP-C2 (CLONTECH). The catalytically inactive mutants, Mst3KR and Mst3KRΔ314 (with Lys53 to Arg53mutation on the full-length and truncated Mst3), as well as caspase-cleavage site mutants (Mst3D301N, Mst3D302N, Mst3D307N, Mst3D309N, Mst3D313N, Mst3D321N, Mst3D324N, and Mst3D333N) of Mst3 were generated by site-directed mutagenesis using Mst3WT and Mst3WTΔ314 as the templates (QuikChangeTM site-directed mutagenesis kit, Stratagene). To induce apoptosis, Jurkat cells were treated with 250 ng/ml anti-Fas monoclonal antibody (clone CH11, Upstate Biotechnology Inc.) (39Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (965) Google Scholar) or 1 μm staurosporine (Calbiochem) (40Na S. Chuang T.H. Cunningham A. Turi T.G. Hanke J.H. Bokoch G.M. Danley D.E. J. Biol. Chem. 1996; 271: 11209-11213Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) at 37 °C for 5 h. To prepare the cell-free extracts, cells were lysed in lysis buffer (20 mm PIPES, pH 7.2, 100 mm NaCl, 1 mm EDTA, 0.1% CHAPS, 10% sucrose, 1 mmphenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 mm Na3VO4, and 10 μg/ml each of leupeptin, aprotinin, chymostatin, and pepstatin) (24Wu Y.M. Huang C.L. Kung H.J. Huang C.Y. J. Biol. Chem. 2001; 276: 17672-17678Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). After incubation at 4 °C for 30 min, cellular debris was removed by centrifugation. The TnT quick coupled transcription/translation system (Promega) was used to synthesize [35S]methionine-labeled proteins. The35S-labeled Mst3 or Mst4 proteins were incubated with various apoptotic extracts (150-μg extracts per reaction) or recombinant caspases (BD PharMingen) at 37 °C for 1 h. For caspase-inhibitor treatment, apoptotic extracts were preincubated with Ac-DEVD-CHO (Calbiochem) at 37 °C for 15 min before35S-labeled Mst3 proteins were added. COS-1 cells were transiently transfected with plasmids encoding various HA-tagged Mst3 proteins. Forty-eight hours after transfection, cells were lysed with modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 6 mm deoxycholate, 1% Igepal CA-630 (Sigma), 1 mm Na3VO4, 2 mm EGTA, 0.5% aprotinin, and 1 mm phenylmethylsulfonyl fluoride). Equal amounts of total lysates were immunoprecipited with anti-HA tag monoclonal antibody (3F10; Roche Molecular Biochemicals) and Protein A-agarose beads (Upstate Biotechnology Inc.) following standard procedures. Immune complexes were resuspended in kinase reaction buffer (20 mm PIPES, pH 7.4, 2 mm MnCl2, 10 mm MgCl2, 20 μm ATP, 10 μCi of [γ-32P]ATP, and 20 μg of histone H1) at 30 °C for 10 min. The kinase reaction mixtures or cell lysates were resolved using 12% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and detected with autoradiography or anti-HA antibody. The complexed IgGs were detected by incubation with secondary antibodies conjugated to horseradish peroxidase, and developed using the ECL system (Amersham Biosciences). The expression of EGFP-Mst3 in living cells was monitored using an inverted fluorescence microscope (Leica). The HEK293, 293T, and HeLa cells were cultured on chamber slides (LAB-TEK, Nagle Nunc) to about 70% confluency and transfected with pEGFP-Mst3 or its mutants. Twelve hours after transfection, the chamber slides were fixed with 5% paraformaldehyde containing 2% sucrose in phosphate-buffered saline at 4 °C for 20 min, followed by staining with 200 ng/ml 4′,6′-diamidino-2-phenylindole at 4 °C for 30 min. The fluorescence was analyzed using 4′,6′-diamidino-2-phenylindole (excitation/emission for 4′,6′-diamidino-2-phenylindole, 372/456 nm) and fluorescein isothiocyanate fluorescence filter cubes (488/510 nm). Photoimages were acquired using a Kodak digital camera, and then processed using Free PLUS software (Media Cybernetics, Silver Spring, MD). The β-galactosidase co-transfection assay for determination of cell death was performed as described earlier (41Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar,42Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Briefly, HEK293 cells in 35-mm culture dishes (about 70% confluency) were transiently transfected with a pCMV/LacZ plasmid (0.5 μg) together with 1 μg of pEGFP-Mst3, pEGFP-Mst3WTΔ314, pEGFP-Mst3KRΔ314, or pEGFP-C2 (vector) constructs. Thirty-six hours after transfection, cell lysates were prepared by incubating with 200 μl of lysis buffer at room temperature for 15 min, as has been described. To