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Mitosis-Dependent Phosphorylation and Activation of LIM-Kinase 1
Biochemical and Biophysical Research Communications 290, 1315–1320 (2002) doi:10.1006/bbrc.2002.6346, available online at http://www.idealibrary.com on
Mitosis-Dependent Phosphorylation and Activation of LIM-Kinase 1 Tomoyuki Sumi, Kunio Matsumoto, and Toshikazu Nakamura 1 Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
Received December 30, 2001
LIM-kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cellcycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements. © 2002 Elsevier Science (USA) Key Words: LIM-kinase; cofilin; actin cytoskeleton; mitosis; cyclins; CDKs; cell cycle; Rho family GTPase; phosphorylation; protein kinase.
The cytoskeletal network composed of actin filaments and microtubules in eukaryotic cells participates in cellular processes, including locomotion, shape changes, cytokinesis, and maintenance of polarity (1, 2). Microtubules play crucial roles in cell division: they form the mitotic spindle, which segregates duplicated chromosomes into a daughter cell (3, 4). Likewise, the actin-based cytoskeleton undergoes dramatic changes throughout the cell-division cycle, however, mechanisms by which actin cytoskeletal rearrangement is coordinated with cell cycle transition and assembly/ disassembly of mitotic apparatus have been unclear. Abbreviations used: LIMK, LIM domain-containing protein kinase; CDKs, cyclin-dependent protein kinases; GST, glutathione S-transferase. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-6-6879-3789. E-mail: [email protected]. osaka-u.ac.jp.
LIM-kinases (LIMK1 and LIMK2) (5– 8) regulate actin cytoskeletal reorganization downstream of Rho family GTPases and LIMKs-induced actin cytoskeletal rearrangement is mediated by cofilin, an actin-depolymerizing factor of actin filaments (9 –11). Activated LIMK catalyzes phosphorylation of an N-terminal 3rd serine residue of cofilin and inhibits its activity to depolymerize actin filaments, thereby leading to stabilization of actin filaments (12, 13). We reported that XLIMKs, the Xenopus counterpart of mammalian LIMKs (14), are critically involved in the progression of progesterone-induced Xenopus oocyte maturation through Xenopus cofilin phosphorylation (15). XLIMKcofilin system was required for the organization of the microtubule-derived precursor of the meiotic spindle. Likewise, using knockout mice of LIMK2, we recently demonstrated that LIMK2 plays an important role in the proper progression of spermatogenesis by regulating of cofilin activity in mammalian germ cells (16). Therefore, LIMKs may be a key component of a fundamental signal transduction system that connects extracellular stimuli to intracellular actin cytoskeletal rearrangements, in a distinct biological context. Although it seems likely that LIMKs may play some roles in coordinated regulation of actin cytoskeletal rearrangement involved in the cell cycle progression during mitosis and/or meiosis, regulation of LIMKs activity during the cell cycle transition has yet to be defined. We now report that LIMK1 activity increases during mitosis and activation of cyclin-dependent kinases (CDKs) is directly or indirectly involved in mitosis-dependent activation of LIMK1. Our results suggest that LIMK1 may participate in the coordination of cell cycle progression and mitosis-dependent actin cytoskeletal organization. MATERIALS AND METHODS Materials. Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgG, and Hoechst 33342 were purchased from Molecular Probes, Inc. (Eugene, OR). Anti-␣-tubulin monoclonal antibody (DM1A) and roscovitine were purchased from
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Sigma (St. Louis, MO) and Calbiochem (Darmstadt, Germany). AntiLIMK1 antibody was purified as described elsewhere (11). Cell culture and synchronization of cell cycle. HeLa cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum and nonessential amino acids. Cells were synchronized at the G1/S boundary by a double thymidine block. In brief, cells were treated with 2 mM thymidine for 24 h, followed by an 8-h release in fresh MEM with 10% fetal bovine serum and successive retreatment with 2 mM thymidine for 14 h. Cells synchronized at the G1/S boundary were released to enter the cell cycle in fresh MEM with 10% fetal bovine serum. At various time points after release, these cells were harvested by trypsinization. Aliquots were subjected to flow cytometric analysis, and the remaining cells were used to prepare whole cell extracts, as described below. For flow cytometric analysis, the cells were fixed with 70% ethanol for 20 min, then stained with propidium iodide (20 g/ml) and treated with RNase A (1 mg/ml) for 10 min at room temperature. Samples of 10,000 cells were then analyzed on a FACSCalibur (Becton–Dickinson, San Diego, CA). To arrest exponentially growing HeLa cells at prometaphase, nocodazole was added to make up a final concentration of 1 g/ml for 16 h, then the cells were collected and extracted as described below. Immunoprecipitation, immunoblot analysis, and protein kinase assay. HeLa cells were lysed with lysis buffer composed of 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 25 mM -glycerophosphate, 10 mM NaF, 1 mM Na 3VO 4, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml leupeptin and aprotinin for 20 min. After centrifugation, the supernatant was incubated for 3 h at 4°C with an anti-LIMK1 antibody and 5 l Protein A–Sepharose (Amersham Pharmacia Biotech, Little Chalfont, UK). Immunoprecipitates were washed three times with lysis buffer, dissolved in the sample buffer for SDS–PAGE, and separated by SDS–PAGE, electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and probed with an anti-LIMK1 antibody. Proteins reacting with antibody were detected