title: Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells.
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.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Zhen Y
authors.initials: ZY
authors.lastname: Jiang
authors.forename: Qiong L
authors.initials: QL
authors.lastname: Zhou
authors.forename: John
authors.initials: J
authors.lastname: Holik
authors.forename: Shraddha
authors.initials: S
authors.lastname: Patel
authors.forename: John
authors.initials: J
authors.lastname: Leszyk
authors.forename: Kerri
authors.initials: K
authors.lastname: Coleman
authors.forename: My
authors.initials: M
authors.lastname: Chouinard
authors.forename: Michael P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D015778:Minor Histocompatibility Antigens; D010455:Peptides / Q000737:chemistry; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D000076204:WNK Lysine-Deficient Protein Kinase 1; D000077191:Wortmannin; D000099293:Protein Kinase C-lambda
chemical_list: D000730:Androstadienes; D005947:Glucose; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D015778:Minor Histocompatibility Antigens; D010455:Peptides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D011972:Receptor, Insulin; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D013936:Thymidine; D014357:Trypsin; D000076204:WNK Lysine-Deficient Protein Kinase 1; D000077191:Wortmannin; D000099293:Protein Kinase C-lambda; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C494922:Akt2 protein, mouse; C498743:WNK1 protein, human; C498744:Wnk1 protein, mouse