Yusoff Permeen, Lao Dieu-Hung, Ong Siew Hwa, Wong Esther Sook Miin, Lim Jormay, Lo Ting Ling, Leong Hwei Fen, Fong Chee Wai, Guy Graeme R
Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Singapore.
J Biol Chem. 2002 Feb 1;277(5):3195-201. doi: 10.1074/jbc.M108368200. Epub 2001 Nov 6.
Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.
果蝇的多项遗传学研究表明，dSprouty（dSpry）蛋白可抑制由各种活化的受体酪氨酸激酶受体诱导的Ras/丝裂原活化蛋白（MAP）激酶途径，最显著的是表皮生长因子受体（EGFR）和成纤维细胞生长因子受体（FGFR）。目前，dSpry的作用方式尚不清楚，抑制点仍存在争议。与EGFR相比，已证明至少有四种哺乳动物Spry异构体优先与FGFR共表达。在本研究中，我们研究了各种哺乳动物Spry异构体对过表达组成型活性FGFR1的细胞中Ras/MAP激酶途径的影响。hSpry2在抑制Ras/MAP激酶途径方面比mSpry1或mSpry4显著更有效。额外的实验表明，全长hSpry2是其充分发挥效力所必需的。hSpry2对JNK或p38途径均无抑制作用，且对FRS2磷酸化、Akt活化和Ras活化均无抑制作用。使用Ras、Raf和Mek的组成型活性突变体来定位hSpry2在活化Ras下游的潜在抑制点。本研究结果表明，hSpry2在Raf水平发挥其抑制作用，这在FGF信号背景下的Raf活化试验中得到了验证。
