Actin filament cross-linking by MARCKS - Characterization of two actin-binding sites within the phosphorylation site domains

We recently identified conformational changes that occur upon phosphorylati on of myristoylated alanine-rich protein kinase C substrate (MARCKS) that p reclude efficient cross-linking of actin filaments (Bubb, M. R,, Lenox, R, H., and Edison, A. S, (1999) J; Biol. Chem. 274, 36472-36478), These result s implied that the phosphorylation site domain of MARCKS has two actin-bind ing sites. We now present evidence for the existence of two actin-binding s ites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin P, and actobindin to bind to actin. The eff ects of substitution of alanine for phenylalanine within a repeated hexapep tide segment suggest that the noncharged region of the domain contributes t o binding affinity, but the binding affinity of peptides corresponding to e ach binding site has a steep dependence on salt concentration consistent wi th 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 eff ect of alanine substitution. However, phosphorylation has a much greater ef fect 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.