| 2007 |
TBC1D1 possesses Rab-GTPase-activating protein (GAP) activity; its GAP domain shares identical Rab substrate specificity with AS160 (TBC1D4). Ectopic expression of TBC1D1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect, demonstrating that this inhibition is GAP-activity-dependent. Insulin treatment leads to phosphorylation of TBC1D1 on a conserved Akt site. |
In vitro GAP activity assay, ectopic overexpression in 3T3-L1 adipocytes, point mutagenesis of catalytic residue, GLUT4 translocation assay |
The Biochemical journal |
High |
17274760
|
| 2008 |
TBC1D1 is identified as the dominant PAS-immunoreactive protein at ~160 kDa in fast-twitch skeletal muscle (tibialis anterior, EDL) but not slow-twitch (soleus). In vivo stimulation by insulin, muscle contraction, and the AMPK activator AICAR each increased TBC1D1 PAS phosphorylation. Mass spectrometry identified multiple novel phosphorylation sites on TBC1D1, the majority being consensus or near-consensus AMPK sites. Purified Akt and AMPK both phosphorylate TBC1D1 in vitro, but AMPK (not Akt) caused a detectable shift in TBC1D1 electrophoretic mobility. |
Immunoprecipitation, mass spectrometry, in vitro kinase assay with purified Akt and AMPK, in vivo stimulation (insulin, contraction, AICAR) with PAS immunoblotting |
The Journal of biological chemistry |
High |
18276596
|
| 2008 |
AMPK activation in HEK-293 cells promotes 14-3-3 binding primarily to phospho-Ser237 of TBC1D1, while IGF-1/EGF/PMA promote 14-3-3 binding primarily via phospho-Thr596. In rat L6 myotubes, AMPK activators (AICAR, phenformin, A-769662) strongly phosphorylate Ser237 and promote 14-3-3 binding, whereas insulin promotes Thr596 phosphorylation but not 14-3-3 binding. In vitro phosphorylation experiments showed regulatory cross-talk among sites (e.g., phospho-Ser235 prevents subsequent phospho-Ser237). |
Site-specific phosphorylation analysis in HEK-293 and L6 cells, 14-3-3 binding assays, in vitro phosphorylation with purified kinases, pharmacological inhibition (LY294002) |
The Biochemical journal |
High |
17995453
|
| 2008 |
Overexpressed TBC1D1 in 3T3-L1 adipocytes inhibits GLUT4 translocation even in response to activated Akt, and endogenous TBC1D1 (which is ~20-fold less abundant than AS160 in adipocytes) does not regulate insulin-stimulated GLUT4 translocation. AMPK activator AICAR partially reversed the inhibition of GLUT4 translocation caused by overexpressed TBC1D1. TBC1D1 is much more highly expressed in skeletal muscle than fat. |
RNAi knockdown of TBC1D1 in 3T3-L1 adipocytes, overexpression with constitutively active Akt, AICAR treatment, GLUT4 translocation assay, quantitative western blotting |
The Journal of biological chemistry |
High |
18258599
|
| 2008 |
TBC1D1 is an Akt substrate phosphorylated at Thr590 in 3T3-L1 adipocytes. RNAi silencing of TBC1D1 elevated basal 2-deoxyglucose uptake (~61%) and strongly increased GLUT1 (but not GLUT4) expression. Loss of TBC1D1 activated the mTOR–p70 S6K pathway, and the GLUT1 upregulation was blocked by rapamycin. Overexpression of the phosphorylation-defective T590A mutant inhibited insulin-stimulated p70 S6K phosphorylation. |
Mass spectrometry-based phosphoproteomic identification of Akt substrates, RNAi knockdown, rapamycin treatment, overexpression of T590A mutant, glucose uptake assay, western blotting |
The Biochemical journal |
High |
18215134
|
| 2008 |
In vivo gene electroporation into mouse tibialis anterior showed that the obesity-associated R125W mutant TBC1D1 significantly decreased insulin-stimulated glucose transport through a mechanism requiring the GAP domain (disrupting GAP activity in the R125W background rescued glucose transport). A TBC1D1 quadruple phosphorylation-site mutant (4P, Ala substitutions at four AMPK/Akt sites) had no effect on insulin-stimulated transport but decreased contraction-stimulated glucose transport via the GAP domain, establishing that insulin- and contraction-regulated glucose transport occur via distinct mechanisms. |
In vivo gene injection/electroporation into mouse tibialis anterior, in vivo glucose transport assay, mutagenesis of phosphorylation sites and GAP domain |
