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J. Biol. Chem., Vol. 279, Issue 51, 53387-53394, December 17, 2004

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Modes of Caldesmon Binding to Actin

SITES OF CALDESMON CONTACT AND MODULATION OF INTERACTIONS BY PHOSPHORYLATION^*

D. Brian Foster¶||, Renjian Huang¶, Victoria Hatch, Roger Craig**, Philip Graceffa, William Lehman, and C.-L. Albert Wang

From the Boston Biomedical Research Institute, Watertown, Massachusetts 02472, the Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, and the ^**Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys⁵⁹⁵, away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

Received for publication, September 2, 2004 , and in revised form, September 15, 2004.

^* This work was supported in part by Diabetes Endocrinology Research Center Grant DK32520 and National Institutes of Health Grants RO1-HL36153 (to W. L.), RO1-HL62468 (to R. C.), and PO1-AR41637 (to C.-L. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both authors contributed equally to this work.

^|| Supported by the Boston Biomedical Research Institute Scholar Program and by an American Heart Association Postdoctoral Fellowship (New England Affiliate). Present address: Institute of Molecular Cardiobiology, Johns Hopkins School of Medicine, Ross Research Bldg., Rm. 858, 720 Rutland Ave., Baltimore, MD 21205.

To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown MA, 02472-2829. Tel.: 617-658-7803; Fax: 617-972-1753; E-mail: wang{at}bbri.org.

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