title: Two distinct actin-binding sites of smooth muscle calponin.
abstract: Amino acid residues 145-163 of calponin have been proposed as a putative actin-binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201] enhanced the Ca2+-induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172-His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172-His187 inhibited the binding of calponin to F-actin in a concentration-dependent manner but not the binding of caldesmon. Gly141-Gly160, including the above-mentioned putative actin-binding site, also competed with intact calponin to the same extent as Lys172-His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141-Gly160. Lys172-His187 and other tested peptides had no effect. However, Gly141-Gly160 and Lys172-His187 reduced the fluorescence intensity of pyrene-labeled F-actin with approximately equal potency. Moreover, Lys172-His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172-His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin-binding activity. These observations suggest the direct involvement of two distinct actin-binding sites, with different regulatory functions, in the interactions of calponin with actin.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Edobashi, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mino
authors.forename: U
authors.initials: U
authors.lastname: Yuasa
authors.forename: F
authors.initials: F
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Naka
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006639:Histidine; D008239:Lysine; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry*; D009219:Myosin-Light-Chain Kinase / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D000096985:Calponins
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; D006639:Histidine; D008239:Lysine; D008840:Microfilament Proteins; D009219:Myosin-Light-Chain Kinase; D010446:Peptide Fragments; D011493:Protein Kinase C; D000096985:Calponins