Tyrosine Phosphorylation Regulates Plasma Membrane Expression and Stability of the Human Bile Acid Transporter ASBT (SLC10A2)

摘要

The human apical sodium-dependent bile acid transporter (hASBT; SLC10A2) is responsible for the reclamation of bile acids from the intestinal lumen, providing a primary mechanism for bile acid and cholesterol homeostasis. However, the regulation of hASBT at the post-translational level is not well understood. In the present study, we investigated the role of Src family kinases (SFKs) and protein tyrosine phosphatases (PTPs) in the regulation of surface expression and function of hASBT. Inhibition of Src family kinases, via treatment with PP2, significantly reduced hASBT function, while the inhibition of PTPs by activated orthovanadate significantly induced function. Src family kinase inhibition by PP2 was associated with a concomitant decrease in maximum transport velocity (J(max)) correlated with a decrease in hASBT surface expression. Interestingly, PP2-mediated suppression of hASBT protein expression was rescued by the proteasome inhibitor MG132, suggesting that dephosphorylation impacts protein stability with the subsequent proteasome-dependent degradation of hASBT. Consequently, single-point mutations were introduced at five intracellular tyrosine residues: Y148F, Y216F, Y308F, Y311F, and Y337F. Although all mutants had significantly altered hASBT function without changes in total cellular expression, sequential tyrosine mutations at the five residues above rendered hASBT nonfunctional with diminished protein expression. Furthermore, orthovanadate-induced transport activity of single-point tyrosine mutants suggested a role for multiple tyrosine residues in the regulation of hASBT function and membrane expression. Overall, our data confirms that tyrosine phosphorylation mediated by Src family kinases (SFKs), in particular, regulates surface expression, function, and stability of hASBT.