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- Regulation of glucose uptake in mammalian cells by protein phosphorylation networksPublication . Henrique, Andreia; Jordan, Peter; Clarke, LukaGlucose uptake is a key mechanism to maintain cell, tissue and body homeostasis. Among others, glucose transporter proteins (GLUTs) are responsible for glucose transport into the cell. Besides their expression level, the GLUT number present at the plasma membrane (PM) is regulated by signaling mechanisms. Previously, protein kinase WNK1 was found to phosphorylate TBC1D4, a Rab-GAP involved in membrane traffic regulation, and to regulate the surface expression of the constitutive glucose transporter GLUT1. In this work the phosphorylation of either TBC1D4 or its paralogue TBC1D1 was studied as a key step in regulatory cascades modulating glucose uptake. First, we showed that downregulation of WNK1 through RNA interference translates in a 2-fold decrease in PM GLUT1 expression and a 60% decrease in glucose uptake. Then, we compared by mass spectrometry (MS) the in vitro phosphorylation of TBC1D1 and 4 by AKT1, WNK1 and SGK1 and 3. We identified two novel WNK1-specific phosphorylation sites at TBC1D1-Ser565 and TBC1D4-Ser704 and showed that transfection of their phosphomimetic or unphosphorylatable mutants affected cell surface abundance of GLUT1. To define new biological pathways regulated by WNK1, we determined the interactome of WNK1 by MS. Interestingly, the bioinformatic and gene ontology analysis pointed to a previously unrecognized function related to mRNA processing. Our studies identified a novel function of WNK1 in alternative splicing using RAC1B in colorectal HT29 cells as a model. In particular, WNK1 acts as a scaffolding protein through complex formation with GSK3β. This complex protects GSK3β from an inhibitory phosphorylation at Ser9. The active GSK3β allows the translocation of kinase SRPK1 and splicing factor SRSF1 to the nucleus, which is important for RAC1B generation. Considering that RAC1B is known to be essential for cell survival and malignant progression, the results establish a new link between WNK kinases and tumorigenesis. Altogether, this work reinforced a role for WNK1 in cell metabolism and uncovered a new function in regulation of alternative splicing, two events that can contribute to tumor development. The data may provide new targets for pharmacological modulation of RAC1B expression and cellular metabolism, with potential impact for the treatment of cancer and type 2 diabetes.