3B), pointing once again toward MAPK dephosphorylation as the molecular event that is targeted by zinc in IL-2- signaling. Our results suggest that zinc release after stimulation with IL-2 conserves ERK phosphorylation by inhibiting phosphatases, and hereby free zinc acts as a permissive signal. Zinc also inhibits protein tyrosine phosphatases, preserving signaling by the insulin and EGF receptors 28–30. The IL-2R itself, as well as JAK1 and 3 and STAT5, are activated by tyrosine phosphorylation 10. However, no activation of the STAT5-pathway by zinc was found in our experiments (Fig. 2A), indicating that zinc in IL-2R signaling primarily acts on phosphatases

that dephosphorylate ERK. Here,

intracellular localization of zinc signals selleck products might be relevant, and should be investigated in more detail, as tyrosine phosphorylation of the IL-2R and JAK occurs at the plasma membrane, whereas MAPK are present in cytosol and nucleus. Alternatively, the binding constants for some protein tyrosine phosphatases are in the low nanomolar concentration range 28, and future experiments should compare these values to the susceptibility of DUSPs and PP2A to zinc inhibition. Notably, there are seven DUSP known to dephosphorylate ERK 31, whereas PP2A also dephosphorylates MEK1/2 in addition to ERK 13. Because zinc had an effect on MEK and ERK in Fig. 2F, it seems likely that PP2A is among the molecular targets of zinc in T cells. Nevertheless, ERK dephosphorylation is completely inhibited by zinc, indicating that all other

ERK dephosphorylating this website enzymes are also susceptible to inhibition by zinc. When the expression of genes specifically triggered by the different pathways was analyzed by PCR (Fig. 3A and B; Supporting Information Fig. 4), i.e. CIS for STAT5 32 and c-fos for ERK 13, corresponding results to the Western blot analysis were found. STAT5-dependent CIS expression was not influenced by chelation or imitation of zinc signals, whereas c-fos induction was significantly decreased by TPEN. ERK signals are involved in proliferation and cellular survival in response to IL-2 10. Hence, we investigated the role of zinc signals in these events. Cells were labeled with (5)6-Carboxyfluorescein diacetate (CFDA) Dolichyl-phosphate-mannose-protein mannosyltransferase to measure proliferation and with propidium iodide to detect cytotoxicity, and analyzed by flow cytometry after growing for 24 h in the presence of various concentrations of TPEN. Concentrations of up to 3 μM TPEN did not lead to a significant reduction of viability, but IL-2-dependent proliferation of CTLL-2 was significantly reduced at TPEN concentrations of 2 μM and above (Fig. 3C), indicating a preferential requirement of zinc signals for IL-2-induced proliferation at concentrations that were not cytotoxic. TPEN can chelate several other metal ions in addition to zinc 33.