Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase.
CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in creating and maintaining the adipocyte phenotype. We have shown previously that insulin stimulates dephosphorylation of C/EBPalpha in 3T3-L1 adipocytes. Studies to identify the insulin-sensitive sites of phosphorylation reveal that a C/EBPalpha peptide (amino acids H215 to K250) is phosphorylated on T222, T226, and S230 in vivo. The context of these phosphoamino acids implicates glycogen synthase kinase 3 (GSK3), whose activity is known to be repressed in response to insulin, as a potential kinase for phosphorylation of T222 and T226. Accordingly, GSK3 phosphorylates the predicted region of C/EBPalpha on threonine in vitro, and GSK3 uses C/EBPalpha as a substrate in vivo. In addition, the effect of pharmacological agents on GSK3 activity correlates with regulation of C/EBPalpha phosphorylation. Treatment of 3T3-L1 adipocytes with the phosphatidylinositol 3-kinase inhibitor wortmannin results in phosphorylation of C/EBPalpha, whereas treatment with the GSK3 inhibitor lithium results in dephosphorylation of C/EBPalpha. Collectively, these data indicate that insulin stimulates dephosphorylation of C/EBPalpha on T222 and T226 through inactivation of GSK3. Since dephosphorylation of C/EBPalpha in response to lithium is blocked by okadaic acid, strong candidates for the T222 and T226 phosphatase are protein phosphatases 1 and 2a. Treatment of adipocytes with insulin alters the protease accessibility of widespread sites within the N terminus of C/EBPalpha, consistent with phosphorylation causing profound conformational changes. Finally, phosphorylation of C/EBPalpha and other substrates by GSK3 may be required for adipogenesis, since treatment of differentiating preadipocytes with lithium inhibits their conversion to adipocytes.
Ross
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SE
Department of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622, USA.
Erickson
R L
RL
Hemati
N
N
MacDougald
O A
OA
D016475:3T3 Cells; D000730:Androstadienes / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007329:Insulin Antagonists / Q000378:metabolism / Q000494:pharmacology; D008094:Lithium / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019606:Phosphoamino Acids; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007328:Insulin; D007329:Insulin Antagonists; D009687:Nuclear Proteins; D019606:Phosphoamino Acids; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D008094:Lithium; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000077191:Wortmannin