The human serine/threonine protein kinases, Mst1 and Mst2, share considerable homology to Ste20 and p21-activated kinase (Pak) throughout their catalytic domains. However, outside the catalytic domains there are no significant homologies to previously described Ste20-like kinases or other proteins. To understand the role of the nonhomologous regions, a structure/function analysis of Mst1 was performed. A series of COOH-terminal and internal deletions indicates that there is an element within a central 63-amino acid region of the molecule that inhibits kinase activity. Removal of this domain increases kinase activity approximately 9-fold. Coimmunoprecipitation assays, the yeast two-hybrid procedure, and in vitro cross-linking analysis indicate that Mst1 homodimerizes and that the extreme COOH-terminal 57 amino acids are required for self-association. Size exclusion chromatography indicates that Mst1 is associated with a high molecular weight complex in cells, suggesting that other proteins may also oligomerize with this kinase. While loss of dimerization alone does not affect kinase activity, a molecule lacking both the dimerization and inhibitory domains is not as active as one that lacks only the inhibitory domain. Comparison of Mst1 and Mst2 indicates that both functional domains lie in regions conserved between the two molecules (Creasy, 1996).
Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. 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. These 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 (Graves, 1998).
The Fas system has been extensively investigated as a model of apoptosis and the caspase cascade has been shown to be a characteristic mechanism of signaling of apoptosis. A kinase has been identified and purified that is activated after the stimulation of Fas on human thymoma-derived HPB-ALL cells. Partial amino acid sequencing of the purified kinase revealed it to be MST/Krs, member of the yeast STE20 family of protein kinases. MST/Krs was activated by proteolytic cleavage and proteolytic activation was blocked by the caspase inhibitor, Z-VAD-FK. A mutant MST with Asp-->Asn replacement at a putative caspase cleavage site is resistant to either the proteolytic cleavage or the activation of the kinase activity. These findings suggest that proteolytic activation is one activation mechanism of MST and plays a role in apoptosis (Lee, 1998).
The serine/threonine kinase Mst1, a mammalian homolog of the budding yeast Ste20 kinase, is cleaved by caspase-mediated proteolysis in response to apoptotic stimuli such as ligation of CD95/Fas or treatment with staurosporine. Furthermore, overexpression of Mst1 induces morphological changes characteristic of apoptosis in human B lymphoma cells. Mst1 may therefore represent an important target for caspases during cell death, which serves to amplify the apoptotic response. Mst1 has two caspase cleavage sites, and evidence is presented indicating that cleavage may occur in an ordered fashion and be mediated by distinct caspases. Caspase-mediated cleavage alone is insufficient to activate Mst1, suggesting that full activation of Mst1 during apoptosis requires both phosphorylation and proteolysis. Another role of phosphorylation may be to influence the susceptibility of Mst1 to proteolysis. Autophosphorylation of Mst1 on a serine residue close to one of the caspase sites inhibited caspase-mediated cleavage in vitro. Finally, Mst1 appears to function upstream of the protein kinase MEKK1 in the SAPK pathway. In conclusion, Mst1 activity is regulated by both phosphorylation and proteolysis, suggesting that protein kinase and caspase pathways work in concert to regulate cell death (Graves, 2001).
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 NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. IEndogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, the caspase cleavage site, AETD(313), was mapped: it is at the junction of the NH(2)-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 (Huang, 2002).
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. MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologs 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 (Lee, 2001).
MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C-terminal regulatory domain, increasing the kinase activity of the MST1 N-terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified. The expression of MST1 results in caspase-3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co-expression of a dominant-negative mutant of JNK inhibits MST1-induced morphological changes as well as caspase activation. In contrast, neither a dominant-negative p38 nor the p38 inhibitor SB203580 inhibit the cellular changes. MST1 induces nucleosomal DNA fragmentation, which is suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase-Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation are not inhibited by caspase inhibitors. These results suggest that MST1 most likely promotes two events through JNK activation: (1) MST1 induces the activation of caspases, resulting in CAD-mediated DNA fragmentation, and (2) MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases (Ura, 2001a).
MST1 is a member of the Sterile-20 family of cytoskeletal, stress, and apoptotic kinases. MST1 is activated by phosphorylation at previously unidentified sites. This study examines the role of phosphorylation at several sites and effects on kinase activation. Thr(183) in subdomain VIII is defined as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII is catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, is also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 are mostly detached, whereas with inactive MST1, adhesion is normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contain all autophosphorylated and essentially all caspase-cleaved MST1. Similar phenotypes were elicited by a caspase-insensitive D326N mutant, suggesting that kinase activity, but not cleavage of MST1, is required. Interestingly, an S327E mutant mimicking Ser(327) autophosphorylation was also caspase-insensitive, but only when expressed in caspase-3-deficient cells. Together, these data suggest a model whereby MST1 activation is induced by existing, active MST kinase, which phosphorylates Thr(183) and possibly Thr(187). Dimerization promotes greater phosphorylation. This leads to induction of the JNK signaling pathway, caspase activation, and apoptosis. Further activation of MST1 by caspase cleavage is best promoted by caspase-3, although this appears to be unnecessary for signaling and morphological responses (Glantschnig, 2002).