using ECL enhanced chemiluminescence (Amersham Pharmacia Biotech). The band densities of immunoblots were analyzed using NIH Image software (Wayne Rasband Analytics, National Institutes of Health). For the measurement of LIMK1 activity, immunoprecipitates were incubated for 20 min at 30°C in 15 l of kinase buffer consisting of 50 mM Hepes–NaOH (pH 7.5), 25 mM -glycerophosphate, 5 mM MgCl 2, 5 mM MnCl 2, 10 mM NaF, and 1 mM Na 3VO 4, 50 M ATP, 5 Ci of [␥- 32P]ATP (6000 Ci/mmol; Perkin–Elmer Life Sciences) and 6 g of GST-fused cofilin as the substrate. Histone H1 kinase assay was done in kinase buffer consisting of 50 mM Tris–HCl (pH 7.5), 10 mM MgCl 2, 2 mM DTT, 1 mM EGTA, 100 M ATP, 5 Ci of [␥- 32P]ATP and 1 g histone H1 together with each cell lysate containing 5 g total protein. After incubation for 20 min at 30°C, the reaction was terminated by heat-treatment (100°C for 3 min) in sample buffer for SDS–PAGE, and then the samples were subjected to SDS–PAGE, and analyzed by autoradiography. Protein phosphatase treatment. Immunoprecipitates prepared using anti-LIMK1 antibody were washed three times with 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, then the supernatant was removed. The reaction buffer for protein phosphatase (New England BioLabs, Inc., Beverly, MA) was added either with or without 100 units of Lambda Protein Phosphatase. Samples were incubated for 30 min at 30°C, reactions were stopped by heat-treatment (100°C for 3 min) in sample buffer for SDS–PAGE, and samples were subjected to SDS–PAGE and subsequent immunoblotting.
RESULTS Phosphorylation and Activation of LIMK1 during Mitosis We previously reported that LIM-kinases, LIMK1 and LIMK2, are involved in the cell cycle progression,
using Xenopus oocyte and LIMK2-knockout mice (15, 16). To further address the cell-cycle-dependent regulation of LIMKs activity, we determined if kinase activity of LIMKs is altered during the cell cycle. HeLa cells were synchronized at the prometaphase by nocodazole-treatment, and the LIMKs activity was measured using GST-fused cofilin as the substrate. As shown in Fig. 1A (top), LIMK1 activity toward cofilin phosphorylation in mitotic cells was about fourfold higher than that in asynchronized cells, whereas LIMK2 activity did not significantly change during cell cycle (data not shown). Similarly, histone H1 kinase activity, a marker for mitosis, in nocodazole-treated cells was much higher than that in asynchronized cells, which indicated that cells were arrested at mitosis by activation of the mitotic checkpoint (Fig. 1A, bottom). In immunoblot analysis of LIMK1, electrophoretic mobility of the LIMK1 in mitotic cells was clearly slower than that in asynchronized cells (Fig. 1A, middle). We speculated that the mobility shift might be due to phosphorylation of LIMK1 during mitosis. To examine such possibility, LIMK1 immunoprecipitated from asynchronized and nocodazole-treated HeLa cells were, respectively, incubated with protein phosphatase then subjected to immunoblotting (Fig. 1B). Without phosphatase-treatment, the slower mobility shift of LIMK1 was observed in mitotic cells; however, the mobility shift of LIMK1 in mitotic cells was diminished by phosphatase-treatment. These results indicate that LIMK1 was specifically phosphorylated and activated in cells arrested at the mitotic checkpoint by nocodazoletreatment. Activation of LIMK1 during Mitosis To determine whether the activation of LIMK1 induced by nocodazole treatment occurred specifically as a result of a spindle disassembly or nonspecifically irrespective of the cell cycle, cell-cycle-dependent regulation of LIMK1 activity was measured in synchronized cells. HeLa cells were synchronized at the G1/S boundary by a double thymidine block method then the cells were released to enter the cell cycle. Cells were harvested at different times after the release from the G1/S arrest and analyzed for LIMK1 activity toward cofilin phosphorylation. In parallel, the DNA content at each time point was analyzed by flow cytometry (Fig. 2A). The LIMK1 activity was low in cells at early stages of the cell cycle (0 – 8 h after release from the G1/S arrest), whereas it thereafter increased and reached a maximal level when cells passed through mitosis (10 –12 h after release from the G1/S arrest) (Fig. 2B, top). By 14 h after release from the G1/S arrest, the bulk of the cell population completed mitosis and reentered G1 phase and this cell cycle transition was associated with a decrease in LIMK1 activity. Consistent with the change in LIMK1 activity, electro-
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were in the G1, S or G2 phase (Fig. 2B, lower middle). To provide a marker for the timing of mitosis, histone H1 kinase activity responsible for activity of cyclindependent kinases (CDKs) (particularly Cdk1 at mitosis) was also determined for each sample (Fig. 2B, bottom). Similar to LIMK1 activity, histone H1 kinase activity reached a maximal level 10 h after release
FIG. 1. Mitosis-specific activation (A) and phosphorylation (B) of LIMK1. (A) HeLa cells were either asynchronized or synchronized with nocodazole in prometaphase of mitosis. Cells were lysed, and protein kinase activity of LIMK1 was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting (middle). Histone H1 kinase activity in each cell lysate was also measured (bottom). Cofilin phosphorylation was estimated using NIH Image software (right). Protein kinase activity of LIMK1 in asynchronized cells was taken as 1.0. Each value represents the mean ⫾ standard error for experiments done in triplicate. (B) Immunoprecipitated LIMK1 prepared from asynchronized and synchronized HeLa cells with nocodazole were, respectively, incubated with or without protein phosphatase. Samples were analyzed by immunoblotting, using an anti-LIMK1 antibody.
