Table 1. Key signalling and trafficking components in GLUT4 translocation. See key at foot of Table p5.
| process | key effectors | insulin regulated | PI3K dependent | Akt dependent |
time scale of activation including phosphorylation (full activation unless otherwise indicated)
Phosphorylation times from ref. [36] |
|---|---|---|---|---|---|
| Overall process | |||||
| Glucose transport | Yes | Yes | Yes | ½ time = 5–10 min | |
| Signalling | |||||
| Formation of the IR–IRS–PI3K complex* | Insulin receptor, IRS, PI3K [36,106,107] | Yes | <1 min InsR and IRS1/2 pTyr in 15 s |
||
| IP3 synthesis at the PM* | PI3K Class 1a [13,33,108–112] | Yes | Yes | <1 min ½ time — 2.4 s [33] |
|
| Akt activation* | Akt, PDK1, mTORC2 [16,36,107] | Yes | Yes Yes |
Yes Yes |
Akt translocation <1 min; Akt phosphorylation <1 min (Akt2 pThr309 in 15 s; pSer474 in 30 s); Akt activation (substrate phosphorylation) <1 min (30 s) [36] mTORC2 multiple phosphosites, full activation within 5 min |
| Actin remodelling | EHD1* [113], EHBP1 [113], EHD2 [106,114] | Yes | Yes | Yes | pTyr453 EHD2 within 5 min [106] EHD1 — pSer456 in 20 min EHBP1 — pSer1061 in 30 s EHD2 — pSer451 in 15 s — only PI3K-dependent |
| CAP, TC10* [74,115,116] | Yes | No No |
No No |
CAP — multiple Ser/Thr phosphosites some, as Ser287, are activated in 5 min and are PI3K- and Akt-dependent perhaps via a feedback loop. TC10 — pSer9 in 1 min No time course of GTP loading reported |
|
| Tropomodulin3 [117] | Yes | Yes No |
Yes No |
pSer71 in 2–5 min | |
| Tropomyosin3.1 [118] | Yes | N.R. No |
N.R. No |
pSer51 in 20–60 min | |
| CLASP2 (microtubules, membrane ruffles) [119,120] | Yes | No | No |
Several Ser phosphosites in 2–60 min
Serine phosphorylation via MAPK [120] |
|
| Rac1 [121,122] | Yes | Yes | No | NPD | |
| Rab13, MICAL-L2, Actinin4* [123] | Yes |
Yes
Yes |
No
No |
2–5 min time course of GTP loading
Rab13 pTyr5 in 15 s pSer178 in 10 min MICAL-L2-pSer143 in 15 s; Actinin4 — pSer424 in 5 min (transient) |
|
| GLUT4 vesicle trafficking | |||||
| Translocation of GSVs to PM and tethering of GSV at PM | TBC1D4* [34,35,58,100,124–127] | Yes | Yes Yes |
Yes Yes |
TBC1D4 phosphorylation 15–30 s on different Ser/Thr residues
Interaction with 14-3-3 [58,125,127], IRAP [100,128] |
| Rab10 (GTP loading) [67] | No | N.R. Yes |
N.R. No† |
pTyr6 in 15 s; Slower pThr73 in 10 min | |
| DENND4c (Rab GEF) [92] | No (GEF activity) | N.R. Yes |
N.R. Yes |
pSer1043, pSer1096 and pSer1321 insulin-sensitive phosphorylation. Multiple phosphosites; pSer 1289 fastest in 2–5 min |
|
| Sec16 [63,129,130] | Yes | N.R. Yes |
Yes No |
Sec16a multiple phosphosites. Fastest pSer2311 in 2 min
Sec16b pSer182/pSer185 Moderate activation 30 s |
|
| TBC1D1 [127,131–133] |
Yes
|
Yes | Yes | 5 phosphosites detected, only pSer231 is insulin sensitive, but not above threshold value of 0.5 | |
| Rab8 and Rab13 [123,134] | Yes |
N.R. No† |
N.R.
