Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells
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ORIGINAL ARTICLE
Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells Jo Suda1 • Don C. Rockey2 • Serhan Karvar2
Received: 11 May 2015 / Accepted: 24 September 2015 / Published online: 24 October 2015 Ó Springer Science+Business Media New York 2015
Results Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. Conclusions These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and function.
Abstract Background The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Aim Here we have investigated the importance of Aktmediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Methods Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function.
Keywords Liver Cytoskeleton Protein kinase B PI3K Bile transporter Bile canaliculus
& Serhan Karvar [email protected]
Introduction
Jo Suda [email protected] Don C. Rockey [email protected] 1
Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
2
Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, 803 CSB, MSC 623, Charleston, SC 29425, USA
The dominant ezrin/radixin/moesin (ERM) protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and virtually absent in other liver cells [1–6]. At 4 weeks of age, radixin knocko
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