Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).
Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.
Lee
Kyung-Kwon
KK
Institute for Virus Research and Graduate School of Biostudies, Kyoto University, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. kklee@virus.kyoto-u.ac.jp
Yonehara
Shin
S
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D020930:MAP Kinase Kinase Kinases; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D000903:Antibiotics, Antineoplastic; D005231:Fatty Acids, Unsaturated; D019319:Okadaic Acid; C038752:leptomycin A; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C093290:MAP3K10 protein, human; C490541:Map3k10 protein, mouse; D010744:Phosphoric Monoester Hydrolases