ERM phosphorylation in lymphocytes

Shaw, James

Abstract

Traditional basophilic kinases in the AGC family (like PKC and ROCK) are widely proposed to be the dominant ERM kinases, but the evidence is less than conclusive and, in particular, genetic evidence is lacking in vertebrates. We exploited findings from our mass spectrometric analysis (BC 010993) and identified a previously obscure kinase LOK (lymphocyte-oriented kinase) as a candidate based on its prominent enrichment in the membrane/microvillus fraction. Immunofluorescence studies demonstrate that LOK is appropriately localized at the plasma membrane to mediate ERM phosphorylation. Peptide specificity analysis shows it to be a basophilic kinase with a predicted optimum sequence resembling the ERM site, including an unusual preference for tyrosine at P 2. Protein phosphorylation analysis demonstrates phosphorylation of ERM that is at least comparable in rate to two favored candidate ERM kinases: PKC and ROCK. 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 more than 50% reduction in ERM phosphorylation. Taken together the findings on localization and specificity argue strongly 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 make sense in the context of current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of major importance in lymphocytes and elucidate a role for it in regulating cell shape and motility. A manuscript on these studies is currently under review. We have also developed a transgenic mouse model as important complementary approach to assessing the roles of ERM phosphorylation. The transgenic mouse expresses a mutant ezrin gene in which the C-terminal phosphorylation site is mutated to an acidic residue to mimic constitutive phosphorylation (T567E) and tagged with a short peptide HA tag at the extreme C-terminus. The gene is selectively expressed in T-cells (by use of a CD2 cassette) and protein expression is at most half of the endogenous ezrin levels. Studies of this mouse are ongoing and include comparisons with a transgenic mouse strain expressing a comparable construct without the mutation. Current findings indicate close to normal immune system development. However, in vivo migration of T-cells in lymph node is about half of normal, and the transgenic mice have a surprisingly early defect in resistance to Toxoplasmosis (given the prevailing view of the role of T-cells in immunity). Ongoing studies are directed at understanding the molecular basis of these defects.