MST, a Physiological Caspase Substrate, Highly Sensitizes Apoptosis Both Upstream and Downstream of Caspase Activation
The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation. The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation. mammalian STE20-like kinase green fluorescence protein polymerase chain reaction monoclonal antibody p21-activated kinase tumor necrosis factor benzyloxycarbonyl- fluoromethylketone phosphate-buffered saline c-Jun N-terminal kinase stress-activated protein kinase polyacrylamide gel electrophoresis amino acids 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside Apoptosis or programmed cell death is a normal cell suicide mechanism that is highly conserved from nematode to human (1Vaux D.L. Haecker G. Strasser A. Cell. 1994; 76: 777-779Abstract Full Text PDF PubMed Scopus (690) Google Scholar,2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2436) Google Scholar). 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MST11 and MST2 belong to a mammalian SPS1/STE20-like kinase family, which is rapidly increasing in number (14Creasy C.L. Chernoff J. Gene (Amst.). 1995; 167: 303-306Crossref PubMed Scopus (119) Google Scholar, 15Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar, 17Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 18Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5662Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, the cellular function of MST as well as that of most other SPS1/STE20-like kinases is largely unknown. MST1 and MST2 have an N-terminal catalytic domain and C-terminal regulatory region, whereas other subfamily members such as p21-activated kinase (PAK) have a long N-terminal regulatory domain containing GTPase binding domain that confers the ability to bind to activated Cdc42 and/or Rac1 (17Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar,19Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 20Tapon N. Nagata K. Lamarche N. Hall A. EMBO J. 1998; 17: 1395-1404Crossref PubMed Scopus (181) Google Scholar). Notably, PAK2 is proteolytically activated by caspase in Fas-mediated apoptosis and has been reported to induce morphological changes of apoptosis (21Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar, 22Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13642-13647Crossref PubMed Scopus (175) Google Scholar). In contrast, the C-terminal regulatory region of MST does not have any known interaction motif, although this region is required for dimerization (23Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Some of the MST subfamily is responsive to cellular stress; inflammatory cytokines such as tumor necrosis factor (TNF) α can trigger activation of GCK, and SOK is responsive to oxidative stress (24Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar, 25Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (137) Google Scholar, 26Pombo C.M. Tsujita T. Kyriakis J.M. Bonventre J.V. Force T. J. Biol. Chem. 1997; 272: 29372-29379Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, MST1 is only activated by non-physiological stresses such as high temperature heat shock and high concentrations of sodium arsenite or staurosporine (16Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar). Previously we identified a protein kinase that is strongly activated in Fas-mediated apoptosis. Biochemical purification of its activity revealed the kinase to be a catalytic fragment of MST1 (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar). Fas-mediated activation of MST resulted from specific the caspase and by caspase suggesting the direct of MST by These that MST and other kinases are in apoptosis. In the study, we investigated the of MST in apoptosis. show that both MST1 and MST2 are cleaved by caspase in vivo and in vitro. The proteolytic reveals the activation mechanism of MST and its cellular we show that MST can induce morphological changes characteristic of apoptosis and the for death receptor-mediated apoptosis in cells. cDNA by K. A. Murakami and A. a from M. antibody and from antibody and antibody from antibody from and from human from MST expressed as a protein MST expressed in Human MST cDNA with a catalytic amino or in into C-terminal into caspase-3, cDNA of human caspase-3 N-terminal amino by and as and and into expressed in and as K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of MST as an N-terminal the in (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google and a The cDNA and by and in into cDNA of MST in into of the amino with and by DNA Human PAK2 cDNA and cDNA by from an cDNA K. S. Yonehara S. J. Biochem. 1998; PubMed Scopus Google and into and and with a of to as K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). containing for of and cloning of cells as K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). from with protein as K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Human human cell and cells in with and human and human cells in with and cells by with of or HeLa cells or before and with in with the HeLa cell with or The and in the of induce apoptosis, cells with (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, S. A. Yonehara M. J. PubMed Scopus Google Scholar), or staurosporine cells with with and in with and with and Cell by for cell with of or for and with protein Cell or by and to The in containing and with temperature for The with or for in and for with with activity an system or with the caspase-3 for The reaction by and with purification of C-terminal into and into cells to for and in containing Cell with of for and the reaction by The with and for The with of containing to the and cleaved gel and to The or with The fragment and by N-terminal (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). MST homologues of mouse and C. identified by and by as identified cDNA homologous to of a cDNA of cDNA a cDNA of mouse cells as cloning of the reported number and The cDNA from the cDNA by a or C. MST from a cDNA by and as The cDNA with a and with the sequences by Gene a The of and C. MST and MST or of MST