Diabetes |
High |
20299473
|
| 2009 |
Insulin-stimulated phosphorylation of multiple Akt sites on TBC1D1 is required for GLUT4 translocation: a TBC1D1 mutant with several Akt sites converted to alanine was considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1 in C2C12 myotubes. AMPK activation partially relieved TBC1D1-mediated inhibition of GLUT4 translocation. Akt sites on TBC1D1 were identified by mass spectrometry from C2C12 myotubes. |
Mass spectrometry identification of Akt phosphorylation sites in C2C12 myotubes, alanine-substitution mutagenesis, GLUT4 translocation assay, AMPK activator treatment |
The Journal of biological chemistry |
High |
19740738
|
| 2009 |
In mouse EDL muscle, both contraction- and insulin-stimulated TBC1D1 Ser237 and Thr596 phosphorylation, and 14-3-3 protein binding to TBC1D1, are abolished in AMPK kinase-dead transgenic mice. AICAR and contraction induced comparable phosphorylation patterns; insulin increased Thr596 but not Ser237 phosphorylation, and insulin-stimulated Thr596 phosphorylation was fully abolished in AMPK KD mice, revealing an unexpected AMPK requirement for insulin-stimulated TBC1D1 Thr596 phosphorylation. |
Genetic epistasis using AMPK kinase-dead transgenic mice, site-specific phospho-antibodies for Ser237 and Thr596, 14-3-3 binding assay, ex vivo muscle contraction/AICAR/insulin stimulation |
American journal of physiology. Endocrinology and metabolism |
High |
19531644
|
| 2009 |
In rat epitrochlearis muscle, contraction-stimulated PAS-TBC1D1 phosphorylation and glucose transport are abolished by the AMPK inhibitor compound C, but are unaffected by Wortmannin (PI3-kinase/Akt inhibitor). Conversely, insulin-stimulated phosphorylation of both AS160 and TBC1D1 requires PI3-kinase/Akt. This establishes that contraction regulates TBC1D1 and glucose transport through an AMPK-dependent, Akt-independent mechanism. |
Pharmacological inhibition (compound C for AMPK, Wortmannin for PI3K) in isolated rat skeletal muscle, PAS immunoblotting, 3-O-methylglucose transport assay |
Diabetes |
High |
19208911
|
| 2010 |
In mouse skeletal muscle, contraction increases TBC1D1 phosphorylation on Ser231 and Ser660 (AMPK sites) but not Thr590 (Akt site). AICAR phosphorylates Ser231, Ser660, and Ser700 but not Thr590; insulin selectively increases Thr590. Contraction-stimulated Ser231, Ser660, and Ser700 phosphorylation is greatly reduced in AMPKα2-inactive transgenic mice, and Akt2-KO mice show blunted insulin-stimulated Thr590 phosphorylation. In vivo overexpression of a TBC1D1 mutated on four AMPK sites decreased glucose uptake in tibialis anterior. |
Site-specific phospho-antibodies, AMPKα2-inactive transgenic mice, Akt2-KO mice, in vivo electroporation/overexpression of phosphosite mutants, glucose uptake assay |
The Biochemical journal |
High |
20701589
|
| 2010 |
Exercise in human skeletal muscle increases TBC1D1 Ser237 phosphorylation (70–230%) and 14-3-3 binding capacity (60–250%) in an exercise-duration-dependent manner. Recombinant AMPK directly phosphorylates Ser237 and induces 14-3-3 binding in vitro. In AMPKα2-knockout (but not α1-knockout) mouse EDL, basal TBC1D1 protein levels and contraction-stimulated Ser237 phosphorylation are reduced, identifying AMPKα2 as the principal isoform regulating TBC1D1 Ser237 in EDL. |
Human exercise biopsy study (30 s, 2 min, 20 min cycling), in vitro AMPK phosphorylation assay, AMPKα1/α2 whole-body knockout mice, site-specific phospho-antibody, 14-3-3 binding overlay assay |
The Journal of physiology |
High |
20837646
|
| 2011 |
Crystal structures of the RabGAP domains of human TBC1D1 (2.2 Å) and TBC1D4/AS160 (3.5 Å) were solved. Both have 16 α-helices and no β-sheet elements. Ala-scanning mutagenesis of inferred Rab-binding interface residues showed only one of five substitutions significantly perturbed catalytic efficiency; substitution of TBC1D1 Met930 outside the canonical yeast interface substantially reduced catalytic activity, and the M930A mutant also failed to promote GLUT4 translocation. Additional residue Leu1019 was predicted to contact Rab; mutants with lowest RabGAP activity confirmed these contacts are required for biological activity. |
