Depolarization Activates ERK and Proline-rich Tyrosine Kinase 2 (PYK2) Independently in Different Cellular Compartments in Hippocampal Slices

Jean-Christophe Corvol,Emmanuel Valjent,Madeleine Toutant,Hervé Enslen,Théano Irinopoulou,Sima Lev,Denis Hervé,Jean-Antoine Girault

doi:10.1074/jbc.m411312200

Abstract

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

Highlights

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity
Depolarization Induces a Rapid and Reversible Phosphorylation of PYK2, Src-family kinases (SFKs), and ERK in Hippocampal Slices—Treatment of rat hippocampal slices with 40 mM KCl for 2 min induced the appearance of two major phosphotyrosine immunoreactive bands at 115 and 42 kDa (Fig. 1A)
Because most previously reported pathways activated by PYK2 and leading to ERK activation involve the phosphorylation of PYK2 by SFKs (37), these results argue against a role of PYK2 in the depolarization-induced activation of ERK

Summary

Introduction

Extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. In non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known We addressed this question using K؉-depolarized rat hippocampal slices. Long term potentiation is prevented by intracellular injection of a catalytically inactive mutant of PYK2 (11) or in the presence of the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059 (12), demonstrating the important functional role of these signaling pathways. Both ERK and PYK2 are activated in the hippocampus following brain ischemia (13–16), and convulsions (15, 17, 18), suggesting their possible role in pathology. PYK2 activation results from its autophosphorylation on Tyr402, which creates an Src homology 2-binding site that recruits

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