Obara Yutaro, Okano Yumiko, Ono Sachiko, Yamauchi Arata, Hoshino Tomohiro, Kurose Hitoshi, Nakahata Norimichi
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Cell Signal. 2008 Jul;20(7):1275-83. doi: 10.1016/j.cellsig.2008.02.016. Epub 2008 Mar 4.
Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA(1) receptor- and G(i/o)-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gbetagamma subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest G(i/o) negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via G(i/o) is not due to inhibition of adenylyl cyclase by Galpha(i/o). However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gbeta(1) and gamma(2) subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gbetagamma subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gbetagamma subunits.
细胞外信号调节激酶（ERK）在细胞增殖、分化和基因表达中发挥着重要的生理作用。ERK5的大小是ERK1/2的两倍，其氨基末端的一半包含与ERK1/2具有同源性的激酶结构域和TEY激活基序，而羧基末端的一半则是独特的。在本研究中，我们研究了G蛋白偶联受体（GPCR）和受体酪氨酸激酶之间的相互作用机制，重点关注ERK1/2和ERK5。用百日咳毒素（PTX）预处理大鼠嗜铬细胞瘤细胞（PC12）可特异性增强表皮生长因子（EGF）诱导的ERK5磷酸化。此外，溶血磷脂酸（LPA）以依赖LPA（1）受体和G（i/o）的方式减弱EGF诱导的ERK5磷酸化。另一方面，单独的LPA通过Gβγ亚基和Ras激活ERK1/2，并在后期增强EGF诱导的ERK1/2磷酸化。这些结果表明，G（i/o）对ERK5起负调节作用，而对ERK1/2起正调节作用。LPA不影响EGF处理后的cAMP水平，促进cAMP产生的试剂如福斯可林和霍乱毒素也减弱了EGF诱导的ERK5磷酸化，这表明LPA通过G（i/o）对ERK5的抑制作用不是由于Gα（i/o）对腺苷酸环化酶的抑制。然而，在稳定过表达GPCR激酶2（GRK2）羧基末端的PC12细胞中，LPA对ERK5的抑制作用被消除，Gβ（1）和γ（2）亚基的过表达也抑制了EGF诱导的ERK5磷酸化。响应LPA，Gβγ亚基以时间依赖性方式与EGF受体相互作用。这些结果强烈表明，LPA通过Gβγ亚基对EGF诱导的ERK5磷酸化起负调节作用。
