Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
Department of Oral and Maxillofacial Functional Rehabilitation, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
FEBS J. 2020 Aug;287(16):3551-3564. doi: 10.1111/febs.15231. Epub 2020 Feb 11.
The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic GnRH neurons, as well as in anterior pituitary gonadotrophs. In our previous study, we found that stimulation of the GnRH receptor activated protein kinase D1 (PKD1), and PKD1 was involved in the Fyn-mediated activation of proline-rich tyrosine kinase 2 (Pyk2) in cultured GnRH neurons (GT1-7 cells). In the present study, we examined the molecular mechanisms of Pyk2 activation and the interaction of Pyk2 and Fyn in GT1-7 cells. Experiments with site-directed mutants of Pyk2 indicated that tyrosine 402 (Tyr402) was phosphorylated both by autophosphorylation and by Fyn, whereas Tyr579 was phosphorylated mainly by Fyn. We found that dasatinib, a Src family inhibitor, enhanced the interaction of Pyk2 and Fyn. Experiments with site-directed mutants of Pyk2 and Fyn indicated that dasatinib enhanced the binding of Pyk2 autophosphorylated at Tyr402 and the Src homology 2 domain in Fyn. Our present data may suggest that fully activated Pyk2 dissociates from Fyn after Fyn-mediated phosphorylation of Pyk2 at sites other than Tyr402 and Tyr579.
促性腺激素释放激素（GnRH）受体在下丘脑 GnRH 神经元以及垂体前叶促性腺细胞中高度表达。在我们之前的研究中，我们发现 GnRH 受体的刺激激活了蛋白激酶 D1（PKD1），PKD1 参与了 Fyn 介导的富含脯氨酸的酪氨酸激酶 2（Pyk2）在培养的 GnRH 神经元（GT1-7 细胞）中的激活。在本研究中，我们研究了 Pyk2 激活的分子机制以及 Pyk2 和 Fyn 在 GT1-7 细胞中的相互作用。使用 Pyk2 的定点突变体的实验表明，酪氨酸 402（Tyr402）通过自身磷酸化和 Fyn 磷酸化，而 Tyr579 主要通过 Fyn 磷酸化。我们发现，Src 家族抑制剂 dasatinib 增强了 Pyk2 和 Fyn 的相互作用。使用 Pyk2 和 Fyn 的定点突变体的实验表明，dasatinib 增强了 Pyk2 在 Tyr402 和 Fyn 的Src 同源 2 结构域处自身磷酸化的结合。我们目前的数据可能表明，完全激活的 Pyk2 在 Fyn 介导的 Tyr402 和 Tyr579 以外的位点磷酸化 Pyk2 后与 Fyn 分离。
