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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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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.
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fig1: 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.

Mentions: To determine the specific insulin signaling pathway responsible for the dissociation of Synip from Syntaxin4, we treated cells with various pharmacological agents and small interfering RNA (siRNA; Fig. 1). As previously observed, after insulin stimulation there was an ∼90% decrease in the amount of Synip protein coimmunoprecipitated with Syntaxin4 (Fig. 1 A). Several studies have suggested that in addition to the PI3 kinase pathway a second PI3 kinase–independent insulin signal pathway is necessary for the efficient translocation of GLUT4 in adipocytes (Watson et al., 2004). This latter pathway involves the function of the Cbl adaptor protein CAP (Cbl-associated protein) as expression of a dominant-interfering CAP blocks insulin-stimulated GLUT4 translocation without significant effect on insulin activation of PI3 kinase signaling and Akt activation (Baumann et al., 2000). However, expression of a dominant-interfering CAP mutant (CAP sorbin) had no significant effect on the insulin-stimulated dissociation of Synip from Syntaxin4 with also an ∼90% reduction in coimmunoprecipitation (Fig. 1 B). Similarly, treatment with a high concentration (10 μM) of PKC inhibitor GF109203X, which inhibits atypical PKCs (Li et al., 1999), resulted in an ∼85% decrease of Synip protein coimmunoprecipitated with Syntaxin4 after insulin stimulation (Fig. 1 C).


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)

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.
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fig1: 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.
Mentions: To determine the specific insulin signaling pathway responsible for the dissociation of Synip from Syntaxin4, we treated cells with various pharmacological agents and small interfering RNA (siRNA; Fig. 1). As previously observed, after insulin stimulation there was an ∼90% decrease in the amount of Synip protein coimmunoprecipitated with Syntaxin4 (Fig. 1 A). Several studies have suggested that in addition to the PI3 kinase pathway a second PI3 kinase–independent insulin signal pathway is necessary for the efficient translocation of GLUT4 in adipocytes (Watson et al., 2004). This latter pathway involves the function of the Cbl adaptor protein CAP (Cbl-associated protein) as expression of a dominant-interfering CAP blocks insulin-stimulated GLUT4 translocation without significant effect on insulin activation of PI3 kinase signaling and Akt activation (Baumann et al., 2000). However, expression of a dominant-interfering CAP mutant (CAP sorbin) had no significant effect on the insulin-stimulated dissociation of Synip from Syntaxin4 with also an ∼90% reduction in coimmunoprecipitation (Fig. 1 B). Similarly, treatment with a high concentration (10 μM) of PKC inhibitor GF109203X, which inhibits atypical PKCs (Li et al., 1999), resulted in an ∼85% decrease of Synip protein coimmunoprecipitated with Syntaxin4 after insulin stimulation (Fig. 1 C).

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