Protein Kinase C-θ Phosphorylation of Moesin in the Actin-binding Sequence

Abstract

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 actinbinding 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 PKCon the basis of immunodepletion of the moesin kinase activity and copurification of PKC-with the enzymic activity. We further demonstrate that PKC-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-in Jurkat cells and purification by Ni 2؉ chelate chromatography yield an active enzyme that phosphorylates moesin. PG vesicle binding experiments with expressed PKC-and moesin demonstrate that both bind to vesicles independently of one another. Thus, PKC-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.Moesin is a member of the ERM 1 family of actin cytoskeletalmembrane linkage proteins. Although closely related, there is evidence that these proteins have distinct functions, based upon their patterns of intracellular and tissue distribution. Moesin is named for its association with extracellular extensions (membrane-organizing-extension spike protein). ERM proteins have N-terminal membrane-binding domains and Cterminal actin-binding domains. Relatively little is known about their intracellular control, although phosphorylation of moesin Thr 558 by an unidentified kinase in activated platelets has recently been described (1). This residue is within the previously identified actin-binding domain of ezrin, which is highly conserved in moesin (2).This laboratory has be...

Effect of merlin phosphorylation on neurofibromatosis 2 (NF2) gene function

“…Phosphorylation represents one mechanism for regulating the function of ERM proteins by modulating the ability of these proteins to form FERM/C-term associations (Nakamura et al, 1995;Matsui et al, 1998;Oshiro et al, 1998;Pietromonaco et al, 1998;Tran Quang et al, 2000;Ng et al, 2001). When phosphorylated at threonine 558 (moesin), 567 (ezrin) or 564 (radixin), ERM proteins assume an open conformation and can interact with F-actin.…”

LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

“…2 A and B). This preference is also shared by PKC-, another candidate ERM kinase (11,23,24). LOK too has a preference for basic residues, but the most prominent feature of LOK's peptide specificity is its strong and unusual preference for Y at P-2 (2 amino acids before the phosphorylation site), which is not shared by ROCK (Fig.…”

Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

“…Activation of ERM proteins occurs by conformational changes triggered by binding of phosphatidylinositol 4,5-biphosphate (PIP 2 ) to the FERM (band 4.1, ERM) domain in the NH 2 -terminal region, termed the NH 2 -terminal ERM-associated domain (N-ERMAD) and phosphorylation of a conserved threonine residue (ezrin-T567, radixin-T564, moesin-T558) in the COOH-terminal domain, termed the COOH-terminal ERMassociated domain (C-ERMAD; Pietromonaco et al, 1998;Barret et al, 2000;Ng et al, 2001;Fievet et al, 2004). This sequence of molecular events releases the intramolecular interaction between the N-and C-termini.…”