Tbc1d4 (AS160, Akt substrate of 160 kDa) is a large cytosolic Rab GTPase-activating protein with two N-terminal phosphotyrosine-binding (PTB) domains and a C-terminal TBC Rab-GAP domain that retains both catalytic fingers. In adipocytes and skeletal muscle it associates with GLUT4 (insulin-responsive) vesicles and, in the unstimulated state, keeps vesicle Rabs of the Rab8A/Rab10/Rab13/Rab14 class in their GDP-bound form, restraining GLUT4 vesicle docking and fusion with the plasma membrane. Insulin-activated Akt (and contraction/AMPK signalling in muscle) phosphorylates Tbc1d4 on multiple sites; phosphorylation and 14-3-3 binding inactivate the GAP, allowing Rab activation, GLUT4 vesicle fusion and increased glucose uptake. It is thus a key signal-to-trafficking node in insulin- and exercise-stimulated glucose transport.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | MODIFY | Summary: Cytoplasm from the ARBA/SubCell combined IEA pipeline. Reason: Correct but general. AS160 is a soluble cytosolic protein that is peripherally and loosely associated with GLUT4 vesicles (lost on centrifugation or high-salt washes); the gene already carries the more specific GO:0005829 cytosol (ISO from human), which is part_of cytoplasm. Refine to cytosol. Proposed replacements: cytosol Supporting Evidence: PMID:21454690 we found that, in rat adipose cells, AS160 is continuously associated with GLUT4 vesicles |
| GO:0005829 cytosol | ISO GO_REF:0000121 | ACCEPT | Summary: Cytosol, transferred from human TBC1D4 (O60343). Reason: Human TBC1D4 is a cytosolic Rab GAP (PAINT IBD at PTN002742831 is seeded by O60343). In rat adipocytes AS160 is a peripheral protein that partitions between cytosol and the cytosolic face of GLUT4 vesicles; cytosol is where the GAP meets its vesicle Rabs. Consistent with the rat biology. Supporting Evidence: PMID:21454690 we found that, in rat adipose cells, AS160 is continuously associated with GLUT4 vesicles |
| GO:0016192 vesicle-mediated transport | ISO GO_REF:0000121 | ACCEPT | Summary: Vesicle-mediated transport, transferred from human TBC1D4. Reason: AS160 acts directly on the GLUT4 vesicle trafficking machinery: as a Rab GAP it holds GLUT4-vesicle Rabs in the GDP-bound state, controlling vesicle docking/fusion. This is the level the PAINT curator placed for the TBC1D4 node; the more specific regulatory claim is captured by the negative regulation of vesicle fusion row. Accepted as a correct, appropriately-scoped process for a Rab GAP acting on vesicle traffic. Supporting Evidence: PMID:21454690 AS160 remains in its GAP-active form, the associated Rab remains in the GDP form, and fusion activity is therefore low PMID:21041651 Constitutively active AS160 lowered basal and insulin-stimulated levels of surface GLUT4, effects that were reversed by overexpressing Rab8A or Rab13, suggesting that both Rabs are targets of AS160-GAP activity in the context of GLUT4 traffic. |
| GO:0031339 negative regulation of vesicle fusion | IMP PMID:21454690 AS160 phosphotyrosine-binding domain constructs inhibit insu... | ACCEPT | Summary: Negative regulation of vesicle fusion, from rat adipocyte GLUT4 vesicle fusion assays. Reason: Full text read. Rat AS160 PTB-domain constructs inhibited insulin-stimulated GLUT4 vesicle fusion in a cell-free assay from rat adipocyte fractions and blocked insulin-stimulated GLUT4 translocation in intact rat adipocytes; the authors interpret this as the constructs preventing 14-3-3 binding so that AS160 remains GAP-active and fusion stays low. Together with the ortholog data (phosphosite mutants inhibit GLUT4 translocation through the GAP function) this supports AS160 as a negative regulator of GLUT4 vesicle fusion. Core regulatory process. Supporting Evidence: PMID:21454690 Protein constructs from rat AS160 incorporating the separate first (AS160(1β290)) and second (AS160(230β532)) PTB domains inhibited vesicle fusion PMID:21454690 AS160 remains in its GAP-active form, the associated Rab remains in the GDP form, and fusion activity is therefore low PMID:12637568 this phosphorylation signals translocation through inactivation of the Rab GAP function |
| GO:0031982 vesicle | IDA PMID:21454690 AS160 phosphotyrosine-binding domain constructs inhibit insu... | MODIFY | Summary: Vesicle; AS160 detected on GLUT4 vesicles immunoisolated from rat adipocytes. Reason: Full text read. The vesicles assayed were GLUT4 vesicles immunoisolated with anti-GLUT4 from rat adipocytes, i.e. the insulin-responsive GLUT4 storage compartment. GO:0032593 insulin-responsive compartment (exact synonym GLUT4 storage compartment) is an is_a descendant of vesicle (via secretory vesicle/cytoplasmic vesicle; checked with OAK), so the IDA supports the more specific term. Proposed replacements: insulin-responsive compartment Supporting Evidence: PMID:21454690 we found that, in rat adipocytes, there was an insulin-dependent increase in the level of AS160 associated with GLUT4 vesicles PMID:21454690 we found that, in rat adipose cells, AS160 is continuously associated with GLUT4 vesicles |
