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1.  Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene. 
Molecular and Cellular Biology  1994;14(9):5812-5819.
Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.
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PMCID: PMC359107  PMID: 8065316
2.  Modulation of platelet-derived growth factor receptor expression in microvascular endothelial cells during in vitro angiogenesis. 
Journal of Clinical Investigation  1994;93(1):131-139.
Microvascular endothelial cells in vivo exhibit a plastic phenotype, forming a nonproliferative, differentiated capillary network, while retaining their ability to respond to injury by proliferation, migration and neovascularization. The presence of PDGF receptors and PDGF responsiveness in microvascular endothelial cells and the significance of PDGF isoforms in the control of endothelial cell growth and differentiation remain controversial. Since culture of microvascular endothelial cells in a three-dimensional (3D) system induced cell differentiation and angiogenesis and inhibited proliferation, the present study investigates the role of different extracellular matrix environments in inducing different microvascular endothelial cell phenotypes on microvascular endothelial cell PDGF receptor expression and PDGF responsiveness. In conventional two-dimensional (2D) culture, microvascular endothelial cells expressed both PDGF receptor alpha and beta chains. Suramin treatment demonstrated continuous downregulation of the alpha receptor surface expression. PDGF BB and, to a lesser extent, PDGF AB were mitogenic in 2D-culture, PDGF AA failed to induce any proliferative response despite inducing receptor autophosphorylation. During in vitro angiogenesis induced by 3D-culture, both PDGF receptors were rapidly downregulated. Assessment of cell proliferation showed quiescent cells and PDGF unresponsiveness. We conclude that the induction of a differentiated phenotype during in vitro angiogenesis (tube formation) driven in part by the spatial organization of the surrounding matrix is associated with a downregulation of PDGF receptors. Identification of the molecular cell-matrix interactions involved in this receptor regulation may allow for targeted manipulation of cell growth in vivo and lead to novel therapeutic applications for PDGF.
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PMCID: PMC293745  PMID: 7506710
3.  Functional dissection of the lck proximal promoter. 
Molecular and Cellular Biology  1992;12(6):2758-2768.
The lck gene encodes a protein tyrosine kinase that participates in lymphocyte-specific signal transduction pathways. Previous studies have established that lck transcription is regulated by two distinct promoter elements termed proximal (or 3') and distal (or 5'). The proximal promoter is active almost exclusively in thymocytes and becomes inactive later during T-cell maturation. To dissect the mechanisms responsible for lck gene regulation, we generated transgenic animals bearing 5' truncations in the proximal promoter element. Sequences between -584 and +37 with respect to the proximal promoter transcription start site act to direct tissue-specific and temporally correct transcription of either a tagged version of the lck gene itself or a heterologous reporter sequence (lacZ). This region contains binding sites for at least five distinct nuclear proteins, of which one is found only in cells that support proximal lck promoter activity and a second appears only in nonexpressing cells. Interestingly, the transcribed region of the lck gene contains positive control elements that can substantially boost expression from minimal (-130 bp) proximal promoter constructs. These results provide a basis for the biochemical dissection of transcriptional regulators that act at defined points during T-cell development.
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PMCID: PMC364470  PMID: 1588967
4.  Selective expression of alternative lck mRNAs in human malignant cell lines. 
Molecular and Cellular Biology  1989;9(7):2983-2988.
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.
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PMCID: PMC362766  PMID: 2779553
5.  Transformation of NIH 3T3 fibroblasts by an activated form of p59hck. 
Molecular and Cellular Biology  1989;9(6):2724-2727.
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.
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PMCID: PMC362345  PMID: 2503711
6.  p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen. 
Journal of Virology  1988;62(12):4673-4679.
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.
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PMCID: PMC253580  PMID: 3184274
7.  Structure of the murine lck gene and its rearrangement in a murine lymphoma cell line. 
Molecular and Cellular Biology  1988;8(8):3058-3064.
The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in neoplastic transformation. We have determined the germ line organization of the murine lck gene and have isolated and characterized a rearranged lck allele in the murine lymphoma cell line LSTRA. The overall exon-intron organization of the normal lck gene is almost identical to that of avian c-src. In LSTRA DNA, an internally rearranged Moloney murine leukemia virus genome is interposed between two distinct promoters that normally generate lck transcripts differing only in 5' untranslated regions. The rearrangement appears to have been selected to permit splicing of transcripts that initiate from the Moloney virus promoter to an acceptor site located within the first exon 3' to the downstream promoter, thus generating an lck mRNA with a novel 5' untranslated region that may be more efficiently translated.
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PMCID: PMC363532  PMID: 2850479
8.  Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). 
Molecular and Cellular Biology  1988;8(2):540-550.
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.
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PMCID: PMC363178  PMID: 3352600
9.  Novel protein-tyrosine kinase gene (hck) preferentially expressed in cells of hematopoietic origin. 
Molecular and Cellular Biology  1987;7(6):2276-2285.
Protein-tyrosine kinases are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes and as components of growth factor receptors. Here we report the characterization of a novel human protein-tyrosine kinase gene (hck) that is primarily expressed in hematopoietic cells, particularly granulocytes. The hck gene encodes a 505-residue polypeptide that is closely related to pp56lck, a lymphocyte-specific protein-tyrosine kinase. The exon breakpoints of the hck gene, partially defined by using murine genomic clones, demonstrate that hck is a member of the src gene family and has been subjected to strong selection pressure during mammalian evolution. High-level expression of hck transcripts in granulocytes is especially provocative since these cells are terminally differentiated and typically survive in vivo for only a few hours. Thus the hck gene, like other members of the src gene family, appears to function primarily in cells with little growth potential.
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PMCID: PMC365352  PMID: 3453117
10.  Hepatitis and aplastic anemia. 
California Medicine  1969;110(2):135-138.
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PMCID: PMC1503305  PMID: 5784116

Results 1-11 (11)