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 phosphorylation 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 phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I 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, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, approximately 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated 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 between the actin cytoskeleton and the plasma membrane.
Nakamura
F
F
Laboratory of Environmental Biochemistry, Department of Environmental Biology, Graduate School of Agricultural Sciences, Tohoku University, Sendai 981-8555, Japan.
Huang
L
L
Pestonjamasp
K
K
Luna
E J
EJ
Furthmayr
H
H
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods; D001792:Blood Platelets / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D000644:Quaternary Ammonium Compounds; D013912:Threonine / Q000378:metabolism
D000199:Actins; D003902:Detergents; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D000644:Quaternary Ammonium Compounds; C071058:moesin; D013912:Threonine; C022806:dodecyltrimethylammonium