Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells.
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.


OBJECTIVE
Here we have investigated the importance of Akt-mediated 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.


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.
Jo
J
Suda
Don C
DC
Rockey
Serhan
S
Karvar
D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D005978:Glutathione; D008565:Membrane Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051057:Proto-Oncogene Proteins c-akt; C000715087:ABCC2 protein, human; C071776:radixin; C494918:AKT1 protein, human; C494921:AKT2 protein, human