Cells were centrifuged at 1000 rpm for 5 min, and excess media were aspirated. TUGUL modifies the kinesin motor protein, KIF5B, and that TUG proteolysis is required to load GLUT4 onto these motors. Insulin stimulates TUG proteolytic processing independently PMPA of phosphatidylinositol 3-kinase. In nonadipocytes, TUG cleavage can be reconstituted by transfection of Usp25m, but not the related Usp25a isoform, together with other proteins present on GLUT4 vesicles. In rodents with diet-induced insulin resistance, TUG proteolysis and Usp25m protein abundance are reduced in adipose tissue. These effects occur soon after dietary manipulation, prior to the attenuation of insulin signaling to Akt. Together with PMPA previous data, these results support a model whereby insulin acts through Usp25m to mediate TUG cleavage, which liberates GLUT4 storage vesicles from the Golgi matrix and activates their microtubule-based movement to the plasma membrane. This TUG proteolytic pathway for insulin action is independent of Akt and is impaired by nutritional excess. in skeletal muscle, similar increases RAF1 in glucose uptake are observed after TUG disruption and after maximal insulin stimulation; there is little or no further effect of insulin in cells with disrupted TUG action (7, 10). In muscle, the mobilization of TUG-bound vesicles results in IRAP translocation, so that glucose uptake is coordinated with inactivation of vasopressin, an IRAP substrate (11). TUG itself is a target of SIRT2-mediated deacetylation, which controls the size of the GSV pool and, consequently, insulin sensitivity (12). Thus, the TUG protein is a PMPA critical regulator of GSV accumulation and release and is a major site of insulin action. To mobilize GSVs, insulin stimulates TUG cleavage. Intact TUG links GSVs to the Golgi matrix by binding GLUT4 and IRAP through its N terminus and Golgin-160 and other matrix proteins through its C terminus (7, 11,C13). Insulin-triggered TUG cleavage separates these N- and C-terminal regions and is required for highly insulin-responsive GLUT4 translocation and glucose uptake (12, 13). Like the formation of an insulin-responsive pool of GSVs, TUG cleavage occurs in fat and muscle cells but is not observed in other cell types. Insulin-stimulated proteolytic processing of intact TUG produces a novel ubiquitin-like protein modifier, TUGUL (for TUG Ubiquitin-Like), but the major target of TUGUL modification (tugulation) has not been identified (13). The TUG proteolytic pathway is thought to act in parallel to insulin signals transduced through phosphatidylinositol 3-kinase (PI3K), Akt, AS160/Tbc1D4, and target Rab proteins, which coordinate overall GLUT4 trafficking (9, 14, 15). It is not known whether attenuated TUG signaling may contribute to insulin resistance, independently of Akt (16). More broadly, how these insulin signaling and vesicle trafficking processes intersect remains to be fully elucidated. Here, we present data to support a model in which the TUG protease is Usp25m, and TUGUL modifies KIF5B (KIF5B, kinesin family member 5B) to load GSVs onto these kinesin motors. Results Previous results support the idea that intact TUG undergoes proteolytic processing, as diagrammed in Fig. 1diagram of TUG processing is shown, based on previous data. Insulin stimulates cleavage of the 60-kDa intact protein to generate 18-kDa N-terminal and 42-kDa C-terminal products. The N-terminal product, TUGUL, is covalently PMPA attached to a substrate protein to make a 130-kDa conjugate. The C-terminal product is modified to a variable extent to make an 54-kDa form. 3T3-L1 cells were lysed at the indicated days after induction of adipocyte differentiation. Lysates were immunoblotted to detect the indicated proteins, including Usp25, intact TUG (60 kDa), TUG C-terminal products (54 and 42 kDa), and TUG N-terminal products (130 kDa). lysates prepared from the indicated mouse tissues were analyzed by SDS-PAGE and immunoblotting using a Usp25 antibody, which distinguished the Usp25a and Usp25m splice forms. Myc-tagged Usp25a and Usp25m proteins expressed together with TUG in transfected 293 cells. Immunoprecipitations (TUG containing a C-terminal biotin tag was stably expressed in 3T3-L1 adipocytes. Vesicles were then purified from homogenates of basal and insulin-stimulated cells using immobilized neutravidin (pulldown). As a negative control, biotin-saturated neutravidin was used. Eluted.