Xu H, Goldfarb M
Department of Biochemistry and Molecular Biology and Graduate Training Program in Molecular, Cellular, Biochemical, and Developmental Sciences, Mount Sinai School of Medicine, Box 1020, New York, New York 10029, USA.
J Biol Chem. 2001 Apr 20;276(16):13049-56. doi: 10.1074/jbc.M009925200. Epub 2001 Jan 12.
Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.
神经元前体细胞对神经营养分化因子作出反应而发生分化的过程中，伴随着膜锚定的SNT信号衔接蛋白的激活。两类分化因子，即神经营养因子和成纤维细胞生长因子，可诱导SNT1（FRS2α）快速发生酪氨酸磷酸化，这进而使SNT1能够募集与Ras GDP/GTP交换因子Sos形成复合物的Shp2酪氨酸磷酸酶和Grb2衔接蛋白。为了确定促进PC12嗜铬细胞瘤细胞神经元分化的SNT效应功能，我们构建了一种嵌合蛋白SNT1（IRS）CX，它带有SNT1的效应区域和IRS2的胰岛素受体识别结构域。胰岛素可促进转染的PC12细胞中SNT1（IRS）CX的酪氨酸磷酸化，同时伴随着ERK1/2丝裂原活化蛋白激酶的持续激活和神经元分化。SNT1（IRS）CX介导的反应依赖于内源性Ras、MEK和Shp2的活性。对SNT1（IRS）CX进行诱变，在SNT中鉴定出了对ERK持续激活和神经元分化都至关重要的三类效应基序：1）介导Grb2募集的四个磷酸酪氨酸基序，2）介导Shp2募集的两个磷酸酪氨酸基序，3）通过帮助募集Sos发挥作用的一个C端基序。我们讨论了三种功能不同的SNT效应基序协同促进下游生化和生物学反应的可能机制。
