The human apical bile acid transporter (hASBT) in the distal ileum reabsorbs bile acids and is responsible for transporting approximately 95 % of bile acids back to liver for recycling in the enterohepatic pathway. ASBT activity has been linked to multiple disease states, including Crohn’s disease, hypercholesterolemia, cholestasis, and type 2 diabetes. ASBT activity is regulated at the post-translational level by glycosylation, ubiquitination, and S-acylation, which control its translocation to the cell surface and protein stability. Although biochemical studies have suggested that phosphorylation of serine, threonine, and tyrosine may play a role in the regulation of ASBT function, no study has determined where phosphorylation of ASBT occurs and how its phosphorylation level correlates with its activity. In this study, we developed a workflow using parallel reaction monitoring (PRM) targeted mass spectrometry to determine and quantify the stoichiometry of ASBT phosphorylation in the presence of various kinase inhibitors and activators. Our findings provide the first evidence of ASBT phosphorylation at multiple sites (Thr330, Ser334, and Ser 335), with Ser 335 being the most abundant phosphosite.Futhermore, we demonstrate that the phosphorylation level of Serine 335 coincides with the bile acid uptake activity of ASBT. Finally, we discovered that PKCalpha, but not other PKC isoforms, regulates ASBT phosphorylation at Ser 335. Taken together, our findings establish the molecular basis of phosphorylation-mediated regulation of ASBT which may include novel therapeutic targets for managing ASBT-linked disease.

The membrane fractions for quantification of phosphorylation of ASBT by targeted PRM mass spectrometry were prepared as follows. The supernatant containing membrane proteins was precipitated using the Methanol/Chloroform method. Proteins were then solubilized with 8M urea in 50 mM ammonium bicarbonate buffer, reduced with 5mM TCEP at 56oC for 30 minutes, and alkylated with 14 mM IAA for 30 minutes in the dark. Urea was diluted to 1M final concentration before adding trypsin at 1:100 (enzyme: substrate ratio by weight) and digestion was carried out overnight at 37o C in a shaker. Digestion was stopped by the addition of 0.5% TFA. The peptide mixture was diluted two times and quantified using the Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific). A background matrix was generated by pooling aliquots of all individual samples from each batch and spiked with SIL peptides to estimate the amount of phosphopeptide and nonphosphopeptide from each batch. 150 microgram peptides from each sample were spiked with an equal amount of SIL nonphosphopeptide or SIL Ser 335 phosphopeptide. After cleaning with a C18 tip, the fractions spiked with nonphosphopeptide SIL were directly used for mass spectrometry analysis, whereas the fractions spiked with SIL Ser335 phosphopeptide were further enriched with the High-Select Fe-NTA Phosphopeptide enrichment kit. The SIL standards for nonphosphopeptide and corresponding pSer 335 phosphopeptides were at high purity (AQUA basic >95% purity). In contrast, the heavy-labeled phosphopeptide standards of pSer 334 and pThr 330 were crude as-synthesized (PEPotec grade 3)(All from Thermo Scientific). Because we predict from preliminary data that Ser 335 is the main phosphorylation residue, the other two SIL peptides were used as crude phosphopeptide for the purpose of determining the coelution time to identify their endogenous phosphoisoforms only. The SIL Ser 335 phosphopeptide was spiked at 2.1 to 12 fmol per 1microgram total peptide whereas the SIL nonphosphopeptide was spike at 300 fmol per 1 microgram of total peptides.

Human ASBT overexpressed in HEK293T.