Effects of O‐GlcNAcylation on insulin signaling on the level of insulin receptor substrate 1 (739.13)

Abstract

β‐O‐Linked N‐acetylglucosamine(O‐GlcNAc) modification (O‐GlcNAcylation) is a dynamic posttranslational modification on Ser/Thr residues and increased O‐GlcNAc is associated with insulin resistance. Insulin receptor substrate 1(IRS1) acts as an adaptor protein transferring signals from insulin to downstream PI3K and Akt. It is O‐GlcNAcylated and highly phosphorylated. Here we mainly focused on how O‐GlcNAcylation affects insulin signaling on IRS1 level. We found that insulin signaling is regulated through pharmacologically modifying the two key enzymes’ activity: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). In addition, we showed phosphorylation status of IRS1 changed accordingly by vertical isoelectrical focusing and two‐dimensional gel electrophoresis. We are performing Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC) on 3T3‐L1 to detect phosphorylation changes on IRS1 as well as other signaling proteins. In addition, We mapped two O‐GlcNAc sites by CID/ETD‐MS/MS, Ser635 and Ser1001. Ser635 is a main phosphorylation site associated with type II diabetes. We found Cycling rate of O‐GlcNAc on Ser635 is fast, phosphorylation and O‐GlcNAcylation compete at Ser635 in an insulin‐responsive manner. Ongoing studies are testing how O‐GlcNAc on Ser635 affects insulin signaling in 3T3‐L1 and whether O‐GlcNAc of Ser635 affects phosphorylation level of nearby sites. These data suggests O‐GlcNAcylation on IRS1 plays an important role in regulating insulin signaling.Grant Funding Source: NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949