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, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 07/2005; 280(22):21622-8. DOI: 10.1074/jbc.M414464200
Source: PubMed

ABSTRACT 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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