|
|
Identification of the In Vivo Phosphorylation Sites in Murine Leukocyte-Specific Protein 1
University of Texas Southwestern Medical Center, Dallas, TX
Abstract
An abundant phosphoprotein was detected in primary B cells and WEHI-231 cells as part of a larger effort to identify ligand-induced changes in protein phosphorylation. This phosphoprotein was identified as leukocyte-specific protein-1 (LSP1), an actin-binding protein that functions in the organization of the actin cytoskeleton and cell movement. LSP1 is known to be phosphorylated in response to numerous stimuli. In this study, the two-dimensional differential gel electrophoresis system (2-D DIGE) and mass spectrometry were used to identify multiple splice variants and phosphorylated forms of LSP1 from murine primary B lymphocytes (B cells) and WEHI-231 cells. By combining 2-D DIGE with 32P-labeling, seven phosphorylated forms of LSP1 were detected. To further characterize the phosphorylation of LSP1 in B cells, the effects of physiological stimuli and phorbol-12-myristate-13-acetate (PMA) were examined. B-cell LSP1 was found to be highly phosphorylated, even under basal conditions. LSP1 phosphorylation was strongly stimulated in response to anti-IgM and by PMA but only weakly stimulated by interleukin-4 (IL-4). Seven in vivo phosphorylation sites were detected in LSP1 using mass spectrometry. The identified phosphorylation sites include putative targets for protein kinase C, glycogen synthase kinase 3, extracellular signal-related kinase 1 (ERK1), calmodulin-dependent protein kinase 2, and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The potential for phosphorylation by multiple kinases suggests that LSP1 serves as an important integration point for regulation of the actin cytoskeleton by multiple signaling pathways.
|
|
