Mammalian STE20-like kinase 2 (MST2), a serine/threonine kinase considerably homologous to STE20 kinase of the budding yeast, has been shown activated in cellular apoptosis induced by a variety of stimuli. In most cases, MST2 activation during apoptosis has been reported to accompany caspase-3 cleavage of the enzyme. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. However, the role of MST2 in apoptosis remains puzzling, the upstream physiological activators, regulatory mechanism and downstream targets of the kinase are not known. In this study, I have shown that the kinase activity of both full-length and caspase-3- truncated MST2 depends on the autophosphorylation of a unique threonine residue, Thr¹⁸⁰, in the activation loop. The endogenous MST2...[ Read more ]

Mammalian STE20-like kinase 2 (MST2), a serine/threonine kinase considerably homologous to STE20 kinase of the budding yeast, has been shown activated in cellular apoptosis induced by a variety of stimuli. In most cases, MST2 activation during apoptosis has been reported to accompany caspase-3 cleavage of the enzyme. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. However, the role of MST2 in apoptosis remains puzzling, the upstream physiological activators, regulatory mechanism and downstream targets of the kinase are not known. In this study, I have shown that the kinase activity of both full-length and caspase-3- truncated MST2 depends on the autophosphorylation of a unique threonine residue, Thr¹⁸⁰, in the activation loop. The endogenous MST2 in proliferating cells is present in an unphosphorylated state, since autophosphorylation of the enzyme is an intermolecular reaction strongly depended on protein concentration. Furthermore, I have identified that heat shock protein 90 (HSP90), as a novel substrate and binding protein of MST2, participates in the regulation of MST2 during apoptosis. The association of unphosphorylated MST2 and HSP90 has been demonstrated predominantly in apoptotic cells. Characterization of the interaction between HSP90 and various MST2 mutants suggest that the HSP90-bound MST2 is more readily proteolyzed by caspase-3 than the free MST2. Upon MST2 autophosphorylation and subsequent phosphorylation of HSP90, the phospho-MST2-HSP90 complex dissociates and active MST2 kinase is then released. Dephosphorylation experiments using MST2 overexpressed in mammalian cells showed that the phosphorylated full-length and truncated forms of MST2 displayed differential susceptibility to protein phosphatases. The full-length phospho-MST2 could be rapidly dephosphorylated by protein phosphatase 1 or 2A, whereas truncated phospho-MST2 is remarkably resistant to the dephosphorylation thus “constitutively” active. Based on these findings, a model of MST2 regulation during apoptosis including phosphorylation, dephosphorylation, proteolysis by caspase-3, and association with HSP90 is postulated. In addition, I demonstrate that staurosporine inhibits MST2 but not as an activator of the kinase used in previous studies. Finally, a few phosphoproteins have been shown to be potential downstream targets of MST2 in this study.