Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor ; chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosinephosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3. 1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this reagent to investigate the effect of tyrosine phosphatase inhibition in a murine T-cell hybridoma. Increasing concentrations of phenylarsine oxide result in an increase in tyrosine phosphate on a number of intracellular substrates in unstimulated T cells, suggesting that a protein-tyrosine kinase is constitutively active in these cells. The effect of phenylarsine oxide on T cells stimulated with an anti-Thy 1 monoclonal antibody is more complex. At low concentrations of drug, there is a synergistic increase in the level of tyrosine phosphate on certain cellular substrates. At higher concentrations, anti-Thy 1-stimulated tyrosine phosphorylation is inhibited. These results indicate that tyrosine phosphorylation in T cells is tightly regulated by tyrosine phosphatases. Partial inhibition of these enzymes results in enhanced substrate phosphorylation. Inhibition of all stimulated tyrosine phosphorylation by high doses of phenylarsine oxide suggests that tyrosine kinase activity is regulated by tyrosine phosphatases.Over the past decade there has been intense study devoted to understanding cellular tyrosine phosphorylation. By investigation of normal and transformed cells, it now appears that protein-tyrosine kinases (PTIK; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) regulate such critical cellular functions as growth, differentiation, and signal transduction (1, 2). Our laboratory has been interested in the function and regulation of tyrosine phosphorylation in T lymphocytes. We demonstrated that activation of T cells by a variety of means results in tyrosine phosphorylation of the T-cell antigen receptor (TCR) on the TCR chain (3-5). Furthermore, we have observed that other cellular substrates are phosphorylated on tyrosine more rapidly than the TCR ; chain (5, 6). Phosphorylation of these proteins can be detected by immunoblotting with specific anti-phosphotyrosine antibodies within seconds of TCR engagement.The PTKs responsible for phosphorylation of TCR r and these other substrates have not been conclusively identified. Since the TCR subunits lack the necessary amino acid sequences that define kinases it has been assumed that the TCR must be activating a nonreceptor kinase such as a member of the src PTK family. In fact, T cells have been shown to express Ick, fyn, and yes PTKs (7,8). The PTK Ick has been demonstrated to be noncovalently associated with the CD4 and CD8 molecules, which are T-cell-specific glycoprot...

The results presented here demonstrate that p53 upregulates estrogen receptor-(ER ) expression in the human breast cancer cell line MCF-7. Two approaches were used to alter the activity of p53 in the cells. In the first approach, stable transfectants expressing an antisense p53 were established. In the stable clones, expression of antisense p53 resulted in a decrease in the expression of ER protein. In the second approach, MCF-7 cells were transiently transfected with wild-type p53. Overexpression of p53 increased the amount of ER . To determine whether the effects of p53 on the expression of ER were due to changes in transcription, deletion mutants of the ER promoter were used. This experimental approach demonstrated that p53 up-regulates ER gene expression by increasing transcription of the gene through elements located upstream of promoter A. Transfection assays using p53 mutants further demonstrated that the p53-induced increase in ER gene transcription was not dependent on the ability of p53 to bind to DNA but on its ability to interact with other proteins.

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G 2 / M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G 2 /M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50 cdc37 , an Hsp90 cochaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

The T cell antigen receptor consists of an antigen-binding heterodimer that is noncovalently associated with at least five CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3-zeta chains are either disulfide-linked homodimers (CD3-zeta 2) or disulfide-linked heterodimers with eta (CD3-zeta eta). Variants of a murine antigen-specific T cell hybridoma that express normal amounts of CD3-zeta 2 but decreased amounts of CD3-zeta eta were isolated. When activated, the parental cell line increased both phosphatidylinositol hydrolysis and serine-specific protein kinase activity to a much greater extent than the variants. In contrast, the activation of a tyrosine-specific kinase after stimulation with a cross-linking antibody to CD3 was similar among these cells. There was a positive linear relation between the expression of CD3-zeta eta and phosphoinositide hydrolysis stimulated by the TCR, suggesting a differential coupling of the T cell alpha beta heterodimer to signal transduction mechanisms due to alpha beta association with either CD3-zeta 2 or CD3-zeta eta.