Identification of calponin as a novel substrate of Rho-kinase.
Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.
Kaneko
T
T
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.
Amano
M
M
Maeda
A
A
Goto
H
H
Takahashi
K
K
Ito
M
M
Kaibuchi
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology / Q000378:metabolism; D010769:Phosphothreonine / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054460:rho-Associated Kinases; D000096985:Calponins
D000199:Actins; D000906:Antibodies; D002135:Calcium-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D010768:Phosphoserine; D010769:Phosphothreonine; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D054460:rho-Associated Kinases; D000096985:Calponins