The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx

Abstract

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxylcytoplasmic domain (residues 464 -509) of the insulinsensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-␤ (T␤RII) receptors (1, 2). The two-hybrid interaction between C-GLUT4 and CDaxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu 489 -Leu 490 ) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with antiDaxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.The cytoplasmic domain of membrane proteins plays important roles in their transport, signal transduction, organization of protein scaffolds, and regulation of their turnover. Trafficking of GLUT4 in adipose and skeletal muscle cells is regulated by insulin and muscle contraction and is critical for the control of glucose levels in blood. Upon increase in insulin levels and muscle contraction the GLUT4 retained in intracellular stores is translocated to the plasma membrane, where it facilitates glucose transport (3). Trafficking of GLUT4 is mediated by motifs localized to the amino and carboxyl-cytoplasmic domains of the protein, though their characterization and the identification of the factors involved in their reading is incomplete.SUMO (also called sentrin, PIC1, and GMP1), a 101-amino acid ubiquitin-like modifier protein that is highly conserved from yeast to human, appears to control protein turnover and compartmentalization (4). Three members of the SUMO family have been described in vertebrates. They show major structural differences in the sequences of their N-extensions, which are absent in ubiquitin. It has been shown recently that Ubc9, the only E2-type SUMO1-conjugating enzyme described in vertebrates, interacts with the carboxyl-cytoplasmic domain of GLUT4 as part of a mechanism that slows its...

The SUMO Conjugating Enzyme Ubc9 is a Regulator of GLUT4 Turnover and Targeting to the Insulin-Responsive Storage Compartment in 3T3-L1 Adipocytes

“…Since wild-type and the catalytically inactive mutant Ubc9 showed indistinguishable effects on GLUT4, Ubc9 would regulate GLUT4 targeting and turnover by a mechanism other than SUMOylation. The observations also contradict the idea that Ubc9 exerts the effects by SUMOylation of GLUT4 itself (19,31). Since knockdown of Ubc9 produced effects opposite to those of its overexpression, Ubc9 may regulate GLUT4 targeting through interaction with a putative target molecule(s).…”

“…Thus, although we do not preclude the possibility that Ubc9 may regulate GLUT4 sorting through the interaction with the transporter, the physiological significance of this interaction has yet to be investigated. On the other hand, Lalioti et al (31) reported the dileucine (L 489 L 490 ) motif-dependent interaction of GLUT4 with Daxx, a Fas binding protein, which also interacts with Ubc9. Since the GLUT4 interacting region and the Ubc9 interacting region of Daxx considerably overlap (amino acid residues 661-740 and 625-740, respectively), it is an intriguing possibility that Ubc9 may regulate GLUT4 trafficking by modifying the interaction of Daxx with GLUT4.…”

SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5

“…SUMOylation may thus be a widespread mechanism of channel regulation. The recent description of SUMOylation of the glucose transporters GLUT1 and GLUT4 (30,40), the two-pore potassium leak channel K2P1 (16), and the G protein-coupled receptor mGluR8 (31) further supports the view that SUMOylation is an important regulatory mechanism for integral membrane proteins. In this report we have demonstrated that disruption of Kv1.5 SUMO modification has a significant effect on the biophysical properties of the channel.…”

“…The effects of SUMO modification in contexts such as transcriptional regulation are likely due to the promotion of protein-protein interactions (27,45,46). Notably, a critical surface in SUMO essential for transcriptional inhibition (28) is the site of interaction for I/L/V rich SUMO-interacting motifs present in multiple proteins (29,47), including some associated with membrane proteins (40). Because Kv1.5 functions as part of a multiprotein/lipid complex comprising proteins with scaffolding, cytoskeletal, and enzymatic activities, SUMO modification of Kv1.5 may function by modulating these interactions.…”

Genes and Proteins for Solute Transport and Sensing

“…On the other hand, rafts seem to be involved in subsequent internalization of GLUT4 (Ros-Baro et al, 2001). Interestingly, GLUT4 interacts physically with ubiquitin-related proteins involved in degradation and stabilization (Lalioti et al, 2002). Finally, transporter activity can be regulated directly by physical factors as e.g.…”