with of and the of as by for (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google or complex kinase complex kinase with of in of kinase reaction with and containing and of for by of and for of the a gel and or by or HeLa cells and with of MST kinase and of a the cells with and with and in for with the cells with and in for and or HeLa cells with in in containing for and in in for for and in The antibody a for with with and in fluorescence of cells, in and the cells for before with and a and to a The Cell with in reaction and and for The reaction by an of and fluorescence cell and by for HeLa cells with or antibody both MST1 and whereas only apoptosis and a cleaved fragment of the the C-terminal fragment of and N-terminal fragment of MST1 and T. N. S. and S. analysis that and whereas only not N-terminal fragment although it in and by or and we the of MST1 and MST2 during Fas-mediated apoptosis in cells, which expressed both MST1 and a fragment of MST the of Fas This fragment by The of MST when apoptosis by the with a caspase and The of cleaved MST with that of the protein kinase from apoptotic that MST is cleaved to a catalytic fragment in apoptosis (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar). MST1 when apoptosis not or However, not MST1 when apoptosis These results suggest that MST1 cleaved to a fragment that not be by only we the of MST induces its activation by complex kinase as in the of by Fas ligation with the as of MST and DNA with MST activation for to Fas expressed MST1 and MST2 and with caspase-3 in vitro. MST1 and MST2 cleaved to the and the by These results suggest that both human MST1 and MST2 are physiological substrates of caspase-3 and are proteolytically activated by caspase in Fas-mediated apoptosis. the of sequences in human MST1 and MST2 and we and C. and and the sequences of MST2 number and MST1 and which show to and to human MST1 and Previously reported cDNA number that a the C-terminal region, which is by However, we not MST2 cDNA with or from a cDNA K.K. Murakawa M. S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). only MST which to mammalian MST1 and The kinase domain most with mammalian the kinase domain of to mammalian MST1 or The C-terminal amino acids required for dimerization highly conserved in homologues (23Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (172) Google The caspase-cleavable conserved in MST and homologues cleaved when apoptosis the for caspase is evolutionarily conserved in MST (16Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google identified a protein kinase responsive to stress which activated by with staurosporine not by Fas purification revealed it as MST (16Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar). However, that Fas ligation activated MST (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google we the MST activation by staurosporine and that by Fas ligation in cells that apoptosis staurosporine and Fas ligation the of MST1 to which the of by caspase-3 is to (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google to a fragment of In the of the fragment in and apoptosis. The for the be that staurosporine a of in which not by Fas investigated the kinase activity of MST1 fragment by as to MST1 by staurosporine and In contrast, Fas ligation not induce of MST1 to and rapidly by although this fragment not in Fas not induce a fragment in or kinase activity of kinase The fragment with kinase activity from not from by staurosporine and Fas ligation These results that staurosporine can MST by two activation and whereas Fas ligation activates MST only in a of to a fragment that in the C-terminal region of and this is the in apoptosis. cleaved to in apoptotic cell the of this we a C-terminal fragment from and N-terminal revealed that the and the to the identified caspase of MST that of both and to and is to proteolytic during Fas-mediated apoptosis. cleaved to the to the fragment not cleaved to a fragment that MST2 is cleaved only during apoptosis. In contrast, MST and cleaved to the and MST1 is cleaved two in a or and the fragment is cleaved to the function of MST in apoptosis, we cells with MST of the MST1 by with not induce MST1 The of with a C-terminal to the MST1 fragment by In with activity activity when MST2 expressed in MST1 changes in The morphological by MST1 from that by of an However, the by MST1 not by the with although that by by the morphological by MST1 with that by PAK2 (21Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar). when PAK2 into cells. N-terminal kinase domain changes in cell that kinase activity of MST1 or PAK2 is required for this morphological of in HeLa cells cells and with of of and resulted in the loss of cells by from This the of the cells in cells in The morphological by be the from the kinase activity or not induce any morphological investigated morphological changes in In HeLa cells the with cells or cells with The morphological of not although the and MST1 and localized in the cytoplasm. the cytoplasmic and cells from the In and fragmentation of by MST1 to in the not with However, cells not for and the characteristic DNA in apoptosis not gel These that not cleaved to by such as that of morphological changes are by of which the activation of MST in apoptosis. MST1 and localized in the when expressed as or N-terminal kinase domain localized in the cytoplasm, in both and nucleus. the cytoplasmic of MST1 is by nuclear HeLa cells MST1 or with an of N. T. Y. M. S. M. Cell 1998; PubMed Scopus Google Scholar, N. N. H. D. M. S. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). MST1 or rapidly cytoplasmic These results that cytoplasmic of MST1 is by a C-terminal regulatory region nuclear by the M. S. T. M. M. M. Nishida E. Nature. 1997; PubMed Scopus Google Scholar, M. M. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Science. 