Inhibition of anti-IgM-induced translocation of protein kinase C beta I inhibits ERK2 activation and increases apoptosis.

Expression of the COOH-terminal residues 179-330 of the LSP1 protein in the LSP1(+) B-cell line W10 increases anti-IgM- or ionomycin-induced apoptosis, suggesting that expression of this LSP1 truncate (B-LSP1) interferes with a Ca(2+)-dependent step in anti-IgM signaling. Here we show that inhibition of Ca(2+)-dependent conventional protein kinase C (cPKC) isoforms with Gö6976 increases anti-IgM-induced apoptosis of W10 cells and that expression of B-LSP1 inhibits translocation of PKCbetaI but not of PKCbetaII or PKCalpha to the plasma membrane. The increased anti-IgM-induced apoptosis is partially reversed by overexpression of PKCbetaI. This shows that the B-LSP1-mediated inhibition of PKCbetaI leads to increased anti-IgM-induced apoptosis. Expression of constitutively active PKCbetaI protein in W10 cells activates the mitogen-activated protein kinase ERK2, whereas expression of B-LSP1 inhibits anti-IgM-induced activation of ERK2, suggesting that anti-IgM-activated PKCbetaI is involved in the activation of ERK2 and that inhibition of ERK2 activation contributes to the increased anti-IgM-induced apoptosis. Pull-down assays show that LSP1 interacts with PKCbetaI but not with PKCbetaII or PKCalpha in W10 cell lysates, while in vitro LSP1 and B-LSP1 bind directly to PKCbetaI. Thus, B-LSP1 is a unique reagent that binds PKCbetaI and inhibits anti-IgM-induced PKCbetaI translocation, leading to inhibition of ERK2 activation and increased apoptosis.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum.

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1(-/-)) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen. Lsp1(-/-) mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5(-) macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in Lsp1(-/-) mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1(-/-) mice are similar to those of wt mice and Lsp1(-/-) mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the Lsp1(-/-) mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1(-) neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis. (Blood. 2000;96:1827-1835)

LSP1 regulates anti-IgM induced apoptosis in WEHI-231 cells and normal immature B-cells.

Expression of LSP1, a 330 amino acid intracellular phosphoprotein, is restricted to lymphocytes, macrophages and neutrophils. In B-lymphoma cell lines LSP1 co-caps with membrane IgM after stimulation with anti-IgM. We used the LSP1+ B-lymphoma cell line WEHI-231/89 and normal lipopolysaccharide treated immature B-cells from Lsp1-/- and wild type mice to determine a role for LSP1 in signaling through membrane IgM. WEHI-231/89 cells were transfected with a truncated LSP1 protein containing the COOH-terminal residues 179-330. The three transfectants expressing the LSP1 truncate were significantly more susceptible to anti-IgM induced apoptosis than the parental cells or G418r control cell lines, while anti-IgM induced growth arrest was not affected. Expression of the LSP1 truncate increased the extent of anti-IgM induced loss of mitochondrial membrane potential, delta(psi)m indicating that LSP1 acts at a early stage in BCR mediated apoptosis. Expression of the LSP1 truncate in WEHI-231/89 cells increased susceptibility to ionomycin induced apoptosis but had no effect on apoptosis induced by nocodazole, sorbitol, C2-ceramide or H2O2. A role for LSP1 in anti-IgM induced apoptosis was confirmed using normal immature B-cells from 129/SvJ-Lsp1-/- mice which were less susceptible to anti-IgM induced apoptosis than those isolated from wild-type 129/SvJ mice. These results suggest that LSP1 regulates a Ca2+-dependent step in the induction phase of anti-IgM mediated apoptosis.

Tli1, a resistance locus for carcinogen-induced T-lymphoma.

Within 180 days after injection with N-methyl-N-nitrosourea (MNU), 83.5% of AKR/J mice and 37.5% of BALB/cJ mice developed T-lymphoma. The high tumor incidence was a dominant trait, as 93% of MNU-injected F1 mice developed T-lymphoma. A genome screen of 285 MNU-injected F2 mice identified a locus, designated T-lymphoma Induced 1 or Tli1, in a approximately 10-cM interval on central Chr 1 between D1Mit87 and D1Mit423 with significant linkage to the incidence of MNU-induced T-lymphoma (P = 0.0004). Injection of BALB/cJ.AKR/J-Tli1 congenic mice with MNU confirmed the presence of Tli1 on central Chr 1. Mice homozygous for the BALB/cJ allele (Tli1bb) were over-represented in the tumor-free F2 mice, while the inheritance of parental alleles of Tli1 in tumor-bearing mice was close to expected. This suggests that the Tli1b allele is recessive and suppresses MNU-induced T-lymphoma development in BALB/cJ mice and in Tli1bb F2 mice. Furthermore, the kinetics of lymphoma development in BALB/cJ and the Tli1 congenic mice suggests that Tli1b acts to suppress lymphomas developing late after injection with MNU. Two known genes that map in the identified genomic interval on central Chr 1 are candidates for Tli1:IL10, encoding the lymphokine IL10, and Cmkar4, encoding the chemokine receptor CXCR4.

