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

Abstract

Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signalng. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) a chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response.In contrast to the growth factor class of receptors that display intrinsic protein-tyrosine kinase (PTK) activity, many immunologically relevant, oligomeric receptors including the T-cell antigen receptor (TCR) and Fc receptors lack intrinsic kinase activity. Nonetheless, stimulation of these receptors results in protein-tyrosine phosphorylation of cellular substrates (reviewed in refs. 1-10), and pharmacologic inhibition of tyrosine kinases prevents both the early biochemical and late functional events that occur after receptor stimulation (11)(12)(13)(14). 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.Vanadate is a well-documented inhibitor of proteintyrosine phosphatases (PlPases), but its effects on intact cells are variable (18)(19)(20). By contrast, the combination of vanadate and H202 generates the compound pervanadate, the efficacy of which has been demonstrated to be far greater than that of vanadate (21)(22)(23) (24). Two-color fluorescence measurements were performed on a FACScan IV flow cytometer (Becton Dickinson) to assess purity. The murine thymoma cell line, BW5147.3, and its CD45-counterpart were grown in RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% fetal bovine serum.Anti-phosphotyrosine [anti-Tyr(P)l Immunobtig. Stock solutions of H202 and sodium orthovanadate, Na3VO4, wer...

PTPN22 and rheumatoid arthritis: Gratifying replication

“…It has been known for more than a decade that tyrosine phosphatase activity is associated with a negative regulatory effect on T cell function. Thus, early experiments showed that generalized phosphatase inhibition results in persistent proliferation of polyclonally activated T cells (15) or can induce spontaneous activation and cytokine release by resting T cells (16). A specific role of PTPN22 in T cell regulation has been confirmed by the results of knocking out the murine homolog of PTPN22 (PEST domainenriched tyrosine phosphatase [PEP]), resulting in lowered thresholds for T cell receptor signaling in these animals (17).…”

Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells

“…Since the mechanisms regulating tyrosine phosphorylation of Bgp have not been clearly de®ned, we chose to arti®cially induce Tyr phosphorylation by treatment with pervanadate, a potent inhibitor of cellular protein tyrosine phosphatases (O'Shea et al, 1992). This choice was based on the observation of others, which showed that vanadate, a less active protein tyrosine phosphatase inhibitor was e cient at provoking phosphorylation of Bgp at Tyr 488 .…”

“…All experiments were performed with a minimum of two clones for each transfectant cell line. Pervanadate treatment of cells was performed by incubating CT51 transfectant cells in a-minimal essential medium for 10 min at 378C with a 10 mM H 2 O 2 and 100 mM sodium vanadate solution (O'Shea et al, 1992). Cells were collected from the dishes by scraping followed by centrifugation (1500 r.p.m.)…”

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

“…Experiments were performed with either cell populations or a minimum of two clones for each transfectant cell line. Pervanadate treatment of cells was performed by incubating transfected CT51 cells in ␣-minimal essential medium for 10 min at 37°C with a solution of 10 mM H 2 O 2 and 100 M sodium vanadate (38). Cells were collected from the dishes by scraping, followed by centrifugation and subsequent washes in the same vanadate-containing medium.…”