title: Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle.
abstract: The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.
authors.affiliation: The Joslin Diabetes Center Section on Metabolism and Proteomics Core and Harvard Medical School, One Joslin Place, Boston, MA 02215, USA.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Taylor
authors.forename: Ding
authors.initials: D
authors.lastname: An
authors.forename: Henning F
authors.initials: HF
authors.lastname: Kramer
authors.forename: Haiyan
authors.initials: H
authors.lastname: Yu
authors.forename: Nobuharu L
authors.initials: NL
authors.lastname: Fujii
authors.forename: Katja S C
authors.initials: KS
authors.lastname: Roeckl
authors.forename: Nicole
authors.initials: N
authors.lastname: Bowles
authors.forename: Michael F
authors.initials: MF
authors.lastname: Hirshman
authors.forename: Jianxin
authors.initials: J
authors.lastname: Xie
authors.forename: Edward P
authors.initials: EP
authors.lastname: Feener
authors.forename: Laurie J
authors.initials: LJ
authors.lastname: Goodyear
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009119:Muscle Contraction / Q000187:drug effects* / Q000502:physiology; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology*
chemical_list: D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012265:Ribonucleotides; C495693:Slc2a4 protein, mouse; C095906:Tbc1d1 protein, mouse; C505647:Tbc1d4 protein, mouse; D000620:Aminoimidazole Carboxamide; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; C031143:AICA ribonucleotide; D005947:Glucose