title: Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1.
abstract: WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.
authors.affiliation: Department of Medicine, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8856, USA.
authors.forename: Chih-Jen
authors.initials: CJ
authors.lastname: Cheng
authors.forename: Chou-Long
authors.initials: CL
authors.lastname: Huang
mesh_terms: D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D018408:Patch-Clamp Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D000076204:WNK Lysine-Deficient Protein Kinase 1; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D017874:Immediate-Early Proteins; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D019869:Phosphatidylinositol 3-Kinases; D024661:Potassium Channels, Inwardly Rectifying; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D000076204:WNK Lysine-Deficient Protein Kinase 1; D000099184:Serum-Glucocorticoid Regulated Kinases; C086989:KCNJ1 protein, human; C494918:AKT1 protein, human; C498743:WNK1 protein, human