Identifying a New Pathway to Regulate Autophagy in HK2 Cells

Diabetic nephropathy (DN) is one of the major diabetic complications, the leading cause of the end stage renal disease (ESRD). Recently autophagy, which is also implicated in diabetes and insulin resistance, was shown to regulate DN. Recently, we demonstrated that Fyn functions as a muscle mass regulator by suppressing macroautophagy through a STAT3-mediated inhibition of the Vps34/Beclin1/ATG14 complex 1 but it is still not known whether Fyn regulates autophagy in kidney and function as a pathogenesis of DN. To investigate this, siRNA mediating knockdown of Fyn in HK2 cells as an in vitro cell model of renal proximal tubular epithelial cells was performed and demonstrating not only decreased p62 expression but also increased LC3-1/2 ratio implicating increased autophagy. To further investigating autophagic flow we utilized ammonium chloride and leupeptin to inhibit the autophagic flow and consistently found that expression of LC3-2 was increased. Phospho-proteomic analysis were performed in vivo and revealed that Fyn phosphorylates Transglutaminase 2 (Tgm2), a known autophagic inhibitor through Beclin1, on Y369 and Y617. To demonstrate that Tgm2 is a direct Fyn substrate, we incubated human purified GST-Tgm2 protein with purified Fyn-CA. In the presence of ATP, Fyn was observed to robustly phosphorylate the Tgm2. Interestingly, Fyn-dependent phosphorylation of Tgm2 was abolished when Y369 and Y617 in Tgm2 was mutated (Tgm2-Y369/617F), suggesting that these sites were specific and unique for Fyn kinase. SiRNA mediating knockdown of Tgm2 in HK2 cells revealed decreased p62 expression and increased LC3-1/2 ratio while overexpressing Tgm2-WT decreased autophagic flow demonstrating Tgm2 could regulate autophagy in the renal tubulus. Taken together, these data suggest that Fyn phorphorylates Tgm2 on Y369 and Y617 and regulates autophagy in the renal tubulus implicating a pathogenesis of DN.