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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 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).
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fig2: 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).

Mentions: Next, we determined whether or not Synip phosphorylation by Akt2 was responsible for the regulation of the Synip–Syntaxin4 interaction. Inspection of the Synip amino acid sequence revealed the presence of an overlapping dual Akt consensus phosphorylation site at serine 97 and 99 (RAKLRSESP) (Fig. 2 A). Because arginine at position −5 is preferred for Akt1 (Alessi et al., 1996; Obata et al., 2000), this suggests that serine 97 would be a preferred Akt1 phosphorylation site. However, to date no Akt2-specific consensus motif has been reported.


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 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).
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fig2: 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).
Mentions: Next, we determined whether or not Synip phosphorylation by Akt2 was responsible for the regulation of the Synip–Syntaxin4 interaction. Inspection of the Synip amino acid sequence revealed the presence of an overlapping dual Akt consensus phosphorylation site at serine 97 and 99 (RAKLRSESP) (Fig. 2 A). Because arginine at position −5 is preferred for Akt1 (Alessi et al., 1996; Obata et al., 2000), this suggests that serine 97 would be a preferred Akt1 phosphorylation site. However, to date no Akt2-specific consensus motif has been reported.

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