Characterization of the Regulatory Domain of Gizzard Calponin

doi:10.1074/jbc.270.15.8867

Characterization of the Regulatory Domain of Gizzard Calponin

INTERACTIONS OF THE 145-163 REGION WITH F-ACTIN, CALCIUM-BINDING PROTEINS, AND TROPOMYOSIN (*)

From the (1) Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, Route de Mende, BP 5051, 34033 Montpellier Cedex, France

^¶ To whom correspondence and reprint requests should be addressed.

Abstract

Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH₂-termi-nal portion of the calponin segment Ala-Tyr(Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr-Ile. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala-Ileis essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln-Ile, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.

Footnotes

↵^§ Recipient of a scholarship from the Moroccan Government and the Association Franaise contre les Myopathies.
↵* This research was supported by grants from CNRS, INSERM, and the Association Franaise contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EDC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

NHS

N-hydroxysuccinimide

HPLC

high performance liquid chromatography

CaT

caltropin

CaM

calmodulin

CaP

calponin

TM

tropomyosin.
↵²M. Mezgueldi, P. Strasser, K. Anderson, M. Jaritz, A. Fattoum, and M. Gimona, submitted for publication.
↵³A. Represa, H. Trabelsi-Terzidis, M. Plantier, A. Fattoum, I. Jorquera, F. Dessi, G. Barbin, Y. Ben-Ari, and E. Der Terrossian, submitted for publication.