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Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles.

Yamada E, Okada S, Saito T, Ohshima K, Sato M, Tsuchiya T, Uehara Y, Shimizu H, Mori M - J. Cell Biol. (2005)

Bottom Line: We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)).Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3.These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.

ABSTRACT
We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.

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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.
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fig3: 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.

Mentions: To determine the functional consequence of this phosphorylation event, we nonspecifically dephosphorylated the Synip protein with alkaline phosphatase before coimmunoprecipitation (Fig. 3 A). Under these conditions, Synip failed to display an insulin-stimulated dissociation from Syntaxin4, suggesting nonphosphorylated Synip preferentially binds to Syntaxin4. Consistent with a complete Synip dephosphorylation, we did not detect any significant difference of Synip–Syntaxin4 complex amount between unstimulated and insulin-stimulated samples. To directly assess the effect of Synip phosphorylation, we examined the in vitro binding of GST-Synip with MBP (maltose binding protein)-Syntaxin4 after Akt2 phosphorylation (Fig. 3 B). In the absence of Akt2, both WT-Synip and S99F displayed identical extents of binding. In contrast, Akt2-dependent phosphorylation of WT-Synip resulted in a 70% reduction in Syntaxin4 binding, whereas there was no effect on the S99F mutant.


Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles.

Yamada E, Okada S, Saito T, Ohshima K, Sato M, Tsuchiya T, Uehara Y, Shimizu H, Mori M - J. Cell Biol. (2005)

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.
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fig3: 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.
Mentions: To determine the functional consequence of this phosphorylation event, we nonspecifically dephosphorylated the Synip protein with alkaline phosphatase before coimmunoprecipitation (Fig. 3 A). Under these conditions, Synip failed to display an insulin-stimulated dissociation from Syntaxin4, suggesting nonphosphorylated Synip preferentially binds to Syntaxin4. Consistent with a complete Synip dephosphorylation, we did not detect any significant difference of Synip–Syntaxin4 complex amount between unstimulated and insulin-stimulated samples. To directly assess the effect of Synip phosphorylation, we examined the in vitro binding of GST-Synip with MBP (maltose binding protein)-Syntaxin4 after Akt2 phosphorylation (Fig. 3 B). In the absence of Akt2, both WT-Synip and S99F displayed identical extents of binding. In contrast, Akt2-dependent phosphorylation of WT-Synip resulted in a 70% reduction in Syntaxin4 binding, whereas there was no effect on the S99F mutant.

Bottom Line: We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)).Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3.These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.

ABSTRACT
We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.

Show MeSH
Related in: MedlinePlus