Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 276, Issue 25, Pages 22351-22358Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M101457200
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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 P, 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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