LSP1 is the major substrate for mitogen-activated protein kinase-activated protein kinase 2 in human neutrophils.

In intact cells, mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 is rapidly activated by various cytokines, stresses, and chemotactic factors. The small heat shock protein p27 has been shown to be a substrate for MAPKAP kinase 2. Recently, we identified a novel substrate, designated p60, for MAPKAP kinase 2 in human neutrophils (Zu, Y.-L., Ai, Y., Gilchrist, A., Labadia, M. E., Sha'afi, R. I., and Huang, C.-K. (1996) Blood 87, 5287-5296). To further understand the signaling pathway of MAPKAP kinase 2, we have purified p60 from a heat-treated neutrophil lysate by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis. Microsequencing of five peptides derived from purified p60 indicates that p60 is lymphocyte-specific protein 1 (LSP1). Furthermore antibodies specific for human and mouse LSP1 react with human and mouse p60. The sequence of human LSP1 indicates two serine residues at positions 204 and 252 as potential phosphorylation sites. The amino acid sequences surrounding these two sites are in agreement with the consensus sequence (Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa) for phosphorylation by MAPKAP kinase 2. Both serine residues in human LSP1 and the corresponding conserved serine residues in mouse LSP1 are in the basic C-terminal F-actin binding domain. Various fusion proteins of wild type and truncated mouse LSP1 with glutathione S-transferase were tested for their capacity to be phosphorylated by MAPKAP kinase 2. The results indicate that LSP1 is a substrate for MAPKAP kinase 2 in vitro and that the phosphorylation sites are located in the basic C-terminal domain of LSP1. Because both the small heat shock proteins and LSP1 are F-actin binding proteins, these results suggest a role for MAPKAP kinase 2 in the regulation of cytoskeletal structure or function.

Low expression of the Lsp1 gene in early mouse T-lymphomas induced by N-methyl-N-nitrosourea.

The mouse Lsp1 gene encodes a 330 amino acid intracellular F-actin binding protein. Previously we showed that the mouse and human Lsp1 genes are expressed in normal B-cells and T-cells, including Thy1+ thymocytes and in normal macrophages and neutrophils. No or little LSP1 RNA and protein was found in a series of transformed mouse and human T-lymphoma cell lines, although normal antigen and lymphokine dependent T-cell lines expressed the Lsp1 gene. Here we show by Northern analysis that three mature antigen independent T-cell lines (CTLL-2, HT-2 and 532.10) which grow in the presence of lymphokine only, do not express LSP1 RNA, while mature resting and activated lymph node T-cells express high levels of LSP1 RNA. To determine whether the down-regulation of LSP1 expression is an early event which occurs in vivo in the tumor, rather than as a consequence of in vitro propagation of T-lymphoma cells, we analyzed LSP1 expression in primary T-lymphomas induced in AKR/J or AKR/J x BALB/cJ mice by a single intraperitoneal (i.p.) injection with 75 mg N-methyl-N-nitrosourea (MNU) per kg body-weight. Two- color Fluorescence Activated Cell Sorter (FACS) analysis showed that all tumors had an immature CD4+/CD8+ double positive (DP) phenotype. Many tumors contained a substantial population of CD4+ single positive (SP) cells. Since these cells may be infiltrating lymphocytes which can be expected to express the Lsp1 gene at a high level these tumors were not included in our analysis. Nineteen tumors were analyzed for Lsp1 gene expression and 13 contained reduced levels of LSP1 RNA, ranging from 4% to 44% of those found in age-matched normal thymus. Six tumors showed either no or only a small reduction in LSP1 RNA. These tumors had developed later than those expressing low levels of LSP1. The level of LSP1 RNA in tumors developing <110 days after injection of MNU was 19.1% +/- 5.2 (mean +/- SEM), while the level of LSP1 in later tumors was 78.4% +/- 13.0 (P = 0.004). Similar data were obtained when the expression of LSP1 protein was analyzed. These findings extend our previous data in several ways. First, they suggest a correlation between the down-regulation of LSP1 expression and abnormal regulation of growth or survival of immature and mature T-lymphocytes. Second, they show that down-regulation of the Lsp1 gene in transformed T-cells is not the result of prolonged in vitro culture, but occurs in the majority of primary tumors. Third, they show that there are two classes of T-lymphoma, which differ in their expression of LSP1 RNA and that the down-regulation of LSP1 is specifically associated with the early appearance of T-lymphoma after injection with MNU. This strongly suggests that the absence or reduced expression of LSP1 contributed to the transformation process and argues against the possibility that loss of LSP1 expression is a mere inconsequential epigenetic event.

