Synip phosphorylation by Akt2. (A) Candidate Akt phosphorylation consensus motif in Synip is presented. (B) Either FLAG-WT-Synip or FLAG-S99F-Synip was expressed in CHOIR cells by electroporation. After 15 min of insulin stimulation, Synip was immunoprecipitated by FLAG antibody and samples were separated on SDS-PAGE. Phosphorylated Synip signal was analyzed by Akt phosphospecific substrate antibody immunoblotting. These are representative experiments independently performed three times. (C) Synthesized oligo peptides were phosphorylated as described in Materials and methods. These were repeated four to five times. Recombinant Akt2 was capable of phosphorylating the Synip peptide compared with Akt1 (*, P < 0.01). A scramble peptide with the same composition as well as mutation of the equivalent position of serine 99 to phenylalanine markedly reduced Akt2-dependent phosphorylation (*, P < 0.01). (D) GST-Synip fusion proteins were phosphorylated as described in Materials and methods. These were repeated five times. Recombinant Akt2 was capable of phosphorylating GST-WT-Synip (*, P < 0.05). However, Akt2 did not phosphorylate GST-S99F-Synip. GST alone showed negligible count. (E) Akt and Synip interaction was estimated by the design of coimmunoprecipitation experiments. FLAG-Synip was expressed in CHO/IR cells by electroporation and either Akt1 or Akt2 was immunoprecipitated by specific antibody. Synip amount associated with Akt was estimated by FLAG immunoblot. These were repeated five times. Synip significantly associates with not Akt1 but Akt2 after insulin stimulation (*, P < 0.01).

Synip phosphorylation is required for glucose uptake and GLUT4 translocation. (A) In 3T3L1 adipocytes, pcDNA3, FLAG-WT-Synip, FLAG-S99F-Synip, or FLAG-S97F-Synip was introduced by electroporation (Min et al., 1999). After 48 h of recovery, serum was removed for 6 h. Cells were stimulated by insulin for 30 min. ³H-labeled 2-deoxyglucose uptake was measured as described in Materials and methods. FLAG-S99F-Synip significantly inhibited ³H-labeled 2-deoxyglucose uptake (*, P < 0.05). (B) In 3T3L1 adipocytes, eGFP-GLUT4 and either pcDNA3, FLAG-WT-Synip, FLAG-S99F-Synip, or FLAG-S97F-Synip were introduced by electroporation (Min et al., 1999). After 48 h of recovery, serum was removed for 6 h. Then, cells were stimulated by insulin for 30 min. EGFP signal was detected by confocal microscopy, and rim formation was considered as completely translocated eGFP-GLUT4 in this assay. FLAG-S99F-Synip significantly inhibited GLUT4 translocation (*, P < 0.05). Experiments were repeated four times. (C) In 3T3L1 adipocytes, eGFP-myc-GLUT4 and either pcDNA3, FLAG-WT-Synip, FLAG-S99F-Synip, or FLAG-S97F-Synip were introduced by electroporation (Min et al., 1999). Myc was inserted in the second loop of GLUT4 to expose to the outside of the plasma membrane after insulin stimulation. When myc signal was detected, docking/fusion step was completed and GLUT4 translocation was accomplished. Myc signal was estimated as described in Materials and methods. FLAG-S99F-Synip significantly inhibited GLUT4 translocation (*, P < 0.05). Experiments were repeated eight times.

Analysis of insulin signal-regulating Synip–Syntaxin4 interaction. FLAG-WT-Synip is expressed in CHOIR (CHO cell with overexpressed human insulin receptor) by electroporation. After 48 h of recovery, 6 h of serum starvation followed. 100 nM insulin was added for 15 min to see Synip–Syntaxin4 interaction. After 4 mg of whole cell lysates were made with NP-40 lysis buffer, 2 μg of Syntaxin4 antibody was added to precipitate Syntaxin4. Coimmunoprecipitated FLAG-WT-Synip was detected by FLAG immunoblotting. In each experiment it was confirmed that same amount of Syntaxin4 was immunoprecipitated by Syntaxin4 immunoblotting, and FLAG-WT-Synip expression was identical by FLAG immunoblotting in each sample (not depicted). Data show representative experiments independently performed and each experiment was repeated three to four times. (A) A typical result of Synip–Syntaxin4 interaction before and after insulin stimulation. Insulin causes Synip dissociation from Syntaxin4 after insulin stimulation, which is recognized as weaker band intensity compared with basal condition. It was statistically significant (*, P < 0.01). (B) The effect of the disruption of CAP/TC10 signal (PI3 kinase–independent pathway) by the design of sorbin overexpression on Synip–Syntaxin4 interaction. After insulin stimulation, Synip dissociation was still observed. The difference in band intensity was statistically significant (*, P < 0.01). (C) The effect of a pharmacological PKC inhibitor such as GF 109203X. Synip dissociation could be still observed. The difference in band intensity was statistically significant (*, P < 0.01). (D) The effect of pharmacological PI3 kinase inhibitor, such as Wortmannin, pretreatment on Synip–Syntaxin4 interaction. Synip dissociation disappeared. (E) The effect of PI3 kinase negative-dominant overexpression. Synip dissociation also disappeared. (F) The effect of siRNA of Akt1. Synip dissociation could be observed. The difference in band intensity was statistically significant (*, P < 0.01). (G) The effect of siRNA of Akt2. Synip dissociation could not be observed.

Synip phosphorylation is required for Synip dissociation from Syntaxin4. (A) In this experiment whole cell lysates were pretreated with alkaline phosphatase first as described previously (Zhao et al., 1998) to dephosphorylate all the phosphorylated molecules. Thereafter, an ordinal coimmunoprecipitation experiment was conducted to see Synip–Syntaxin4 interaction. These are representative experiments independently performed two times. (B) Either GST-WT-Synip or GST-S99F-Synip was phosphorylated by Akt2 first and then incubated with MBP-Syntaxin4. Samples were separated on SDS-PAGE and GST-Synip associated with MBP-Syntaxin4 was estimated by GST immunoblot (*, P < 0.01). These were repeated five times. In addition, we stripped the GST antibody from filters and reprobed them with phosphospecific Akt substrate antibody. We confirmed that there was no signal (not depicted) and that nonphosphorylated Synip binds to Syntaxin4. (C–E) FLAG-WT-Synip or FLAG-S99F-Synip or FLAG-S97F-Synip was electroporated to CHOIR cells and 3T3L1 adipocytes and the cells stimulated with or without insulin for 15 min. In these experiments, the amount of expressed Synip protein was adjusted to match the expression levels in 3T3L1 adipocytes that have a lower level of transfection efficiency and protein expression than CHOIR cells (Min et al., 1999). In comparison to Fig. 1, 8 mg of detergent whole cell lysates were immunoprecipitated with 4 μg of Syntaxin4 antibody. Coimmunoprecipitated Synip amount was estimated by FLAG immunoblotting. FLAG-WT-Synip and FLAG-S97F-Synip dissociation could be observed in CHOIR cells and 3T3L1 adipocytes. The difference in band intensity was statistically significant (*, P < 0.05). These are representative experiments independently performed four times. (F and G) FLAG-WT-Synip or FLAG-S99F-Synip was electroporated to CHOIR. After Syntaxin4 was immunoprecipitated, coimmunoprecipitated Synip amount was estimated by FLAG immunoblotting. FLAG-WT-Synip dissociation appeared 5 min after insulin stimulation and continued up to 30 min. The difference in band intensity was statistically significant (*, P < 0.05; **, P < 0.01). These are representative experiments independently performed three times. In this design, insulin stimulation was done at 5, 15, and 30 min.