| GO:0032869 cellular response to insulin stimulus | IDA PMID:21454690 AS160 phosphotyrosine-binding domain constructs inhibit insu... | ACCEPT | Summary: Cellular response to insulin stimulus; insulin increases AS160 and 14-3-3 on rat adipocyte GLUT4 vesicles. Reason: Full text read. In rat adipocytes insulin increased the amount of AS160 on GLUT4 vesicles and its 14-3-3gamma association, and AS160 PTB constructs blocked insulin-stimulated GLUT4 vesicle fusion and translocation. AS160 is a direct Akt substrate whose inactivation transduces the insulin signal to GLUT4 traffic, so it participates in the cellular response to insulin. Core process. Supporting Evidence: PMID:21454690 The levels of AS160 were also higher on GLUT4 vesicles from insulin-treated cells compared with basal cells PMID:21454690 The tandem PTB domain construct potently inhibited insulin-stimulated (but not basal) GLUT4 translocation to the cell surface in the intact adipose cell PMID:21454690 insulin-stimulated 14-3-3Ξ³ interaction, but not AS160 phosphorylation alone, generates a fusion-activating GAP-inactive form of AS160 |
| GO:0032869 cellular response to insulin stimulus | IEA GO_REF:0000117 | ACCEPT | Summary: Cellular response to insulin stimulus, ARBA machine-learning prediction. Reason: Same claim as the IDA row from rat adipocytes (PMID:21454690), which independently establishes it for this gene; consistent action. Supporting Evidence: PMID:21454690 The levels of AS160 were also higher on GLUT4 vesicles from insulin-treated cells compared with basal cells PMID:21454690 The tandem PTB domain construct potently inhibited insulin-stimulated (but not basal) GLUT4 translocation to the cell surface in the intact adipose cell |
| GO:0032869 cellular response to insulin stimulus | ISO GO_REF:0000121 | ACCEPT | Summary: Cellular response to insulin stimulus, transferred from mouse Tbc1d4 (MGI:2429660). Reason: Same claim as the rat IDA row; the mouse source reflects knock-in/knockout phenotypes with impaired insulin-stimulated GLUT4 traffic. Consistent with rat experimental data, so accepted. Supporting Evidence: PMID:21454690 The tandem PTB domain construct potently inhibited insulin-stimulated (but not basal) GLUT4 translocation to the cell surface in the intact adipose cell |
| GO:0032869 cellular response to insulin stimulus | ISO GO_REF:0000121 | ACCEPT | Summary: Cellular response to insulin stimulus, transferred from human TBC1D4 (O60343). Reason: Same claim as the rat IDA row; human AS160 is the insulin/Akt-regulated Rab GAP controlling GLUT4 translocation. Consistent with rat data. Supporting Evidence: PMID:12637568 this phosphorylation signals translocation through inactivation of the Rab GAP function PMID:21454690 The levels of AS160 were also higher on GLUT4 vesicles from insulin-treated cells compared with basal cells |
| GO:0005096 GTPase activator activity | ISS PMID:15971998 AS160, the Akt substrate regulating GLUT4 translocation, has... | NEW | Summary: Proposed: Rab GTPase-activating protein activity of the TBC domain, transferred from human AS160 and supported by rat cell data. Reason: The rat entry has no molecular-function row, although Rab GAP activity is the activity through which Tbc1d4 acts. The recombinant human AS160 TBC domain stimulates GTP hydrolysis by Rab2A, Rab8A, Rab10 and Rab14 and is inactivated by mutation of the catalytic arginine; the arginine (ILVDLGR, R919) and glutamine (YCQ, Q956) fingers are conserved in rat Tbc1d4 within the PS50086 TBC domain (local motif check). In rat L6 myotubes constitutively active AS160 lowered surface GLUT4 in a manner reversed by Rab8A or Rab13, and in rat adipocytes the GAP-active form keeps the vesicle Rab GDP-bound. Molecular function, not a process, so the process NEW bar does not apply; not an ancestor or descendant of any term on the gene. Supporting Evidence: PMID:15971998 The GAP domain was active against Rabs 2A, 8A, 10 and 14. PMID:15971998 recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive file:rat/Tbc1d4/Tbc1d4-bioinformatics/RESULTS.md arginine finger IxxDxxR: motif ILVDLGR at 913-919; catalytic residue R919 PMID:21041651 Constitutively active AS160 lowered basal and insulin-stimulated levels of surface GLUT4, effects that were reversed by overexpressing Rab8A or Rab13, suggesting that both Rabs are targets of AS160-GAP activity in the context of GLUT4 traffic. PMID:21454690 AS160 remains in its GAP-active form, the associated Rab remains in the GDP form, and fusion activity is therefore low |
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Download this section (compressed HTML)Q: Should rat Tbc1d4 carry a 14-3-3 protein binding MF row? PMID:21454690 shows insulin-dependent binding of rat adipocyte AS160 to GST-14-3-3 isoforms and to 14-3-3gamma on GLUT4 vesicles.
Q: Which Rab is the physiological AS160 target on rat adipocyte GLUT4 vesicles (Rab10, as in 3T3-L1) versus rat muscle (Rab8A/Rab13)?
Experiment: Reconstitute the rat adipocyte cell-free GLUT4 vesicle fusion assay with vesicles from cells expressing GAP-dead (arginine-finger R919K) versus wild-type rat Tbc1d4, and measure Rab10/Rab8A GTP loading alongside fusion.
Hypothesis: Tbc1d4 GAP activity, not only its phosphorylation, controls GLUT4 vesicle fusion in rat adipocytes.
Type: in vitro reconstitution with mutagenesis
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