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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 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.
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fig4: 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.

Mentions: Having established a biochemical consequence of Synip phosphorylation, we next examined the physiological requirement of this event. Expression of WT-Synip or S97F-Synip in 3T3L1 adipocytes had no significant effect on insulin-stimulated glucose uptake compared with empty vector-transfected cells (Fig. 4 A). However, expression of S99F-Synip significantly suppressed insulin-stimulated glucose uptake. To determine if the reduction of insulin-stimulated glucose uptake was due to an inhibition of GLUT4 translocation, we compared the cell surface localization of GLUT4 by confocal fluorescent microscopy (Fig. 4 B). Expression of S99F-Synip but not WT-Synip or S97F-Synip reduced insulin-stimulated GLUT4 translocation compared with control cells (Fig. 4 B). Finally, a quantitative colorimetric assay for the translocation of a myc epitope-tagged GLUT4 (Konrad et al., 2002) demonstrated that expression of S99F-Synip reduced insulin-stimulated GLUT4 translocation by 52% compared with control, WT-Synip, and S97F-Synip–expressing cells (Fig. 4 C). In this particular assay, because the myc epitope tag is located on the extracellular loop between transmembrane domains 1 and 2, only the GLUT4 proteins that have undergone plasma membrane fusion are detected.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171785&req=5

fig4: 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.
Mentions: Having established a biochemical consequence of Synip phosphorylation, we next examined the physiological requirement of this event. Expression of WT-Synip or S97F-Synip in 3T3L1 adipocytes had no significant effect on insulin-stimulated glucose uptake compared with empty vector-transfected cells (Fig. 4 A). However, expression of S99F-Synip significantly suppressed insulin-stimulated glucose uptake. To determine if the reduction of insulin-stimulated glucose uptake was due to an inhibition of GLUT4 translocation, we compared the cell surface localization of GLUT4 by confocal fluorescent microscopy (Fig. 4 B). Expression of S99F-Synip but not WT-Synip or S97F-Synip reduced insulin-stimulated GLUT4 translocation compared with control cells (Fig. 4 B). Finally, a quantitative colorimetric assay for the translocation of a myc epitope-tagged GLUT4 (Konrad et al., 2002) demonstrated that expression of S99F-Synip reduced insulin-stimulated GLUT4 translocation by 52% compared with control, WT-Synip, and S97F-Synip–expressing cells (Fig. 4 C). In this particular assay, because the myc epitope tag is located on the extracellular loop between transmembrane domains 1 and 2, only the GLUT4 proteins that have undergone plasma membrane fusion are detected.

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