Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1

Abstract

, a member of the transient receptor potential (TRP) superfamily, is a Ca 2ϩ -permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNF␣, or ␤-amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H 2O2 or TNF␣. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca 2ϩ ]i observed after H2O2 or TNF␣ 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-Stransferase 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 2ϩ ]i and the loss of cell viability, which follow H2O2 or TNF␣ 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 2ϩ ]i following H2O2 treatment, and enhanced susceptibility to H 2O2-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 2ϩ 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.

TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration

“…Specifically, inhibition of hematopoietic PTP (HePTP) expressed in U937 cells 33 might be involved, as HePTP inhibition has been reported to trigger Erk activation without Mek activation 32 . In addition, PTP inhibition positively regulates H 2 O 2 -evoked TRPM2 activation 34 . Therefore, some Erk activation through HePTP inhibition may be responsible for the residual nuclear trans-location of NF-κB and CXCL8 production independent of TRPM2 and Mek.…”

PTPL1: a large phosphatase with a split personality

“…Endogenous PTPL1 and TAPP1 co-localized predominantly in the cytoplasm, while an H 2 O 2 -stimulated increase of intracellular PIP2 levels led to higher levels of co-localization between endogenous TAPP1 and PTPL1 at the cell membrane [25]. In addition to the TAPP1/2 interactions, PDZ1 has been shown to bind the inhibitor of nuclear factor kappa-B alpha (IκBα) [26] as well as the transient receptor potential (TRP) superfamily member TRPM2 [27].…”

“…And lastly, EphrinB1, a transmembrane ligand for Bclass Ephrin receptor tyrosine kinases also binds PDZ4 [33]. PTPL1's final PDZ domain (PDZ5) has thus far only been shown to bind to TRPM2, which also binds PDZ1 [27]. Although a number of interacting molecules have been described, few have been definitively identified as PTPL1 substrates.…”

“…Tyrosine phosphorylation of p75 NTR induces its ubiquitination [56] and its potential proteosomal degradation, while dephosphorylation of p75 NTR by PTPL1 may stabilize the protein leading to an increase in its intracellular level, thus promoting cell survival. TRPM2, a member of the transient receptor potential (TRP) superfamily that binds to PDZ1 and/or PDZ5 within PTPL1, is phosphorylated in response to either oxidative stress or TNFα treatment [27]. Phosphorylation then results in an influx of extracellular Ca +2 and leads to apoptosis [27].…”

“…TRPM2, a member of the transient receptor potential (TRP) superfamily that binds to PDZ1 and/or PDZ5 within PTPL1, is phosphorylated in response to either oxidative stress or TNFα treatment [27]. Phosphorylation then results in an influx of extracellular Ca +2 and leads to apoptosis [27]. Cells transfected with PTPL1 display reduced TRPM2 phosphorylation and a corresponding reduction in Ca +2 influx leading to cell survival [27].…”