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R M Perlmutter

Publications and source records attributed to R M Perlmutter.

At least 91 records · Page 5Linked to original sources
PubMed1990

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

Animals↗
PubMed1990

Structure and evolution of mammalian VH families.

Antibodies are encoded by a limited number of germline gene segments that undergo somatic diversification through rearrangement and mutation. Because these mutation processes are efficient, it is widely believed that there is little environmental selection pressure for the maintenance of specific antibody gene sequences. We have performed pairwise comparisons of known germline (as opposed to somatically generated) antibody VH elements with the hope of identifying conserved structural features common to sets of VH gene segments. These studies reveal that VH families arose prior to the mammalian radiation and have since been conserved, that this conservation appears to reflect selection at the level of protein sequence, and that the conserved regions are discretely localized on a solvent-exposed face of the heavy chain, at some distance from the antibody combining site. A family-specific region was also identified within the recombinase recognition sequences. Our results provide a context for theories that address the physiological significance of variations in VH family utilization during the development of the immune repertoire.

Animals↗
PubMed1990

Disruption of thymocyte development and lymphomagenesis induced by SV40 T-antigen.

The lck gene encodes a membrane-associated protein tyrosine kinase that is expressed specifically in lymphoid cells, especially thymocytes. Structural analysis of the murine and human lck genes previously identified conserved 5' flanking sequences that were proposed to represent transcriptional regulatory elements. Here we demonstrate that a murine lck promoter construct containing these sequences directs the expression of the SV40 T-antigen gene in lymphoid cells. Remarkably, expression of SV40 T-antigen in transgenic animals dramatically disturbs thymic development, resulting in preferential loss of CD4+CD8+ thymocytes. In contrast, immature cells lacking both CD4 and CD8 markers are present in near-normal numbers. Thus SV40 T-antigen expression appears partially to arrest thymopoiesis. Mice bearing the lck-SV40 transgene develop readily explantable thymic tumors at 12-18 weeks of age. Fluorocytometric analyses of lck-SV40 tumor cells reveal that immature thymocytes are frequently immortalized. The lck-SV40 mouse may therefore provide materials for the in vitro investigation of thymocyte differentiation.

Animals↗
PubMed1990

Translational regulation of the lymphocyte-specific protein tyrosine kinase p56lck.

The lck gene encodes a lymphocyte-specific membrane-associated protein tyrosine kinase that is implicated in signal transduction processes that regulate T-cell growth. Previous studies demonstrate that lck transcripts, unlike most other vertebrate mRNAs, contain 5' proximal AUG sequences upstream relative to the major open reading frame (5' AUGs) that serve as satisfactory targets for recognition by 40S ribosomal subunits. There exist two distinct lck transcripts, generated through alternative promoter utilization, that differ only in their 5' untranslated region sequences. Both transcripts are present in murine and human lymphocytes, and the structures of the two distinct 5' untranslated regions have been closely conserved since the divergence of these species. Moreover, both transcripts contain 5'-AUG elements. Intriguingly, inspection of primary sequence data for other src-family transcripts reveals that virtually all contain 5'AUG triplets, and many contain a large number of these potential initiators. By analogy with the well-studied translational regulation of the yeast GCN4 gene, I propose that control of ribosomal reinitiation permits tight regulation of expression of the lck gene product and of other src-like protein tyrosine kinases.

Animals↗
PubMed1990

Autoantibodies and the fetal antibody repertoire.

Developing fetal B cells preferentially rearrange a restricted subset of the encoded antibody gene segments. There are striking structural similarities between elements expressed early in man and in mouse, most evident on comparison of murine VH elements from the VH7183 family to human VH elements of the VH3 family. The similarity is pronounced in two framework regions which together encode a possible binding site that is distinct from the classical antigen-combining site. By comparing all known human and murine VH gene sequences, we have demonstrated that these regions have been conserved in a family-specific manner throughout the mammalian radiation. The "non-conserved" spacer of the recombinase recognition signal is also highly conserved in a family-specific manner, suggesting a mechanism by which the expression of family-dependent features may be regulated. The evidence that such features contribute to the high incidence of self- and poly-specificity in the fetal antibody repertoire is reviewed.

Amino Acid Sequence↗
PubMed1989

Heterogeneity in the human Ig VH locus.

