Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation.
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. We define Thr(183) in subdomain VIII as a primary site of phosphoactivation. Thr(187) is also critical for kinase activity. Phosphorylation of MST1 in subdomain VIII was catalyzed by active MST1 via intermolecular autophosphorylation, enhanced by homodimerization. Active MST1 (wild-type or T183E), but not inactive Thr(183)/Thr(187) mutants, was also highly autophosphorylated at the newly identified Thr(177) and Thr(387) residues. Cells expressing active MST1 were mostly detached, whereas with inactive MST1, adhesion was normal. Active MKK4, JNK, caspase-3, and caspase-9 were detected in the detached cells. These cells also contained 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
Helmut
H
Department of Bone Biology and Osteoporosis, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Rodan
Gideon A
GA
Reszka
Alfred A
AA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010010:Osteoclasts / Q000201:enzymology; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D000099317:Hippo Kinases
D053148:Caspase 3; D020169:Caspases; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins; C490660:Stk4 protein, mouse; D000099317:Hippo Kinases; C095566:STK4 protein, human; C482530:MAP2K3 protein, human; C482531:Map2k3 protein, mouse; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse