Two distinct actin‐binding sites of smooth muscle calponin

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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, 20 400−20 403]. 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.

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