Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments.
To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.
Inada
H
H
Division of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Chikusa-ku, Nagoya, Aichi 464-8681, Japan.
Togashi
H
H
Nakamura
Y
Y
Kaibuchi
K
K
Nagata
K
K
Inagaki
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D003893:Desmin / Q000276:immunology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008387:Marine Toxins; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014746:Vimentin / Q000276:immunology / Q000378:metabolism*; D054460:rho-Associated Kinases
D003893:Desmin; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008387:Marine Toxins; D010080:Oxazoles; D011994:Recombinant Proteins; D014746:Vimentin; D019319:Okadaic Acid; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases