Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells
Program in Molecular Medicine; Department of Biochemistry and Molecular Pharmacology
1-Phosphatidylinositol 3-Kinase; 3T3-L1 Cells; Adipocytes; Androstadienes; Animals; Apoptosis; Binding Sites; Biological Transport; Cell Membrane; Cell Proliferation; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; *Gene Expression Regulation, Enzymologic; Glucose; Humans; Immunoblotting; Immunoprecipitation; Insulin; Isoenzymes; Mass Spectrometry; Mice; Peptides; Phosphorylation; Protein Kinase C; Protein Structure, Tertiary; Protein Transport; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Receptor, Insulin; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymidine; Time Factors; Transfection; Trypsin
Life Sciences | Medicine and Health Sciences
Insulin signaling through protein kinase Akt/protein kinase B (PKB), a downstream element of the phosphatidylinositol 3-kinase (PI3K) pathway, regulates diverse cellular functions including metabolic pathways, apoptosis, mitogenesis, and membrane trafficking. To identify Akt/PKB substrates that mediate these effects, we used antibodies that recognize phosphopeptide sites containing the Akt/PKB substrate motif (RXRXX(p)S/T) to immunoprecipitate proteins from insulin-stimulated adipocytes. Tryptic peptides from a 250-kDa immunoprecipitated protein were identified as the protein kinase WNK1 (with no lysine) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, consistent with a recent report that WNK1 is phosphorylated on Thr60 in response to insulin-like growth factor I. Insulin treatment of 3T3-L1 adipocytes stimulated WNK1 phosphorylation, as detected by immunoprecipitation with antibody against WNK1 followed by immunoblotting with the anti-phosphoAkt substrate antibody. WNK1 phosphorylation induced by insulin was unaffected by rapamycin, an inhibitor of p70 S6 kinase pathway but abolished by the PI3K inhibitor wortmannin. RNA interference-directed depletion of Akt1/PKB alpha and Akt2/PKB beta attenuated insulin-stimulated WNK1 phosphorylation, but depletion of protein kinase C lambda did not. Whereas small interfering RNA-induced loss of WNK1 protein did not significantly affect insulin-stimulated glucose transport in 3T3-L1 adipocytes, it significantly enhanced insulin-stimulated thymidine incorporation by about 2-fold. Furthermore, depletion of WNK1 promoted serum-stimulated cell proliferation of 3T3-L1 preadipocytes, as evidenced by a 36% increase in cell number after 48 h in culture. These data suggest that WNK1 is a physiologically relevant target of insulin signaling through PI3K and Akt/PKB and functions as a negative regulator of insulin-stimulated mitogenesis.
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Citation: J Biol Chem. 2005 Jun 3;280(22):21622-8. Epub 2005 Mar 30. Link to article on publisher's site
The Journal of biological chemistry
Jiang, Zhen Y.; Zhou, Qiong L.; Holik, John; Patel, Shraddha; Leszyk, John D.; Coleman, Kerri; Chouinard, My T.; and Czech, Michael P., "Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells" (2005). Open Access Articles. 690.