TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNFalpha, or beta-amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H(2)O(2) or TNFalpha. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca(2+)](i) observed after H(2)O(2) or TNFalpha treatment in TRPM2-expressing cells, suggesting that phosphorylation is important in TRPM2 activation. Utilizing a TransSignal PDZ domain array blot to identify proteins which interact with TRPM2, we identified PTPL1 as a potential binding protein. PTPL1 is a widely expressed tyrosine phosphatase, which has a role in cell survival and tumorigenesis. Immunoprecipitation and glutathione-S-transferase pull-down assays confirmed that TRPM2 and PTPL1 interact. To examine the ability of PTPL1 to modulate phosphorylation or activation of TRPM2, PTPL1 was coexpressed with TRPM2 in human embryonic kidney-293T cells. This resulted in significantly reduced TRPM2 tyrosine phosphorylation, and inhibited the rise in [Ca(2+)](i) and the loss of cell viability, which follow H(2)O(2) or TNFalpha treatment. Consistent with these findings, reduction in endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a significantly greater rise in [Ca(2+)](i) following H(2)O(2) treatment, and enhanced susceptibility to H(2)O(2)-induced cell death. Endogenous TRPM2 and PTPL1 was associated in U937-ecoR cells, confirming the physiological relevance of this interaction. These data demonstrate that tyrosine phosphorylation of TRPM2 is important in its activation and function and that inhibition of TRPM2 tyrosine phosphorylation reduces Ca(2+) influx and protects cell viability. They also suggest that modulation of TRPM2 tyrosine phosphorylation is a mechanism through which PTPL1 may mediate resistance to cell death.