128-OR: DOC2b Boosts Insulin Secretion via a Yes Kinase-Dependent Mechanism

Abstract

Exocytosis regulatory protein DOC2b is a crucial regulator of SNARE assembly in β-cells and enrichment of DOC2b enhances glucose-stimulated insulin secretion (GSIS). However, the underlying mechanism of how DOC2b boosts GSIS remains to be explored. We hypothesize that tyrosine-phosphorylation of DOC2b’s functionally indispensable C2AB domain is required for DOC2b’s beneficial function on GSIS. Biochemical analysis revealed that DOC2b is tyrosine-phosphorylated specifically by glucose within 1-2 min of stimulation in MIN6 β-cells and human islets. Pervanadate (pV) treatment, which inhibits tyrosine-specific phosphatases, validated the phosphorylation event to be tyrosine-specific. Further, the Src family kinases (SFKs) were implicated in this phosphorylation event, based upon the ability of SFK inhibitor SU-6656 to markedly attenuate the level of tyrosine-phosphorylated DOC2b under pV treated conditions. Phosphorylated/activated SFKY416 co-immunoprecipitated with DOC2b from pV treated β-cells; SU-6656 abrogated the interaction. YES kinase, an SFK, was also co-immunoprecipitated with DOC2b following glucose stimulation or pV treatment, suggesting YES kinase to be the specific SFK that phosphorylates DOC2b in β-cells. All-atom molecular dynamics simulations identified a specific tyrosine residue present in DOC2b’s C2B domain, Y301, as solvent-exposed and available for modification. Our results using phosphodeficient and phosphomimetic point mutants validated the involvement of the Y301 phosphorylation in for the boosting effect of DOC2b on GSIS. Taken together, these results resolve the first glucose-induced post-translational modification of DOC2b in β-cells, pinpointing the kinase and the site of action. These findings support a new model for DOC2b in GSIS: 1) glucose stimulation of the β-cell rapidly phosphorylates DOC2b on residue Y301 via YES kinase at the plasma membrane, yielding 2) an activated form of DOC2b that regulates insulin release. Disclosure D. Chatterjee bhowmick: None. A. Aslamy: None. S. Bhattacharya: None. E. Oh: None. M. Ahn: None. D. C. Thurmond: Consultant; Self; Immutics. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233 to D.C.T.); JDRF (3-PDF-2020-934-A-N to D.C.B.)