Corvol Jean-Christophe, Valjent Emmanuel, Toutant Madeleine, Enslen Hervé, Irinopoulou Théano, Lev Sima, Hervé Denis, Girault Jean-Antoine
Signal Transduction and Plasticity in the Nervous System Unit, INSERM/Université Pierre et Marie Curie U536, Institut du Fer à Moulin, 75005 Paris, France.
J Biol Chem. 2005 Jan 7;280(1):660-8. doi: 10.1074/jbc.M411312200. Epub 2004 Nov 10.
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.
在海马体中，细胞外信号调节激酶（ERK）和非受体蛋白富含脯氨酸的酪氨酸激酶2（PYK2）可通过去极化被激活，并参与突触可塑性。在缺血、惊厥或电惊厥休克后的病理条件下，它们也会被激活。尽管在非神经元细胞中，PYK2通过募集Src家族激酶（SFK）来激活ERK，但海马体中这些信号通路之间的联系尚不清楚。我们使用钾离子去极化的大鼠海马体切片来解决这个问题。去极化增加了PYK2、SFK和ERK的磷酸化。这些效应是由钙离子通过电压门控钙通道内流引起的，并被蛋白激酶C抑制剂GF109203X减弱。用PP2抑制SFK可显著降低PYK2的酪氨酸磷酸化，但不影响其在Tyr-402位点的自身磷酸化。此外，在fyn基因敲除小鼠中，PYK2的自身磷酸化和总酪氨酸磷酸化发生了深刻变化，揭示了海马体中Fyn和PYK2之间重要的功能关系。相比之下，PP2、Fyn基因敲除或磷脂酰肌醇-3-激酶抑制剂LY294002对ERK激活没有影响。MEK抑制剂可阻止ERK激活，而对PYK2没有影响。海马体切片的免疫荧光显示，PYK2和ERK分别在树突状区域和神经末梢的不同细胞区室中被激活，几乎没有重叠。ERK激活对于去极化后突触小泡蛋白突触素I的再磷酸化至关重要，突显了其在神经末梢中的功能重要性。因此，在海马体切片中，与细胞系不同，去极化诱导的非受体酪氨酸激酶和ERK激活在不同的细胞区室中独立发生，在这些区室中它们似乎具有不同的功能作用。