detect the β-galactosidase activity, 50 μl of cell lysates were mixed with 200 μl of PM2 buffer (0.1 m Na3PO4, 1 mm MgSO4, and 0.2 mmMnSO4) and 200 μl ofo-nitrophenyl-β-d-galactopyranoside (4 mg/ml) for 3 h, or until a yellow color developed, at 37 °C. The reaction was stopped by adding 0.5 ml of 1 m Tris base (pH 7.0) and the β-galactosidase activity was determined by reading the optical density at 420 nm. DNA fragmentation analysis of the HEK293 cells was performed as described previously (43Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (309) Google Scholar). Total DNA of the HEK293 (3 × 106) cells, with or without transfection of the various pEGFP-Mst3 plasmids, was isolated using a Puregene DNA Isolation Kit (Gentra Systems), as described in the manufacturer's instructions. The DNA pellet was redissolved in 50 μl of DNA-hydration solution. Equal amounts of each isolated DNA sample were separated on a 2% agarose gel and visualized under UV light. To examine whether Mst3 might serve as a caspase substrate during apoptosis, cell-free apoptotic extracts were first prepared from Jurkat T cells as a source of caspases. It has been demonstrated that Jurkat T cells are hypersensitive to a wide variety of apoptotic inducers, such as anti-Fas antibody (11Cross T.G. Scheel-Toellner D. Henriquez N.V. Deacon E. Salmon M. Lord J.M. Exp. Cell Res. 2000; 256: 34-41Crossref PubMed Scopus (614) Google Scholar, 39Enari M. Talanian R.V. Wong W.W. Nagata S. Nature. 1996; 380: 723-726Crossref PubMed Scopus (965) Google Scholar) and staurosporine (40Na S. Chuang T.H. Cunningham A. Turi T.G. Hanke J.H. Bokoch G.M. Danley D.E. J. Biol. Chem. 1996; 271: 11209-11213Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Treatment of Jurkat T cells with either anti-Fas antibody (CH11) or staurosporine for 5 h resulted in ∼30–40% cell death (Fig. 1), with a concurrent elevation of the activities of caspase 3, 6, 8, and 9 (3–5-fold; data not shown). The in vitro translated [35S]methionine-labeled Mst3 was proteolytically degraded in both apoptotic extracts, but remained intact when untreated extracts were used (Fig. 1). In addition, Mst3 cleavage followed the same time course as that for the activation of the caspases (data not shown), suggesting that caspase activity in these apoptotic extracts was likely responsible for the Mst3 cleavage. To test whether the cleavage of endogenous Mst3 also occurs in apoptotic cells, Jurkat T cells were treated with either anti-Fas antibody or staurosporine to induce apoptosis. Cell lysates were prepared, and immunoblot analysis was performed using an antibody against the Mst3 COOH terminus. Compared with the untreated Jurkat cell lysates, the data revealed a decrease in the amount of full-length Mst3 and the appearance of Mst3/C in the anti-Fas antibody and staurosporine-treated cells (Fig. 2). We were unable to detect the Mst3 NH2 terminus cleavage product because of the poor specificity of antibody against the Mst3 NH2 terminus. Similar cleavage patterns were also observed when HeLa cells were treated with staurosporine or tumor necrosis factor-α plus cycloheximide (data not shown). Several lines of evidence suggest that caspases are capable of Mst3 cleavage. First, the cleavage of [35S]methionine-labeled Mst3 in anti-Fas antibody (Fig. 3A) or staurosporine-induced apoptotic extracts (data not shown) was inhibited by Ac-DEVD-CHO, an inhibitor for caspase 3-like caspases. Second, proteolytic cleavage of the endogenous Mst3 was also partially blocked in the presence of Ac-DEVD-CHO when Jurkat T cells were treated with anti-Fas antibody (Fig. 3B) or staurosporine (not shown). Finally, [35S]methionine-labeled Mst3 was cleaved by purified recombinant caspases 3, 7, and 8 (Fig. 3C), generating two fragments (Mst3/N and Mst3/C) that mirror those observed for the apoptotic extracts. These results suggest that proteolytic cleavage of Mst3 is a general feature of apoptotic cells. It has been shown that the phosphorylation of presenilin-2 (45Walter J. Schindzielorz A. Grunberg J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1391-1396Crossref PubMed Scopus (109) Google Scholar) and Mst1 (46Graves J.D. Draves K.E. Gotoh Y. Krebs E.G. Clark E.A. J. Biol. Chem. 2001; 276: 14909-14915Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) regulates their cleavage by caspases. To verify if the kinase activity of Mst3 