phoretic mobility shift of LIMK1 was observed in cells 10 h after release from the G1/S arrest, whereas the mobility shift of LIMK1 was not detected when cells
FIG. 2. Cell cycle-dependent activation of LIMK1. (A) Flow cytometric analysis of cell cycle transition. Exponentially growing HeLa cells were synchronized at the G1/S boundary by a double thymidine block. Cells were harvested at 0, 4, 8, 10, 12, and 14 h after the release from the G1/S arrest, and cell cycle profiles were obtained by flow cytometry. (B) Cell cycle-dependent changes in LIMK1 activity and histone H1 kinase activity. At the indicated time points, cells were lysed, and LIMK1 activity was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. Cofilin phosphorylation was estimated using NIH Image software and protein kinase activity of LIMK1 in cells at 0 h after the release was taken as 1.0 (upper middle). The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting, using an anti-LIMK1 antibody (lower middle). Histone H1 kinase activity in each cell lysate was also measured (bottom).
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from the G1/S arrest (the time when most of cells were in mitosis) and thereafter decreased. Taken together, these results indicate that phosphorylation and activation of LIMK1 occurs when cells enter into mitosis, similar to the profile of Cdk1 activation during cell cycle transition. CDKs Kinase Activity Is Required for LIMK1 Activation during Mitosis Since the protein complex of cyclin B and its partner Cdk1 plays a crucial role in the regulation of mitosis (17–19), we addressed whether activation of Cdk1 is required for LIMK1 activation during mitosis. Cells in G2/M-phase were treated with the CDKs inhibitor roscovitine, and LIMK1 and CDKs activities were measured using GST-cofilin and histone H1 as the substrate, respectively. Although roscovitine inhibits Cdk1, Cdk2, and Cdk5 (20, 21), Cdk1 is the major activity present in M-phase cells (17–19). To exclude effects of roscovitine on cell cycle progression, treatment of cells with roscovitine was done 8 h after the release from G1/S arrest. At 8 h after the release, most of cell population had reached at the G2/M phase, then passed through mitosis during 10 –14 h after the release (Fig. 3A). Consistent with above results, activities of LIMK1 and CDKs displayed a similar cell cycle dependency (Fig. 3B). Both activities reached a maximal level 10 h after the release, then decreased after cell division. Cells treated with roscovitine were arrested at the G2/M phase after a 10 h release and cell cycle transition into G1 phase was not observed (Fig. 3A). Under this condition, condensed chromosomes were seen in roscovitine-treated cells, thereby suggesting that roscovitine arrested at prophase on the cell cycle (data not shown). Importantly, roscovitine-treatment diminished increases in LIMK1 activity as well as CDKs activity during mitosis (Fig. 3B). These observations suggest that CDKs kinase activity is directly or indirectly required for activation of LIMK1 at mitosis. DISCUSSION We earlier reported that the activities of LIMkinases, LIMK1 and LIMK2, are distinctly regulated by Rho family GTPases (11). ROCK, a Rho-dependent protein kinase, specifically activates LIMK2 (22), whereas MRCK␣, a downstream effector of Cdc42, activates both LIMK1 and LIMK2 (23). LIMK1 is also activated by Pak downstream of Rac1 (24, 25). Although the possibility remains that cell-cycle-dependent activation of LIMK1 is mediated by MRCK␣ and/or Pak during mitosis, cell-cycle-dependent regulation of both MRCK␣ and Pak has not been reported. On the other hand, we found herein that CDKs kinase activity is required for the activation of LIMK1 during mitosis. This would suggest that CDKs, particularly
FIG. 3. Requirement of CDK kinase activity for LIMK1 activation at mitosis. (A) Flow cytometric analysis of cell cycle transition. Exponentially growing HeLa cells were synchronized at the G1/S boundary by a double thymidine block. Cells were harvested at 0, 4, 8, 10, 12, and 14 h after the release from the G1/S arrest, and cell cycle profiles were obtained by flow cytometry. Cells were nontreated or treated with 100 M roscovitine at 8 h after the release. (B) Cell cycle-dependent changes in LIMK1 activity and histone H1 kinase activity. At the indicated time points, cells were lysed, and LIMK1 activity was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. Cofilin phosphorylation was estimated using NIH Image software and protein kinase activity of LIMK1 in cells at 0 h after the release was taken as 1.0 (upper middle). The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting, using an anti-LIMK1 antibody (lower middle). Histone H1 kinase activity in each cell lysate was also measured (bottom).