X-ray crystallography (2.2 Å for TBC1D1 GAP domain), Ala-scanning mutagenesis, in vitro RabGAP activity assay, GLUT4 translocation assay |
The Journal of biological chemistry |
High |
21454505
|
| 2012 |
The Rab-GTPase-activating proteins TBC1D1 and TBC1D4 together are essential for insulin-stimulated glucose uptake; double-knockout mice (D1/4KO) showed almost complete abolition of insulin-stimulated glucose uptake in skeletal muscle and adipocytes, whereas single knockouts showed only partial impairment. GLUT4 protein (but not mRNA) was substantially reduced in D1/4KO skeletal muscle and white adipose tissue. Cell surface labeling indicated that RabGAP deficiency impairs intracellular GLUT4 retention in the basal state. |
Generation of double-knockout (TBC1D1-/-/TBC1D4-/-) mice, hyperinsulinemic clamp, isolated skeletal muscle glucose uptake, cell surface GLUT4 labeling, GLUT4 protein/mRNA quantification |
Diabetes |
High |
25249576
|
| 2013 |
Conventional Tbc1d1-knockout mice show severely impaired insulin- and AICAR-stimulated glucose uptake in EDL (glycolytic) but not soleus muscle, and substantially increased fatty acid oxidation in oxidative soleus muscle. These mice had moderately reduced body weight, decreased respiratory quotient, and elevated resting metabolic rate, establishing that TBC1D1 plays a major role in glucose and lipid substrate preference in skeletal muscle. |
Conventional Tbc1d1-knockout mice, ex vivo glucose uptake assay (EDL, soleus), fatty acid oxidation assay, indirect calorimetry, body weight measurement |
Endocrinology |
High |
23892475
|
| 2013 |
AICAR treatment prior to insulin stimulation enables TBC1D1 to acquire insulin responsiveness, triggering GLUT4 trafficking. This regulatory mode shift requires Ser237 phosphorylation and an intact phosphotyrosine-binding 1 (PTB1) domain of TBC1D1. Mutations in PTB1, including the obesity-associated R125W, abolish the acquisition of insulin responsiveness while leaving AICAR-responsive GLUT4-liberation activity intact. |
GLUT4 nanometry in cell-based reconstitution model, AICAR and insulin stimulation, site-directed mutagenesis of Ser237 and PTB1 domain (including R125W), temporal GLUT4 tracking |
Molecular biology of the cell |
High |
23325788
|
| 2014 |
TBC1D1 is phosphorylated by AKT and AMPK in response to insulin and muscle contraction, and both phosphorylation events increase 14-3-3 binding but do not alter intrinsic RabGAP activity on Rab8a, Rab10, and Rab14. Full-length TBC1D1 shows markedly higher catalytic activity toward Rab8a, Rab10, and Rab14 than the isolated GAP domain. TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic tail of insulin-regulated aminopeptidase (IRAP), a resident of GLUT4 storage vesicles, and this interaction is disrupted by AKT or AMPK phosphorylation, suggesting that TBC1D1 recruitment to GLUT4 vesicles (not GAP activity per se) is regulated by insulin/contraction signaling. |
Baculovirus/Sf9 expression of recombinant full-length TBC1D1, in vitro RabGAP assay, in vitro phosphorylation by AKT/AMPK, 14-3-3 binding assay, co-immunoprecipitation with IRAP cytoplasmic tail, PTB domain pulldown |
The Journal of biological chemistry |
High |
30275018
|
| 2015 |
TBC1D1-knockout mice show impaired exercise-induced 2-deoxyglucose uptake specifically in white (non-oxidative) but not red skeletal muscle, and GLUT4 protein levels are reduced ~40% in white TBC1D1-/- muscle. TBC1D1-/- mice also display impaired exercise endurance. Normal body weight and glucose/insulin tolerance indicate TBC1D1 is dispensable for basal and insulin-stimulated glucose homeostasis under normal conditions. |
TBC1D1-knockout mouse generation, in vivo exercise-stimulated 2-deoxyglucose uptake, glucose/insulin tolerance tests, GLUT4 protein quantification, treadmill endurance test |
Diabetes |
High |
25576050
|
| 2015 |
