C2 Domain-Containing Phosphoprotein CDP138 Regulates GLUT4 Insertion into the Plasma Membrane

Abstract

Summary The protein kinase Bβ (Akt2) pathway is known to mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138-kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca2+ and lipid membranes. CDP138 mutants lacking the Ca2+-binding sites in the C2 domain or Akt2 phosphorylation site Ser197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle - PM fusion.

Insulin elicits a ROS-activated and an IP3-dependent Ca2+ release; both impinge on GLUT4 translocation

“…Indeed, there are reports that GLUT4 exocytosis requires intracellular Ca 2+ in adipocytes, allowing activation of Akt (Whitehead et al, 2001) and CDP138, a Ca 2+ -dependent protein downstream of Akt (Xie et al, 2011). In skeletal muscle, however, direct evidence showing intracellular Ca 2+ -dependent GLUT4 translocation was missing, although results have suggested that it occurs (Lanner et al, 2006;Wijesekara et al, 2006), and localized, sub-membrane changes in Ca 2+ levels have been reported in response to the hormone (Bruton et al, 1999).…”

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice

“…Recently, it has been reported in live adipocytes that the 138-kDa C2 domain-containing phosphoprotein (CDP138) is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4-containing vesicles with the plasma membrane (63). The purified C2 domain of CDP138 binds Ca 2ϩ and lipid membranes (63).…”

“…Recently, it has been reported in live adipocytes that the 138-kDa C2 domain-containing phosphoprotein (CDP138) is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4-containing vesicles with the plasma membrane (63). The purified C2 domain of CDP138 binds Ca 2ϩ and lipid membranes (63). In addition, studies in vitro have shown that the cytoplasmic protein Doc2b, which possesses two homologous C2 domains, promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium-and membrane-bending-dependent manner (65).…”

Tollip, an Intracellular Trafficking Protein, Is a Novel Modulator of the Transforming Growth Factor-β Signaling Pathway

“…Subcellular Fractionation with Continuous Iodixanol Gradients-Subcellular fractionation was performed using a method as previously reported (24,25). In short, the cells were washed with cold PBS and homogenized in buffer (20 mM Hepes, pH 7.4, 1 mM EDTA, 250 mM sucrose with protease and phosphatase inhibitors) 20 times with a 22-gauge needle and 5 times with a 26 gauge needle.…”

“…According to previous reports (24,25), the lower density fractions mainly contains plasma membrane, whereas the middle density fractions contain large organelles including Golgi apparatus and the higher density fractions mainly consist of small organelles including endosomal structures. When caT␤RI was expressed alone, it was mainly distributed in the fractions containing large subcellular organelles (Fig.…”