Cdk1 during mitosis, might be directly or indirectly involved in the regulation of LIMK1 activity. Alternatively, other kinase(s), which involved in the mitotic
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progression, may regulate LIMK1 activity (17–19, 26 – 28). Whether or not LIMK1 is activated by these potential protein kinases remains to be addressed. Mitosis-dependent activation of LIMK1 led to the hypothesis that LIMK1 has a significant role in the mitosis. This thesis is supported by the following: The ectopic expression of XLIMK, the Xenopus counterpart of mammalian LIMK (14) in oocyte impaired progression of the meiotic process, and XLIMK was functionally involved in organization, maintenance, and migration of meiotic spindle (15). A similar finding was seen during spermatogenesis in a Drosophila mutant twinstar, wherein the mutated gene encodes cofilin, the substrate of LIMKs (29). Spermatocytes in the mutant fly showed defects in aster migration and separation at prophase/prometaphase during meiotic divisions by an unusual accumulation of actin at centrosomes. Furthermore, recent studies showed that the actin cytoskeleton is tightly associated with the spindle microtubule, and is involved in the poleward flux of tubulin in metaphase kinetochore microtubules (30 –32). In light of all these findings we propose that the actin cytoskeletal dynamics mediated by LIMK1-cofilin system may play a role in organization and proper functions of the mitotic apparatus. In summary, we provided the first evidence that LIMK1 is activated during mitosis and activation of CDKs is directly or indirectly involved in LIMK1 activation. Our results suggest that LIMK1 has an important role in progression of mitosis and mitosis-dependent actin cytoskeletal rearrangement. Future studies to identify detailed functions of LIMK1 during mitosis should facilitate understanding of an orchestrated interrelationship between cell cycle transition and actin cytoskeletal organization.
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ACKNOWLEDGMENTS This work was supported by a Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan. We are also grateful to M. Ohara for helpful comments and for language assistance.
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Miyata, T., and Nakamura, T. (1994) Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif. Oncogene 9, 1605–1612. Okano, I., Hiraoka, J., Otera, H., Nunoue, K., Ohashi, K., Iwashita, S., Hirai, M., and Mizuno, K. (1995) Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs. J. Biol. Chem. 270, 31321–31330. Koshimizu, U., Takahashi, H., Yoshida, M. C., and Nakamura, T. (1997) cDNA cloning, genomic organization, and chromosomal localization of the mouse LIM motif-containing kinase gene, Limk2. Biochem. Biophys. Res. Commun. 241, 243–250. Arber, S., Barbayannis, F. A., Hanser, H., Schneider, C., Stanyon, C. A., Bernard, O., and Caroni, P. (1998) Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393, 805– 809. Yang, N., Higuchi, O., Ohashi, K., Nagata, K., Wada, A., Kangawa, K., Nishida, E., and Mizuno, K. (1998) Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809 – 812. Sumi, T., Matsumoto, K., Takai, Y., and Nakamura, T. (1999) Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2. J. Cell Biol. 147, 1519 –1532. Agnew, B. J., Minamide, L. S., and Bamburg, J. R. (1995) Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. J. Biol. Chem. 270, 17582–17587. Moriyama, K., Iida, K., and Yahara, I. (1996) Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. Genes Cells 1, 73– 86. Takahashi, T., Aoki, S., Nakamura, T., Koshimizu, U., and Matsumoto, K. (1997) Xenopus LIM motif-containing protein kinase, Xlimk1, is expressed in the developing head structure of the embryo. Dev. Dyn. 209, 196 –205. Takahashi, T., Koshimizu, U., Abe, H., Obinata, T., and Nakamura, T. (2001) Functional involvement of Xenopus LIM kinases in progression of oocyte maturation. Dev. Biol. 229, 554 –567. Takahashi, H., Koshimizu, U., Miyazaki, J., and Nakamura, T. (2002) Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene. Dev. Biol. 241, 259 –272, doi:10.1006/dbio.2001.0512. Nigg, E. A. (1995) Cyclin-dependent protein kinases: Key regulators of the eukaryotic cell cycle. BioEssays 17, 471– 480. Nigg, E. A. (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32. O’Farrell, P. (2001) Triggering the all-or-nothing switch into mitosis. Trends Cell Biol. 11, 512–519. Meijer, L., and Kim, S. H. (1997) Chemical inhibitors of cyclindependent kinases. Methods Enzymol. 283, 113–128. Meijer, L., Borgne, A., Mulner, O., Chong, J. P., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J. G., and Moulinoux, J. P. (1997) Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243, 527–536. Sumi, T., Matsumoto, K., and Nakamura, T. (2001) Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase. J. Biol. Chem. 276, 670 – 676. Sumi, T., Matsumoto, K., Shibuya, A., and Nakamura, T. (2001) Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha. J. Biol. Chem. 276, 23092–23096. Edwards, D. C., Sanders, L. C., Bokoch, G. M., and Gill, G. N. (1999) Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signaling to actin cytoskeletal dynamics. Nat. Cell Biol. 1, 253–259.
