12-O-Tetradecanoylphorbol-13-acetate activates the Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of SOS involving serine phosphorylation of Shc in NIH3T3 cells.
We investigated the activation of the Ras/ERK signaling pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) in NIH3T3 fibroblasts. Interestingly, the activation was suppressed not only by dominant negative Raf-1 but also by dominant negative Ras and SOS. Further analysis revealed that TPA treatment induced, dependently on protein kinase C, the mobility shift of p66(shc) in SDS-polyacrylamide gel electrophoresis, which could be prevented by treatment of the Shc immunoprecipitate with serine/threonine-specific protein phosphatase 1 (PP1) or 2A (PP2A). Phosphoamino acid analysis of Shc showed that unlike growth factor-induced Shc phosphorylation, where Shc is mainly phosphorylated at tyrosine residues, TPA-induced phosphorylation was only at serine residues. Like growth factor-induced Shc phosphorylation, which leads to the association of Shc with Grb2, TPA also induced this association, but, correspondingly to the above results, the TPA-induced association was disrupted by in vitro treatment of the Shc immunoprecipitate with PP1. Taken together, these results suggest that the TPA signal was fed at or upstream of Shc to activate the Ras/ERK signaling pathway involving serine phosphorylation of Shc.
El-Shemerly
M Y
MY
Friedrich Miescher Institute, CH-4002 Basel, Switzerland.
Besser
D
D
Nagasawa
M
M
Nagamine
Y
Y
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005786:Gene Expression Regulation; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051745:Protein Kinase C-delta; D011506:Proteins / Q000378:metabolism; D013014:SOS Response, Genetics; D012694:Serine / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016222:Fibroblast Growth Factor 2; D012694:Serine; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate