Phosphorylation and Dimerization Regulate Nucleocytoplasmic Shuttling of Mammalian STE20-like Kinase (MST)
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m108138200
ISSN1083-351X
AutoresKyung-Kwon Lee, Shin Yonehara,
Tópico(s)Heat shock proteins research
ResumoMammalian 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. 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. MST 1The abbreviations used are: MSTmammalian STE20-like kinasePAKp21-activated kinaseGFPgreen fluorescence proteinNESnuclear export signalPBSphosphate-buffered salineLMBleptomycinNLSnuclear localization signalOAokadaic acidSTRstaurosporineMAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKKMAPK kinase kinase kinaseMAPKAP-2MAPK-activated protein kinase 2 1The abbreviations used are: MSTmammalian STE20-like kinasePAKp21-activated kinaseGFPgreen fluorescence proteinNESnuclear export signalPBSphosphate-buffered salineLMBleptomycinNLSnuclear localization signalOAokadaic acidSTRstaurosporineMAPKmitogen-activated protein kinaseMAPKKMAPK kinaseMAPKKKKMAPK kinase kinase kinaseMAPKAP-2MAPK-activated protein kinase 2 is a member of a subfamily of kinases that share high similarity in the catalytic domain with STE20. STE20 is a yeast mitogen-activated protein kinase kinase kinase kinase (MAPKKKK) (1.Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 2.Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar, 3.Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5662Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, the cellular function of MST as well as that of most other STE20-like kinases is largely unknown (3.Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5662Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 4.Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (262) Google Scholar). The non-catalytic C-terminal region of MST does not show any significant similarity with that of other family members and may function as a negative regulatory domain, because its removal markedly increases the kinase activity (5.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 6.Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar). It was reported that MST1 could be activated by stresses such as high temperature heat shock and high concentrations of sodium arsenite (2.Taylor L.K. Wang H.C. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar). In some mammalian cells, MST appears to function as an upstream kinase and be involved in c-Jun N-terminal kinase and p38 pathways (7.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar). mammalian STE20-like kinase p21-activated kinase green fluorescence protein nuclear export signal phosphate-buffered saline leptomycin nuclear localization signal okadaic acid staurosporine mitogen-activated protein kinase MAPK kinase MAPK kinase kinase kinase MAPK-activated protein kinase 2 mammalian STE20-like kinase p21-activated kinase green fluorescence protein nuclear export signal phosphate-buffered saline leptomycin nuclear localization signal okadaic acid staurosporine mitogen-activated protein kinase MAPK kinase MAPK kinase kinase kinase MAPK-activated protein kinase 2 MST1/MST2 is cleaved and activated by various apoptotic stimuli, including death receptor triggering and chemical apoptotic inducers such as staurosporine (STR), etoposide, and ceramide (6.Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar, 7.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar). In addition, bisphosphonate, a pharmacological drug for osteoporosis, and cytotrienin A, an anti-tumor drug, have been reported to induce cleavage of MST in osteoclast and leukemia cells (8.Reszka A.A. Halasy-Nagy J.M. Masarachia P.J. Rodan G.A. J. Biol. Chem. 1999; 274: 34967-34973Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 9.Watabe M. Kakeya H. Onose R. Osada H. J. Biol. Chem. 2000; 275: 8766-8771Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). MST1/MST2 is a direct substrate of caspase, and truncated MST is greatly activated by the removal of its regulatory region (6.Lee K.K. Murakawa M. Nishida E. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (117) Google Scholar, 7.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (321) Google Scholar, 10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Truncated MST strongly induces cell death, suggesting that some apoptotic events are mediated by cleavage-dependent activation of MST (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Interestingly, other kinases of the STE20 family such as PAK2, HPK1, and SLK, have been reported as caspase substrates, further implying that some of the family play an important role in apoptosis (11.Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (601) Google Scholar, 12.Chen Y.R. Meyer C.F. Ahmed B. Yao Z. Tan T.H. Oncogene. 1999; 18: 7370-7377Crossref PubMed Scopus (66) Google Scholar, 13.Sabourin L.A. Seale P. Wagner J. Rudnicki M.A. Mol. Cell. Biol. 2000; 20: 684-696Crossref PubMed Scopus (91) Google Scholar). Small molecules rapidly diffuse through the nuclear pore complex. It is reported that the upper size limit for macromolecules to diffuse through the nuclear pore complex is 50–60 kDa (14.Paine P.L. Moore L.C. Horowitz S.B. Nature. 1975; 254: 109-114Crossref PubMed Scopus (570) Google Scholar, 15.Feldherr C.M. Akin D. Int. Rev. Cytol. 1994; 151: 183-228Crossref PubMed Scopus (35) Google Scholar). One of the major mechanisms for the cytoplasmic localization of proteins is nuclear export system. Many cytoplasmic proteins involved in cellular signaling are transported actively to the cytoplasm by nuclear export signal (NES) (16.Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. 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Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar, 25.Wozniak R.W. Rout M.P. Aitchison J.D. Trends Cell Biol. 1998; 8: 184-188Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The classic NLS motif consists of short sequences (5–20 residues) containing several lysine and arginine residues (26.Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1843) Google Scholar, 27.Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1238) Google Scholar). In this study, we investigated the subcellular localization and analyzed the molecular mechanism that regulates the nucleocytoplasmic translocation of MST. MST is a cytoplasmic protein that has functional NES and NLS. Using a phospho-specific antibody, we directly show that MST is phosphorylated in an activation loop, and, importantly, the phosphorylated MST is rapidly translocated to the nucleus. We propose a molecular mechanism for the regulation of nucleocytoplasmic localization and suggest that MST directly transmits signals into the nucleus. Anti-MST monoclonal antibodies were prepared as described previously (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Anti-FLAG M2 antibody was obtained from Sigma Chemical Co., and Texas Red-conjugated anti-mouse IgG, Alexa Fluor 488-conjugated anti-mouse IgG, and Alexa Fluor 594-conjugated anti-rabbit IgG were from Molecular Probes. Rabbit phospho-PAK1 (Thr423)/PAK2 (Thr402) antibody was purchased from Cell Signaling Technology. Anti-Myc monoclonal antibody (9E10) was purified from hybridoma using a column of protein G-Sepharose (Amersham Biosciences, Inc.). Okadaic acid, staurosporine, and Hoechst 33342 were purchased from Calbiochem. Leptomycin was a generous gift from Dr. M. Yoshida (Tokyo University). Expression constructs of MST were prepared as N-terminal FLAG-tagged or GFP-fused forms. We introduced FLAG-tag after the initiation codon in pME18S vector as described previously (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28.Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). For the GFP fusion constructs, cDNA of human MST1 or human MST2 was subcloned in-frame into pCMX-SAH/Y145F (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Substitution of amino acid residues was performed with a QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing. pGEG plasmid, expressing two tandem GFP proteins with a central nuclear export signal (NES) sequence derived from Xenopus MAPKK (16.Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), was generated by subcloning in-frame into pCMX-SAH/Y145F. For the cloning of PAK2, total mRNA from HPB-ALL cells was prepared. Single-stranded cDNAs were synthesized by reverse transcription, using a First Strand cDNA Synthesis kit (Invitrogen), and then used as a template in polymerase chain reactions. Two primers, 5′-ggcctcgagatgtctgataacggagaactg-3′ and 5′-ggactgcagttaacggttactcttcattgc-3′, were used to specifically amplify the entire coding region of human PAK2. The amplified products were cloned into pME18S-FLAG vector. For kinase-deficient PAK2, Lys299 was changed to Arg by QuikChange site-directed mutagenesis. HeLa and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 μg/ml kanamycin. Cells were seeded on glass chamber slides (Nalge Nunc) or plates 24 h before transfection and then transfected with