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25. Dan, C., Kelly, A., Bernard, O., and Minden, A. (2001) Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J. Biol. Chem. 276, 32115– 32121. 26. Nigg, E. A. (1998) Polo-like kinases: Positive regulators of cell division from start to finish. Curr. Opin. Cell Biol. 10, 776 –783. 27. Giet, R., and Prigent, C. (1999) Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J. Cell Sci. 112, 3591–3601. 28. Glover, D. M., Hagan, I. M., and Tavares, A. A. (1998) Polo-like kinases: A team that plays throughout mitosis. Genes Dev. 12, 3777–3787. 29. Gunsalus, K. C., Bonaccorsi, S., Williams, E., Verni, F., Gatti, M., and Goldberg, M. L. (1995) Mutations in twinstar, a Dro-
sophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis. J. Cell Biol. 131, 1243– 1259. 30. Sider, J. R., Mandato, C. A., Weber, K. L., Zandy, A. J., Beach, D., Finst, R. J., Skoble, J., and Bement, W. M. (1999) Direct observation of microtubule-f-actin interaction in cell free lysates. J. Cell Sci. 112, 1947–1956. 31. Goode, B. L., Drubin, D. G., and Barnes, G. (2000) Functional cooperation between the microtubule and actin cytoskeletons. Curr. Opin. Cell Biol. 12, 63–71. 32. Silverman-Gavrila, R. V., and Forer, A. (2000) Evidence that actin and myosin are involved in the poleward flux of tubulin in metaphase kinetochore microtubules of crane-fly spermatocytes. J. Cell Sci. 113, 597– 609.
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Mitosis-Dependent Phosphorylation and Activation of LIM-Kinase 1 Tomoyuki Sumi, Kunio Matsumoto, and Toshikazu Nakamura 1 Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
Received December 30, 2001
LIM-kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cellcycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements. © 2002 Elsevier Science (USA) Key Words: LIM-kinase; cofilin; actin cytoskeleton; mitosis; cyclins; CDKs; cell cycle; Rho family GTPase; phosphorylation; protein kinase.
The cytoskeletal network composed of actin filaments and microtubules in eukaryotic cells participates in cellular processes, including locomotion, shape changes, cytokinesis, and maintenance of polarity (1, 2). Microtubules play crucial roles in cell division: they form the mitotic spindle, which segregates duplicated chromosomes into a daughter cell (3, 4). Likewise, the actin-based cytoskeleton undergoes dramatic changes throughout the cell-division cycle, however, mechanisms by which actin cytoskeletal rearrangement is coordinated with cell cycle transition and assembly/ disassembly of mitotic apparatus have been unclear. Abbreviations used: LIMK, LIM domain-containing protein kinase; CDKs, cyclin-dependent protein kinases; GST, glutathione S-transferase. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-6-6879-3789. E-mail: [email protected]. osaka-u.ac.jp.
