Thr 649 Ala-AS160 knock-in mutation does not impair contraction/ AICAR-induced glucose transport in mouse muscle
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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.
Original language | English |
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Journal | American Journal of Physiology - Endocrinology and Metabolism |
Volume | 302 |
Issue number | 9 |
Pages (from-to) | E1036-E1043 |
ISSN | 0193-1849 |
DOIs | |
Publication status | Published - 1 May 2012 |
Externally published | Yes |
- 14-3-3, 5-ami-noimidazole-4-Carboxamide-1-β-D-Ribofuranoside, Akt Substrate of 160 kDa, Contraction, Glucose Transport, Phosphorylation
Research areas
ID: 239567469