We have used synthetic oligonucleotides corresponding to human VH sequences to analyze sequence variation in human genomic DNA. By using probes 20 to 24 bp long and conditions of hybridization and washing under which identity in 17 to 21 consecutive bp is required for hybridization, it has been possible to dramatically reduce the complexity of hybridization patterns. We have been able to identify unambiguously individual VH elements. Concomitant with the reduction in overall complexity of hybridization patterns has been a marked increase in the variation between hybridization patterns when different individuals are compared. Variation between individuals was detected using probes corresponding to both framework and complementarity determining regions and depended in part on the complexity of the corresponding VH gene family. Probes corresponding to a cDNA clone belonging to the single-member VH6 family, hybridized to a single, invariant, band in all individuals tested. An oligonucleotide probe corresponding to CDR2 of one member of the VH3 family also detected a single, invariant, band in all individuals tested. However, an oligonucleotide probe corresponding to framework region 2 revealed variants of more than 40% of the 22 VH elements it detects. In addition, a panel of 5 oligonucleotide probes corresponding to a second member of the VH3 family revealed variants of 10 of 14 elements detected. The patterns of variation suggest that some VH elements have multiple alleles, whereas some elements are remarkably conserved. The number of variant elements we have detected is evidence that the haplotype arrangement of the human VH locus is probably extremely complex. Importantly, this heterogeneity may contribute directly to disease susceptibility in man.

Antibody Diversity↗
PubMed1989

Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase (p56lck).

The protein tyrosine kinase p56lck is implicated in the control of lymphocyte growth by virtue of its overexpression in some lymphoid malignancies and its transforming activity in heterologous systems. Previous studies have demonstrated that levels of lck mRNA and of p56lck decline rapidly after T cell activation. The disappearance of p56lck results primarily from post-translational conversion of p56lck to more slowly migrating forms with apparent sizes of approximately 60 kDa. This modification can be provoked by treatment of lymphocytes with PMA, and has been associated with increased serine phosphorylation of the p56lck molecule. Here we demonstrate that conversion of p56lck to p60lck is a feature of the physiologic activation of T lymphocytes by antigen-presenting cells. In addition, we show that the PMA-induced modification of p56lck proceeds via a mechanism distinct from conventional protein kinase C activation. The rapid conversion of p56lck to p60lck after antigenic stimulation is consistent with the view that this membrane-associated protein tyrosine kinase regulates some aspects of the lymphocyte activation sequence.

Adult↗
PubMed1989

Transformation of NIH 3T3 fibroblasts by an activated form of p59hck.

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

Amino Acid Sequence↗
PubMed1989

Selective expression of alternative lck mRNAs in human malignant cell lines.

The lck protein tyrosine kinase is normally expressed in a cell type-specific fashion, with mRNA being confined to cells of lymphoid lineage. Despite this highly specific pattern of expression in normal tissues, lck mRNA has also been detected in selected cell lines derived from human nonlymphoid neoplasms. In this study we explored the mechanisms underlying the expression of lck mRNA within human nonlymphoid neoplastic cell lines. We determined that lck mRNA expression was correlated with transcriptional activation and that there was no evidence for genomic rearrangement or amplification within the lck coding region to account for the expression of lck mRNA in the nonlymphoid neoplastic cell lines. The lck gene has previously been shown to contain two distinct promoter elements. In this study, we demonstrated that lck-producing cell lines derived from human nonlymphoid neoplasms expressed transcripts initiated exclusively from the 3'-most promoter element (3' promoter). In contrast, lymphoid cell lines derived from nonmalignant sources expressed lck transcripts exclusively initiated from the 5'-most promoter element (5' promoter). Most cell lines derived from human lymphoid neoplasms express lck transcripts initiated from both the 5' and 3' promoters in various ratios. Thus, lck expression in a variety of malignant cell lines results from a selective induction of transcription from the 3' promoter.

Base Sequence↗
PubMed1989

Expression of a novel form of the fyn proto-oncogene in hematopoietic cells.

The fyn proto-oncogene encodes a 59-kD membrane-associated protein-tyrosine kinase that is implicated in the control of cell growth. We report here that there are two distinct fyn-encoded transcripts that exhibit mutually exclusive expression patterns; one form is found in thymocytes, splenocytes, and some hematolymphoid cell lines, while a second form accumulates principally in brain. The thymocyte and brain forms of p59fyn differ in their kinase domains, and are generated by mutually exclusive alternative splicing of a single exon. Immunoprecipitation followed by in vitro autophosphorylation demonstrates that both forms of p59fyn are enzymatically active. Our results define a novel form of the p59fyn protein-tyrosine kinase, with an altered catalytic domain, that apparently participates in a specialized signal transduction pathway in hematopoietic cells.

