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J Jongstra

Publications and source records attributed to J Jongstra.

At least 19 recordsLinked to original sources
PubMed1996

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.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

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)

Animals↗
PubMed1994

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.

Actins↗
PubMed1992

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.

Actins↗
PubMed1991

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1988

A new lymphocyte-specific gene which encodes a putative Ca2+-binding protein is not expressed in transformed T lymphocyte lines.

The LSP1 gene is a new lymphocyte-specific gene which is expressed in normal mouse B and T lymphocytes and in transformed B cells but not (or in much smaller amounts) in nine T lymphoma lines tested. No LSP1 mRNA is found in myeloid cells or in liver, kidney, or heart tissue. Inspection of the predicted LSP1 protein sequence reveals the presence of two putative Ca2+-binding domains in the LSP1 protein. Southern blotting analysis of genomic DNA from mouse liver suggests that the LSP1 gene is present as one copy per haploid genome. Similar analysis of genomic DNA extracted from three transformed B cell lines and five transformed T cell lines shows that the absence of LSP1 mRNA in T cell lines is not due to deletion or gross rearrangements of the LSP1 locus. With the use of the mouse LSP1 cDNA as a probe we can detect a cross-hybridizing RNA species in four normal human functional T cell lines but not in three transformed human T cell lines. This suggests that at least part of the DNA sequence and the expression pattern of the LSP1 gene is conserved between mouse and man. These conserved features, together with the particular expression pattern and the protein sequence homologies, suggest that the LSP1 protein is involved in a Ca2+-dependent aspect of normal T cell growth.

Amino Acid Sequence↗
PubMed1988

A human T cell-specific molecule is a member of a new gene family.

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.

Amino Acid Sequence↗
PubMed1988

The in vitro translation product of the murine lambda 5 gene contains a functional signal peptide.

We have isolated and sequenced a novel lambda 1 constant region related cDNA clone which might represent an allelic variant of the recently described lambda 5 gene. This lambda 5 transcript is present in pre-B cell lines and bone marrow cells, but not in B cell lines, plasma cell lines or in spleen cells. In vitro translation studies show that the translation product contains a signal peptide of approx. 30 amino acids at its N-terminus.

Amino Acid Sequence↗
PubMed1987

The isolation and sequence of a novel gene from a human functional T cell line.

Using a subtractive hybridization procedure we have constructed a cDNA library enriched for sequences present in functional human T cell lines, but not in human EBV-transformed B cell lines. We have isolated a cDNA clone, AH2-519, representing a novel gene, designated 519. This novel gene is expressed in functional human cytolytic and Th cell lines but not in a variety of other cell lines, including several long-term human T cell tumor lines. The expression of gene 519 is inducible in cultures of normal human PBL using antigenic or mitogenic stimulation. Neither the DNA sequence determined from a full-length cDNA clone overlapping with clone AH2-519 nor the amino acid sequence of its predicted protein product has significant homology to published sequences in the GenBank or NBRF databases. The restricted expression of gene 519 suggests that its gene product is involved in the growth and/or differentiation of normal T cells. The data also show that normal, nontransformed, functional T cells express gene products that can not be readily identified in long-term tumor lines of the same cell lineage.

Amino Acid Sequence↗