Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle.
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(649)Ala 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(649)Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.
Medical Rersearch Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Scotland, United Kingdom.
Serge
S
Ducommun
Hong Yu
HY
Wang
Kei
K
Sakamoto
Carol
C
MacKintosh
Shuai
S
Chen
D048948:14-3-3 Proteins / Q000378:metabolism; D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D019943:Amino Acid Substitution; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives; D000704:Analysis of Variance; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D007328:Insulin / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D012265:Ribonucleotides / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D048948:14-3-3 Proteins; D001786:Blood Glucose; D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009687:Nuclear Proteins; D012265:Ribonucleotides; C495693:Slc2a4 protein, mouse; C095906:Tbc1d1 protein, mouse; C505647:Tbc1d4 protein, mouse; D000620:Aminoimidazole Carboxamide; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide