Abstract
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 beta(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.
Highlights
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 alaninerich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments
Since phosphorylated MARCKS phosphorylation site domain (PSD) did not cross-link actin filaments as well as nonphosphorylated MARCKS PSD, we speculated that either steric and ionic effects of phosphorylation resulted in a loss of site-specific affinity at either of two postulated actin-binding sites or that the conformation changes in the PSD resulted in a dynamic structure that had an inaccessible actin-binding site
Summary
Materials—Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers AR) in buffer G (5.0 mM Tris-HCl, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl2, and 0.01% sodium azide, pH 7.8) [24], and pyrenyl-labeled actin was prepared with 0.7– 0.95 mol of label/mol of protein using the method of Kouyama and Mihashi [23]. The direct binding assay is used to determine a nonsaturating amount of actin that will bind about two-thirds of the labeled peptide, and the anisotropy is measured as a function of concentration of competing substance. The data were analyzed as previously described [25], assuming only that the concentration of free labeled peptide, [L], and unlabeled peptide [P] satisfy the relation, [L]/KdL ϽϽ (1 ϩ [P]/KdP), where KdL and KdP are the respective equilibrium dissociation constants for labeled and unlabeled peptide-actin complex. Bound fluorescently labeled peptide was assumed to have the same anisotropy, rb, independent of which and how many sites were occupied In this case, these binding constants were fixed, and the data were fit to an additional parameter, , the ratio of equilibrium dissociation constants for binding at one site relative to that for the same site when the other site is occupied. Data were processed using NMRPipe [33] and analyzed using NMRView [34]
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