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Phosphorylation of Glial Fibrillary Acidic Protein at the Same Sites by Cleavage Furrow Kinase and Rho-associated Kinase

1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês

10.1074/jbc.272.16.10333

ISSN

1083-351X

Autores

Hidetaka Kosako, Mutsuki Amano, Maki Yanagida, Kazushi Tanabe, Yoshimi Nishi, Kozo Kaibuchi, Masaki Inagaki,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895–2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635–641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5′-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed. Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895–2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635–641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5′-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed. Intermediate filaments (IFs), 1The abbreviations used are: IF, intermediate filament; GFAP, glial fibrillary acidic protein; CF, cleavage furrow; MBS, myosin-binding subunit; GST, glutathione S-transferase; GTPγS, guanosine 5′-(3-O-thio)-triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Rho-kinase, Rho-associated kinase. 1The abbreviations used are: IF, intermediate filament; GFAP, glial fibrillary acidic protein; CF, cleavage furrow; MBS, myosin-binding subunit; GST, glutathione S-transferase; GTPγS, guanosine 5′-(3-O-thio)-triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Rho-kinase, Rho-associated kinase.major components of the cytoskeleton and the nuclear envelope in most eukaryotic cells, undergo dramatic reorganization of their structure during cell signaling and cell cycle (for review, see Refs. 1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1988; 57: 593-625Crossref PubMed Scopus (1118) Google Scholar, 2Eriksson J.E. Opal P. Goldman R.D. Curr. Opin. Cell Biol. 1992; 4: 99-104Crossref PubMed Scopus (125) Google Scholar, 3Fucks E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1264) Google Scholar). This IF reorganization is thought to be regulated by site-specific phosphorylation of IF proteins at serine and threonine residues, and several protein kinases have been shown to act as IF kinases in vivo (for review, see Ref. 4Inagaki M. Matsuoka Y. Tsujimura K. Ando S. Tokui T. Takahashi T. Inagaki N. BioEssays. 1996; 18: 481-487Crossref Scopus (160) Google Scholar). Site- and phosphorylation state-specific antibodies that recognize a phosphorylated serine/threonine residue and its flanking sequence can visualize site-specific IF phosphorylation and thereby IF kinase activitiesin situ by immunocytochemistry (Ref. 5Nishizawa K. Yano T. Shibata M. Ando S. Saga S. Takahashi T. Inagaki M. J. Biol. Chem. 1991; 266: 3074-3079Abstract Full Text PDF PubMed Google Scholar; for review, see Ref.6Inagaki M. Inagaki N. Takahashi T. Takai Y. J. Biochem. (Tokyo). 1997; 121: 407-417Crossref PubMed Scopus (51) Google Scholar). Recently, we reported two distinct types of mitotic phosphorylation of glial fibrillary acidic protein (GFAP), an IF protein expressed in the cytoplasm of astroglia, using antibodies that react with four distinct phosphorylated sites on GFAP (7Matsuoka Y. Nishizawa K. Yano T. Shibata M. Ando S. Takahashi T. Inagaki M. EMBO J. 1992; 11: 2895-2902Crossref PubMed Scopus (90) Google Scholar, 8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar). One type is the phosphorylation of Ser-8 on GFAP, which appeared at G2-M phase transition in the entire cytoplasm. The other type is the phosphorylation of Thr-7, Ser-13, and Ser-34, which appeared at metaphase-anaphase transition at the cleavage furrow. This GFAP phosphorylation specifically localized at the cleavage furrow was observed not only in astroglial cells but also in other cultured cells transfected with GFAP cDNA (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar). These findings suggested the existence of a protein kinase specifically activated at the cleavage furrow and its important role in cytokinesis. We tentatively termed this kinase cleavage furrow (CF) kinase (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar). However, the molecular identity, regulation, and function of CF kinase remained to be examined. The small GTP-binding protein Rho is implicated in the control of cytoskeletal structures, cell adhesions, and cell morphology (for review, see Ref. 9Machesky L.M. Hall A. Trends Cell Biol. 1996; 6: 304-310Abstract Full Text PDF PubMed Scopus (252) Google Scholar). Upon stimulation with certain signals, the GDP-bound inactive form of Rho may be converted to the GTP-bound active form, which binds to specific targets and thereby exerts its biological functions. We have identified three putative targets for Rho, p128 protein kinase N (10Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (392) Google Scholar, 11Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (346) Google Scholar), p138 myosin-binding subunit (MBS) of myosin phosphatase (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2399) Google Scholar), and p164 Rho-kinase (13Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (923) Google Scholar), which is also named ROK (14Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). Rho-kinase phosphorylates MBS and consequently inactivates myosin phosphatase (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2399) Google Scholar). Rho-kinase also phosphorylates myosin light chain and thereby activates myosin ATPase (15Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1647) Google Scholar). Other putative targets for Rho include rhophilin (11Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (346) Google Scholar), p160 Rho-associated coiled-coil containing protein kinase (16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (783) Google Scholar), and citron (17Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (147) Google Scholar). Recently, Rho was shown to play a critical role in cytokinesis by inducing and maintaining the contractile ring, an actin-based cytoskeletal structure (18Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. Cell Biol. 1993; 120: 1187-1195Crossref PubMed Scopus (308) Google Scholar, 19Mabuchi I. Hamaguchi Y. Fujimoto H. Morii N. Mishima M. Narumiya S. Zygote. 1993; 1: 325-331Crossref PubMed Scopus (212) Google Scholar). In addition, Rho was reported to be translocated from the cytosol to the cleavage furrow during cytokinesis (20Takaishi K. Sasaki T. Kameyama T. Tsukita S. Tsukita S. Takai Y. Oncogene. 1995; 11: 39-48PubMed Google Scholar). These results raise the possibility that Rho may also be implicated in the regulation of CF kinase and thereby in the efficient separation of IFs to daughter cells. As a first step toward defining this possibility, we have examined whether protein kinase N and/or Rho-kinase can phosphorylate GFAP at the same sites as CF kinase. Protein kinase N was found to phosphorylate GFAP mainly at Ser-8, a site that is not phosphorylated by CF kinase. 2K. Matsuzawa, H. Kosako, N. Inagaki, M. Amano, Y. Nishi, H. Mukai, Y. Ono, Y. Matsuura, K. Kaibuchi, I. Azuma, and M. Inagaki, manuscript in preparation. 2K. Matsuzawa, H. Kosako, N. Inagaki, M. Amano, Y. Nishi, H. Mukai, Y. Ono, Y. Matsuura, K. Kaibuchi, I. Azuma, and M. Inagaki, manuscript in preparation. In this report, we show that GFAP can serve as an excellent substrate for Rho-kinase and that the GFAP phosphorylation by Rho-kinase prevents its filament formation in vitro. Furthermore, we present evidence that Rho-kinase phosphorylates GFAP at Thr-7, Ser-13, and Ser-34 in vitro, the same sites that are phosphorylated by CF kinase in vivo. Recombinant human GFAP was prepared fromEscherichia coli as described previously (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar). Mouse monoclonal antibodies YC10, KT13, KT34, and MD389 were prepared as described previously (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar, 21Yano T. Taura C. Shibata M. Hirono Y. Ando S. Kusubata M. Takahashi T. Inagaki M. Biochem. Biophys. Res. Commun. 