Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase.
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.
Kosako
H
H
Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464, Japan.
Amano
M
M
Yanagida
M
M
Tanabe
K
K
Nishi
Y
Y
Kaibuchi
K
K
Inagaki
M
M
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001254:Astrocytoma; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D054460:rho-Associated Kinases
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005904:Glial Fibrillary Acidic Protein; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases