DOC2b Protects Functional Beta‐cell Mass via A Novel Tyrosine Phosphorylation Dependent Mechanism

Abstract

Type 2 diabetes (T2D) has reached the stage of a global pandemic. Approximately 10% of the total US population (~30 million people) are impacted by T2D and 86 million more are in the stage of prediabetes. Accumulating evidence suggests that intrinsic beta‐cell stresses, secretory dysfunction, and inflammation are the early players during the progression of the T2D and leads to the loss of functional beta‐cell mass. Hence, reduction of cell stress and enhancement of beta‐cell secretory function hold promise towards delayed disease progression and prevention. Our lab has revealed the crucial role of the SNARE‐regulator protein DOC2b (double C2 domain‐containing protein) on stimulating the glucose‐stimulated insulin secretion (GSIS) and protecting the beta‐cell from the diabetogenic stresses. The objective of this study is to develop DOC2b as a therapeutic target for the treatment of T2D by elucidating the mechanism by which DOC2b preserves functional beta‐cell mass. We hypothesize that tyrosine phosphorylation of DOC2b’s functionally indispensable C2AB domain is required for DOC2b’s beneficial function during the pathological conditions related to T2D.To test this hypothesis we have used human and rodent clonal beta‐cell lines and primary human islets procured from non‐diabetic and T2D donors. Our results showed a marked loss of DOC2b mRNA and protein (~50% reduction) in the T2D human islets and a resurrected first phase of GSIS in otherwise dysfunctional T2D islets following DOC2b overexpression. Multiple studies confirmed elevated expressions of the CXCL9 and 10 (chemokine receptor 3 ligands) in diabetics. Our qPCR and biochemical analysis revealed significant attenuation of cytokine‐induced elevation of CXCL9 and 10 gene expressions following DOC2b overexpression in the clonal and primary beta‐cells. These results again signified the beneficial effect of DOC2b enrichment in the beta‐cell. In islet beta‐cells, glucose‐stimulated tyrosine phosphorylation events regulate SNARE complex assembly and GSIS. Using biochemical approaches, we determined that DOC2b is phosphorylated following glucose stimulation and pervanadate (a tyrosine phosphatase inhibitor) treatment in the clonal beta‐cells. In silico evaluation from the crystal structure of the C2B domain of DOC2b protein revealed three putative tyrosine phosphorylation sites: Y301 (within the loop region), and Y305, Y309 (within the ordered domain) and our results using point mutants of each putative site (Y to F) indicated the requirement for all three phosphorylation sites for the boosting effect of DOC2b on GSIS in the beta‐cells. We further identified that the mutations in at least Y301 and Y305 residues significantly blunted DOC2b’s ability to protect beta‐cells from cytokine‐induced beta‐cell stress and apoptosis. These results unveil a novel mechanism by which DOC2b enrichment boosts GSIS and protects beta‐cells against diabetogenic stimuli, the knowledge of which would be crucial for pursuing DOC2b as a novel drug target to prevent and/treat diabetes.Support or Funding Information5R01DK112917‐02