TBC1D1 deficiency in EDL but not soleus muscle impairs insulin-, AICAR-, and contraction-stimulated glucose transport. In TBC1D1-deficient (Nob1.10SJL) congenic mice, suppression of hepatic glucose production during hyperinsulinemic clamp was increased. A 50% reduction in GLUT4 protein in EDL from TBC1D1-deficient mice was identified as a mechanism for reduced glucose transport, with proximal AMPK/Akt signaling unaltered. |
TBC1D1-deficient congenic mouse model, euglycemic hyperinsulinemic clamp, isolated EDL/soleus glucose transport, GLUT4 protein quantification, proximal signaling analysis |
American journal of physiology. Endocrinology and metabolism |
High |
22693207
|
| 2018 |
AMPK heterotrimers containing the α1 (but not α2) catalytic subunit form a stable, direct association with TBC1D1 but not its paralogue AS160. The interaction involves both PTB domains of TBC1D1 and is enhanced by AMPK activators (AICAR, A769662). This AMPKα1-TBC1D1 complex increases the efficiency of AMPK-mediated Ser237 phosphorylation. The obesity-associated R125W mutation in PTB1 reduces AMPKα1 binding and concomitantly reduces Ser237 phosphorylation. |
Co-immunoprecipitation, GST pulldown with purified proteins (direct interaction), pharmacological AMPK activation, site-directed mutagenesis (R125W, PTB domain mutants), in vitro kinase assay, quantitative phosphorylation analysis |
The Biochemical journal |
High |
30135087
|
| 2018 |
In cells with both TBC1D1 and AS160, TBC1D1 functionally dominates AS160 in controlling GLUT4 release. AICAR and intracellular Ca2+ serve as proximal stimuli for TBC1D1-governed GLUT4 release. AS160 modulates sensitivity to external stimuli in TBC1D1-mediated GLUT4 release. The synergistic cooperative actions depend on TBC1D1 PTB1 domain, calmodulin-binding domain, and phosphorylation of AS160 Thr642 and TBC1D1 Ser237/Thr596. |
GLUT4 nanometry in cell-based reconstitution models with varying TBC1D1:AS160 expression ratios, site-directed mutagenesis (PTB1, calmodulin-binding domain, phosphosites), AICAR and Ca2+ stimulation |
The Journal of biological chemistry |
High |
30482843
|
| 2016 |
A Tbc1d1 Ser231Ala knock-in mutation attenuates AICAR-induced glucose lowering and reduces AICAR-stimulated glucose uptake and GLUT4 cell surface content in isolated skeletal muscle. However, the Ser231Ala mutation does not impair exercise-induced muscle glucose uptake or exercise capacity, demonstrating that Ser231 phosphorylation is specifically required for AMPK-activator- but not exercise-stimulated glucose uptake. |
Tbc1d1 Ser231Ala knock-in mice, AICAR stimulation in vivo and ex vivo, exercise-stimulated glucose uptake, cell surface GLUT4 labeling, glucose tolerance test |
Diabetologia |
High |
27826658
|
| 2014 |
APPL2 interacts with TBC1D1 through its BAR domain. Insulin stimulates TBC1D1 phosphorylation on Ser235, which enhances APPL2 binding; this APPL2–TBC1D1 interaction suppresses insulin-evoked TBC1D1 Thr596 phosphorylation. Substitution of Ser235 with alanine diminishes APPL2-mediated inhibition of Thr596 phosphorylation and reverses the suppressive effects of TBC1D1 on insulin-induced GLUT4 translocation and glucose uptake in myotubes. |
Co-immunoprecipitation of APPL2 and TBC1D1, site-directed mutagenesis (S235A), overexpression and knockdown in C2C12 myotubes, GLUT4 translocation assay, glucose uptake assay, conditional muscle-specific APPL2 KO mice |
Diabetes |
High |
24879834
|
| 2016 |
Rab28 is identified as an in vitro substrate for the GAP domains of both TBC1D1 and TBC1D4. Rab28 GTP-loading state is acutely regulated by insulin in skeletal muscle. siRNA-mediated knockdown of Rab28 in isolated mouse skeletal muscle decreases basal glucose uptake, and constitutively active Rab28-Q72L in adipocytes increases basal cell surface HA-GLUT4. |
In vitro GAP assay with purified Rab28 and TBC1D1/TBC1D4 GAP domains, Rab28-GTP loading assay in muscle, siRNA knockdown of Rab28 in skeletal muscle, overexpression of Rab28-Q72L in adipocytes, cell surface GLUT4 labeling |
FEBS letters |
High |
27929607
|
| 2019 |
WNK1 kinase phosphorylates TBC1D1 on Ser565 (identified by mass spectrometry). Phosphomimetic and unphosphorylatable mutants of TBC1D1 at S565 both affected GLUT1 cell surface abundance, indicating a regulatory role for WNK1-mediated TBC1D1 phosphorylation in constitutive GLUT1 trafficking. |
