Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.
TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.
Vichaiwong
Kanokwan
K
Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, U.S.A.
Purohit
Suneet
S
An
Ding
D
Toyoda
Taro
T
Jessen
Niels
N
Hirshman
Michael F
MF
Goodyear
Laurie J
LJ
D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000494:pharmacology; D016384:Consensus Sequence; D004041:Dietary Fats / Q000494:pharmacology; D020690:GTPase-Activating Proteins; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology
D045163:Antibodies, Phospho-Specific; D004041:Dietary Fats; D020690:GTPase-Activating Proteins; D007328:Insulin; D050505:Mutant Proteins; D009687:Nuclear Proteins; D012265:Ribonucleotides; C095906:Tbc1d1 protein, mouse; D010768:Phosphoserine; D000620:Aminoimidazole Carboxamide; D051057:Proto-Oncogene Proteins c-akt; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D005947:Glucose