or its autophosphorylation were required for caspase-mediated cleavage, the invariant lysine (Lys53) in the ATP-binding pocket of Mst3 was mutated to arginine. The resultant mutant, Mst3KR, has neither kinase activity nor autophosphorylation ability, as determined by an in vitrokinase activity assay (will be discussed later). This mutant was transcribed/translated in vitro and treated with either apoptotic extracts or recombinant caspases. The kinetics of Mst3KR degradation were identical to that of the Mst3WT (data not shown), indicating that the kinase activity and/or autophosphorylation of Mst3 were not required for caspase-mediated cleavage. Previous studies have demonstrated that caspase 3 cleaves Mst1 (25Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar, 27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar, 28Reszka A.A. Halasy-Nagy J.M. Masarachia P.J. Rodan G.A. J. Biol. Chem. 1999; 274: 34967-34973Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 44Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (47) Google Scholar) and Mst2 (26Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar) at the consensus caspase-cleavage sites, DEMD326and DELD307, respectively (Fig.4, A and B). We have noticed that Mst3 also contains a potential caspase 3-recognition motif, DSGD324/W, in a similar region (Fig. 4, Aand B; the slash indicates the potential cleavage site in the amino acid sequence), suggesting that Mst3 may serve as a substrate for caspase 3 (or a caspase 3-like activity) during apoptosis. All the DXXD motifs of the Mst family members are located within the regulatory domain (Fig. 4A). Based on the size of the Mst3-cleavage products, it seems probable that D324/W is the candidate cleavage site (Fig. 4, A and B). The Asp324 site was therefore mutated to asparagine, and its susceptibility to cleavage by recombinant caspases was tested. Both the [35S]methionine-labeled Mst3WT and Mst3D324N mutants were cleaved by recombinant caspases 3, 7, and 8, indicating that D324/W was not an acceptor site for these caspases (Fig. 4E). Furthermore, [35S]methionine-labeled Mst4 was not cleaved by any of the recombinant caspases, including caspase 3, 7, and 8 (Fig.4C). These results suggest that the DXXD motif was not always the preferred cleavage site for caspases (especially the caspase 3) on their substrates, and that the cleavage properties of Mst3 and Mst4 (the GCK-III subfamily) are intrinsically different from that of Mst1 and Mst2 (the GCK-II subfamily). It was noted that there are several aspartic acids, such as Asp301, Asp302, Asp307, Asp309, Asp313, Asp321, and Asp333 (Fig.4D), in the vicinity of DSGD324. To identify the cleavage site for Mst3, these aspartic acids were individually mutated to asparagines, and their potential for caspase cleavage was tested by recombinant caspases 3, 7, and 8. Of all the mutants investigated, the Asp313 mutation blocked cleavage by recombinant caspases 3 (Fig. 4E), 7 (data not shown), and 8 (data not shown). Similar to Mst3WT, the rest of the mutants were cleaved by these recombinant caspases (Fig. 4E and data not shown). Similar experiments were carried out using cell-free apoptotic extracts and confirmed our findings (data not shown). Therefore, it is demonstrated that the caspase-cleavage site of Mst3 is at AETD313/G. Interestingly, there is no homologous site in Mst4, which may explain why Mst4 is not cleaved by caspases (Fig.4C). Caspase-mediated cleavage of Mst3 generates two fragments (Fig. 3C). To test whether Mst3 caspase cleavage changes its kinase activity, COS-1 cells were transiently transfected with HA-tagged Mst3 constructs encoding Mst3WTΔ314, Mst3KRΔ314, Mst3WT, or Mst3KR. Each sample was immunoprecipitated with anti-HA antibody and the immunocomplexes were subjected to a kinase-activity assay using histone H1 as the substrate. The kinase activity of Mst3WTΔ314 was about 10-fold higher than that of Mst3WT and no detectable kinase activity was observed for the catalytically inactive mutants of Mst3 (Fig.5). The most plausible explanation for these results is that caspase cleavage removes an inhibitory COOH-terminal regulatory domain from Mst3, thereby generating an active kinase. This is consistent with earlier data showing that deletion of the regulatory domain leads to a constitutively active Mst1 (25G
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