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

Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4707-12. doi: 10.1073/pnas.0805963106. Epub 2009 Mar 2.

Abstract

ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Cell Polarity / drug effects
  • Chemokines / pharmacology
  • Chemotaxis / drug effects
  • Cytoskeletal Proteins / metabolism*
  • Cytoskeleton / enzymology*
  • Humans
  • Jurkat Cells
  • Lymphocytes / cytology
  • Lymphocytes / drug effects
  • Lymphocytes / enzymology*
  • Lymphocytes / ultrastructure
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Knockout
  • Microfilament Proteins / metabolism*
  • Molecular Sequence Data
  • Peptides / metabolism
  • Phosphorylation / drug effects
  • Protein Transport / drug effects
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism*
  • Substrate Specificity / drug effects

Substances

  • Chemokines
  • Cytoskeletal Proteins
  • Membrane Proteins
  • Microfilament Proteins
  • Peptides
  • ezrin
  • moesin
  • radixin
  • Protein-Serine-Threonine Kinases
  • STK10 protein, human
  • Stk10 protein, mouse