PROTEIN-KINASE C-THETA PHOSPHORYLATION OF MOESIN IN THE ACTIN-BINDINGSEQUENCE

Moesin, a member of the ezrin-radixin-moesin (ERM) family of membrane/ cytoskeletal linkage proteins, is known to be threonine-phosphorylated at Thr(558) in activated platelets within its conserved putative acti n-binding domain, The pathway leading to this phosphorylation step and its control have not been previously elucidated, We have detected and characterized reactions leading to moesin phosphorylation in human le ukocyte extracts, In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphati dylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG), Analysis of cha rge shifts, phosphoamino acid analysis, and stoichiometry was consiste nt with a single phosphorylation site, By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosph orylation site was identified as KYKTLRQIR (where * indicates the pho sphorylation site) (Thr(558)), which is conserved in the ERM family, R ecombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein, The phosphoryla tion site sequence of moesin displays a high degree of conservation wi th the pseudosubstrate sequences of the protein kinase C (PKC) family, We identified the kinase activity as PKC-theta on the basis of immuno depletion of the moesin kinase activity and copurification of PKC-thet a with the enzymic activity, We further demonstrate that PKC-theta dis plays a preference for PG vesicles over PI or PS/DAG, with minimal act ivation by DAG, as well as specificity for moesin compared with myelin basic protein, histone H1, or other cellular proteins, Expression of a human His(6)-tagged PKC-theta in Jurkat cells and purification by Ni 2+ chelate chromatography yield an active enzyme that phosphorylates m oesin, PG vesicle binding experiments with expressed PKC-theta and moe sin demonstrate that both bind to vesicles independently of one anothe r, Thus, PKC-theta is identified as a major kinase within cells with s pecificity for moesin and with activation under non-classical PKC cond itions, It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as o ther ERM proteins.