Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP: MOLECULAR BASIS FOR ZN2+-INDUCED ERK MAPK ACTIVATION

Ranjana Poddar,Sathyanarayanan Rajagopal,C William Shuttleworth,Surojit Paul

doi:10.1074/jbc.m115.663468

Ranjana Poddar, Sathyanarayanan Rajagopal + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m115.663468

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Abstract

Excessive release of Zn(2+) in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn(2+)-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn(2+) led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn(2+)-mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn(2+)-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn(2+)-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn(2+). The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn(2+)-induced sustained activation of ERK2.

Highlights

A growing body of evidence indicates that Zn2ϩ, the second most abundant transition metal in the brain, is released at high concentrations during excitotoxic neurological conditions and can contribute to neuronal injury [1,2,3,4,5,6,7]
Consistent with these findings we show that brain-derived neurotropic factor (BDNF)/tropomyosin receptor kinase (Trk)/protein kinase A (PKA) mediated signaling is required for Zn2؉-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of striatal-enriched phosphatase (STEP) that is involved in Zn2؉-dependent neurotoxicity
A key finding of the present study is that exposure of cultured neurons to high concentrations of exogenous Zn2ϩ leads to hyperphosphorylation of STEP61 involving multiple PKA sites

Summary

Experimental Procedures

Materials—Pregnant female Sprague-Dawley rats (16-day gestation) were obtained from Harlan Laboratories. Cells were washed twice with minimum essential medium (MEM) followed by treatment with ZnCl2, BDNF, or NGF for the indicated times at 37 °C. Some cultures were treated with thapsigargin for the specified times without stimulation with Zn2ϩ, BDNF, or NGF. Cells were harvested at the specified times after stimulation and processed for immunoblot analysis. After 24 – 48 h, the cells were treated with forskolin (40 ␮M) or phorbol ester (10 ng/ml) at 37 °C Both treated and untreated cells were harvested at the specified times after stimulation and either processed for immunoprecipitation with anti-ERK2 antibody or immunoblot analysis. Proteins were eluted using SDS-sample buffer and processed for SDS/PAGE and immunoblotting analysis with antibodies, as described in the individual experiments. Differences were considered statistically significant when p Ͻ 0.05

Results

Immunoblot analysis showed that phosphorylation of ERK

Discussion