Thr 649 Ala-AS160 knock-in mutation does not impair contraction/ AICAR-induced glucose transport in mouse muscle
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Thr 649 Ala-AS160 knock-in mutation does not impair contraction/ AICAR-induced glucose transport in mouse muscle. / Ducommun, Serge; Wang, Hong Yu; Sakamoto, Kei; MacKintosh, Carol; Chen, Shuai.
In: American Journal of Physiology - Endocrinology and Metabolism, Vol. 302, No. 9, 01.05.2012, p. E1036-E1043.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Thr 649 Ala-AS160 knock-in mutation does not impair contraction/ AICAR-induced glucose transport in mouse muscle
AU - Ducommun, Serge
AU - Wang, Hong Yu
AU - Sakamoto, Kei
AU - MacKintosh, Carol
AU - Chen, Shuai
PY - 2012/5/1
Y1 - 2012/5/1
N2 - AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr 649, and promotes its binding to 14-3-3 proteins through phospho-Thr 649. We recently provided genetic evidence that AS160-Thr 649 phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr 649. In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ~ 150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ~150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr 649 phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr 649Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ~ 150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr 649Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.
AB - AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr 649, and promotes its binding to 14-3-3 proteins through phospho-Thr 649. We recently provided genetic evidence that AS160-Thr 649 phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr 649. In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ~ 150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ~150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr 649 phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr 649Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ~ 150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr 649Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.
KW - 14-3-3
KW - 5-ami-noimidazole-4-Carboxamide-1-β-D-Ribofuranoside
KW - Akt Substrate of 160 kDa
KW - Contraction
KW - Glucose Transport
KW - Phosphorylation
U2 - 10.1152/ajpendo.00379.2011
DO - 10.1152/ajpendo.00379.2011
M3 - Journal article
C2 - 22318952
AN - SCOPUS:84860635058
VL - 302
SP - E1036-E1043
JO - A J P: Endocrinology and Metabolism (Online)
JF - A J P: Endocrinology and Metabolism (Online)
SN - 1522-1555
IS - 9
ER -
ID: 239567469