Negative regulation of insulin-stimulated mitogen-activated protein kinase signaling by Grb10.
Grb10 is a Pleckstrin homology and Src homology 2 (SH2) domain-containing protein that binds to the tyrosine-phosphorylated insulin receptor in response to insulin stimulation. Loss of Grb10 function in mice results in fetal and placental overgrowth; however, the molecular mechanism remains unknown. In the present study, we show that overexpression of Grb10 in Chinese hamster ovary cells expressing the insulin receptor or in 3T3-L1 adipocytes reduced insulin-stimulated phosphorylation of MAPK. Overexpression of Grb10 in rat primary adipocytes also inhibited insulin-stimulated phosphorylation of the MAPK downstream substrate Elk1. To determine the mechanism by which Grb10 inhibited insulin-stimulated MAPK signaling, we examined whether Grb10 affects the phosphorylation of MAPK upstream signaling components. We found that overexpression of Grb10 inhibited the insulin-stimulated phosphorylation of Shc, a positive regulator of the MAPK signaling pathway. The inhibitory effect was diminished when the SH2 domain of Grb10 was deleted. The negative role of Grb10 in insulin signaling was established by suppression of endogenous Grb10 by RNA interference in HeLa cells overexpressing the insulin receptor, which enhanced insulin-stimulated phosphorylation of MAPK, Shc, and Akt. Taken together, our findings suggest that Grb10 functions as a negative regulator in the insulin-stimulated MAPK signaling pathway. In addition, the inhibitory effect of Grb10 on the MAPK pathway is most likely due to a direct block of insulin-stimulated Shc tyrosine phosphorylation.
Langlais
Paul
P
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA.
Dong
Lily Q
LQ
Ramos
Fresnida J
FJ
Hu
Derong
D
Li
Yunhua
Y
Quon
Michael J
MJ
Liu
Feng
F
D048868:Adaptor Proteins, Signal Transducing / Q000187:drug effects / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D051383:GRB10 Adaptor Protein; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D050807:ets-Domain Protein Elk-1; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; C494113:Elk1 protein, mouse; C494114:Elk1 protein, rat; C495959:Grb10 protein, mouse; D007328:Insulin; D011506:Proteins; D011518:Proto-Oncogene Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D051383:GRB10 Adaptor Protein; D014443:Tyrosine; D011972:Receptor, Insulin; D020928:Mitogen-Activated Protein Kinases