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Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1

1998; Springer Nature; Volume: 17; Issue: 8 Linguagem: Inglês

10.1093/emboj/17.8.2224

1460-2075

Jonathan D. Graves,

Heat shock proteins research

Article15 April 1998free access Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1 Jonathan D. Graves Jonathan D. Graves Present address: Department of Immunology, University of Washington Medical Center, Seattle, WA, 98109 USA Department of Pharmacology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Yukiko Gotoh Yukiko Gotoh Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, 606-01 Japan Present address: Department of Neurology, Children's Hospital, 300 Longwood Avenue, Enders 260, Boston, MA, 02115 USA Search for more papers by this author Kevin E. Draves Kevin E. Draves Department of Microbiology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Diane Ambrose Diane Ambrose Department of Pathology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author David K. M. Han David K. M. Han The Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA, 19111 USA Search for more papers by this author Michael Wright Michael Wright The Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA, 19111 USA Search for more papers by this author Jonathan Chernoff Jonathan Chernoff Department of Pathology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Edward A. Clark Edward A. Clark Department of Microbiology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Edwin G. Krebs Edwin G. Krebs Department of Pharmacology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Jonathan D. Graves Jonathan D. Graves Present address: Department of Immunology, University of Washington Medical Center, Seattle, WA, 98109 USA Department of Pharmacology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Yukiko Gotoh Yukiko Gotoh Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, 606-01 Japan Present address: Department of Neurology, Children's Hospital, 300 Longwood Avenue, Enders 260, Boston, MA, 02115 USA Search for more papers by this author Kevin E. Draves Kevin E. Draves Department of Microbiology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Diane Ambrose Diane Ambrose Department of Pathology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author David K. M. Han David K. M. Han The Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA, 19111 USA Search for more papers by this author Michael Wright Michael Wright The Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA, 19111 USA Search for more papers by this author Jonathan Chernoff Jonathan Chernoff Department of Pathology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Edward A. Clark Edward A. Clark Department of Microbiology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Edwin G. Krebs Edwin G. Krebs Department of Pharmacology, University of Washington Medical Center, Seattle, WA, 98109 USA Search for more papers by this author Author Information Jonathan D. Graves1,2, Yukiko Gotoh3,4, Kevin E. Draves5, Diane Ambrose6, David K. M. Han7, Michael Wright7, Jonathan Chernoff6, Edward A. Clark5 and Edwin G. Krebs2 1Present address: Department of Immunology, University of Washington Medical Center, Seattle, WA, 98109 USA 2Department of Pharmacology, University of Washington Medical Center, Seattle, WA, 98109 USA 3Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, 606-01 Japan 4Present address: Department of Neurology, Children's Hospital, 300 Longwood Avenue, Enders 260, Boston, MA, 02115 USA 5Department of Microbiology, University of Washington Medical Center, Seattle, WA, 98109 USA 6Department of Pathology, University of Washington Medical Center, Seattle, WA, 98109 USA 7The Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA, 19111 USA ‡D.Weitz and M.Zoche contributed equally to this work The EMBO Journal (1998)17:2224-2234https://doi.org/10.1093/emboj/17.8.2224 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mst1 is a ubiquitously expressed serine–threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment. CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA. Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1. Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response. Introduction Apoptosis, or programmed cell death, is an active process that is fundamental to the development and homeostasis of multicellular organisms (reviewed in Jacobson et al., 1997). It is characterized by dramatic cellular alterations, particularly membrane blebbing, cell shrinkage, chromosome condensation and fragmentation of DNA (reviewed in Dive et al., 1992). Apoptosis can be triggered by a wide variety of cellular stresses, including DNA damage, UV radiation, ionizing radiation, heat shock and oxidative stress, as well as by extracellular stimuli acting through cell-surface receptors (reviewed in Nagata, 1997). Central to the apoptotic execution pathway are a family of cysteine proteases, termed caspases, which are homologous to the Caenorhabditis elegans death gene ced-3 and are expressed as inactive zymogens (Alnemri et al., 1996; reviewed in Henkart, 1996). Ten mammalian caspases have now been identified and classified according to structure and substrate specificity. Overexpression studies have shown caspases to be capable of inducing all the characteristic features of apoptosis. Furthermore caspase inhibitors, such as the cowpox viral serpin CrmA, and cell permeable peptides such as ZVAD-fmk, block cell death induced by a wide variety of apoptotic agents (Ray et al., 1992; Enari et al., 1995, 1996; Los et al., 1995; Tewari and Dixit, 1995). The mechanism of activation of caspases is probably best understood for the Fas receptor. Fas (CD95/APO-1) is a transmembrane receptor belonging to the tumor necrosis factor (TNF) receptor family (Itoh et al., 1991; Oehm et al., 1992). Cross-linking Fas with Fas-ligand or agonistic antibodies results in rapid apoptosis of many cell types (reviewed in Nagata and Golstein, 1995). Although the role of Fas in other tissues is not clear, Fas-induced apoptosis plays an important role in the development of the lymphoid system and the maturation of the immune response (Brunner et al., 1995; Dhein et al., 1995; Rathmell et al., 1996). The intracellular domain of the Fas receptor contains a death domain, which is required for the induction of programmed cell death (Itoh and Nagata, 1993). The activated Fas receptor recruits other death domain-containing proteins including the adapter protein FADD/MORT-1, which in turn recruits caspase 8 via another dimerization domain termed the death effector domain (Chinnaiyan et al., 1995; Boldin et al., 1996; Muzio et al., 1996). Upon recruitment to activated Fas receptors, caspase 8 undergoes autoproteolytic activation. Active caspase 8 then cleaves and activates downstream caspases including caspases 1, 4 and 5, which in turn activate terminal or effector caspases such as caspases 3, 6 and 7 (Enari et al., 1996; Srinivasula et al., 1996). This cascade of sequential autoproteolytic events apparently transmits and amplifies the apoptotic signal (reviewed in Salvesen and Dixit, 1977). Identification of caspase substrates is essential in order to understand how these proteases induce the phenotypes associated with apoptosis. Caspase substrates include components of cellular DNA repair mechanisms such as poly ADP-ribose polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) (Lazebnik et al., 1994; Casciola Rosen et al., 1995; Nicholson et al., 1995); structural proteins such as actin, fodrin, lamin and gelsolin, which are likely to contribute to alterations in nuclear and cellular morphology (Cryns et al., 1996; Orth et al., 1996; Takahashi et al., 1996; Kothakota et al., 1997; Mashima et al., 1997); and IκB, an endogenous inhibitor of nuclear factor kB (NF-κB), suggesting one mechanism by which caspases may influence transcriptional events (Barkett et al., 1997). Furthermore, caspases can cleave components of signal transduction pathways such as D4-GDI, a regulator of Rho family GTPases (Na et al., 1996), the p21 activated kinase-2 (PAK2) (Rudel and Bokoch, 1997), the δ and θ isoforms of protein kinase C (PKC) (Emoto et al., 1995; Datta et al., 1997), PKC-related kinase 2 (PRK2) (Cryns et al., 1997), focal adhesion kinase (FAK) (Crouch et al., 1996) and MEKK1 (Cardone et al., 1997). The fact that caspase cleavage of several of these protein kinases results in stimulation of their kinase activity suggests that, as with other cell fate decisions, protein phosphorylation/ dephosphorylation mechanisms may play an important role in the initiation and progression of apoptosis. Activation of the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways has been observed to correlate with apoptosis induced by a variety of agents, including nerve growth factor withdrawal, B-cell receptor cross-linking and Fas ligation (Xia et al., 1995; Cahill et al., 1996; Graves et al., 1996; Wilson et al., 1996; Juo et al., 1997). Caspase inhibitors can block SAPK and p38 MAPK activation by Fas cross-linking, indicating that these pathways may function downstream of caspase activation (Cahill et al., 1996; Juo et al., 1997). However, overexpression of upstream components of the SAPK and p38 MAPK pathway such as MEKK1, which functions in the SAPK pathway, and MKK6b, an activator of p38 MAPK, can also induce caspase activity and cell death (Cardone et al., 1997; Huang et al., 1997). Although these results suggest that the SAPK and p38 MAPK pathways may function both upstream and downstream of caspases in the apoptotic response, the mechanism by which these pathways might influence caspase activity is unknown. Here we show that the ubiquitously expressed serine–threonine kinase Mst1, a mammalian homolog of the budding yeast Ste20 kinase, is cleaved and activated by caspase-mediated proteolysis in response to apoptotic stimuli. Overexpression of Mst1 induces caspase activity, morphological changes characteristic of apoptosis, and activates the SAPK and p38 MAPK pathways. Thus, Mst1 may function in a positive feedback pathway that amplifies the apoptotic response. Results During our analysis of CD95/Fas-induced apoptosis in the human B-lymphoma cell line BJAB, using myelin basic protein (MBP) in-gel kinase assays, we observed the induction of a 36 kDa kinase activity (Figure 1, upper panel). This kinase activity was first stimulated between 1 and 2 h after anti-Fas treatment, increased in a time-dependent fashion and persisted for at least 10 h. The kinetics of induction of this 36 kDa kinase paralleled the onset of apoptosis as determined by annexin V binding. In addition, the appearance of the 36 kDa activity correlated with the disappearance of a 63 kDa MBP in-gel kinase activity. Based on these observations, we hypothesized that the 36 kDa in-gel kinase activity resulted from cleavage of a 63 kDa kinase by an apoptotic protease. Consistent with this theory, pretreatment of the cells with the cell-permeable cysteine protease inhibitor ZVAD-fmk blocked both the induction of the 36 kDa kinase activity and the decrease in the 63 kDa activity. Staurosporine, a potent inducer of apoptosis in many cells, also stimulated the appearance of a 36 kDa kinase activity in BJAB cells (Figure 2 upper panel). As with the response to anti-Fas, induction of the 36 kDa kinase activity correlated temporally with the onset of apoptosis and could be inhibited by pretreatment with ZVAD-fmk. However, in contrast to the effects of anti-Fas treatment, staurosporine led to an increase in the 63 kDa kinase activity prior to its degradation. This increase in activity was detectable within 1 h of staurosporine treatment and decreased in parallel with the appearance of the 36 kDa kinase activity. In the presence of ZVAD-fmk, persistent activation of the 63 kDa kinase was observed in response to staurosporine treatment. Figure 1.Cleavage of Mst1 during Fas-induced apoptosis. BJAB cells were treated with 1 μg/ml anti-Fas. Where indicated, cells were pre-treated for 3 h with 50 μM ZVAD-fmk. At the indicated times cell extract was prepared and MBP in-gel kinase assays (upper panel) and Western blotting with anti-Mst1 antiserum (lower panel) performed as described in Materials and methods. The percentage of cells undergoing apoptosis was determined at each timepoint by annexin V binding assay. Download figure Download PowerPoint Figure 2.Cleavage of Mst1 during staurosporine-induced apoptosis. BJAB cells were treated with 100 nM staurosporine. Where indicated, cells were pre-treated for 3 h with 50 μM ZVAD-fmk. At the indicated times cell extract was prepared and MBP in-gel kinase assays (upper panel) and Western blotting with anti-Mst1 antiserum (lower panel) performed as described in Materials and methods. The percentage of cells undergoing apoptosis was determined at each timepoint by annexin V binding assay. Download figure Download PowerPoint In order to identify the 63 kDa kinase, we looked for a known kinase that migrated at this molecular mass, was renaturable, stimulated by staurosporine and sensitive to proteolysis yielding a catalytically active fragment of ∼36 kDa. One candidate was Mst1, a ubiquitously expressed 63 kDa serine–threonine kinase, which was originally cloned by virtue of its homology to the yeast Ste20 kinase (Creasy and Chernoff, 1995a; Taylor et al., 1996). Extract from BJAB cells was subjected to Western blotting using a polyclonal antibody raised against a peptide from the N-terminus of Mst1. This antibody recognized a single 63 kDa band in extract from unstimulated BJAB cells (Figure 1, lower panel). Upon anti-Fas treatment, the 63 kDa band diminished in intensity in parallel with the accumulation of a 36 kDa immunoreactive band. These results suggest that Mst1 is rapidly cleaved to generate a 36 kDa fragment upon treatment of BJAB cells with