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.
"Upon activation by the binding of VEGF, the VEGFR2 tyrosine kinase phosphorylates downstream kinases, such as the phosphoinositide-dependent protein kinase (PI3 kinase), which then phosphorylates and activates the protein kinase Akt/PKB1. Multiple Akt/PKB substrates have been discovered, and WNK1 is a novel Akt/PKB substrate in insulin-stimulated 3T3-L1 adipocytes . "
[Show abstract][Hide abstract] ABSTRACT: The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in
situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression.
PLoS ONE 08/2014; 9(8):e106129. DOI:10.1371/journal.pone.0106129 · 3.23 Impact Factor
"The activated IRK phosphorylates substrates, including IRS-1 to -4, which bind to effector molecules such as phosphatidylinositol 3-kinase (PI3K), resulting in their activation (reviewed in 4). Downstream events involve the activation of protein kinase B (Akt), protein kinase A (PKA), and WNK1 (5), leading ultimately to branching intracellular pathways, inducing glucose uptake and regulating cell metabolism, growth, and differentiation. In endosomes, IRK is dephosphorylated and either sent to lysosomes for degradation or recycled to the plasma membrane for another round of binding, activation, and internalization (6). "
[Show abstract][Hide abstract] ABSTRACT: Neuraminidases (sialidases) catalyze the removal of sialic acid residues from sialylated glycoconjugates. We now report that mammalian neuraminidase 1 (Neu1), in addition to its catabolic function in lysosomes, is transported to the cell surface where it is involved in regulation of insulin signaling. Insulin binding to its receptor rapidly induces interaction of the receptor with Neu1, which hydrolyzes sialic acid residues in the glycan chains of the receptor and, consequently, induces its activation. Cells from sialidosis patients with a genetic deficiency of Neu1 show impairment of insulin-induced phosphorylation of downstream protein kinase AKT, while treatment of these cells with purified Neu1 restores signaling. Genetically-modified mice with ∼10% of the normal Neu1 activity exposed to a high-fat diet develop hyperglycemia and insulin resistance twice as fast as their wild type counterparts. Together, these studies identify Neu1 as a novel component of the signaling pathways of energy metabolism and glucose uptake.
"The threonine-58 of WNK1 is a phosphorylation site for Akt/protein kinase B, a downstream kinase of insulin/IGF pathway (Vitari et al., 2004; Xu et al., 2005b). Insulin and IGF1 cause phosphorylation of endogenous WNK1 at the threonine residue (Vitari et al., 2004: Jiang et al., 2005). Activation of ENaC by WNK1 via serum and glucocorticoid-inducible kinase (SGK1) depends on basal phosphorylation of threonine-58 (Xu et al., 2005b). "
[Show abstract][Hide abstract] ABSTRACT: WNKs (with-no-lysine [K]) are a family of serine-threonine protein kinases with an atypical placement of the catalytic lysine relative to all other protein kinases. The roles of WNK kinases in regulating ion transport were first revealed by the findings that mutations of two members cause a genetic hypertension and hyperkalemia syndrome. More recent studies suggest that WNKs are pleiotropic protein kinases with important roles in many cell processes in addition to ion transport. Here, we review roles of WNK kinases in the regulation of ion balance, cell signaling, survival, and proliferation, and embryonic organ development.
Experimental and Molecular Medicine 11/2007; 39(5):565-73. DOI:10.1038/emm.2007.62 · 3.45 Impact Factor
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