Introduction

Leukocyte-specific protein 1 (also known as lymphocyte-specific protein 1, LSP1, pp52, and p50; AfCS ID A002813) is an actin-binding protein specifically expressed in B lymphocytes (B cells), T lymphocytes (T cells), macrophages, and neutrophils(1,2). LSP1 functions in the organization of the actin cytoskeleton and in cell movement. LSP1 assembles F-actin into thick, multi-branched bundles in vitro and coaggregates into caps with surface IgM in stimulated B cells(3). During chemotaxis in neutrophils, LSP1 colocalizes with F-actin in filopodia, lamellipodia, and membrane ruffles. Neutrophils from LSP1 knockout mice are impaired in their ability to perform chemotaxis(4), and ectopic expression of LSP1 modulates the motility of the U937 leukocyte cell line(5). LSP1 also appears to play a role in lymphocyte apoptosis(6).

The predicted size of LSP1 is 37-kDa, but the protein migrates as a 47- to 52-kDa species during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(7). Two splice variants of LSP1 that migrate with apparent molecular masses of 50- and 52-kDa during SDS-PAGE are expressed in murine B cells. The shorter protein (324 amino acids) differs from the longer one (330 amino acids) due to deletion of a short peptide (HLIRHQ) in the central region of the molecule. Each splice variant of LSP1 migrates as multiple spots during two-dimensional gel electrophoresis, with isoelectric points ranging from pH 4.4 to 4.7 (8). There is an additional splice variant, named S37, that differs in its N-terminal amino acid sequence and is expressed in non-hematopoietic tissues(9).

LSP1 is phosphorylated in response to numerous stimuli. Phosphorylation of murine T-cell LSP1 on serine and threonine residues occurs in response to stimulation with concanavalin A or phorbol-12-myristate-13-acetate (PMA) but not with calcium ionophore(8). Peptide mapping showed that several sites phosphorylated in response to concanavalin A are phosphorylated by protein kinase C (PKC) in vitro(10). LSP1 is the major PKC substrate in B cells(11) and can interact directly with PKCb. PKC-mediated phosphorylation of LSP1 causes dissociation from PKCb and translocation of LSP1 from the plasma membrane to the cytosol(6,10). LSP1 is also a major substrate for mitogen-activated protein-kinase-activated protein kinase 2 (MAPKAP kinase 2) in neutrophils (12,13). Phosphorylation of LSP1 by MAPKAP kinase 2 may be part of the signaling pathway involving Rac1/Cdc42h, p21-activated kinase (PAK), p38 MAP kinase, and MAPKAP kinase 2 that regulates actin organization (14). The functional consequences of LSP1 phosphorylation are not known.

During the course of experiments to identify ligand-induced changes in protein phosphorylation in B cells, a major phosphoprotein was found in the murine WEHI-231 B lymphoma cell line and in primary murine B cells. By two-dimensional electrophoresis and mass spectrometry, this protein was identified as LSP1. Because of its potential importance as a downstream target of multiple signaling pathways, we mapped the in vivo phosphorylation sites on LSP1. A total of seven phosphorylation sites were detected in murine LSP1. Four of these sites were mapped to specific serine residues, and the remaining three sites were localized to specific peptide regions. This work establishes, within the Alliance for Cellular Signaling (AfCS) laboratories, the methodology for efficient identification of phosphorylation sites that may be pursued in future studies.