LIM-kinases (LIMK1 and LIMK2) (5– 8) regulate actin cytoskeletal reorganization downstream of Rho family GTPases and LIMKs-induced actin cytoskeletal rearrangement is mediated by cofilin, an actin-depolymerizing factor of actin filaments (9 –11). Activated LIMK catalyzes phosphorylation of an N-terminal 3rd serine residue of cofilin and inhibits its activity to depolymerize actin filaments, thereby leading to stabilization of actin filaments (12, 13). We reported that XLIMKs, the Xenopus counterpart of mammalian LIMKs (14), are critically involved in the progression of progesterone-induced Xenopus oocyte maturation through Xenopus cofilin phosphorylation (15). XLIMKcofilin system was required for the organization of the microtubule-derived precursor of the meiotic spindle. Likewise, using knockout mice of LIMK2, we recently demonstrated that LIMK2 plays an important role in the proper progression of spermatogenesis by regulating of cofilin activity in mammalian germ cells (16). Therefore, LIMKs may be a key component of a fundamental signal transduction system that connects extracellular stimuli to intracellular actin cytoskeletal rearrangements, in a distinct biological context. Although it seems likely that LIMKs may play some roles in coordinated regulation of actin cytoskeletal rearrangement involved in the cell cycle progression during mitosis and/or meiosis, regulation of LIMKs activity during the cell cycle transition has yet to be defined. We now report that LIMK1 activity increases during mitosis and activation of cyclin-dependent kinases (CDKs) is directly or indirectly involved in mitosis-dependent activation of LIMK1. Our results suggest that LIMK1 may participate in the coordination of cell cycle progression and mitosis-dependent actin cytoskeletal organization. MATERIALS AND METHODS Materials. Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgG, and Hoechst 33342 were purchased from Molecular Probes, Inc. (Eugene, OR). Anti-␣-tubulin monoclonal antibody (DM1A) and roscovitine were purchased from
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Sigma (St. Louis, MO) and Calbiochem (Darmstadt, Germany). AntiLIMK1 antibody was purified as described elsewhere (11). Cell culture and synchronization of cell cycle. HeLa cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum and nonessential amino acids. Cells were synchronized at the G1/S boundary by a double thymidine block. In brief, cells were treated with 2 mM thymidine for 24 h, followed by an 8-h release in fresh MEM with 10% fetal bovine serum and successive retreatment with 2 mM thymidine for 14 h. Cells synchronized at the G1/S boundary were released to enter the cell cycle in fresh MEM with 10% fetal bovine serum. At various time points after release, these cells were harvested by trypsinization. Aliquots were subjected to flow cytometric analysis, and the remaining cells were used to prepare whole cell extracts, as described below. For flow cytometric analysis, the cells were fixed with 70% ethanol for 20 min, then stained with propidium iodide (20 g/ml) and treated with RNase A (1 mg/ml) for 10 min at room temperature. Samples of 10,000 cells were then analyzed on a FACSCalibur (Becton–Dickinson, San Diego, CA). To arrest exponentially growing HeLa cells at prometaphase, nocodazole was added to make up a final concentration of 1 g/ml for 16 h, then the cells were collected and extracted as described below. Immunoprecipitation, immunoblot analysis, and protein kinase assay. HeLa cells were lysed with lysis buffer composed of 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 25 mM -glycerophosphate, 10 mM NaF, 1 mM Na 3VO 4, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml leupeptin and aprotinin for 20 min. After centrifugation, the supernatant was incubated for 3 h at 4°C with an anti-LIMK1 antibody and 5 l Protein A–Sepharose (Amersham Pharmacia Biotech, Little Chalfont, UK). Immunoprecipitates were washed three times with lysis buffer, dissolved in the sample buffer for SDS–PAGE, and separated by SDS–PAGE, electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and probed with an anti-LIMK1 antibody. Proteins reacting with antibody were detected using ECL enhanced chemiluminescence (Amersham Pharmacia Biotech). The band densities of immunoblots were analyzed using NIH Image software (Wayne Rasband Analytics, National Institutes of Health). For the measurement of LIMK1 activity, immunoprecipitates were incubated for 20 min at 30°C in 15 l of kinase buffer consisting of 50 mM Hepes–NaOH (pH 7.5), 25 mM -glycerophosphate, 5 mM MgCl 2, 5 mM MnCl 2, 10 mM NaF, and 1 mM Na 3VO 4, 50 M ATP, 5 Ci of [␥- 32P]ATP (6000 Ci/mmol; Perkin–Elmer Life Sciences) and 6 g of GST-fused cofilin as the substrate. Histone H1 kinase assay was done in kinase buffer consisting of 50 mM Tris–HCl (pH 7.5), 10 mM MgCl 2, 2 mM DTT, 1 mM EGTA, 100 M ATP, 5 Ci of [␥- 32P]ATP and 1 g histone H1 together with each cell lysate containing 5 g total protein. After incubation for 20 min at 30°C, the reaction was terminated by heat-treatment (100°C for 3 min) in sample buffer for SDS–PAGE, and then the samples were subjected to SDS–PAGE, and analyzed by autoradiography. Protein phosphatase treatment. Immunoprecipitates prepared using anti-LIMK1 antibody were washed three times with 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, then the supernatant was removed. The reaction buffer for protein phosphatase (New England BioLabs, Inc., Beverly, MA) was added either with or without 100 units of Lambda Protein Phosphatase. Samples were incubated for 30 min at 30°C, reactions were stopped by heat-treatment (100°C for 3 min) in sample buffer for SDS–PAGE, and samples were subjected to SDS–PAGE and subsequent immunoblotting.