anti-Fas mAb (Figure 1, lower panel). The kinetics of Mst1 proteolysis, as well as the molecular mass of Mst1 and its cleavage product, match those of the 63 kDa and 36 kDa in-gel kinase activities. A similar pattern of Mst1 cleavage was also observed in extract prepared from staurosporine-treated BJAB cells (Figure 2, lower panel). An antibody raised against the C-terminus of Mst1 recognized only the 63 kDa fragment indicating that cleavage of Mst1 results in removal of the C-terminus (data not shown). Pretreatment of cells with ZVAD-fmk, a broad specificity inhibitor of caspases, blocked proteolysis of Mst1 in response to either anti-Fas or staurosporine treatment (Figures 1 and 2, lower panels). The fact that Mst1 exibited many of the characteristics of the 63 and 36 kDa in-gel kinase activities suggests that they might be identical. Additional studies, have provided confirmation that the 63 and 36 kDa in-gel kinase activities are Mst1 and a 36 kDa proteolytic product. Analysis of anion exchange chromatographic fractions by Western blotting with anti-Mst1 and MBP in-gel kinase assay revealed that immunoreactive Mst1 precisely co-eluted with the 63 and 36 kDa kinase activities (data not shown). Furthermore, the N-terminal Mst1 antibody specifically immunoprecipitates and depletes the 63 and 36 kDa MBP in-gel kinase activities from anti-Fas-treated BJAB extracts (data not shown). Having established the identity of the MBP in-gel activities as Mst1 and implicated caspases in its proteolysis during apoptosis, we further investigated the role of caspases in Mst1 cleavage. The cowpox virus serpin CrmA, which binds to and inhibits caspases 1 and 8, has previously been employed to implicate caspases in the cell death program (Ray et al., 1992; Tewari and Dixit, 1995; Tewari et al., 1995; Srinivasula et al., 1996). BJAB cells transfected with either wild-type CrmA, an inactive point mutant termed CrmA p14, or a vector control were treated with anti-Fas. As previously reported, cells expressing CrmA, but not CrmA p14 or vector control, were resistant to anti-Fas-induced apoptosis (Tewari and Dixit, 1995; Figure 3A). Mst1 cleavage induced by Fas cross-linking, as determined by in-gel kinase assay and Western blotting, was also blocked in the CrmA-expressing cells but not those expressing CrmA p14 or vector control (Figure 3). The use of peptide caspase inhibitors that are more selective than ZVAD-fmk has allowed further characterization of the caspases responsible for specific apoptotic events. Such inhibitors include DEVD-CHO, which is relatively selective for caspase 3 and its related subfamily (Nicholson et al., 1995), and YVAD-CHO, which is relatively selective for caspase 1 and its subfamily (Thornberry et al., 1992). Preincubation of BJAB cells with 100 μM DEVD-CHO almost completely blocked Fas-induced apoptosis and Mst1 cleavage as determined by both in-gel kinase activity and Western blotting (Figure 4). By comparison, YVAD-CHO was an inefficient inhibitor of Mst1 cleavage. Even at a concentration of 100 μM, YVAD-CHO only partially inhibited Mst1 cleavage induced by anti-Fas (Figure 4). These results suggest that Mst1 is cleaved in vivo by a DEVD-sensitive caspase such as caspase 3. Figure 3.CrmA blocks Mst1 cleavage. BJAB cells expressing either vector control, CrmA p14 or CrmA were treated with 1 μg/ml anti-Fas for the indicated time. Cell extract was prepared and MBP in-gel kinase assays (upper panel) and Western blotting with anti-Mst1 antiserum (lower panel) performed as described in Materials and methods. The percentage of cells undergoing apoptosis was determined at each timepoint by annexin V binding assay. Download figure Download PowerPoint Figure 4.Caspase inhibitors selective for the caspase 3 subfamily block Mst1 cleavage. BJAB cells were treated with the indicated concentration of either DEVD-CHO or YVAD-CHO for 3 h prior to the addition of 1 μg/ml anti-Fas. Cell extract was prepared and MBP in-gel kinase assays (upper panel) and Western blotting with anti-Mst1 antiserum (lower panel) performed as described in Materials and methods. The percentage of cells undergoing apoptosis was determined at each timepoint by annexin V binding assay. Download figure Download PowerPoint Analysis of the primary structure of Mst1 revealed an amino acid sequence that corresponds to a potential consensus caspase recognition site (DEMD326S) at the junction of the N-terminal catalytic domain and C-terminal regulatory domain (Figure 5A). To determine whether this site is the main caspase cleavage site in Mst1 we constructed a D326N mutant (DEMN326S) and used an in vitro assay