1991; 175: 1144-1151Crossref PubMed Scopus (57) Google Scholar). GST-RhoA was purified and loaded with guanine nucleotides (22Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). Rho-kinase was purified from bovine brain (13Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (923) Google Scholar). Constitutively active GST-Rho-kinase, a GST fusion protein of the catalytic fragment of Rho-kinase, was purified from Sf9 cells as described previously (15Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1647) Google Scholar). The catalytic subunit of cAMP-dependent protein kinase (protein kinase A) was prepared from bovine heart by the method of Beavo et al. (23Beavo J.A. Bechtel P.J. Krebs E.G. Methods Enzymol. 1974; 38: 299-308Crossref PubMed Scopus (389) Google Scholar). Cdc2 kinase was prepared from FM3A cells by the method of Kusubata et al. (24Kusubata M. Tokui T. Matsuoka Y. Okumura E. Tachibana K. Hisanaga S. Kishimoto T. Yasuda H. Kamijo M. Ohba Y. Tsujimura K. Yatani R. Inagaki M. J. Biol. Chem. 1992; 267: 20937-20942Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were measured according to Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (210811) Google Scholar) using bovine serum albumin as a standard. The phosphorylation reaction for Rho-kinase was performed for 30 min at 25 °C in 20 μl of 25 mm Tris-Cl (pH 7.5), 0.2% CHAPS, 4 mmMgCl2, 3.6 mm EDTA, 100 μm[γ-32P]ATP (5 μCi), 0.1 μm calyculin A, 130 μg/ml GFAP, and 0.5 μg/ml purified Rho-kinase in the presence of GST, GDP·GST-RhoA, or GTPγS·GST-RhoA (each 1 μm). The phosphorylation reaction for GST-Rho-kinase, protein kinase A, or Cdc2 kinase was performed for 30 min at 25 °C in 100 μl of the reaction mixture (25 mm Tris-Cl (pH 7.5), 0.4 mm MgCl2, 100 μm ATP, 0.1 μm calyculin A, and 130 μg/ml GFAP) in the presence of 8.5 μg/ml GST-Rho-kinase, 5 μg/ml protein kinase A, or 0.5 μg/ml Cdc2 kinase. The reaction was stopped by the addition of Laemmli's sample buffer and boiling. GFAP (130 μg) was incubated with GST-Rho-kinase (8.5 μg) and [γ-32P]ATP (50 μCi) for 120 min at 25 °C in 1 ml of the reaction mixture described above. The phosphorylated GFAP was precipitated with 10% trichloroacetic acid and digested with 5 μg of lysyl endopeptidase (Wako) in 100 μl of 50 mm Tris-Cl (pH 8.0) and 4m urea at 30 °C for 2 h. The phosphorylated head domain of GFAP was isolated by reverse-phase HPLC and treated with 1/50 (w/w) l-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma), as described by Tsujimura et al. (26Tsujimura K. Tanaka J. Ando S. Matsuoka Y. Kusubata M. Sugiura H. Yamauchi T. Inagaki M. J. Biochem. (Tokyo). 1994; 116: 426-434Crossref PubMed Scopus (39) Google Scholar). The obtained peptides were separated by HPLC on a Zorbax C8 (0.46 × 25 cm) column equilibrated with 5% (v/v) 2-propanol/acetonitrile (7:3) containing 0.1% trifluoroacetic acid. Elution was carried out with a 60-min linear gradient of 5–50% 2-propanol/acetonitrile followed by a further 10-min linear gradient of 50–80% 2-propanol/acetonitrile at a flow rate of 0.8 ml/min. All the procedures for immunoblotting have been described elsewhere in detail (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar, 21Yano T. Taura C. Shibata M. Hirono Y. Ando S. Kusubata M. Takahashi T. Inagaki M. Biochem. Biophys. Res. Commun. 1991; 175: 1144-1151Crossref PubMed Scopus (57) Google Scholar). Immunofluorescence microscopy was performed as described previously (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar). Amino acid sequences were analyzed with an ABI 476A gas-phase sequencer. Two-dimensional phosphoamino acid analysis was performed as described (27Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1270) Google Scholar). Electron microscopy was carried out as described (28Inagaki M. Gonda Y. Nishizawa K. Kitamura S. Sato C. Ando S. Tanabe K. Kikuchi K. Tsuiki S. Nishi Y. J. Biol. Chem. 1990; 265: 4722-4729Abstract Full Text PDF PubMed Google Scholar). We recently developed four monoclonal antibodies, YC10 (21Yano T. Taura C. Shibata M. Hirono Y. Ando S. Kusubata M. Takahashi T. Inagaki M. Biochem. Biophys. Res. Commun. 