Determination of Site-Specific Phosphorylation Occupancy Using Targeted Mass Spectrometry Reveals the Regulation of Human Apical Bile Acid Transporter, ASBT

Abstract

The human apical bile acid transporter (hASBT, SLC10A2) reabsorbs bile acids in the distal ileum, facilitating their recycling to the liver and resecretion. Its activity has been implicated in various disease states, including Crohn's disease, hypercholesterolemia, cholestasis, and type-2 diabetes. Post-translational modifications such as N-glycosylation, ubiquitination, and S-acylation regulate ASBT function by controlling its translocation and stability. However, the precise role of phosphorylation and its relationship with activity remains unknown. Here, we employed parallel reaction monitoring targeted mass spectrometry to investigate ASBT phosphorylation in the presence of various kinase inhibitors and activators. Our study ascertains phosphorylation at multiple sites (Thr330, Ser334, and Ser335), with Ser335 being the predominant phosphosite. We further demonstrate the critical involvement of PKC in regulating ASBT activity by phosphorylation at Ser335. Importantly, we establish a proportional relationship between the phosphorylation level of Ser335 and ASBT bile acid uptake activity. Collectively, our findings shed light on the molecular mechanisms underlying phosphorylation-mediated regulation of ASBT.

Figure 1

Diagnostic spectra identifying phosphorylation of ASBT at Ser335, Thr330, and Ser334. Phosphopeptides were enriched with an Fe-NTA spin column before being analyzed with nanoLC-MS/MS using DDA and PRM. The b- and y-ion series confirming phosphorylation of ASBT are shown for Ser335 (A), Thr330 (B), and Ser334 (C).

Figure 2

Targeted high-resolution MS/MS combined with spiked-in SIL phosphopeptides identifies ASBT is phosphorylated at three sites Ser335, Thr330, and Ser334 with Ser335 being the most abundant. Phosphopeptides were enriched with an Fe-NTA spin column before being analyzed with nanoLC-MS/MS using PRM. Endogenous phosphopeptides (upper panel) were coeluted with their corresponding spiked-in stable isotope-labeled phosphoisoforms (lower panel), respectively, for Ser 335 (A,B), Thr330 (C,D), and Ser 334 (E,F). The y- and b-ion series, shown at the top of each image, further confirm the phosphorylation of ASBT at these three residues.

Figure 3

Quantification of phosphorylation occupancy shows kinase inhibitors inhibit ASBT phosphorylation and its TCA uptake activity. Amount of Ser 335 phosphopeptide and its nonphosphopeptide counterpart were quantified as fmol/μg total peptide based on the known amount of spiked-in stable isotope labeling peptides. Amount of phosphorylated ASBT at Ser335 (A), and its corresponding nonphosphorylated peptide (B), and Ser335 phosphorylation occupancy (C), and TCA uptake assay of ASBT after 6 h of treatment with various kinase inhibitors compared to the vehicle control DMSO. Uptake of TCA was measured in a 12 min assay using 10 μM cold nonradiolabeled TCA spiked with 1 μCi/mL [³H]-TCA. Uptake assay data are presented as percentage of control. Values are mean ± SD with n = 3 for A,B,C, n = 12 for D. Significance levels are indicated as follows ****p-value <0.0001, ***p-value <0.0003, **p-value <0.006 (Dunnett’s post hoc test), indicating statistical significance compared to the control.

Figure 4

PKC activation or specific isoform overexpression increases ASBT phosphorylation and its TCA uptake activity. PKC activation by PMA (A–D). Amount of Ser335 phosphopeptide (A) and its nonphosphopeptide counterpart (B) were quantified as fmol/μg total peptide based on the known amount of spiked-in stable isotope-labeled peptides; phosphorylation occupancy of Ser335 (C); and TCA uptake assay of ASBT (D) upon treatment with different dose of PMA for 15 min. PKC activation by PMA ± PKC inhibitor BIM1 (E,F). Amount of Ser335 phosphopeptide (E) and TCA uptake assay of ASBT (F) in the presence of 0.1 μM PMA with or without 5 μM BIM1. Overexpression of PKC isoforms (G–I). Amount of phosphorylated Ser335 (G) and its corresponding nonphosphorylated peptide (H), and Ser335 phosphorylation occupancy (I) for empty vector (EV), human PRKCε (hPKCE), rat PRKCζ (rPKCZ), and bovine PRKCα (bPKCA). Uptake of TCA was measured in a 12 min assay using 10 μM cold nonradiolabeled TCA spiked with 1 μCi/mL [³H]-TCA. Uptake assay data are presented as percentage of control. Values are mean ± SD with n = 3 for A,B,C,E, n = 8 for D, n = 12 for F, and n = 2–3 for G,H,I. Significance levels are indicated as follows ****p-value <0.0001, **value <0.007, *p-value <0.03 (Dunnett’s post hoc test), indicating statistical significance compared to the control.

Figure 5

Model for regulation of ASBT activity by phosphorylation at the C-terminal domain. Under basal conditions, ASBT is phosphorylated at Ser 335 by PKC, specifically, the isoform PKCα. Stimulation of PKC activity increases the phosphorylation of Ser 335 and the total activity of ASBT. This is a reversible process because kinase inhibitors reduce the level of phosphorylation at Ser 335 and inhibit its activity.