Identification of the human T-lymphocyte protein-tyrosine kinase by peptide-specific antibodies

Trevillyan, James M.; Canna, Cheral; Maley, Derrick; Linna, T. Juhani; Phillips, Chris

doi:10.1016/0006-291x(86)91103-4

Cited by 16 publications

(14 citation statements)

References 12 publications

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“…Indeed, a family of putative kinases in B and T lymphocytes at =55 kDa have recently been described (19)(20)(21)(22)(23)(24)(25). In particular, a tyrosine kinase (PTK) of 58 kDa has been reported that appears to be expressed specifically in human T cells (28,29). The cDNA for this T-cell kinase encodes a human analogue of pp56LSRTA/Ick with extensive homology to the catalytic domain of pp60c-src (28).…”

Section: Resultsmentioning

confidence: 99%

“…Murine T cells possess relatively high levels of PTK activity, and distinct patterns of tyrosine phosphorylation have been noted in T and B cells (19)(20)(21)(22)(23)(24)(25). In particular, a protein kinase termed pp56LSTRA/lck has been identified in murine T cells (26,27) and, recently, in human T lymphocytes (28)(29)(30). The rather dramatic decrease of ppS6lck within T cells after activation is suggestive of an importance ofthis enzyme in the activation process (31).…”

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confidence: 99%

“…Lastly, the anti-PTK antiserum was generated against a synthetic peptide of the sequence Cys-Lys-GluArg-Pro-Glu-Asp-Arg-Pro-Thr-Phe-Asp-Tyr-Leu-Arg-SerVal-Leu-Glu-Asp-Phe-Phe-Thr-Ala-Thr-Glu-Gly-Gln-TyrGln-Pro-Gln-Pro, as described (29). The peptide was coupled to bovine serum albumin through the N-terminal cysteine at position 33 (Cys-Pro) by using 3-(2-pyridyldithio)proprionic acid N-hydroxysuccinimide ester as described (35 (36).…”

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confidence: 99%

“…The cells were initially lysed by 1% (vol/vol) Nonidet P-40; however, in later experiments, 3% (vol/vol) detergent was found to be a more efficient method of extraction of the kinase. The lysate was centrifuged at 12,000 x g and precleared for 30 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] min at 25°C, the reaction mixture was subjected to NaDodSO4/PAGE and autoradiography (38). For the analysis of reprecipitated antigen, the reaction mixture was supplemented to 1.0-2.0% (wt/vol) NaDodSO4, boiled for 5 min, and then diluted to 0.1% NaDodSO4 with a 1:10 or 1:20 dilution of lysis buffer.…”

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The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

Rudd

Trevillyan

Dasgupta

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

656

324

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The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface ofhelper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells.The PTK is the human analogue of the murine pp56ISTRA (ppS6Ick) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PIK at 55-60 kDa.The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.The CD4 (T4) antigen is a polypeptide of 55 kDa that is expressed predominately on the surface of helper T cells and has been implicated in association with the T3 (CD3)-Ti complex in the recognition of class II antigens of the major histocompatibility complex (1, 2). The CD4 antigen also appears to serve as a receptor for the human immunodeficiency virus (HIV) [the acquired immunodeficiency syndrome (AIDS) virus] (3-5). Structurally, the CD4 antigen is a member of the immunoglobulin superfamily (6) and, as such, appears capable of regulating the proliferation of the CD4 subset of T cells (7)(8)(9)(10)(11). Monoclonal antibodies to the CD4 antigen were initially reported to inhibit specific T-cell function by reducing the strength of interaction of the T cell with its target (1, 2, 7). However, it also appears that monoclonal antibodies to the CD4 receptor can inhibit or potentiate the activation of T lymphocytes in a manner independent of the recognition of HLA-D region antigens (8)(9)(10)(11). The inhibitory effect of antibodies to CD4 appears to preferentially influence the T4+2H4+ subset within the CD4 population, a subset defined by the differential expression of the L-C/T200 (CD45) antigens (12,13 Tyrosine phosphorylation is thought to be a relatively rare modification to proteins; however, the past few years have seen a rapidly expanding number of retroviral oncogenes and cellular receptors within the src family. These include the retroviral oncogenes such as v-abl, v-erb, v-fes/...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

Rudd

Trevillyan

Dasgupta

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

656

324

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“…Interestingly, the expression pattern of the lyn gene contrasts with that of lck. The lck gene is highly expressed in mature T lymphocytes (22,57), whereas the lyn gene is expressed in B cells, macrophages/monocytes, and platelets but only at low levels in T lymphocytes (67). However, the lyn gene is expressed in T-cell lines infected with and producing human T-cell leukemia virus type I (HTLV-I) (67).…”

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Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.

et al. 1992

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The src family gene lyn is expressed preferentially in B lymphocytes but very little in normal T lymphocytes. Transcription of the lyn gene in T lymphocytes was shown to be induced by the p40tax protein encoded by human T-cell lymphotropic virus type I. For determination of the mechanism of p40tax-mediated trans activation, the transcriptional promoter region of the lyn gene was characterized. By endonuclease S1 mapping, the transcriptional initiation sites were identified within the 770-bp EcoRI-SacI fragment of the 5'-terminal portion of the human lyn gene. This fragment showed promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into various cell lines. Nucleotide sequence analysis revealed that the lyn promoter region contained four GC box-like sequences but not a TATA or CCAAT box. In addition, it contained sequences characteristic of a cyclic AMP-responsive element, octamer-binding motif, PEA3-like motifs, and NF kappa B-binding motif-like sequence. Mutational analysis suggested that the octamer-binding motif sequence is of primary importance for the lyn promoter activity but that the other elements are not. Cotransfection of various chloramphenicol acetyltransferase constructs containing different length of the lyn promoter together with p40tax expression plasmids into Jurkat T cells showed that the sequence responsible for p40tax-induced transcription is present around the transcription initiation sites.

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Biochemical identification of a direct physical interaction between the CD4: p56^lck and T_i(TcR)/CD3 complexes

et al. 1991

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The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.

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Identification of the human T-lymphocyte protein-tyrosine kinase by peptide-specific antibodies

Cited by 16 publications

References 12 publications

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.

Biochemical identification of a direct physical interaction between the CD4: p56^lck and T_i(TcR)/CD3 complexes

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Identification of the human T-lymphocyte protein-tyrosine kinase by peptide-specific antibodies

Cited by 16 publications

References 12 publications

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.

Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.

Biochemical identification of a direct physical interaction between the CD4: p56lck and Ti(TcR)/CD3 complexes

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Biochemical identification of a direct physical interaction between the CD4: p56^lck and T_i(TcR)/CD3 complexes