title: Disruption of ShcA signaling halts cell proliferation--characterization of ShcC residues that influence signaling pathways using yeast.
abstract: Shc adapter proteins are thought to regulate cellular proliferation, differentiation and apoptosis by activating the SOS-Grb2-RAS-MAPK signaling cascade. Using the small hairpin RNA (shRNA) technique, we found that decreasing ShcA mRNA reduced the proliferative ability of HEK293 mammalian culture cells. We then recapitulated phosphorylation-dependent Shc-Grb2 complex formation in Saccharomyces cerevisiae. Immunoprecipitation followed by Western analysis demonstrated that activated TrkB, composed of the intracellular domain of TrkB fused to glutathione S-transferase (GST-TrkB(ICD)), promoted the association of ShcC and Grb2 in yeast. The Ras-recruitment system (RRS), in which a myristoylated (Myr)-bait and son of sevenless (hSOS)-prey are brought together to complement the defective Ras-cAMP pathway in a thermosensitive cdc25H mutant yeast strain, was used to validate a phenotypic assay. Yeast cells transformed with both Myr-ShcC and hSOS-Grb2 (referred to as scheme 1) or Myr-Grb2 and hSOS-ShcC (scheme 2) did not grow at non-permissive temperature; the additional transformation of GST-TrkB(ICD) enabled growth. GST-TrkB(ICD) also enabled growth with hSOS-Grb2 and either Myr-ShcA or Myr-SHP2. Mutational analysis of TrkB showed that its kinase activity was essential for complementation, while its docking site for Shc proteins was not. Mutational analysis of ShcC showed that the PTB and SH2 domains were not essential for complementation but phosphorylation at Y304 in the CH1 domain was. Phosphorylation at Y304 could not be substituted by an acidic amino acid. The RRS provides a genetic system to probe Shc proteins and potentially identify member specific protein partners and pharmacological reagents.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, 865 Ridge Road, Monmouth Junction, New Jersey 08852-2718, USA. heinrij@wyeth.com
authors.forename: Julia N
authors.initials: JN
authors.lastname: Heinrich
authors.forename: Seung P
authors.initials: SP
authors.lastname: Kwak
authors.forename: David S
authors.initials: DS
authors.lastname: Howland
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Stephen
authors.initials: S
authors.lastname: Sturner
authors.forename: Kelly
authors.initials: K
authors.lastname: Sullivan
authors.forename: Kerri
authors.initials: K
authors.lastname: Lipinski
authors.forename: Ke-Yi
authors.initials: KY
authors.lastname: Cheng
authors.forename: Yijin
authors.initials: Y
authors.lastname: She
authors.forename: Frederick
authors.initials: F
authors.lastname: Lo
authors.forename: Afshin
authors.initials: A
authors.lastname: Ghavami
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D005656:Fungal Proteins / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D006801:Humans; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020813:Receptor, trkB / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000235:genetics; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071427:Src Homology 2 Domain-Containing, Transforming Protein 3
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; D051380:GRB2 Adaptor Protein; D009479:Neuropeptides; C528024:SHC1 protein, human; C122029:SHC3 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000071427:Src Homology 2 Domain-Containing, Transforming Protein 3; D020813:Receptor, trkB