Surrogate light chain-dependent selection of Ig heavy chain V regions.

Bone marrow B cell precursors frequently rearrange the Ig heavy chain variable (VH) gene segment VH81X. It is puzzling, therefore, that mature B cells in adult mice rarely express mu-heavy chains bearing this VH gene segment. We show in this work in transformed pre-B cell lines that two VH81X/mu-chains that differ in their VH-D-JH joining sequences are not assembled covalently with the B cell precursor-specific surrogate light (SL) chain and are not expressed on the cell surface. From these findings, we propose that a B cell precursor clonally expands and proceeds to the next developmental stage only if it expresses a mu-chain with a VH domain that, together with the SL chain, directs the formation of a signal-transducing mu/SL chain membrane complex. Therefore, a checkpoint exists early in B cell development, at which SL chain not only screens B cell precursors for the presence of a full-length mu-chain, but also for a VH domain that promotes the assembly of a mu/SL chain complex.

Expression of mouse LSP1/S37 isoforms. S37 is expressed in embryonic mesenchymal cells.

Mouse LSP1 is a 330 amino acid intracellular F-actin binding protein expressed in lymphocytes and macrophages but not in non-hematopoietic tissues. A 328 amino acid LSP1-related protein, designated S37, is expressed in murine bone marrow stromal cells, in fibroblasts, and in a myocyte cell line. The two proteins differ only at their N termini, the first 23 amino acid residues of LSP1 being replaced by 21 different residues in S37. The presence of different amino termini suggests that the LSP1 and S37 proteins are encoded by transcripts arising through alternative exon splicing. Here we report the genomic organization of the Lsp1 gene and show that the distinct N termini of LSP1 and S37 are encoded by two alternatively used exons, each containing a translational start codon. We also demonstrate that alternative 3' acceptor sites are used in the splicing of exon 5. This results in LSP1 and S37 transcripts that either do or do not contain 18 bp encoding the 6 amino acids HLIRHQ of the acidic domain. Therefore, the Lsp1 gene encodes four protein isoforms: full-length LSP1 and S37 proteins, designated LSP1-I and S37-I and the same proteins without the HLIRHQ sequence, designated LSP1-II and S37-II. By in situ hybridization analysis we show that the S37 isoforms are expressed in mesenchymal tissue, but not in adjacent epithelial tissue, of several developing organs during mouse embryogenesis. This, together with our finding that S37 is an F-actin binding protein, suggests that S37 is a cytoskeletal protein of mesenchymal cells, which may play a role in mesenchyme-induced epithelial differentiation during organogenesis.

The LSP1 gene is expressed in cultured normal and transformed mouse macrophages.

The mouse LSP1 protein is an F-actin binding protein initially isolated as a cDNA from the BALB/c pre-B cell line 220.2. Its expression pattern is highly restricted. Only lymphocytes and lymphoma cell lines were reported to express LSP1. Non-lymphoid cell lines or normal mouse tissues such as brain, lung, liver, skeletal muscle, kidney or testis do not express LSP1. Here we report that mouse macrophage cell lines also express LSP1 mRNA and protein. DNA sequence analysis shows that the coding sequence of LSP1 RNA expressed in the macrophage cell line P388D1 is identical to the sequence of LSP1 RNA from the pre-B cell line 220.2. To determine the expression of LSP1 RNA in normal macrophages derived from fetal liver and adult bone marrow and in other hematopoietic cells we used a recently described technique to make representative amplified polyA cDNA samples from small numbers of cells or isolated hematopoietic colonies. Analysis of these cDNA samples with an LSP1 cDNA probe showed that eight of nine macrophage samples expressed LSP1 RNA. One of two neutrophil samples but none of eight other non-lymphoid colonies was positive for LSP1 RNA. From these results it appears that the expression of LSP1 RNA in the hematopoietic system is restricted to the lymphocyte, macrophage and neutrophil lineages.

Distinct p53/56lyn and p59fyn domains associate with nonphosphorylated and phosphorylated Ig-alpha.

Among the earliest detectable events in B-cell antigen receptor-mediated signal transduction are the activation of receptor-associated Src-family tyrosine kinases and the tyrosine phosphorylation of Ig-alpha and Ig-beta receptor subunits. These kinases appear to interact with resting B-cell antigen receptor complexes primarily through the Ig-alpha chain antigen receptor homology 1 (ARH1) motif. Recent studies showed a dramatic increase in the amount of Src-family kinase p59fyn bound to Ig-alpha when ARH1 motif tyrosines were phosphorylated. To explore the submolecular basis of these interactions, we conducted mutational analysis to localize sites in p53/56lyn and p59fyn that bind nonphosphorylated and phosphorylated Ig-alpha. Here we report that distinct regions within these kinases bind nonphosphorylated and phosphorylated Ig-alpha ARH1 motifs. The N-terminal 10 residues mediate binding to the nonphosphorylated Ig-alpha ARH1 motif. Association with the phosphorylated Ig-alpha ARH1 motif is mediated by Src homology 2 domains. These findings suggest a mechanism whereby ligand-induced Ig-alpha tyrosine phosphorylation initiates a change in the orientation of an associated kinase that may alter its activity and/or access to substrates and other effectors.

Monoclonal anti-lambda 5 antibody FS1 identifies a 130 kDa protein associated with lambda 5 and Vpre-B on the surface of early pre-B cell lines.

mAbs specific for mouse lambda 5 protein were prepared by fusion of spleen cells from a hamster immunized with recombinant lambda 5 protein synthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14. Here we report the characteristics of the antibodies produced by the FS1 hybridoma. FS1 antibody stains a variety of mouse pre-B cell lines but not B cell lines or T cell lines. The staining of the pre-B cell lines A-1 and C-7 by phycoerythrin (PE)-conjugated FS1 (FS1-PE) can be blocked by preincubation of these cells with unconjugated FS1 antibody or with affinity purified polyclonal lambda 5 specific Ig but not with normal hamster or mouse IgG or with affinity purified polyclonal anti-Mb-1 Ig. From these experiments we concluded that FS1 specifically recognizes lambda 5 protein. We used FS1-PE to probe for surface (s) lambda 5+ cells in normal BALB/c mouse bone marrow. Such cells were undetectable when total bone marrow or FACS sorted subpopulations were analyzed. However, when B220+, CD43+, s lambda 5-bone marrow cells were cultured for 4 days on the stromal cell line FLST2 in the presence of IL-7, s lambda 5 expression became apparent. Further expansion of these cells in IL7 alone augmented the s lambda 5 expression to readily detectable levels. This modulation may indicate that s lambda 5 expression on normal bone marrow cells in vivo is transient and that at any given moment only a small fraction of bone marrow cells expresses low levels of lambda 5 protein on the surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.

The immunoglobulin light chain related protein lambda 5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transducing molecule.

Abelson Leukemia Virus-transformed mouse cell lines with an early pre-B phenotype carry partially rearranged or unrearranged Ig-H genes and consequently do not express intact IgM-H protein (mu protein). Such early mu- pre-B cells express an intracellular protein complex of the pre-B cell specific 22 kDa protein lambda 5 and a 16 kDa protein designated p16. Late pre-B cell lines which carry a rearranged IgM-H chain gene in which a continuous translational reading frame has been established in the fused V-D-J element express intact mu-protein, which forms an intracellular complex with lambda 5 and p16. We show here that both the lambda 5/p16 or the mu/lambda 5/p16 complexes can be immunoprecipitated from lysates of cells surface labeled with 125I. Thus early pre-B cells express the lambda 5/p16 complex on the cell surface in the absence of mu protein, while mu+ late pre-B cells express a surface mu/lambda 5/p16 complex. To investigate a possible signal transduction function of the lambda 5/p16 and mu/lambda 5/p16 complexes on the surface of pre-B cell lines we measured the changes in intracellular free Ca2+ after treatment of cells with anti-lambda 5 or anti-mu antibodies. Two mu- early pre-B cell lines showed a rapid and transient increase in intracellular free Ca2+ when incubated with anti-lambda 5 antibodies but not when incubated with anti-mu, while the mu+ late pre-B cell line CB32 showed a rapid and transient increase in intracellular Ca2+ after incubation with anti-lambda 5 or anti-mu. These results show that both the lambda 5/p16 and the mu/lambda 5/p16 cell surface protein complexes can transduce an external signal to the inside of the cell, which implicates these complexes in the regulation of pre-B cell physiology.

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM.

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein.

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.

Human and mouse LSP1 genes code for highly conserved phosphoproteins.

With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.

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

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 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 LSP1 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 LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.