Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing Recruitment of p52-Shc to the Grb2/Shc Complex.
Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1-24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275-1286, 2017. © 2016 Wiley Periodicals, Inc.
Department of Physiology, Alcohol and Drug Abuse Center of Excellence, LSU Health New Orleans, New Orleans, Louisiana.|Department of Genetics, LSU Health New Orleans, New Orleans, Louisiana.|Stanley S. Scott Cancer Center, LSU Health New Orleans, New Orleans, Louisiana.
Matthew
M
Dean
Stanley S. Scott Cancer Center, LSU Health New Orleans, New Orleans, Louisiana.
Adam
A
Lassak
University of South Alabama Mitchell Cancer Institute, Mobile, Alabama.
Anna
A
Wilk
Stanley S. Scott Cancer Center, LSU Health New Orleans, New Orleans, Louisiana.
Adriana
A
Zapata
Morphology and Imaging Core, LSU Health New Orleans, New Orleans, Louisiana.
Luis
L
Marrero
Department of Physiology, Alcohol and Drug Abuse Center of Excellence, LSU Health New Orleans, New Orleans, Louisiana.
Patricia
P
Molina
Stanley S. Scott Cancer Center, LSU Health New Orleans, New Orleans, Louisiana.
Krzysztof
K
Reiss
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D051379:Mice; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1 / Q000378:metabolism*; D013997:Time Factors
D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D055504:Insulin Receptor Substrate Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D000431:Ethanol; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D048049:Extracellular Signal-Regulated MAP Kinases