Browsing by Author "Henriques, Andreia F.A."
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- Ibuprofen disrupts a WNK1/GSK3β/SRPK1 protein complex required for expression of tumor-related splicing variant RAC1B in colorectal cellsPublication . Gonçalves, Vânia; Henriques, Andreia F.A.; Matos, Paulo; Jordan, PeterA major risk factor promoting tumor development is chronic inflammation and the use of nonsteroidal anti-inflammatory drugs (NSAID), including ibuprofen, can decrease the risk of developing various types of cancer, including colorectal cancer (CRC). Although the molecular mechanism behind the antitumor properties of NSAIDs has been largely attributed to inhibition of cyclooxygenases (COXs), several studies have shown that the chemopreventive properties of ibuprofen also involve multiple COX-independent effects. One example is its ability to inhibit the alternative splicing event generating RAC1B, which is overexpressed in a specific subset of BRAF-mutated colorectal tumors and sustains cell survival. Here we describe the mechanism by which ibuprofen prevents RAC1B alternative splicing in a BRAF mutant CRC cell line: it leads to decreased translocation of SRPK1 and SRSF1 to the nucleus and is regulated by a WNK1/GSK3β/SRPK1 protein kinase complex. Surprisingly, we demonstrate that ibuprofen does not inhibit the activity of any of the involved kinases but rather promotes disassembly of this regulatory complex, exposing GSK3β serine 9 to inhibitory phosphorylation, namely by AKT, which results in nuclear exclusion of SRPK1 and SRSF1 hypophosphorylation. The data shed new light on the biochemical mechanisms behind ibuprofen's action on alternative spliced RAC1B and may support its use in personalized approaches to CRC therapy or chemoprevention regimens.
- WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1Publication . Henriques, Andreia F.A.; Matos, Paulo; Carvalho, Ana Sofia; Azkargorta, Mikel; Elortza, Felix; Matthiesen, Rune; Jordan, PeterGlucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.