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 558threonine 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 α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼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.

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.