Two distinct actin-binding sites of smooth muscle calponin

Eur J Biochem. 1998 Jan 15;251(1-2):262-8. doi: 10.1046/j.1432-1327.1998.2510262.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Binding, Competitive / drug effects
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / drug effects
  • Calcium-Binding Proteins / metabolism*
  • Histidine
  • Lysine
  • Microfilament Proteins
  • Molecular Sequence Data
  • Muscle, Smooth / chemistry*
  • Myosin-Light-Chain Kinase / drug effects
  • Myosin-Light-Chain Kinase / metabolism
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / metabolism
  • Peptide Fragments / pharmacology
  • Phosphorylation
  • Protein Kinase C / metabolism

Substances

  • Actins
  • Calcium-Binding Proteins
  • Microfilament Proteins
  • Peptide Fragments
  • calponin
  • Histidine
  • Protein Kinase C
  • Myosin-Light-Chain Kinase
  • Lysine