Expression of v-src in a murine T-cell hybridoma results in constitutive T-cell receptor phosphorylation and interleukin 2 production.

Abstract

Ligand binding to the T-cell antigen receptor results in phosphatidylinositol hydrolysis and the resultant activation of protein kinase C, as well as the activation of a receptor-coupled protein-tyrosine kinase. As a model for tyrosine kinase activation in T cells, we used retroviral gene transfer to express the v-src oncogene in an antigen-specific murine T-cell hybridoma. Clones that expressed v-src mRNA demonstrated constitutive tyrosine phosphorylation of several cellular substrates, including the {chain of the T-cell receptor, and constitutive interleukin 2 production. Thus, expression of a constitutively active protein-tyrosine kinase such as pp6Ovsrc appears to be sufficient to induce the expression of at least one gene critical to the process of T-cell activation.The biochemical events that occur following perturbation of the T-cell antigen receptor (TCR) include the activation of protein kinase C (PKC) (reviewed in ref.

The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations.

“…Furthermore, two drugs that antagonize PTK activity also inhibit activation-induced increases in inositol phosphates and [Ca2+]i (2,3). More support for a direct role of PTKs in T cell activation comes from studies in which v-src was introduced into T cell hybridomas (4). Cells expressing the active pp60 v-~ PTK spontaneously produced IL-2, even after depletion of protein kinase C. Moreover, their [Ca a +]i 1Abbreviations used in this paper: GAPDH, glyceraldehyde phosphate dehydrogenase; PI, phosphoinositide; PTK, protein tyrosine kinase.…”

Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation.

“…Thus, PTK activity appears to be necessary for T-cell activation, but is PTK function alone sufficient to initiate any or all of the events involved in this process? Expression of PTKs in T-cell lines can enhance TCRmediated interleukin 2 (IL-2) induction or in the case of v-src can induce constitutive but suboptimal IL-2 secretion (15)(16)(17). However, the limitation oftransfection studies is that the consequences of acute alteration of the levels of tyrosine phosphorylation in normal T cells cannot be addressed.…”

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

“…To expand IP-FCM to a multi-colour technique, we stimulated the murine 2B4 T-cell line [33] , [34] with the phosphatase inhibitor pervanadate at multiple time points. After cell lysis, the TCR-CD3 complex was purified with anti-TCRβ-coupled beads.…”

“…The murine cell line 2B4 [33] , [34] was maintained in complete RPMI 1640 medium supplemented with 5% fetal bovine serum [42] . OT-1 mice [35] , which contain transgenes for the OVA-specific OT-1 TCRα and TCRβ chains were killed and single cell suspensions of spleen and thymus were prepared.…”