1991; 175: 1144-1151Crossref PubMed Scopus (57) Google Scholar), KT13, KT34, and MO389 (8Sekimata M. Tsujimura K. Tanaka J. Takeuchi Y. Inagaki N. Inagaki M. J. Cell Biol. 1996; 132: 635-641Crossref PubMed Scopus (44) Google Scholar) against four distinct phosphopeptides corresponding to the partial amino acid sequences of porcine GFAP. YC10, KT13, and KT34 react with GFAP phosphorylated at Ser-8, Ser-13, and Ser-34, respectively. MO389 reacts with both the phosphorylated and unphosphorylated GFAPs and stains filamentous structures in both mitotic and interphase cells. MO389 immunostained an intricate mesh of glial filaments in the entire cytoplasm of both metaphase and anaphase cells, but YC10 stained filamentous structures throughout the cytoplasm of metaphase but not anaphase cells (Fig.1 A). In contrast, the immunoreactivities of KT13 and KT34 were observed specifically between the daughter nuclei and at the cleavage furrow of anaphase cells (Fig. 1 A). Immunocytochemical studies with KT13 (Fig. 1 B) and KT34 (data not shown) using confocal laser scanning microscopy revealed that GFAP phosphorylated at Ser-13 and Ser-34 is associated with the cleavage furrow to form a ring-like structure but not a disc-like structure, such as the telophase disc reported by Andreassen et al. (29Andreassen P.R. Palmer D.K. Wener M.H. Margolis R.L. J. Cell Sci. 1991; 99: 523-534Crossref PubMed Google Scholar). To search for the putative CF kinase responsible for the cleavage furrow-specific phosphorylation described above, we examined whether Rho-kinase purified from bovine brain can phosphorylate GFAP in vitro. The results indicated clearly that Rho-kinase phosphorylated GFAP in a GST-RhoA-dependent manner (Fig.2 A). GDP-bound GST-RhoA enhanced the phosphorylation of GFAP by Rho-kinase 13-fold, and GTPγS-bound GST-RhoA enhanced it 291-fold (Fig. 2 A). We then examined the phosphorylation sites on GFAP by Rho-kinase using the anti-phosphoGFAP antibodies described above. After the phosphorylation reaction, samples were resolved by SDS-PAGE and immunoblotted with MO389, YC10, KT13, or KT34. As shown in Fig.2 B, Rho-kinase phosphorylated GFAP at Ser-13 and Ser-34 but not at Ser-8 in a GTPγS·GST-RhoA-dependent manner. We also used the constitutively active GST-Rho-kinase, a fusion protein between GST and the catalytic fragment of Rho-kinase produced in Sf9 cells by recombinant baculovirus infection. Analyses with the anti-phosphoGFAP antibodies revealed that GST-Rho-kinase also phosphorylated GFAP at Ser-13 and Ser-34 but not at Ser-8 (Fig.2 C). These results suggest that catalytic characteristics of GST-Rho-kinase are similar to those of native Rho-kinase activated by GTPγS·GST-RhoA. In contrast, the catalytic subunit of cAMP-dependent protein kinase phosphorylated all three serine residues, and Cdc2 kinase weakly phosphorylated only Ser-8 (Fig.2 C). To confirm phosphorylation sites on GFAP by Rho-kinase, GFAP (130 μg) was phosphorylated by GST-Rho-kinase in the presence of [γ-32P]ATP to approximately 2.7 mol of phosphate/mol of GFAP (Fig. 3 A). The radioactive GFAP was then digested with lysyl endopeptidase to generate about a 6.5-kDa fragment consisting mainly of the amino-terminal head domain. Tricine-SDS-PAGE analysis (30Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10393) Google Scholar) revealed that all radioactivity associated with GFAP was retained in this 6.5-kDa head domain (Fig. 3 B). This phosphorylated head domain was isolated by reverse-phase HPLC, digested with trypsin, and then again subjected to reverse-phase HPLC. As shown in Fig. 4 A, three major radioactive peaks, R1 to R3, were obtained. Phosphoamino acid analysis showed the presence of phosphothreonine in R1 and phosphoserine in both R2 and R3 (Fig.4 B). Amino acid sequence analysis revealed that R1 was the peptide containing Thr-7, R2 was the peptide containing Ser-34, and R3 was the peptide containing Ser-13 (Fig. 4 A). Ethanethiol treatment of R2 and R3, a procedure that converts specifically phosphoserine into S-ethylcysteine (31Meyer H.E. Hoffmann-Posorske E. Korte H. Heilmeyer Jr., L.M.G. FEBS Lett. 1986; 204: 61-66Crossref PubMed Scopus (218) Google Scholar), suggested that phosphates were located on Ser-34 and Ser-13, respectively (data not shown). Therefore, GFAP was shown to be phosphorylated at Thr-7, Ser-13, and Ser-34 by GST-Rho-kinase. By using a rabbit polyclonal antibody recognizing phosphorylated Thr-7, we have previously reported that Thr-7 is also phosphorylated at