RESULTS Phosphorylation and Activation of LIMK1 during Mitosis We previously reported that LIM-kinases, LIMK1 and LIMK2, are involved in the cell cycle progression,
using Xenopus oocyte and LIMK2-knockout mice (15, 16). To further address the cell-cycle-dependent regulation of LIMKs activity, we determined if kinase activity of LIMKs is altered during the cell cycle. HeLa cells were synchronized at the prometaphase by nocodazole-treatment, and the LIMKs activity was measured using GST-fused cofilin as the substrate. As shown in Fig. 1A (top), LIMK1 activity toward cofilin phosphorylation in mitotic cells was about fourfold higher than that in asynchronized cells, whereas LIMK2 activity did not significantly change during cell cycle (data not shown). Similarly, histone H1 kinase activity, a marker for mitosis, in nocodazole-treated cells was much higher than that in asynchronized cells, which indicated that cells were arrested at mitosis by activation of the mitotic checkpoint (Fig. 1A, bottom). In immunoblot analysis of LIMK1, electrophoretic mobility of the LIMK1 in mitotic cells was clearly slower than that in asynchronized cells (Fig. 1A, middle). We speculated that the mobility shift might be due to phosphorylation of LIMK1 during mitosis. To examine such possibility, LIMK1 immunoprecipitated from asynchronized and nocodazole-treated HeLa cells were, respectively, incubated with protein phosphatase then subjected to immunoblotting (Fig. 1B). Without phosphatase-treatment, the slower mobility shift of LIMK1 was observed in mitotic cells; however, the mobility shift of LIMK1 in mitotic cells was diminished by phosphatase-treatment. These results indicate that LIMK1 was specifically phosphorylated and activated in cells arrested at the mitotic checkpoint by nocodazoletreatment. Activation of LIMK1 during Mitosis To determine whether the activation of LIMK1 induced by nocodazole treatment occurred specifically as a result of a spindle disassembly or nonspecifically irrespective of the cell cycle, cell-cycle-dependent regulation of LIMK1 activity was measured in synchronized cells. HeLa cells were synchronized at the G1/S boundary by a double thymidine block method then the cells were released to enter the cell cycle. Cells were harvested at different times after the release from the G1/S arrest and analyzed for LIMK1 activity toward cofilin phosphorylation. In parallel, the DNA content at each time point was analyzed by flow cytometry (Fig. 2A). The LIMK1 activity was low in cells at early stages of the cell cycle (0 – 8 h after release from the G1/S arrest), whereas it thereafter increased and reached a maximal level when cells passed through mitosis (10 –12 h after release from the G1/S arrest) (Fig. 2B, top). By 14 h after release from the G1/S arrest, the bulk of the cell population completed mitosis and reentered G1 phase and this cell cycle transition was associated with a decrease in LIMK1 activity. Consistent with the change in LIMK1 activity, electro-
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were in the G1, S or G2 phase (Fig. 2B, lower middle). To provide a marker for the timing of mitosis, histone H1 kinase activity responsible for activity of cyclindependent kinases (CDKs) (particularly Cdk1 at mitosis) was also determined for each sample (Fig. 2B, bottom). Similar to LIMK1 activity, histone H1 kinase activity reached a maximal level 10 h after release
FIG. 1. Mitosis-specific activation (A) and phosphorylation (B) of LIMK1. (A) HeLa cells were either asynchronized or synchronized with nocodazole in prometaphase of mitosis. Cells were lysed, and protein kinase activity of LIMK1 was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting (middle). Histone H1 kinase activity in each cell lysate was also measured (bottom). Cofilin phosphorylation was estimated using NIH Image software (right). Protein kinase activity of LIMK1 in asynchronized cells was taken as 1.0. Each value represents the mean ⫾ standard error for experiments done in triplicate. (B) Immunoprecipitated LIMK1 prepared from asynchronized and synchronized HeLa cells with nocodazole were, respectively, incubated with or without protein phosphatase. Samples were analyzed by immunoblotting, using an anti-LIMK1 antibody.
phoretic mobility shift of LIMK1 was observed in cells 10 h after release from the G1/S arrest, whereas the mobility shift of LIMK1 was not detected when cells
FIG. 2. Cell cycle-dependent activation of LIMK1. (A) Flow cytometric analysis of cell cycle transition. Exponentially growing HeLa cells were synchronized at the G1/S boundary by a double thymidine block. Cells were harvested at 0, 4, 8, 10, 12, and 14 h after the release from the G1/S arrest, and cell cycle profiles were obtained by flow cytometry. (B) Cell cycle-dependent changes in LIMK1 activity and histone H1 kinase activity. At the indicated time points, cells were lysed, and LIMK1 activity was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. Cofilin phosphorylation was estimated using NIH Image software and protein kinase activity of LIMK1 in cells at 0 h after the release was taken as 1.0 (upper middle). The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting, using an anti-LIMK1 antibody (lower middle). Histone H1 kinase activity in each cell lysate was also measured (bottom).
