Role of guanine nucleotide exchange factors for Rab10 in insulin-regulated GLUT4 trafficking

Abstract

The insulin regulation of glucose uptake via translocation of the glucose transporter, GLUT4, is essential for maintenance of whole-body glucose homeostasis. GLUT4 traffic is a strikingly sophisticated biochemical process. The transporter must navigate its way throughout diverse endomembranous systems and multiple nodes along this path are exquisitely regulated by the concerted actions of Rab GTPases and their effectors. Understanding precisely how insulin orchestrates regulation of Rab activity appropriately at the various transport steps in the GLUT4 itinerary, however, has been a longstanding puzzle and the identities of key regulatory players have remained elusive. In pursuit of novel regulatory proteins functioning in GLUT4 traffic, I examined datasets from global quantitative mass spectrometric analyses of insulin-regulated protein phosphorylation in 3T3-L1 adipocytes, L6 skeletal muscle myotubes and murine liver tissue. This search uncovered DENND4A and DENND4C, two closely related members of the novel differentially expressed in normal versus neoplastic (DENN) domain-containing protein family of Rab guanine nucleotide exchange factors (GEFs), as highly insulin-sensitive phosphoproteins. Both DENND4A and DENND4C target Rab10, a Rab thought to regulate a single distal step in GLUT4 traffic. Using affinity pull-down and protein overlay methods, I demonstrated that DENND4A is an insulin-regulated 14-3-3 binding protein downstream of mTORC1 kinase. Point mutation of 35 serine/threonine phosphorylation sites in the DENND4A C-terminus eliminated its 14-3-3 binding capacity and enhanced the GEF activity of DENND4A towards Rab10 in vivo, as assessed by Rab effector pull-down. Live-cell microscopic analysis revealed that DENND4A inhibits insulin-stimulated GLUT4 translocation when overexpressed in adipocytes, whereas the phospho-dead '35P' mutant, in which 14-3-3 binding is lost, has no effect. DENND4C, previously implicated as a positive regulator of GLUT4 exocytosis, was found to localise to a distinct subpopulation of GLUT4 storage vesicles (GSVs) immunoisolated from 3T3-L1 adipocytes that exclude the well-characterised Rab10 GTPase activating protein (GAP), AS160. My findings indicate that there is at least one additional insulin-regulated step than previously thought in GLUT4 traffic involving DENND4A phosphorylation. I propose a model where multiple Rab10 GEFs have opposing actions in GLUT4 traffic and the simultaneous inhibition of DENND4A and AS160 by insulin-mediated phosphorylation confers net GLUT4 transfer to the adipocyte plasma membrane. Collectively, my work illuminates the potential of DENND4 proteins as novel therapeutic targets for insulin resistance and Type II diabetes mellitus.