It is known that activation of Akt2 is required for insulin-stimulated GLUT4 translocation, and that Akt2 acts by regulating mobilization of GSV and fusion between GSV and the PM (Zaid et al., 2008
). It has been reported previously that Akt2 controls GLUT4 retention and trafficking by phosphorylating the RabGAP AS160. However, the molecular mechanism by which Akt2 regulates GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation, remains unclear. In the present study, we used a SILAC quantitative phosphoproteomic approach to identify CDP138, a previously unknown C2 domain-containing phosphoprotein, and confirmed that it is an Akt2 substrate. RNAi-based functional assays revealed that CDP138 is required for maximal
insulin-stimulated glucose transport and GLUT4 translocation to the PM, but not for GLUT4 movement to the periphery in adipocytes. We used both pH-sensitive IRAP-pHluorin and GLUT4-EGFP as molecular probes to demonstrate in live adipocytes that CDP138 is critical for optimal membrane fusion between GSV and the PM, but not for GSV trafficking to the TIRF zone. Collectively, these complementary functional analyses demonstrate that the novel phosphoprotein CDP138 is involved in regulating GLUT4 translocation, most likely at the GSV – PM fusion step. Thus, CDP138 represents a novel link between Akt2 activation and GLUT4 insertion into the PM. It is possible that Akt2 regulates the GLUT4 trafficking and membrane fusion steps in adipocytes through the RabGAP AS160 and CDP138, respectively ().
Our results are consistent with the hypothesis that CDP138 is a downstream target of Akt2 and is involved in the regulation of GLUT4 translocation. First, insulin stimulated phosphorylation of CDP138 in cultured cells, and the phosphorylation was partially blocked by the PI3K inhibitors. We also detected several phosphorylation sites in CDP138 by mass spectrometry, which suggests that insulin might induce CDP138 phosphorylation at different sites through both PI3K-dependent and -independent pathways. Second, we showed that constitutively active Akt2 induced CDP138 phosphorylation at a Ser197 residue within a consensus Akt substrate motif. Over-expression of a mutant CDP138 lacking the Ser197 phosphorylation site, but not Ser200, significantly inhibited insulin-stimulated myc-GLUT4-GFP translocation to the cell surface and GSV - PM fusion in adipocytes, suggesting that Ser197 phosphorylation is important to the glucose transporter system. Third, overexpressed human WT but not S197A or 5DA mutant CDP138 rescues the mouse CDP138 siRNA-induced inhibitory effect on the membrane fusion in 3T3-L1 adipocytes. Fourth, our results showed that CDP138 co-localizes with phospho-Akt in insulin-stimulated cells. We also observed that CDP138 interacts with Akt2 upstream kinase PDK1 in a proteomics study and this was confirmed in a co-immunoprecipitation study (Data not shown). These observations suggest the interesting possibility that the CDP138-PDK1 interaction might bring CDP138 and phospho-Akt2 in close proximity at the PM. This might be mediated through PI3K-derived PI(3,4,5)P3, which interacts with the PH domains of both PDK1 and Akt2 in insulin-stimulated cells. If this occurs, CDP138 would become accessible to phosphorylation by active Akt2. Furthermore, RNAi-induced gene specific knockdown of CDP138 did not affect insulin-stimulated Akt phosphorylation but significantly inhibited GLUT4 translocation induced by constitutively active Akt2, suggesting this novel phosphoprotein functions downstream of the Akt2 pathway.
To understand the molecular mechanism by which CDP138 regulates GLUT4 translocation, we also analyzed the biochemical and functional interactions of the C2 domain-containing protein. Deletion of the C2 domain from CDP138 significantly inhibited insulin-stimulated GLUT4 translocation, suggesting the C2 domain is crucial for this process. The C2 domain of CDP138 is similar to those of known membrane fusion proteins such as synaptotagmin. Biophysical analyses revealed that the purified C2 domain of CDP138 is able to bind Ca2+ and liposomes with a lipid composition that mimics the cytoplasmic face of plasma membranes. It is interesting to note that the C2 domain contains two Ca2+-binding sites of differing affinity, presumably one each in loop 1 and 3. The mutant C2 domain lacking five aspartate residues in loop1 and 3 regions is unable to bind Ca2+ or membrane lipids, suggesting that interaction of the C2 domain with lipids is Ca2+-dependent. It is possible that Ca2+-binding to the C2 domain results in exposure of nonpolar residues that mediate membrane binding. Alternatively, Ca2+ ions may serve as ionic bridges between acidic residues of the protein and negatively charged membranes. Interestingly, in the studies using GLUT4 vesicles and density gradient fractionation of membrane compartments, we also observed that CDP138 associated with GLUT4 vesicles and the lower density PM-containing fractions within 10 min of treatment of adipocytes with insulin. Surprisingly, CDP138 dissociated from the vesicles and redistributed in the PM-containing fractions in adipocytes after 30 min. These dynamic interactions further support the notion that CDP138 may be involved in the regulation of GLUT4 vesicle fusion with the PM either directly or indirectly. Since CDP138 is a highly regulated phosphoprotein, it is unique among proteins known to be involved in membrane fusion processes. Further studies are needed to understand the molecular basis by which CDP138 regulates membrane fusion.