Regulation of F-actin binding to platelet moesin in vitro by both phosphorylation of threonine 558 and polyphosphatidylinositides

Activation of human platelets with thrombin transiently increases phosphory lation at (558)threonine of moesin as determined with phosphorylation state -specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphata se inhibitor calyculin A, making it possible to purify the two forms of moe sin to homogeneity. Blot overlay assays with F-actin probes labeled with ei ther [P-32]ATP or I-125 show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified, in the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonio nic, or amphoteric detergents. The interaction affinity is high (K-d, simil ar to 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This inte raction is also observed in platelets extracted with cationic but not with nonionic detergents. in 0.1% Triton X-100, F-actin interacts with phosphory lated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high- affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages be tween the actin cytoskeleton and the plasma membrane.