1997; PubMed Scopus Google Scholar). the of MST is in Fas-mediated apoptosis, we HeLa cell caspase-cleavable and MST1 the C-terminal of for of morphological and cell MST not show morphological with cell although expressed MST morphological In the cell and MST1 a cytoplasmic before of However, in apoptotic cells that of their nuclear MST1 and into the nucleus although MST1 and in the and In contrast, in the although the apoptosis as by the apoptotic of In apoptotic cells with nuclear cytoplasmic and in the the translocation the of the nuclear that MST1 is cytoplasmic protein and the cleaved MST1 translocates into the nucleus before the nuclear fragmentation is the function of MST in apoptosis, we investigated the of HeLa cell stably MST1 in and apoptosis. The of and by with or with antibody as a for and to or apoptosis HeLa cells or cells not or or apoptosis. the activation of in Fas-mediated apoptosis suggesting that MST1 can caspase-3 activation. this we investigated the of caspase-3 in in the of caspase-3 in cell and in cells. Furthermore, the of with that of caspase-3 the activation of caspase-3, MST1 to be cleaved by activated caspase-3 not the of other apoptosis by such as and that stably expressed MST1 sensitizes death receptor-mediated apoptosis the of caspase-3 activation. reported that during Fas-mediated apoptosis MST1 and MST2 are proteolytically activated by specific However, it MST1 and MST2 are in apoptosis. In this study, we show that MST1 and MST2 are activated by and in apoptosis. sequences of MST1 and MST2 are conserved and of other protein kinases known to be activated proteolytically by including and (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, J. 1998; PubMed Scopus Google Scholar). This only MST as a protein kinase that activated in Fas-mediated apoptosis (27Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar). sequences in MST1 and MST2 are conserved not only in their mammalian in the nematode strongly suggesting their including a role in apoptotic cell death The of MST be a in apoptosis apoptotic induce MST including from Fas and and apoptotic such as and for and such as have been reported to induce of MST in and cells J.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. R. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The of MST is with its activation. MST has kinase activity in cells, MST induces and activation of the catalytic Furthermore, has kinase activity the activation by In human in this which a catalytic fragment of The fragment is in the kinase whereas the catalytic fragment is only a by staurosporine or Fas that and of the fragment to the the we that human MST1 is cleaved and in apoptosis. However, it is not that the is a for MST1 activation is not conserved in In is to the fragment with kinase a specific function of the in apoptotic be it shows distinct catalytic activity from the show that MST is localized in the cytoplasm. analysis shows that the C-terminal region of MST is for cytoplasmic that is with of MST be regulated by nuclear although the that other nuclear or cytoplasmic protein MST MST by caspase results in the of catalytic In Fas-mediated apoptosis, cleaved MST1 translocates into the nucleus before nuclear fragmentation is This nuclear functions of cleaved MST1 in apoptosis. is in mammalian apoptotic cells and is with in apoptosis K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is of to cleaved MST in apoptosis is an and other functions of MST1 and MST2 reported to or the C-terminal region (23Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and of MST to the of N-terminal catalytic fragments. of MST during apoptosis results in and translocation of catalytic fragment into the nucleus. These regulatory in signaling be in the apoptotic function of of MST or MST induces morphological changes in cells. These changes are to the characteristic of apoptotic cells such as cell from and the fragmentation and of nucleus. However, we not other apoptotic such as of DNA or of apoptotic These morphological changes by MST are to by PAK2 (21Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar, 22Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13642-13647Crossref PubMed Scopus (175) Google Scholar). of of MST1 and MST2 does not Fas-mediated This that other members are in apoptosis. kinase, reported to be cleaved by caspase-3 and to induce M.A. Oncogene. 1999; PubMed Scopus Google Scholar, P. J. M.A. Cell. Biol. PubMed Scopus Google Scholar, E. K. S. Y. Y. H. PubMed Scopus Google Scholar). and apoptotic of and suggest that kinases are in apoptosis, Previously it that of MST1 activates caspase the kinase J.D. Y. Ambrose D. M. Chernoff J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). However, J.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google not the activation. the activation the caspase activation by of and caspase not cleaved in In the morphological of the cell by with caspase and morphological changes in the of caspase we that morphological changes by of MST not the activation of and not be in the morphological stably expressed in the MST1 highly sensitizes HeLa cells to or receptor-mediated apoptosis. to apoptosis by activation of This the of that caspase activation is not required for the morphological is that MST has two direct of morphological changes and to death receptor-mediated apoptosis. The apoptotic by expressed MST MST function downstream of This is by the that MST1 is a of and MST induces morphological changes that are stably MST caspase activation the of or or be that MST the of apoptotic or it is largely unknown MST is in apoptosis, shows that it as both an upstream and a downstream of MST death caspase activation and morphological to the and function of MST in cell physiological are the activation of MST by staurosporine that a mechanism of which is regulated by upstream or critical to the function of MST be from the analysis of protein kinases are regulated in K. A. M. EMBO J. 1998; 17: PubMed Scopus Google Scholar, Wang Y. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Nishida E. J. Cell Biol. PubMed Scopus (175) Google Scholar). the of as a mechanism cellular function of The of physiological to a of MST function in apoptosis or other cell Murakawa for of the MST