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J. Biol. Chem., Vol. 276, Issue 25, 22351-22358, June 22, 2001

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Actin Filament Cross-linking by MARCKS
CHARACTERIZATION OF TWO ACTIN-BINDING SITES WITHIN THE PHOSPHORYLATION SITE DOMAIN^*

Elena G. Yarmola, Arthur S. Edison, Robert H. Lenox^§, and Michael R. Bubb^¶

From the Departments of Medicine and  Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, the ^§ Departments of Psychiatry, Pharmacology, and Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the ^¶ Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin ₄ and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

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