Discrimination between phosphotyrosine-mediated signaling properties of conventional and neuronal Shc adapter molecules.
The phosphotyrosine (pTyr) adapter Shc/ShcA is a major connector in various tyrosine kinase signalings following a variety of stimulation such as growth factor/neurotrophin, as well as in those following calcium influx and integrin activation. As in other tissues, Shc has been implicated in neuronal signalings; however, recent evidence suggests that N-Shc/ShcC and Sck/ShcB would take over most of the roles of Shc in mature central neurons, and switching phenomena between Shc and N-Shc expression were observed in several neuronal paradigms. Little is, however, known as to the signal-output differences between Shc and N-Shc. Here we determined the efficacy of Shc and N-Shc toward Erk activation in NGF-treated PC12 cells, and found that N-Shc transduced Grb2/Sos/Ras-dependent Erk activation less efficiently than Shc. This was mainly because N-Shc has only one high-affinity Grb2-binding site, whereas Shc has two such sites. Phosphopeptide mapping revealed that N-Shc has novel tyrosine-phosphorylation sites at Y259/Y260 and Y286; in vivo-phosphorylation of these tyrosines was demonstrated by site-specific anti-pTyr antibodies. Phosphorylated Y286 bound to several proteins, of which one was Crk. The pY221/pY222 site, corresponding to one of the Grb2-binding sites of Shc, also preferentially bound to Crk. The phosphorylation-dependent interaction between N-Shc and Crk was demonstrated in vitro and in vivo. These results indicate that N-Shc has specific features of signal-output, and further suggest that the switching between Shc and N-Shc during neural development and regeneration would lead to differentiation of downstream signalings.
Nakamura
Takeshi
T
Department of Molecular Genetics, National Institute for Longevity Sciences, Oobu, Aichi 474-8522, Japan.
Komiya
Misako
M
Gotoh
Noriko
N
Koizumi
Shinichi
S
Shibuya
Masabumi
M
Mori
Nozomu
N
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D020935:MAP Kinase Signaling System / Q000502:physiology*; D046911:Macromolecular Substances; 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; D009363:Neoplasm Proteins / Q000502:physiology; D009414:Nerve Growth Factors / Q000494:pharmacology; D009479:Neuropeptides / Q000737:chemistry / Q000502:physiology*; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071427:Src Homology 2 Domain-Containing, Transforming Protein 3; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C493872:Crk protein, rat; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D009479:Neuropeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; C485683:Shc3 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071427:Src Homology 2 Domain-Containing, Transforming Protein 3; D019000:Phosphotyrosine; D011494:Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases