Abstract
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.
Highlights
Within the developing vertebrate nervous system, proliferating progenitor cells differentiate into post-mitotic neurons under the control of specific secreted polypeptide growth factors
To test whether SNT1(IRS) could promote neuronal differentiation, Myc-tagged SNT1(IRS) or SNT1 were transiently transfected into PC12 cells, which were subsequently challenged for 2 days with the neurotrophin nerve growth factor (NGF), FGF1, or insulin
PC12 cells transfected with SNT1 or SNT1(IRS) underwent neuronal differentiation in response to NGF or fibroblast growth factors (FGFs) (Fig. 2B), analogous to untransfected cells, insulin failed to induce differentiation of SNT1(IRS)-transfected cells (Fig. 2B)
Summary
Immunological Reagents—Mouse monoclonal antibodies used were 4G10 anti-phosphotyrosine (pY) (Upstate Biotechnology Inc.), anti-Ras and anti-Shp (PTP1D) (Transduction Laboratories), 12CA5 anti-HA tag and 9E10 anti-Myc tag Expression of Proteins in PC12 Cells—PC12 cells were transiently transfected with pSR␣-Myc-tagged SNT-derived constructs or with corresponding HA-epitope-tagged constructs in which the triple Myc tag was replaced with annealed oligonucleotides encoding two tandem copies of the HA-epitope sequence (YDVPDYAS). For PC12-stable transfections, each Myc-tagged SNT1(IRS)CX open reading frame was first shuttled into the expression vector pMIRB [21] After blocking in 10% bovine calf serum/PBS for 1 h, cells were incubated with 0.3 g/ml 9E10 anti-Myc for 1 h, with 1:5000 AP-conjugated secondary anti-mouse IgG1 for 0.5 h, and stained with nitro blue tetrazolium (440 g/ml) ϩ 5-bromo-4chloro-3-indolyl phosphate (165 g/ml) in alkaline substrate buffer (0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 1 mM levamisole) for 15– 60 min. Representative images were scanned with a Leica TCS-CP confocal laser scanning microscope
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