system. Incubation of in vitro-translated, 35S-labeled, wild-type Mst1 with either apoptotic extract or caspase 3 resulted in the generation of a 36 kDa fragment that corresponded to the Mst1 fragment observed in vivo (Figure 5B left panel). In contrast, the D326N mutant was completely resistant to cleavage in vitro by either apoptotic extract or recombinant caspase 3 (Figure 5B right panel). These results establish the caspase cleavage site in Mst1 as being between aspartic acid 326 and serine 327 in the sequence DEMDS. The similarity between the cleavage patterns obtained in vivo and in vitro with recombinant caspase 3 lends further support to the idea that Mst1 cleavage is mediated by caspase 3 or a member of its related subfamily during apoptosis in vivo. Figure 5.Mst1 is cleaved in vitro by recombinant caspase 3. (A) A diagram showing the functional organization of Mst1. The location of the predicted caspase cleavage site, which resembles the consensus sequence for caspase 3 substrates, is indicated. The amino acid sequence surrounding the cleavage sites of several known caspase 3 substrates is provided for comparison. (B) Mst1 was transcribed and translated in vitro in the presence of 35S-labeled methionine. Labeled Mst1 was then incubated with either extract from untreated or anti-Fas-treated cells (left panel) or recombinant, purified, caspase 3 (right panel). Where indicated, 1 μM ZVAD-fmk was added simultaneously. Download figure Download PowerPoint A major question concerns the effect of cleavage at this site on Mst1 catalytic activity. Interestingly, deletion analysis of Mst1 has shown that removal of the regulatory domain results in an activated kinase fragment. Although our in-gel data appear to suggest that the Mst1 cleavage product is more active than full-length Mst1, we wanted to determine more directly whether Mst1 is activated by caspase-mediated cleavage. In order to do this, we immunoprecipitated Mst1 from BJAB cells that were either unstimulated or treated with anti-Fas and performed an immune-complex kinase assay with histone H1 as substrate. An increase in immunoprecipitated Mst1 activity was detected within 2 h of anti-Fas treatment and increased up to at least 8 h after stimulation (Figure 6, lower panel). This increase in Mst1 activity correlated temporally with Mst1 breakdown and induction of apoptosis (Figure 6, upper panel). In addition, preincubation of the cells with ZVAD-fmk blocked Mst1 cleavage and the increase in Mst1 immune-complex kinase activity. These results suggest that caspase-mediated cleavage during apoptosis results in an increase in the catalytic activity of Mst1. Figure 6.Caspase-mediated cleavage stimulates Mst1 kinase activity. BJAB cells were treated with 1 μg/ml anti-Fas. Where indicated, cells were pre-treated for 3 h with 50 μM ZVAD-fmk. At the indicated times cell extract was prepared and anti-Mst1 immune-complex kinase assays performed using histone H1 as substrate as described in Materials and methods (lower panel). Western blotting with anti-Mst1 antiserum was performed in parallel (upper panel). The percentage of cells undergoing apoptosis was determined at each time point by annexin V binding assay. Download figure Download PowerPoint Although the above studies establish that Mst1 is specifically cleaved and activated by caspases during apoptosis, the contribution of Mst1 to the cell death program is unclear. To address this question we transiently transfected BJAB human B lymphoma cells with Myc-tagged wild-type Mst1, Mst1 Δ330 or kinase dead Mst1 (K59R). Twelve hours post-transfection the cells were fixed for immunostaining and analyzed by fluorescent microscopy. Cells expressing either Myc-tagged wild-type Mst1 or Mst1 Δ330 displayed a profoundly shrunken morphology and nuclear condensation as determined by Hoechst 33342 (Figure 7A). In contrast, cells expressing Myc-tagged kinase-dead Mst1 were morphologically indistinguishable from cells that did not express Mst1. In repeated experiments, 60–70% of wild-type Mst1 transfectants and 70–80% of Mst1 Δ330 transfectants exhibited morphological changes characteristic of apoptosis (Figure 7A, right panel). Furthermore, analysis of transfectants at later times following transfection revealed that the proportion of cells positive for either wild-type Mst1 or Mst1 Δ330 was very significantly reduced relative to those staining for kinase-dead Mst1 (data not shown). Western blotting of extract prepared from cells between 6 and 48 h after transfection revealed that the expression level of both Myc-tagged wild-type Mst1 and the truncated Δ330 mutant declined rapidly with time