Determination of\nSite-Specific Phosphorylation Occupancy\nUsing Targeted Mass Spectrometry Reveals the Regulation of Human Apical\nBile Acid Transporter, ASBT

The human apical bile acid transporter (hASBT, SLC10A2)\nreabsorbs\nbile acids in the distal ileum, facilitating their recycling to the\nliver and resecretion. Its activity has been implicated in various\ndisease states, including Crohn’s disease, hypercholesterolemia,\ncholestasis, and type-2 diabetes. Post-translational modifications\nsuch as <i>N</i>-glycosylation, ubiquitination, and <i>S</i>-acylation regulate ASBT function by controlling its translocation\nand stability. However, the precise role of phosphorylation and its\nrelationship with activity remains unknown. Here, we employed parallel\nreaction monitoring targeted mass spectrometry to investigate ASBT\nphosphorylation in the presence of various kinase inhibitors and activators.\nOur study ascertains phosphorylation at multiple sites (Thr330, Ser334,\nand Ser335), with Ser335 being the predominant phosphosite. We further\ndemonstrate the critical involvement of PKC in regulating ASBT activity\nby phosphorylation at Ser335. Importantly, we establish a proportional\nrelationship between the phosphorylation level of Ser335 and ASBT\nbile acid uptake activity. Collectively, our findings shed light on\nthe molecular mechanisms underlying phosphorylation-mediated regulation\nof ASBT.