Mammalian STE20-like kinase 2 (MST2), a member of the STE20-like kinase family, has been shown in previous studies to undergo proteolytic activation by caspase-3 during cell apoptosis. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. This study examined the mechanism of MST2 regulation with an emphasis on the relationship between caspase-3 cleavage and protein phosphorylation. Both the full-length MST2 and the caspase-3-truncated form of MST2 overexpressed in 293T cells exist in a phosphorylated state. However, the endogenous full-length MST2 from rat thymus or from proliferating cells is mainly unphosphorylated whereas the caspase-3-truncated endogenous MST2 from apoptotic cells is highly phosphorylated. Cell transfection studies using mutant MST2 constructs indicate that MST2 depends on the autophosphorylation of a unique threonine residue, Thr(180), for kinase activity. The autophosphorylation reaction shows strong dependence on MST2 concentration, suggesting that it is an intermolecular reaction. While both the full-length MST2 and the caspase-3-truncated form of MST2 undergo autophosphorylation, the two forms of the phosphorylated MST2 display marked differences in susceptibility to protein phosphatases. The full-length phospho-MST2 is rapidly dephosphorylated by protein phosphatase 1 or protein phosphatase 2A whereas the truncated MST2 is remarkably resistant to the dephosphorylation. Based on the present results, a novel molecular mechanism for MST2 regulation in apoptotic cells is postulated. In normal cells, because of the low concentration and the ready reversal of the autophosphorylation by protein phosphatases, MST2 is present mainly in the unphosphorylated and inactive state. During cell apoptosis, MST2 is cleaved by caspase-3 and undergoes irreversible autophosphorylation, thus resulting in the accumulation of active MST2 (Deng, 2003).
The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. All three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. It is concluded that MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V (Khokhlatchev, 2002).
MST1, mammalian STE20-like kinase 1, is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. MST1 is capable of inducing apoptotic morphological changes such as chromatin condensation upon overexpression. MST1 contains two functional nuclear export signals (NESs) in the C-terminal domain, which is released from the N-terminal kinase domain upon caspase-mediated cleavage. Full-length MST1 is excluded from the nucleus and localized to the cytoplasm. However, either truncation of the C-terminal domain, point mutation of the two putative NESs, or treatment with leptomycin B, an inhibitor of the NES receptor, results in nuclear localization of MST1. Staurosporine treatment induces chromatin condensation, MST1 cleavage, and nuclear translocation. Staurosporine-induced chromatin condensation is partially inhibited by expressing a kinase-negative mutant of MST1, suggesting an important role of MST1 in this process. Significantly, MST1 is more efficient at inducing chromatin condensation when it is constitutively localized to the nucleus by mutation of its NESs. Moreover, inhibition of MST1 nuclear translocation by mutation of its cleavage sites reduces its ability to induce chromatin condensation. Taken together, these results suggest that truncation of the C-terminal domain of MST1 by caspases may result in translocation of MST1 into the nucleus, where it promotes chromatin condensation (Ura, 2001b).
The protein kinase MST1 is proapoptotic when overexpressed in an active form, however, its physiologic regulation and cellular targets are unknown. An overexpressed inactive MST1 mutant associates in COS-7 cells with an endogenous 761-amino acid polypeptide known as 'death-associated protein 4' (DAP4). The DAPs are a functionally heterogeneous array of polypeptides isolated in a screen for elements involved in the interferon gamma-induced apoptosis of HeLa cells. DAP4, which is encoded by a member of a vertebrate-only gene family, contains no identifiable domains, but is identical over its amino-terminal 488 amino acids to p52(rIPK), a putative modulator of protein kinase R. DAP4 is a widely expressed, constitutively nuclear polypeptide that homodimerizes through its amino terminus and binds MST1 through its carboxyl-terminal segment. MST1 is predominantly cytoplasmic, but cycles continuously through the nucleus, as evidenced by its rapid accumulation in the nucleus after addition of the Crm1 inhibitor, leptomycin B. Overexpression of DAP4 does not cause apoptosis, however, coexpression of DAP4 with a submaximal amount of MST1 enhances MST1-induced apoptosis in a dose-dependent fashion. DAP4 is not significantly phosphorylated by MST1 nor does it alter MST1 kinase activity in vivo or in vitro. MST1-induced apoptosis is suppressed by a dominant interfering mutant of p53. MST1 is unable to directly phosphorylate p53, however, DAP4 binds endogenous and recombinant p53. DAP4 may promote MST1-induced apoptosis by enabling colocalization of MST with p53 (Lin, 2002).
DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. A 34 kDa apoptosis-induced H2B kinase has been identified as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provides insights into a previously unrecognized physiological substrate for Mst1 kinase. The data provide evidence for a potential apoptotic 'histone code' (Cheung, 2003).
Originally identified in Drosophila, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis reveals that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction is observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis ( Chan, 2005 ).
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