Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells.
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.
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.
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D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology* / Q000502:physiology*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017354:Point Mutation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; C106534:FRS2 protein, human; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C509248:Ptpn6 protein, mouse; C509250:Ptpn6 protein, rat