the cleavage furrow (7Matsuoka Y. Nishizawa K. Yano T. Shibata M. Ando S. Takahashi T. Inagaki M. EMBO J. 1992; 11: 2895-2902Crossref PubMed Scopus (90) Google Scholar).Figure 4Phosphorylation of Thr-7, Ser-13, and Ser-34 on GFAP by GST-Rho-kinase. A, the phosphorylated 6.5-kDa fragment derived from GFAP was digested with trypsin and subjected to reverse-phase HPLC as described under "Experimental Procedures." Three radioactive peaks (R1, R2, andR3) were analyzed with a gas-phase sequencer. The determined amino acid sequences of R1 (residues 5–11), R2 (residues 37–41), and R3 (residues 13–29) are indicated on the right. Note that we describe Ser-38 of human GFAP as Ser-34 because Ser-38 of human GFAP corresponds to Ser-34 of porcine GFAP. B, aliquots of R1, R2, and R3 in A were subjected to two-dimensional phosphoamino acid analysis.View Large Image Figure ViewerDownload (PPT) We then examined the effect of phosphorylation of GFAP by Rho-kinase on the filament forming ability of GFAP. Soluble GFAP was preincubated with or without GST-Rho-kinase for 30 min, and the samples were incubated under conditions of polymerization (25 mmimidazole-HCl, pH 6.75, and 100 mm NaCl at 37 °C) (28Inagaki M. Gonda Y. Nishizawa K. Kitamura S. Sato C. Ando S. Tanabe K. Kikuchi K. Tsuiki S. Nishi Y. J. Biol. Chem. 1990; 265: 4722-4729Abstract Full Text PDF PubMed Google Scholar) for a further hour. Then the NaCl- and pH-dependent filament formation of GFAP in these samples was analyzed by centrifugation (Fig. 5 A) and electron microscopy (Fig. 5 B). As shown in Fig. 5, the phosphorylation of GFAP by GST-Rho-kinase resulted in a nearly complete inhibition of its filament formation. These results increase the possibility that GFAP phosphorylation at Thr-7, Ser-13, and Ser-34 during cytokinesis may induce the fragmentation of glial filaments at the cleavage furrow. In the present study, we obtained evidence that GFAP can serve as an excellent substrate for Rho-kinase in a GTP·Rho-dependent manner. So far, MBS (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2399) Google Scholar) and myosin (15Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1647) Google Scholar) were the only preferred substrates for Rho-kinase. The phosphorylated GFAP lost its ability to form filaments in vitro. The in vitrophosphorylation sites on GFAP by Rho-kinase were Thr-7, Ser-13, and Ser-34, which are the same sites that CF kinase phosphorylates at the cleavage furrow during cytokinesis. We are considering that Rho-kinase may be CF kinase itself, and if so it may play an important role in the cleavage furrow-specific reorganization of IFs during cytokinesis. Because Rho-kinase was recently reported to act downstream of Rho in the regulation of myosin phosphorylation (12Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2399) Google Scholar, 15Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1647) Google Scholar) and the formation of stress fibers and focal adhesion complexes (32Leung T. Chen X.-Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 33Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (942) Google Scholar), Rho-kinase may also mediate the regulation of cytokinesis by Rho (18Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. Cell Biol. 1993; 120: 1187-1195Crossref PubMed Scopus (308) Google Scholar, 19Mabuchi I. Hamaguchi Y. Fujimoto H. Morii N. Mishima M. Narumiya S. Zygote. 1993; 1: 325-331Crossref PubMed Scopus (212) Google Scholar). Whether Rho-kinase is activated during cytokinesis is the subject of ongoing studies. Because Rho-kinase belongs to a family of related serine/threonine kinases including myotonic dystrophy kinase, these kinases may phosphorylate the similar sites on GFAP. Further investigations are necessary to elucidate the relationship between CF kinase and Rho-kinase or its family members. ACKNOWLEDGEMENTS We are grateful to Dr. H. Goto (our laboratory) for kind help with the reverse-phase HPLC and Dr. K. Tsujimura (Aichi Cancer Center Research Institute) for helpful discussions and comments. M. Ohara provided critical comments on the manuscript.

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