WNK1 RNAi in HEK293 cells, mass spectrometry identification of Ser565 phosphosite, transfection of phosphomimetic/unphosphorylatable TBC1D1 mutants, cell surface GLUT1 quantification |
Archives of biochemistry and biophysics |
Medium |
31816312
|
| 2020 |
TBC1D1 interacts with VPS13A and VPS13C, the Rab-binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1 in C2C12 myotubes. These interactions are mediated through the PTB domains of TBC1D1 and are not affected by AMPK activation, distinguishing them from the AMPK-regulated AMPKα1–TBC1D1 interaction. Depletion of VPS13A or VPS13C post-transcriptionally increases cellular GLUT4 protein and enhances cell surface GLUT4 in response to AMPK activation. |
Unbiased quantitative proteomics/co-immunoprecipitation to identify TBC1D1 interactors in C2C12 myotubes, siRNA depletion of VPS13A/VPS13C, cell surface GLUT4 assay, AMPK activation |
Scientific reports |
Medium |
33087848
|
| 2013 |
TBC1D1 is expressed in pancreatic beta-cells and is phosphorylated in response to glucose. siRNA knockdown of TBC1D1 in beta-cells increased basal and glucose-stimulated insulin secretion and decreased beta-cell proliferation without affecting apoptosis. |
TBC1D1 expression analysis in human and rat beta-cells, phosphorylation assay (glucose stimulation), siRNA knockdown, insulin secretion assay, proliferation and apoptosis assays |
FEBS letters |
Medium |
24239544
|
| 2022 |
AMPK-TBC1D1 signaling regulates GTP-loading of Rab2A: nutrition repletion suppresses AMPK-TBC1D1 phosphorylation, increasing GTP-bound Rab2A, which stabilizes PPARγ protein and promotes hepatic lipid accumulation. In TBC1D1-S231A knock-in mice, increased GTP-Rab2A and fatty liver were observed. Inhibition of Rab2A expression alleviated hepatic lipid deposition in obese mice. |
TBC1D1-S231A knock-in mice, GTP-Rab2A pulldown assay, siRNA knockdown of Rab2A in hepatic cells and mice, western blotting for PPARγ stability, in vitro and in vivo lipid accumulation assays |
PLoS biology |
Medium |
35061665
|
| 2014 |
Overexpression of TBC1D1 in mouse soleus muscle decreased basal and AICAR-stimulated palmitate oxidation by ~18–22% and increased glucose oxidation, without altering FAT/CD36, mitochondrial content, CPT1, AMPK, or ACC. TBC1D1-mediated reduction of fatty acid oxidation was associated with reduced β-hydroxyacyl-CoA dehydrogenase (β-HAD) enzyme activity. |
Electrotransfection of TBC1D1 cDNA into mouse soleus, palmitate oxidation assay, glucose oxidation assay, β-HAD enzyme activity measurement, protein expression analysis |
American journal of physiology. Regulatory, integrative and comparative physiology |
Medium |
25163918
|
| 2017 |
Ablation of TBC1D1 in rats impairs contraction-induced sarcolemmal GLUT4 redistribution and glucose uptake specifically in white gastrocnemius muscle but does not alter insulin-induced GLUT4 trafficking or whole-body insulin tolerance. TBC1D1-KO rats show increased skeletal muscle fatty acid oxidation and increased maximal ADP-stimulated mitochondrial respiration, but reduced exercise run time to exhaustion. |
Rat TBC1D1 KO model, sarcolemmal GLUT4 fractionation after contraction/insulin, in vivo insulin tolerance test, ex vivo fatty acid oxidation, permeabilized fiber respiration, treadmill exercise |
The Journal of biological chemistry |
High |
28808062
|
| 2008 |
The SJL mouse strain carries a naturally occurring mutation in Tbc1d1 resulting in a truncated protein lacking the TBC RabGAP domain. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, while overexpression had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation, and reduced glucose uptake in isolated skeletal muscle. |
Identification of naturally occurring Tbc1d1 truncation mutation in SJL strain, siRNA knockdown and overexpression in skeletal muscle cells, fatty acid uptake and oxidation assays, isolated muscle glucose uptake, indirect calorimetry in recombinant congenic mice |
Nature genetics |
High |
18931681
|