Protein kinase C-θ phosphorylation of moesin in the actin-binding sequence

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 actin-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 leukocyte extracts, In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphatidylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG), Analysis of charge shifts, phosphoamino acid analysis, and stoichiometry was consistent with a single phosphorylation site, By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosphorylation site was identified as KYKT*LRQIR (where * indicates the phosphorylation site) (Thr(558)), which is conserved in the ERM family, Recombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein, The phosphorylation site sequence of moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family, We identified the kinase activity as PKC-theta on the basis of immunodepletion of the moesin kinase activity and copurification of PKC-theta with the enzymic activity, We further demonstrate that PKC-theta displays a preference for PG vesicles over PI or PS/DAG, with minimal activation 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 Ni2+ chelate chromatography yield an active enzyme that phosphorylates moesin, PG vesicle binding experiments with expressed PKC-theta and moesin demonstrate that both bind to vesicles independently of one another, Thus, PKC-theta is identified as a major kinase within cells with specificity for moesin and with activation under non-classical PKC conditions, It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as other ERM proteins.