ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.

The tyrosine-based activation motif is a 20-to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59'" and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of . Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fi" association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59&f binding was not disrupted.Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding.Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We motif, known as the antigen receptor homology motif (2) or tyrosine-based activation motif (TAM) (21), is not known.Because the T-cell receptor does not itself encode a tyrosine kinase, one possible function of the activating motif, which we will refer to as the TAM, is that it binds and/or activates a tyrosine kinase. Three candidate cytoplasmic tyrosine kinases, p561ck, p59f'3" and ZAP-70, have all been implicated in the activation of T cells (51). pS6lck, a member of the Src family of tyrosine kinases, associates with the T-cell receptor coreceptors CD4 and CD8 (38,47) and, in some cells, has been shown to be essential for T-cell receptor activation (20,44). Although it functions in the T-cell receptor signalling pathway, there is little evidence to support a direct interaction of p56kck with the T-cell receptor. p591", another member of the Src family, is the only kinase that has been shown to be physically associated with the unactivated T-cell receptor complex (39, 41). Reconstitution experiments demonstrate that this association is specific and that p59fy' can associate directly with multiple CD3 subunits (13). Importantly, p59y'3 kinase activity is stimulated by engagement of the T-cell receptor (46), and p59O' can mediate calcium mobilization and phospholipase C phosphorylation when coexpressed with a 4...

Background Heterogeneity in bladder cancer results in variable clinical outcomes, posing challenges for clinical management of this malignancy. Recent studies suggest both tumor suppressive and oncogenic role of PPARγ in bladder cancer. The fuction of PPARγ signaling pathway in modulating carcinogenesis is controversial. Methods The expression of PPARγ and association with overall survival were analyzed in patients from two cohorts. The effect of PPARγ activation on cell proliferation, cell cycle, and cell apoptosis were determined with the agonists (rosiglitazone and pioglitazone), the inverse agonist (T0070907), and the antagonist (GW9662) in Umuc-3 and 5637 bladder cancer cells. The correlation of PPARγ activation with PI3K-Akt pathway was evaluated with RNA sequencing data from the TCGA cases and 30 human bladder cancer cell lines. The effect of PPARγ activation on tumor growth was validated with subcutaneous tumor models in vivo. The effect of PPARγ activation on PI3K-Akt signaling transduction was determined with multiple assays including immunohistochemistry, flow cytometry, proteomic array, and western blotting. Results We showed that PPARγ was a favorable prognostic factor in patients with bladder cancer. PPARγ activation by rosiglitazone and pioglitazone markedly induced cell cycle G2 arrest and apoptosis in bladder cancer cells, which resulted in inhibition of cell proliferation in vitro and suppression of tumor growth in vivo. The underlying mechanism involved marked inhibition of PI3K-Akt pathway. Conclusions This study reported the tumor-suppressive effect of PPARγ agonists in bladder cancer, suggesting that transactivation of PPARγ could be served as a potential strategy for the chemoprevention and therapeutic treatment of bladder cancer. Electronic supplementary material The online version of this article (10.1186/s12885-019-5426-6) contains supplementary material, which is available to authorized users.

The present evidence demonstrates that retroperitoneal adrenalectomy is better than transperitoneal approach for patients with adrenal tumor in short-term outcomes. However, extended follow-ups and further randomized controlled trials should be required to analysis.