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from the G1/S arrest (the time when most of cells were in mitosis) and thereafter decreased. Taken together, these results indicate that phosphorylation and activation of LIMK1 occurs when cells enter into mitosis, similar to the profile of Cdk1 activation during cell cycle transition. CDKs Kinase Activity Is Required for LIMK1 Activation during Mitosis Since the protein complex of cyclin B and its partner Cdk1 plays a crucial role in the regulation of mitosis (17–19), we addressed whether activation of Cdk1 is required for LIMK1 activation during mitosis. Cells in G2/M-phase were treated with the CDKs inhibitor roscovitine, and LIMK1 and CDKs activities were measured using GST-cofilin and histone H1 as the substrate, respectively. Although roscovitine inhibits Cdk1, Cdk2, and Cdk5 (20, 21), Cdk1 is the major activity present in M-phase cells (17–19). To exclude effects of roscovitine on cell cycle progression, treatment of cells with roscovitine was done 8 h after the release from G1/S arrest. At 8 h after the release, most of cell population had reached at the G2/M phase, then passed through mitosis during 10 –14 h after the release (Fig. 3A). Consistent with above results, activities of LIMK1 and CDKs displayed a similar cell cycle dependency (Fig. 3B). Both activities reached a maximal level 10 h after the release, then decreased after cell division. Cells treated with roscovitine were arrested at the G2/M phase after a 10 h release and cell cycle transition into G1 phase was not observed (Fig. 3A). Under this condition, condensed chromosomes were seen in roscovitine-treated cells, thereby suggesting that roscovitine arrested at prophase on the cell cycle (data not shown). Importantly, roscovitine-treatment diminished increases in LIMK1 activity as well as CDKs activity during mitosis (Fig. 3B). These observations suggest that CDKs kinase activity is directly or indirectly required for activation of LIMK1 at mitosis. DISCUSSION We earlier reported that the activities of LIMkinases, LIMK1 and LIMK2, are distinctly regulated by Rho family GTPases (11). ROCK, a Rho-dependent protein kinase, specifically activates LIMK2 (22), whereas MRCK␣, a downstream effector of Cdc42, activates both LIMK1 and LIMK2 (23). LIMK1 is also activated by Pak downstream of Rac1 (24, 25). Although the possibility remains that cell-cycle-dependent activation of LIMK1 is mediated by MRCK␣ and/or Pak during mitosis, cell-cycle-dependent regulation of both MRCK␣ and Pak has not been reported. On the other hand, we found herein that CDKs kinase activity is required for the activation of LIMK1 during mitosis. This would suggest that CDKs, particularly
FIG. 3. Requirement of CDK kinase activity for LIMK1 activation at mitosis. (A) Flow cytometric analysis of cell cycle transition. Exponentially growing HeLa cells were synchronized at the G1/S boundary by a double thymidine block. Cells were harvested at 0, 4, 8, 10, 12, and 14 h after the release from the G1/S arrest, and cell cycle profiles were obtained by flow cytometry. Cells were nontreated or treated with 100 M roscovitine at 8 h after the release. (B) Cell cycle-dependent changes in LIMK1 activity and histone H1 kinase activity. At the indicated time points, cells were lysed, and LIMK1 activity was determined by immune complex kinase assay, using GST-cofilin as the substrate (top). Arrowheads, phosphorylated LIMK1; arrow, phosphorylated cofilin. Cofilin phosphorylation was estimated using NIH Image software and protein kinase activity of LIMK1 in cells at 0 h after the release was taken as 1.0 (upper middle). The amount of LIMK1 protein in the immunoprecipitates was analyzed by immunoblotting, using an anti-LIMK1 antibody (lower middle). Histone H1 kinase activity in each cell lysate was also measured (bottom).
Cdk1 during mitosis, might be directly or indirectly involved in the regulation of LIMK1 activity. Alternatively, other kinase(s), which involved in the mitotic
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progression, may regulate LIMK1 activity (17–19, 26 – 28). Whether or not LIMK1 is activated by these potential protein kinases remains to be addressed. Mitosis-dependent activation of LIMK1 led to the hypothesis that LIMK1 has a significant role in the mitosis. This thesis is supported by the following: The ectopic expression of XLIMK, the Xenopus counterpart of mammalian LIMK (14) in oocyte impaired progression of the meiotic process, and XLIMK was functionally involved in organization, maintenance, and migration of meiotic spindle (15). A similar finding was seen during spermatogenesis in a Drosophila mutant twinstar, wherein the mutated gene encodes cofilin, the substrate of LIMKs (29). Spermatocytes in the mutant fly showed defects in aster migration and separation at prophase/prometaphase during meiotic divisions by an unusual accumulation of actin at centrosomes. Furthermore, recent studies showed that the actin cytoskeleton is tightly associated with the spindle microtubule, and is involved in the poleward flux of tubulin in metaphase kinetochore microtubules (30 –32). In light of all these findings we propose that the actin cytoskeletal dynamics mediated by LIMK1-cofilin system may play a role in organization and proper functions of the mitotic apparatus. In summary, we provided the first evidence that LIMK1 is activated during mitosis and activation of CDKs is directly or indirectly involved in LIMK1 activation. Our results suggest that LIMK1 has an important role in progression of mitosis and mitosis-dependent actin cytoskeletal rearrangement. Future studies to identify detailed functions of LIMK1 during mitosis should facilitate understanding of an orchestrated interrelationship between cell cycle transition and actin cytoskeletal organization.
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ACKNOWLEDGMENTS This work was supported by a Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan. We are also grateful to M. Ohara for helpful comments and for language assistance.
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