and was barely detectable by 48 h after transfection (Figure 7C). In contrast, expression levels of the kinase-dead Mst1 were relatively stable and no evidence of cleavage was observed. Significantly, overexpression of wild-type Mst1 resulted in cleavage of exogenous Mst1 to generate a fragment that corresponded precisely to the 36 kDa fragment observed upon caspase-mediated proteolysis in vitro and apoptosis in vivo. This observation suggests that overexpression of Mst1 results in activation of caspases and is consistent with the morphological changes observed in such cells being a consequence of apoptosis. These findings also imply that overexpression of either wild-type Mst1 or Mst1 Δ330 is sufficient to induce caspase activity and apoptosis in BJAB cells and that Mst1 kinase activity is required for this process. Figure 7.Overexpression of Mst1 induces caspase activity and morphological changes characteristic of apoptosis. BJAB cells were transiently transfected with either wild-type Mst1, truncated Mst1 Δ330 or kinase-dead Mst1 K59R. All constructs were Myc epitope-tagged. (A) Twelve h after transfection the cells were fixed and stained with anti-Myc to determine Mst1 expression and Hoechst 33342 to allow analysis of nuclear morphology. Cell staining of a typical field is shown in the left panel and the percentage of cells staining positive for Mst1 that also exhibit a shrunken morphology with nuclear condensation is shown in the right panel. (B) Extract was prepared from transfected cells at the indicated times following transfection and subjected to Western blotting with anti-myc antibody. Download figure Download PowerPoint The downstream targets for Mst1 that might function in cell death pathways are unknown. However, since Ste20 functions in yeast MAPK pathways, Mst1 may regulate mammalian MAPK pathways (reviewed in Sells and Chernoff, 1997). In order to test this hypothesis we transiently co-transfected 293T cells with either wild-type Mst1, kinase-dead Mst1, or Mst1 Δ330 and MAPK, SAPK or p38 MAPK. 293T cells, which have been widely used for this type of study, have the advantages of being transfectable to high efficiencies and of expressing exogenous proteins at high levels. The MAPK constructs used were epitope-tagged with hemagglutinin to allow immunoprecipitation and measurement of activity by immune-complex kinase assay with the appropriate exogenous substrate. Expression of either wild-type Mst1 or Mst1 Δ330 in 293T cells induced morphological changes characteristic of apoptosis and similar to those observed upon Mst1 overexpression in BJAB cells (Y.Gotoh and J.Graves, unpublished observations). Co-expression of wild-type Mst1 or Mst1 Δ330 induced 7- and 5-fold activation of SAPK and p38 MAPK respectively while co-expression of kinase-dead Mst1 was without effect (Figure 8). The level of activation of SAPK and p38 MAPK by Mst1 was comparable to that induced by sorbitol treatment. In contrast, no detectable activation of MAPK in response to co-expression of wild-type or truncated Mst1 was observed. Western blotting with anti-HA confirmed that the expression level of the various MAPKs was not significantly influenced by co-expression of Mst1 constructs. In a continuation of these studies, the relationship between Mst1 and various MAPKKs was investigated using the same techniques. Consistent with its effect on SAPK and p38 MAPK, Mst1 activated MKK6 and MKK7 but had little effect on MKK4 and MKK3 (Figure 9). As might be predicted, Mst1 had no detectable effect on MKK1 activity. To investigate the role of the p38 MAPK pathway in Mst1-induced apoptotic pathways, the p38 MAPK inhibitor, SB203580, was added at a final concentration of 2 μM immediately after transfection with wild-type Mst1. In three repeat experiments, SB203580 inhibited Mst1-induced morphological changes in 293T cells, observed 18 h after transfection, by 60–70% relative to untreated control cells. These results suggest that p38 MAPK may constitute one pathway by which Mst1 mediates apoptotic changes. Figure 8.Mst1 activates SAPK and p38 MAPK. 293T cells were transfected with either wild-type Mst1, truncated Mst1 Δ330 or kinase-dead Mst1 K59R in addition to epitope-tagged MAPK, SAPK or p38 MAPK. Kinase activities were determined by immune-complex kinase assay with the appropriate substrate as described in Materials and methods (upper panel). Western blotting with anti-epitope tag antibodies shows the expression level of exogenous MAPKs (lower panel). The effect of either 10% FCS or 0.5 M sorbitol treatment on MAPK activities is shown for comparison. Download figure Download PowerPoint Figure 9.