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

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.