In adipocytes and myocytes insulin signaling causes intracellular vesicles carrying the GLUT4 glucose transporter to translocate to the plasma membrane, allowing the cells to take up glucose from the bloodstream (reviewed in Zaid et al. 2008, Leney and Tavare 2009, Bogan and Kandror 2010, Foley et al. 2011, Hoffman and Elmendorf 2011, Kandror and Pilch 2011). In myocytes muscle contraction alone can also cause translocation of GLUT4. Though the entire pathway leading to GLUT4 translocation has not been elucidated, several steps are known. Insulin activates the kinases AKT1 and AKT2. Muscle contraction activates the kinase AMPK-alpha2 and possibly also AKT. AKT2 and, to a lesser extent, AKT1 phosphorylate the RAB GTPase activators TBC1D1 and TBC1D4, causing them to bind 14-3-3 proteins and lose GTPase activation activity. As a result RAB proteins (probably RAB8A, RAB10, RAB14 and possibly RAB13) accumulate GTP. The connection between RAB:GTP and vesicle translocation is unknown but may involve recruitment and activation of myosins. Myosins 1C, 2A, 2B, 5A, 5B have all been shown to play a role in translocating GLUT4 vesicles near the periphery of the cell. Following docking at the plasma membrane the vesicles fuse with the plasma membrane in a process that depends on interaction between VAMP2 on the vesicle and SNAP23 and SYNTAXIN-4 at the plasma membrane.
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AS160 (TBC1D4) phosphorylated on serines 318, 341, 570, 588, and 751 and threonine 642 binds to all 14-3-3 proteins, although binding to 14-3-3 delta is comparatively low (Ramm et al. 2006, Howlett et al. 2007, Ngo et al. 2009, Treebak et al. 2009, Koumanov et al. 2011). As inferred from mouse, binding to 14-3-3 does not interfere with the interaction between AS160 and IRAP (LNPEP).
As inferred from rat L6 muscle cells, TBC1D1 is located in the perinuclear cytosol (Chen et al. 2008). TBC1D1 is observed throughout the cytosol and, based on its homology to TBC1D4 and its interaction with membrane-bound RAB proteins, TBC1D1 is expected to be concentrated near vesicle membranes.
As inferred from rat L6 muscle cells, TBC1D1 is located in the perinuclear cytosol (Chen et al. 2008). TBC1D1 is observed throughout the cytosol and, based on its homology to TBC1D4 and its interaction with membrane-bound RAB proteins, TBC1D1 is expected to be concentrated near vesicle membranes.
As inferred from rat L6 muscle cells, TBC1D1 is located in the perinuclear cytosol (Chen et al. 2008). TBC1D1 is observed throughout the cytosol and, based on its homology to TBC1D4 and its interaction with membrane-bound RAB proteins, TBC1D1 is expected to be concentrated near vesicle membranes.
AS160 (TBC1D4) phosphorylated on serines 318, 341, 570, 588, and 751 and threonine 642 binds to all 14-3-3 proteins, although binding to 14-3-3 delta is comparatively low (Ramm et al. 2006, Howlett et al. 2007, Ngo et al. 2009, Treebak et al. 2009, Koumanov et al. 2011). As inferred from mouse, binding to 14-3-3 does not interfere with the interaction between AS160 and IRAP (LNPEP).
TBC1D1 phosphorylated on serine-237 binds 14-3-3 proteins in assays with yeast 14-3-3 proteins BMH1 and BMH2 (Chen et al. 2008, Frosig et al. 2010). Binding with human 14-3-3 proteins is inferred.
RALA releases GDP and binds GTP, producing the active form of RALA. The reaction is accelerated by guanine nucleotide exchange factors (GEFs) and opposed by GTPase-activating proteins (GAPs) which enhance the conversion of RALA:GTP back to RALA:GDP by activating the GTPase activity of RALA.
RALA is a guanine nucleotide binding protein that hydrolyzes bound GTP to yield GDP and phosphate. RGC1 and RGC2 are GAPs (GTPase-activating proteins) that activate the GTPase activity of RALA. Insulin activates AKT, which phosphorylates RGC2, inactivating the GAP activity of RGC1:RGC2 and allowing RALA:GTP to accumulate.
RAB8A/10/13/14 release GDP and bind GTP to yield the active complex. Guanine nucleotide exchange factors (GEFs) stimulate the reaction. GTPase-activating proteins (GAPs) oppose the reaction by stimulating the intrinsic GTPase activity of the RAB proteins.
