Phosphorylation and Dimerization Regulate Nucleocytoplasmic Shuttling of Mammalian STE20-like Kinase (MST)

Abstract

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, kinasedeficient 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.

Prognostic significance of mammalian sterile20-like kinase 1 in colorectal cancer

“…This is consistent with the finding that endogenous Mst1 accumulates in the nucleus upon treatment with leptomycin Ba specific inhibitor of nuclear export-suggesting that Mst1 shuttles continuously between cytoplasm and nucleus. 7,29 Univariate analysis in the MMR-proficient group showed that loss of cytoplasmic Mst1 was associated with higher T and N stages, vascular invasion and worse survival. These results are consistent with Mst1 function in induction of apoptosis and tumor suppression.…”

“…The pro-apoptotic function of Mst1-at least in part-is attributed to caspase-3 mediated cleavage of Mst1, although subsequent studies demonstrated that this is not essential for signaling and morphological features of apoptosis. 7 In fact, Mst1 has also shown to be activated by phosphorylation of Thr 183 and Thr 187 in its activation loop which is promoted by dimerization. Mst1 phosphorylation results in caspase activation and apoptosis 7,8 indicating that Mst1 might function both upstream and downstream of caspases.…”

“…7 In fact, Mst1 has also shown to be activated by phosphorylation of Thr 183 and Thr 187 in its activation loop which is promoted by dimerization. Mst1 phosphorylation results in caspase activation and apoptosis 7,8 indicating that Mst1 might function both upstream and downstream of caspases. Recent findings that most Ste20 group kinases activate mitogen-activated protein kinase (MAPK) and more specifically stressed-induced p38 MAPK and JNK (c-Jun N-terminal kinase) support this model ( Figure 1).…”

“…1,[8][9][10] Cellular localization of Mst1 is also regulated by caspase-mediated cleavage as well as threonine phosphorylation. 7,8,11 Under resting conditions, Mst1 is localized predominantly in the cytoplasm but cleavage of Mst1 by caspases or phosphorylation in its activation loop lead to Mst1 translocation to the nucleus. 7,8,11 Nuclear retention of Mst1 results in chromatin condensation and DNA fragmentation indicating that nuclear entry of Mst1 may be important in its pro-apoptotic function.…”

“…7,8,11 Under resting conditions, Mst1 is localized predominantly in the cytoplasm but cleavage of Mst1 by caspases or phosphorylation in its activation loop lead to Mst1 translocation to the nucleus. 7,8,11 Nuclear retention of Mst1 results in chromatin condensation and DNA fragmentation indicating that nuclear entry of Mst1 may be important in its pro-apoptotic function. 11,12 Extensive studies to identify potential Mst1 substrates in the nucleus have led to identification of Histone H2B as a physiologic substrate for the catalytic domain of Mst1, the phosphorylation of which leads to chromatin condensation and apoptosis.…”