Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.

Abstract

The gene for LSPI is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSPI mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lynrp}iocytes. To study the expression of the mouse LSPI protein, we prepared a polyclonal antiserum spetific for the LSPI protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSPI protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSPI protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.The lymphocyte-specific gene for LSP1 has been isolated by us in the course of a systematic search for cDNA clones which are expressed specifically in all or certain lymphocyte populations but not in nonlymphoid cells (10,12). Such cell type-specific genes are presumably involved in cell typespecific effector functions or, alternatively, they are involved in the regulation of growth or differentiation of the specific cell types in which they are expressed. Northern (RNA) blot analysis has shown that mouse LSP1 mRNA is present in normal B lymphocytes and transformed B-lymphoma cell lines. LSP1 is also expressed in normal thymocytes and in normal functional helper and cytotoxic T-cell lines. However, no LSP1 RNA is present in nine transformed T-lymphoma cell lines tested. The gene for LSP1 is also not expressed in a variety of myeloid cell lines or in nonlymphoid tissues, such as liver, kidney, and heart tissues. A gene with a similar expression pattern can be detected in a series of human cell lines by using the mouse LSP1 cDNA clone as a hybridization probe (12). This lymphocyte-specific expression pattern and the absence of any detectable LSP1 mRNA in transformed T-lymphoma cell lines suggest a role for the LSP1 protein in the regulation of normal lymphocyte growth. Sequence analysis of an LSP1 cDNA clone predicts that the LSP1 protein consists of 330 amino acids with an acidic amino-terminal domain which contains two putative Ca2'-binding sites (12). Given the well-established role of Ca2' as an intracellular mediator of signal transduction (14, 18), ...

Modulation of Mac-1 (CD11b/CD18)-Mediated Adhesion by the Leukocyte-Specific Protein 1 Is Key to Its Role in Neutrophil Polarization and Chemotaxis

“…LSP1 mRNA and protein is expressed in mature and immature B and T cells, macrophages, and neutrophils (31)(32)(33). Human and mouse LSP1 share the same expression patterns (26, 34 -37) and the amino acid identity between the two species is 85% for the basic C-terminal half containing multiple F-actin binding domains and 53% for the acidic N-terminal half.…”

Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton

“…LSP1 is an F-actin binding protein that accumulates on the peripheral actin filaments underneath the plasma membrane, often referred to as the membrane skeleton or cortical cytoskeleton (Jongstra-Bilen et al, 1992;Klein et al, 1990;Klein et al, 1989). To test the hypothesis that LSP1 targets KSR, MEK1 and ERK2 to the membrane skeleton, we used confocal microscopy to localize KSR, MEK1 and ERK2 in the LSP1 neg T-lymphoma cell line BW5147 and an LSP1 + transfectant of BW5147, designated T22.…”

“…Goat anti-PKCβI, goat anti-MEK1, goat anti-ERK2, goat anti-KSR and goat anti-caspase 1 were from SantaCruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-LSP1 serum was prepared as described previously (Klein et al, 1989). The MEK1 inhibitor U0126 (Favata et al, 1998) was purchased from Calbiochem (La Jolla, CA), was dissolved in DMSO and added 20 minutes before addition of anti-IgM.…”

Inhibition of Anti-IgM-induced Translocation of Protein Kinase C βI Inhibits ERK2 Activation and Increases Apoptosis

“…Evidence for a role of one or more of the Ca 2ϩ -dependent cPKC isoforms ␣, ␤I, ␤II, or ␥ in this protection comes from experiments showing that the protective action of phorbol esters on immature B-lymphocytes (13) is abrogated by a cPKC-specific inhibitor. This inhibitor also renders mature Blymphocytes susceptible to anti-IgM-induced apoptosis (13), suggesting that anti-IgM-induced activation of cPKC isoforms plays an important role in regulating susceptibility of different B-lymphocyte lineage cells to anti-IgM-induced apoptosis.The mouse leukocyte-specific protein 1 (LSP1) is a 330-amino acid residue intracellular protein expressed in B-and T-lymphocytes and in macrophages and neutrophils (15)(16)(17)(18)(19). Transfection experiments using the LSP1 ϩ B-lymphoma cell line WEHI-231/89 or a single cell subclone, W10, showed that expression of an LSP1 truncate containing residues 179 -330 (designated B-LSP1) significantly increased the extent of apoptosis induced by anti-IgM or by ionomycin but not by sorbitol, nocodazole, C 2 -ceramide, or H 2 O 2 (20).…”