As inferred from mouse, AKT2 and, to a lesser extent, AKT1 phosphorylate AS160 (TBC1D4) in response to insulin signaling (Howlett et al. 2007, Karlsson et al 2005). AS160, a RAB GTPase activating protein, interacts with IRAP (LNPEP) and is associated with cytoplasmic vesicles containing GLUT4.
As inferred from mouse, GLUT4 initially translocates from endosomes to insulin-responsive vesicles (IRVs, GSVs). RAB11 appears to play a role in this process. IRVs bearing GLUT4 are then translocated across the cortical actin network to the plasma membrane. Myosins 2A, 2B, 5A, and 5B contribute to translocation and are presumed to be involved in this step. Myosin 1C appears to act close to the plasma membrane and may facilitate fusion of the vesicle with the plasma membrane. RAB:GTP complexes coupled to the vesicles may interact with myosins to regulate their activity.
After docking at the membrane VAMP2 on the vesicle interacts with SYNTAXIN-4 and SNAP23 on the plasma membrane to catalyze fusion of the vesicle with the plasma membrane. STXBP3 (MUNC18C) bound to STX4 prevents fusion until STXBP3 is phosphorylated.
As inferred from mouse adipocytes, insulin signals via PKC-lambda to cause Rab4 to load GTP and associate with Kif3, which then has higher affinity for microtubules. Motor activity of Kif3 along microtubules is believed to transport vesicles containing Glut4 across the cytosol to the cortical actin network.
As inferred from mouse, insulin causes phosphorylation and inactivation of the Ral GTPase activating complex RGC, causing RALA:GTP to accumulate and associate with the unconventional myosin MYO1C. MYO1C, with calmodulin as a light chain, motors across cortical actin and interacts with the exocyst complex to tether vesicles at the plasma membrane (Chen et al. 2007).
RAB proteins have intrinsic weak GTPase activity that is enhanced by RAB-GTPase activating proteins (RAB-GAPs, Sano et al. 2007). The GTPase activity of RAB13 is inferred from other RAB proteins. AS160 (TBC1D4) and TBC1D1 are GAPs that activate the GTPase activity of RAB8A/10/13. Insulin signaling activates AKT, which phosphorylates and inactivates AS160 and TBC1D1, allowing GTP-bound (active) RABs to accumulate.
As inferred from mouse, AKT2 (PKB-beta) phosphorylates RBC2 (RALGAPA2) on serine-486, serine-696, and threonine-715 in response to insulin. The phosphorylation prevents RBC1:RBC2 from activating RALA GTPase and allows RALA:GTP to accumulate.
In response to muscle contraction and insulin signaling, AMPK-alpha2 phosphorylates TBC1D1 on serine 237 and probably other residues (Frosig et al. 2010, Vichaiwong et al. 2010). As inferred from rat L6 muscle cells TBC1D1 colocalizes with perinuclear vesicles bearing GLUT4 and may be involved in an early step that mobilizes them (Chen et al. 2008). Human TBC1D1 appears cytosolic and is believed to be concentrated near vesicle membranes (Park et al. 2011).
As inferred from mouse, AKT2 phosphorylates Myosin 5A on serine-1652. The phosphorylation promotes association of Myosin 5A with actin and ATPase activity of Myosin 5A.
Though the entire pathway leading to GLUT4 translocation has not been elucidated, several steps are known. Insulin activates the kinases AKT1 and AKT2. Muscle contraction activates the kinase AMPK-alpha2 and possibly also AKT. AKT2 and, to a lesser extent, AKT1 phosphorylate the RAB GTPase activators TBC1D1 and TBC1D4, causing them to bind 14-3-3 proteins and lose GTPase activation activity. As a result RAB proteins (probably RAB8A, RAB10, RAB14 and possibly RAB13) accumulate GTP. The connection between RAB:GTP and vesicle translocation is unknown but may involve recruitment and activation of myosins.
Myosins 1C, 2A, 2B, 5A, 5B have all been shown to play a role in translocating GLUT4 vesicles near the periphery of the cell. Following docking at the plasma membrane the vesicles fuse with the plasma membrane in a process that depends on interaction between VAMP2 on the vesicle and SNAP23 and SYNTAXIN-4 at the plasma membrane.
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=1445148
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AMPK-beta AMPK-gamma
AMP14-3-3
IRAPGTP KIF3
microtubuleGTP MYO1C Calmodulin
F-actinGTP MYO1C
ExocystSTX4
SNAP23Annotated Interactions
AMPK-beta AMPK-gamma
AMPGTP MYO1C Calmodulin
F-actinGTP MYO1C Calmodulin
F-actinSTX4
SNAP23