Amino Acid Sequence↗
PubMed1988

Augmented expression of a myeloid-specific protein tyrosine kinase gene (hck) after macrophage activation.

Protein tyrosine kinases are thought to participate in signal transduction pathways in a variety of cell types. Recent studies have identified a new src family protein tyrosine kinase (hck) that is preferentially expressed in myeloid cells. To examine the hypothesis that this kinase may regulate myeloid cell activity, antisera were generated that define the 59-kD product of the hck gene. Functional activation of human cultured macrophages with LPS augmented the expression of hck transcripts and of p59hck, but decreased the level of transcripts encoded by the closely related c-fgr protooncogene. Thus these two structurally similar src family kinases almost certainly subserve distinct functions. Reasoning from the known properties of the src family protein tyrosine kinases, it is likely that the products of these two protooncogenes assist in regulating the behavior of activated phagocytes.

Blotting, Northern↗
PubMed1988

Translational activation of the lck proto-oncogene.

The lck proto-oncogene, a member of the src gene family, encodes a lymphocyte-specific protein tyrosine kinase (p56lck) that is implicated in the pathogenesis of lymphoid neoplasia. We report here that 5' lck sequence elements, containing AUG codons, significantly reduce the in vivo efficiency of p56lck translation from the normal messenger RNA. This result provides a quantitative explanation for the overexpression of p56lck in two retrovirally-induced murine lymphomas that express abnormal lck transcripts from which the physiological 5' translational control region has been deleted and non AUG-containing viral sequences substituted. In contrast to other mammalian genes, most proto-oncogenes contain AUG codons 5' to the authentic initiation codon, suggesting that cells can regulate translational start sites in these mRNAs. Abrogation of translational control may be a general mechanism of proto-oncogene activation in malignancy.

Animals↗
PubMed1988

Structure and expression of lck transcripts in human lymphoid cells.

The murine lck gene encodes a membrane-associated protein tyrosine kinase that has been implicated in lymphocyte oncogenesis. Here we report the structure of normal human lck transcripts and the pattern of expression of these transcripts in developing thymus and in peripheral T cell subsets. The human lck gene encodes a 509 amino acid polypeptide that is closely related to the murine lck-encoded protein throughout its length. Analysis of the deduced amino acid sequence of human p56lck demonstrates that an amino-terminal domain, widely divergent among the seven known src family members, has been conserved between murine and human p56lck, and thus probably includes sequences crucial to the lymphocyte-specific function of this molecule. Human lck transcripts were detected in CD4+ and CD8+ T cells, in partially purified B cells, and in Epstein-Barr virus-immortalized B cell lines, but not in monocytes, granulocytes, or in nonhematopoetic cell types. Human lck transcripts are readily detectable in fetal thymocytes at 70 days of gestation, but not at 57 days of gestation, indicating that lck expression appears coordinately with the appearance of lymphoid cells in the developing thymus. Thus lck gene expression is a marker for cells of the lymphocyte lineage in man. We conclude that the lck gene probably participates in a signal transduction pathway uniquely present in lymphoid cells.

Base Sequence↗
PubMed1988

Physical linkage of a human immunoglobulin heavy chain variable region gene segment to diversity and joining region elements.

Antibody genes are assembled from a series of germ-line gene segments that are juxtaposed during the maturation of B lymphocytes. Although diversification of the adult antibody repertoire results in large part from the combinatorial joining of these gene segments, a restricted set of antibody heavy chain variable (VH), diversity (DH), and joining (JH) region gene segments appears preferentially in the human fetal repertoire. We report here that one of these early-expressed VH elements (termed VH6) is the most 3' VH gene segment, positioned 77 kilobases on the 5' side of the JH locus and immediately adjacent to a set of previously described DH sequences. In addition to providing a physical map linking human VH, DH, and JH elements, these results support the view that the programmed development of the antibody VH repertoire is determined in part by the chromosomal position of these gene segments.

B-Lymphocytes↗
PubMed1988

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.

Animals↗
PubMed1988

Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck).

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.

Animals↗