Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells

Differentiation of neuronal precursor cells in response to neurotrophic dif ferentiation 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 phospho rylation of SNT1(FRS2 alpha), which in turn enables SNT1 to recruit Shp2 ty rosine phosphatase and GrbB adaptor protein in complex with the Res GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote n euronal differentiation of PC12 pheochromocytoma cells, we engineered a chi meric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the ins ulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosph orylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differ entiation. The SNT1 (IRS)CX-mediated response was dependent on endogenous R es, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three c lasses of effector motifs within SNT critical for both sustained ERK activa tion and neuronal differentiation: 1) four phosphotyrosine motifs that medi ate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruit ment of Shp2, and 3) a C-terminal motif that functions by helping to recrui t Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and bio logical response.