No† |
2–5 min time course of GTP loading
Rab8: Transient phosphorylation pSer181/185 30 s to 1 min Rab13 see above |
|
| MyoVA [68,135,123,134] | Yes | Yes No |
Yes No |
Akt substrate pSer1650 in 5–10 min pSer600 and pSer1650 in 10 min |
|
| Synip [69] | Yes | Yes No† |
Yes No† |
Akt substrate, phosphorylation in 5–10 min Only pSer12 at 60 min |
|
| RGC2/AS250 (RalGAP)* [71] | Yes | Yes Yes |
Yes Yes |
Akt substrate, phosphorylation within 5 min Multiple phosphosites. Fastest Thr715 in 15 s + pSer486/696/765 in 30 s |
|
| RalA [71,75] | Yes | Yes | Yes | <5 min for GTP loading NISPD |
|
| Exocyst (Exo70, Exo 84 [77,96,136], Sec6 [137], Sec8 [138]) | Yes | No No |
N.R. No |
Exo 84 is dependent on transient phosphorylation (2–5 min) of TBK1. Exo70 pSer 242/245/250 at 60 min; Sec 8 pSer32 in 5–10 min. Exo84 NISPD |
|
| Myo1C [75,139–141,142] | Yes | Yes No† |
No No† |
Phosphorylation 2–5 min Small activation at 2 min pSer375 |
|
| CAMKII [141] | Yes | N.R. No† |
No No† |
Several phosphosites activated in 60 min | |
| Rab3(B,D), Noc2 [73] | Yes | No | No | <5 min for GTP loading NPD Rab3GAP1 and Rab3IP are phosphorylated within 30 s |
|
| GSV docking, fusion with PM and dispersal of GLUT4 in the PM | Munc18c* [72,86,90,91,106] | Yes | No No |
No No |
pTyr 5 min pSer588 in 15 s to 2 min |
| Doc2B [143] | Yes | N.R. | N.R. | NPD | |
| Tomosyn1/2* [70,97,144,145] | Yes | Yes No |
Yes No |
Akt substrate Ser783[70]. Timing <5 min pSer724/786/783 all within 15–30 s |
|
| Syntaxin4* [79,146] | Yes | Yes Yes |
Yes Yes |
Stx4 pY [36,106] 30 s for full activation pTyr251 30 s; Multiples phosphosites: Ser208 in 15 s; pSer248 transient 2–5 min but no PI3K or Akt dependence |
|
| SNAP23* [80,81] | N.R. | N.R. No† |
N.R. No† |
pThr24 transient 30 s to 2 min | |
| Vamp2 [57,78,147] | N.R. | N.R. Yes |
N.R. Yes |
pSer48 in 5–10 min | |
| Complexin-2 [148] | Yes |
N.R.
No |
No
No |
pSer93 in 30 s | |
| NSF and αSNAP* [93,95,149,150] | N.R. | N.R. No |
N.R. No |
NSF: pSer207 in 15 s
αSNAP: Ser24 in 20–60min |
|
| EFR3 and PI 4-kinase IIIα* [66] | N.R. | N.R. Yes |
N.R. Yes |
EFR3: very low Ser360/363 in 15 s | |
| GLUT4 internalisation (endocytosis) | Clathrin [98,151] | Yes | N.R. No |
N.R. No |
Heavy chain: pTyr434 in 15 s |
| Dynamin-2* [152–154] | Yes | N.R. No |
N.R. No |
NPD but Dynamin-1 (Dnm) detected with multiple phosphosites — some in 15 s or 30 s | |
| CIP4/2 [74] N-WASp, CIP4* [155] |
Yes Yes |
N.R. No |
N.R. No |
CIP4 Multiple phosphosites pSer296 in 15 s
N-WASP: pSer426 1–2 min |
|
| Cholesterol, nystatin endocytosis [101] | Yes | N.R. | N.R. | ||
| EEA1, Rab5, GAPEX5, Rab31 [156–158] | Yes | N.R. Yes |
N.R. No |
EEA1: pSer720 in 10 and 60 min;
Rab5a: pSer193 — 60min GAPEX5: Multiple phosphosites pSer742, pSer1017 in 5–10 min Rab31: NPD |
|
| Ub, USP25* [159,160] | N.R. | N.R. | N.R. | USP25: Transient pThr61 at 30 s | |
| Caveolae* [116,161–163] | No | N.R. Yes |
N.R. No |
Multiple phosphosites on Caveolin-1 pTyr phosphorylated within 30 s | |
| GLUT4 return to GSV compartment and sorting away from recycling endosomes | LRP1 [164] | Yes | N.R. Yes |
N.R. Yes |
pSer4524 and pThr4525 at 60 min |
| IRAP [165–169] | Yes | Yes Yes |
Yes Yes |
pSer51 in 5 min; pTyr70 high 1–10 min transient | |
| Sortilin [170–173] | Yes | N.R. No |
N.R. No |
pSer824 marginal (∼0.5) 15–20 min | |
| GGA, AP1* [174–177] | N.R. | N.R. Yes |
N.R. No |
GGA: pSer414/418 in 5 min LY only
AP1(subunit mu): pThr152/154 in 30 s and 15 s resp. LY only Other AP1 subunit NPD |
|
| ESCRT [178] | N.R. | N.R. No |
N.R. No |
Chmp3 and Vps4 NPD
Chmp2b phosphorylated in 15 s |
|
| Retromer [166,179,180] | N.R. | N.R. Yes |
N.R. Yes |
Vps35: pSer7 at 60 min
Vps26a NPD Vps26b: multiple phosphosites and pSer302 in 2–5 min Vps29 NPD Snx3 and 27: some phosphorylation in 60 min. Not PI3K- or Akt-dependent |
|
| Golgin97 and Vti1a [170] | N.R. | N.R. | N.R. | NISPD | |
| Syntaxin 6* [181], Syntaxin 16 [182], mVps45 [183] | Yes (Stx16) | N.R. No† |
N.R. No† |
Stx16 dephosphorylation within 30min [181] Stx16 some phosphorylation detected at long timepoints 60 min. Dephosphorylation for Ser35 detected since 15 s |
|
| Rab11, Rip11* [184,185] | Yes | Yes Yes |
No Yes |
Rab11b: Multiple phosphosites some activated at 2–10 min
Rip11: Multiple phosphosites, some pSer188 and 480 very fast 15 s |
|
| Rab4, KIF13 (motor protein) [135,186–188] | Yes | Yes Yes |
N.R. No |
Slow GTP loading over 45 min Rab4 NPD Kif13: Fast 30 s multiple sites pThr1717 |
|
| Rab14 [55,189] | N.R. | N.R. No |
N.R. Yes |
2 min pSer97 | |
| TUG [190–192] | N.R. | N.R. Yes |
N.R. No |
Proteolytic cleavage in 10 min pSer279 at 5 min |
|
| TRARG1(TUSC5) [193,194] | N.R. | N.R. Yes |
N.R. Yes |
Multiple phosphosites Ser56, 70 and 72 in 20–60 min | |
| TBC1D13, Rab35 [195] | N.R. | N.R. No |
N.R. Yes |
TBC1D13: Ser184 in 20–60 min | |
| CHC22 [196] | N.R. | N.R. | N.R. | NPD | |
| Budding of GSV, PLD [61,62] | Yes | N.R. | N.R. | PLD1 — NISPD | |
| Delivery of newly synthesised GLUT4 to GSVs and maintaining insulin responsiveness | Sortilin [170–172] | Yes | N.R. | N.R. | See above |
| syntaxin 16, mVps45, syntaxin 6 [181–183] | Yes (Stx16) | N.R. | N.R. | Stx16 dephosphorylation within 30 min [181]. See above | |
| GGA, AP1 [174–177] | N.R. | N.R. | N.R. | See above | |
| ACAP1, Arf6 [197] | Yes | N.R. | N.R. | NPD | |
| VAMP7 (TI-VAMP), VAMP4 [198] | N.R. | N.R. Yes |
N.R. No |
Vamp4 — Multiple phosphosites pSer17/20 in 1–5 min | |
| Axin, TNKS [199–201] | N.R. | N.R. | N.R. | Axin1 — multiple sites but below 0.5 | |
| P115 [202] | N.R. | N.R. No |
N.R. No |
pSer717 slow 60 min | |
| Sec16 [63,129,130] | N.R. | N.R. | N.R. | See above | |
| CHC22 [196] | N.R. | N.R. | N.R. | NPD | |
| Transfected GLUT4 becoming insulin responsive | N.R. | N.R. | N.R. | 6–9 h [174] 3.5 h in primary adipocytes [152] |
|
Key molecules reported to modulate GLUT4 trafficking and their activation time scale and phosphorylation status in response to insulin stimulation and sensitivity to PI3K/Akt inhibitors (where known).
Italics text —proteins reported to have a role in skeletal muscle; N.R. — not reported.
Bold text — phosphorylation times and PI3K and Akt inhibitors data from Humphrey et al. [36]. Only values above the threshold of 0.5 log2 of the ratio Insulin-stimulated/unstimulated cells were reported.
NISPD — no insulin-sensitive phosphosites detected (protein listed in the database as a phosphorylated protein but no kinetic data available).
NPD — no phosphorylation detected (protein not listed in the database).
*
Fast phosphorylation, less than 1 min, according to Humphrey et al. [36].
†
As the inhibition of phosphorylation in [36] was assessed only at a time point of 20 min the effect of the inhibitors on transient phosphorylation at earlier timepoints or phosphorylation events occurring at later timepoints >20 min cannot be assessed.