LipofectAMINE (Invitrogen) in accordance with the manufacturer's recommendation. HeLa and NIH 3T3 cells on chamber slides were washed with PBS and fixed in 3.7% formaldehyde in PBS for 10 min. Fixed cells were permeabilized in PBS containing 0.1% Triton X-100 for 10 min, and blocked in PBS containing 2% bovine serum albumin for 10 min. Cells were treated with anti-MST monoclonal antibody (1 μg/ml), anti-FLAG antibody (1 μg/ml), or anti-phospho antibody for 1 h. Texas Red-conjugated anti-mouse IgG, Alexa Fluor 488-conjugated anti-mouse IgG, and Alexa Fluor 594-conjugated anti-rabbit IgG were used at a dilution of 1:500 for 45 min. Cells were washed with PBS and incubated with Hoechst 33342 (0.2 μg/ml) for 5 min. Samples were observed, and images were collected using Axioplan2 microscope (Carl Zeiss) connected to charge-coupled device camera. The figures were prepared using Photoshop software (Adobe). After 16-h transfection, cells were washed once with PBS and suspended in cold lysis buffer, 10 mm Tris·HCl, pH 7.4, 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 50 mm NaF, 20 mm β-glycerol phosphate, 1 mm EDTA, and 1 mm EGTA with the protease inhibitor mixture, Complete (Roche Molecular Biochemicals). Cell lysates were cleared by centrifugation at 15,000 × g for 20 min. For immunoprecipitation, the lysate was incubated with 2 μg of anti-FLAG or anti-Myc antibody for 2 h and incubated with protein G-Sepharose (Amersham Biosciences, Inc.) for 2 h to overnight. Cell lysates or immunoprecipitates were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked in TBST buffer (20 mm Tris·HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween 20) with 5% skim milk at room temperature for 1 h. The membrane was incubated with antibody for 1 h, washed in TBST, and then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences, Inc.). After further washing with TBST, peroxidase activity was detected on x-ray films using an enhanced chemiluminescence detection system (DuPont). Cell lysates were centrifuged at 15,000 × g for 20 min and loaded onto a column of Superdex 200 HR 10/30. Chromatography was performed with fast protein liquid chromatography protein purification (Amersham Biosciences, Inc.) at 4 °C. The column was eluted with cold lysis buffer described above. The column was calibrated with protein standards (Amersham Biosciences, Inc.), including blue dextran, thyroglobulin, ferritin, catalase, bovine serum albumin, and ovalbumin. The eluted fractions were analyzed by Western blotting with anti-FLAG or anti-Myc antibody. HeLa cells were transfected with vectors encoding FLAG-MST1 and FLAG-PAK2, and cell lysates were immunoprecipitated with anti-FLAG antibody. Equal amounts of immunoprecipitates as analyzed by immunoblotting were used for immune-complex kinase assay. Immunoprecipitates were incubated with 2 μg of histone H2B in 20 μl of kinase reaction buffer, 40 mm Hepes, pH 7.5, 20 mm MgCl2, 20 mm β-glycerol phosphate, and 0.1 mm vanadate containing 25 μm ATP and 2.5 μCi of [γ-32P] ATP. Samples were incubated for 20 min at 30 °C, and reactions were terminated by adding 7 μl of Laemmli sample buffer and boiling for 5 min. A portion of the sample (15 μl) was separated on a 15% SDS-polyacrylamide gel and autoradiographed or analyzed by phosphorimaging. Our previous study revealed that overexpressed MST was localized exclusively in the cytoplasm (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). However, overexpressed proteins are not always localized in the same way as the endogenous proteins. To examine the subcellular localization of endogenous MST, we immunostained NIH 3T3 cells with two antibodies that recognize different epitopes on MST. The cytoplasm was specifically stained by indirect immunostaining with either J7B, which recognized the C-terminal region of MST, or G2B, which recognized the N-terminal catalytic domain of MST (Fig. 1A, top row) (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). A similar pattern of distribution was observed with fluorescein- or Texas Red-conjugated G2B or J7B. 2K. K. Lee and S. Yonehara, unpublished data. To investigate whether the cytoplasmic localization of MST is regulated by a nuclear export pathway, we treated NIH 3T3 cells with leptomycin (LMB), a specific inhibitor of nuclear export mediated by leucine-rich NES (29.Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (834) Google Scholar). MST rapidly lost its cytoplasmic localization within 20 min after LMB treatment (Fig. 1A, bottom row). Furthermore, prolonged incubation with LMB for 60 min resulted in the nuclear accumulation of MST, indicating that MST is localized to the cytoplasm in an LMB-dependent manner. Then, we examined whether MST has a polypeptide region responsible for cytoplasmic localization. Various deletion constructs of MST1 and MST2 were prepared as GFP-fused forms, and their subcellular localization was observed (Fig. 1B). The deletion of 320 N-terminal amino acid residues, including the kinase domain or 60 C-terminal residues contributing to dimer formation did not change the cytoplasmic localization, indicating that the kinase activity and dimerization of MST were not involved in its cytoplasmic localization. However, further deletion of a central region (323–381) of MST2 resulted in a diffused localization in the cytoplasm and nucleus. When this region was fused to GFP, only a cytoplasmic localization was observed, indicating that a peptide sequence within this region is capable of functioning as a signal for cytoplasmic localization. A closer look at the region corresponding to 323–381 in MST2 revealed that leucine-rich NES-like sequences were present in MST homologues in human, mouse, rat, and even Caenorhabditis elegans, suggesting evolutionary significance (10.Lee K.K. Ohyama T. Yajima N. Tsubuki S. Yonehara S. J. Biol. Chem. 2001; 276: 19276-19285Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). This region contained several conserved hydrophobic residues with typical and characteristic spacing, like the well-established NES (Fig. 2, A and B). To test whether the conserved residues are able to function as an NES, we mutated the hydrophobic residues to alanine (361A/365A and 368A/370A), and investigated the subcellular localization. Unexpectedly, FLAG-MST1361A/365A and FLAG-MST1361A/365A were still localized exclusively in the cytoplasm. The cytoplasmic localization did not change even when four residues were replaced with alanine (361A/365A/361A/365A). These results strongly raised the possibility that some other C-terminal region of MST is involved in the localization. Actually, MST1 and MST2 have long clusters of hydrophobic residues in C-terminal proximal regions (430–460) (Fig. 2A). Therefore, we introduced mutations in these hydrophobic residues and investigated the localization. As shown in Fig. 2C, when two residues in the C-terminal proximal region were mutated to alanine (439A/441A or 441A/444A), MST1439A/441A and MST1441A/444A still showed a cytoplasmic localization. However, when hydrophobic residues in both regions were mutated (MST1368A/370A/439A/441A and MST1368A/370A/441A/444A), the localization was changed to the nucleus (Fig. 2C). These results indicate that MST has two functional NES sequences (NES1 and NES2, see Fig. 2, A and B), and either NES1 or NES2 is sufficient for the cytoplasmic localization of full-length MST. The result that MST1 with mutations in both NES sequences (368A/370A/441A/444A, designated MST1-ΔNES) localized only in the nucleus prompted us to investigate whether MST has potential nuclear-localizing activity. This possibility was further supported by the data of Fig. 1A showing that endogenous MST was rapidly accumulated in the nucleus when cells were treated with LMB. We noticed peptide sequences near the C-terminal tail of MST1 and MST2, which is conserved throughout MST homologues and similar to the bipartite NLS consensus sequence (Fig. 2, A and B). To test whether this NLS-like sequence is capable of functioning as a NLS, the C-terminal 25 residues containing this sequence were fused to GFP (GFP-C25), and its subcellular localization was observed. As shown in Fig. 2D, GFP-C25 showed an intense nuclear localization while GFP was distributed throughout the cytoplasm and nucleus. MST1-ΔNES, which is exclusively nuclear (Fig. 2C, M9), was no longer accumulated in the nucleus and showed a diffused localization when lysine residues of the putative NLS were further replaced with asparagine (480N/481N), or when the C-terminal 25 residues were further removed (Fig. 2D). MST1-ΔC25, deleted of the 25 C-terminal residues, did not accumulate in the nucleus when cells were treated with LMB for over 3 h.2 Thus, the C-terminal NLS-like sequence functions as a NLS and induces the nuclear accumulation of MST. The apparent molecular mass of 60 kDa determined by SDS-PAGE and the value of 55 kDa calculated from the peptide sequence of MST are close to the limit of diffusion through the nuclear pore complex. Because MST was reported to form a dimer (5.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), we examined the relationship between dimer formation and the kinetics of the nuclear translocation. HeLa cells were co-transfected with wild-type Myc-MST1 and FLAG-MST1444P, mutated at Leu444 to proline, which is not capable of forming a dimer (5.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Because the cyclic structure of the proline reside could markedly influence the protein architecture, resulting in a misfolding, we co-transfected wild-type Myc-MST1 and other FLAG-MST1 mutants in which Leu444 was replaced with alanine (444A) or neighboring Leu448 with alanine (448A). The interaction of Myc-MST1 and FLAG-tagged mutant proteins was analyzed by immunoprecipitation with anti-Myc antibody (Fig. 3A). Wild-type FLAG-MST1 and FLAG-MST1448A were co-immunoprecipitated, whereas FLAG-MST1444P was not detected in the immunoprecipitates. FLAG-MST1444A was co-immunoprecipitated but less efficiently than wild-type FLAG-MST1. We then compared the elution profile of wild-type MST1 and MST1444P by size-exclusion chromatography (Fig. 3B). Wild-type MST1 was eluted at a peak fraction of 250 kDa as calculated from an elution profile of protein standards. However, MST1444P was eluted at the fraction around 130 kDa, being about half the molecular size of wild-type MST1. Next, we examined the LMB-induced translocation of these mutants in HeLa cells (Fig. 3C). MST1444P lost its cytoplasmic localization within 5 min, and most cells expressing MST1444P showed a nuclear accumulation of MST within 10 min of LMB treatment. In contrast, wild-type MST1 still showed a cytoplasmic localization at 30 min (Fig. 3C) and prolonged incubation induced localization change.2 The nuclear translocation of MST1444P was much faster than that of wild-type MST1 or endogenous MST1 (Figs. 1A and 3C). MST1448A, which showed strong activity to bind wild-type MST1, was translocated to the nucleus with much slower kinetics than MST1444P. MST1444A, with weak but significant binding activity for wild-type MST1, showed intermediate translocation kinetics between MST1444P and MST1448A. We conclude that monomeric MST is translocated into the nucleus more rapidly upon treatment with LMB, and dimerization facilitates the cytoplasmic retention of MST. A general method to measure the phosphorylation of protein is in vitro kinase assay, but some members of the STE20 kinase family are markedly activated by autophosphorylation in vitro (1.Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 30.Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (203) Google Scholar, 31.Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (133) Google Scholar). Therefore, the results of this assay greatly depend on the incubation time, significantly masking any difference between control and stimulated samples. PAK shares a nearly identical activation loop sequence in the kinase domain with MST, and the phosphorylation of the conserved threonine (Thr423 in PAK1, Thr402 in PAK2, and Thr183 in MST1) is required for autoactivation (32.Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 33.Zenke F.T. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 32565-32573Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34.Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (133) Google Scholar). In addition, anti-phosphopeptide antibody against the activation loop of PAK was reported to cross-react in vitro with phosphorylated MST (34.Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (133) Google Scholar). We tested whether a commercially available anti-phosphopeptide antibody to PAK1/PAK2 could cross-react with activated MST. We chose as an inducer for the activation of MST, okadaic acid (OA), a specific inhibitor of protein phosphatase 2A, because it activates many protein kinases and because phosphatase 2A is involved in various signaling pathways (35.Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). When HeLa cells were treated with OA, the anti-phosphopeptide antibody specifically recognized a band of around 60 kDa from cell lysates expressing FLAG-MST1 but not from untreated cell lysates (Fig. 4A). Unexpectedly, this band was not detected in cell lysates expressing FLAG-PAK2, whereas both forms of the protein were equally detected with anti-FLAG antibody. When Thr183 in MST1 was mutated to Ala, no band could be detected by OA treatment,2 indicating that Th
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