PKB-Mediated Thr649 Phosphorylation of AS160/TBC1D4 Regulates the R-Wave Amplitude in the Heart

The Rab GTPase activating protein (RabGAP), AS160/TBC1D4, is an important substrate of protein kinase B (PKB), and regulates insulin-stimulated trafficking of glucose transporter 4. Besides, AS160/TBC1D4 has also been shown to regulate trafficking of many other membrane proteins including FA translocase/CD36 in cardiomyocytes. However, it is not clear whether it plays any role in regulating heart functions in vivo. Here, we found that PKB-mediated phosphorylation of Thr⁶⁴⁹ on AS160/TBC1D4 represented one of the major PAS-binding signals in the heart in response to insulin. Mutation of Thr⁶⁴⁹ to a non-phosphorylatable alanine increased the R-wave amplitude in the AS160^Thr649Ala knockin mice. However, this knockin mutation did not affect the heart functions under both normal and infarct conditions. Interestingly, myocardial infarction induced the expression of a related RabGAP, TBC1D1, in the infarct zone as well as in the border zone. Together, these data show that the Thr⁶⁴⁹ phosphorylation of AS160/TBC1D4 plays an important role in the heart’s electrical conduction system through regulating the R-wave amplitude.     

Exercise increases TBC1D1 phosphorylation in human skeletal muscle

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% Vo(2 max)). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser(711) (AMPK), TBC1D1 Ser(231) (AMPK), TBC1D1 Ser(660) (AMPK), TBC1D1 Ser(700) (AMPK), and TBC1D1 Thr(590) (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser(711), TBC1D1 Ser(231), and TBC1D1 Ser(660) but had no effect on TBC1D1 Ser(700). Exercise did not increase TBC1D1 Thr(590) phosphorylation or TBC1D1/AS160 PAS phosphorylation, consistent with the lack of Akt activation. These data demonstrate that a single bout of exercise regulates TBC1D1 and AS160 phosphorylation on multiple sites in human skeletal muscle.     

Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery

Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKalpha2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased ( approximately 35%) during postexercise recovery (P 相似文献   

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.     

Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle

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.     

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb     

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP)³ known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4–7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of AS160 (4). TBC1D1 is predominantly expressed in skeletal muscle; its expression in adipocytes is very low (5, 6). Nevertheless, 3T3-L1 adipocytes are a convenient cell type in which to examine the role of proteins in GLUT4 translocation, because insulin causes an ∼10-fold increase in GLUT4 at the cell surface. Previously, we examined the role of TBC1D1 in GLUT4 translocation by overexpressing it in 3T3-L1 adipocytes. Surprisingly, even though insulin led to phosphorylation of TBC1D1 on Akt site(s), ectopic TBC1D1 potently inhibited GLUT4 translocation (4, 5). By contrast, overexpression of AS160 did not inhibit GLUT4 translocation (8). This difference suggested that the regulation of TBC1D1 might be fundamentally different from that of AS160. In the present study, we show that this is not the case. By reducing the level of ectopic TBC1D1, we have obtained evidence that phosphorylation of TBC1D1 on several likely Akt sites relieves the inhibitory effect on GLUT4 translocation. In addition, we have examined the effect of a variant of TBC1D1 genetically associated with obesity on GLUT4 translocation and determined the relative levels of TBC1D1 in muscle biopsies from healthy and type 2 diabetic individuals.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney

TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na(+)-K(+)-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman's capsule, the thin and thick limbs of Henle's loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na(+)-K(+)-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.     

Regulation of glucose transporter 4 translocation by the Rab guanosine triphosphatase-activating protein AS160/TBC1D4: role of phosphorylation and membrane association

Insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane in muscle and fat cells depends on the phosphatidylinositide 3-kinase/Akt pathway. The downstream target AS160/TBC1D4 is phosphorylated upon insulin stimulation and contains a TBC domain (Tre-2/Bub2/Cdc16) that is present in most Rab guanosine triphosphatase-activating proteins. TBC1D4 associates with GLUT4-containing membranes under basal conditions and dissociates from membranes with insulin. Here we show that the association of TBC1D4 with membranes is required for its inhibitory action on GLUT4 translocation under basal conditions. Whereas the insulin-dependent dissociation of TBC1D4 from membranes was not required for GLUT4 translocation, its phosphorylation was essential. Many agonists that stimulate GLUT4 translocation failed to trigger TBC1D4 translocation to the cytosol, but in most cases these agonists stimulated TBC1D4 phosphorylation at T642, and their effects on GLUT4 translocation were inhibited by overexpression of the TBC1D4 phosphorylation mutant (TBC1D4-4P). We postulate that TBC1D4 acts to impede GLUT4 translocation by disarming a Rab protein found on GLUT4-containing-membranes and that phosphorylation of TBC1D4 per se is sufficient to overcome this effect, allowing GLUT4 translocation to the cell surface to proceed.     

Cellular distribution and contribution of cyclooxygenase COX-2 to diabetogenesis in NOD mouse

Unlike most other mammalian cells, beta-cells of Langerhans constitutively express cyclooxygenase (COX)-2 rather than COX-1. COX-2 is also constitutively expressed in type 1 diabetes (T1D) patients' periphery blood monocytes and macrophage. To understand the role of COX-2 in the beta-cell, we investigated COX-2 expression in beta-cells and islet infiltrates of NOD and BALB/c mice using fluorescence immunohistochemistry and cytochemical confocal microscopy and Western blotting. Immunostaining showed that COX-2 is expressed in islet-infiltrating macrophages, and that the expression of insulin and COX-2 disappeared concomitantly from the beta-cells when NOD mice progressed toward overt diabetes. Also cultured INS-1E cells coexpressed insulin and COX-2 but clearly in different subcellular compartments. Treatment with celecoxib increased insulin release from these cells in a dose-dependent manner in glucose concentrations ranging from 5 to 17 mM. Excessive COX-2 expression by the islet-infiltrating macrophages may contribute to the beta-cell death during insulitis. The effects of celecoxib on INS-1E cells suggest that PGE(2) and other downstream products of COX-2 may contribute to the regulation of insulin release from the beta-cells.     

A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.     

TBC proteins: GAPs for mammalian small GTPase Rab?

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.     

Cell surface trafficking of Fas in NIT-1 cells and dissection of surface and total Fas expression.

The appearance of Fas receptor at the surface of pancreatic beta-cells affected by progressive insulitis strongly suggests that Fas-mediated beta-cell apoptosis plays an important role in the pathogenesis of type 1 diabetes. In support of this concept, the present study has shown that islet cells from NOD mice and the beta-cell line NIT-1 respond to the proinflammatory cytokines IL-1beta and IFN-gamma with Fas surface expression in a dose- and time-dependent manner. Moreover, the prevention of cytokine-induced surface Fas expression by actinomycin D, cycloheximide, and brefeldin A demonstrated that trafficking of Fas to the beta-cell surface requires RNA and protein synthesis and, in addition is critically dependent on intracellular protein transport. Compared with total cellular Fas protein, the amount of Fas at the cell surface was relatively small and indicated that Fas is preferentially expressed in cytoplasmic compartments of NIT-1 cells. It is concluded that inflammatory insults specifically induce translocation of Fas to the beta-cell surface and that interference with cell surface Fas expression is a new strategy to improve beta-cell survival in inflamed islets.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.     

The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an Atypical Phosphotyrosine-Binding Domain That Interacts with Plasma Membrane Phospholipids To Facilitate GLUT4 Trafficking in Adipocytes

The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the present study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodes a phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved in other related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a nonoverlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs and not with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting of AS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the docking and/or fusion of GSVs with the PM.     

AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.     

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation

In humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.     

Extracts of Rehmanniae radix, Ginseng radix and Scutellariae radix improve glucose-stimulated insulin secretion and β-cell proliferation through IRS2 induction

Recent studies have revealed that beta-cell dysfunction is an important factor in developing type 2 diabetes. beta-cell dysfunction is related to impairment of the insulin/IGF-1 signaling cascade through insulin receptor substrate-2 (IRS2). The induction of IRS2 in beta-cells plays an important role in potentiating beta-cell function and mass. In this study, we investigated whether herbs used for treating diabetes in Chinese medicine-Galla rhois, Rehmanniae radix, Machilus bark, Ginseng radix, Polygonatum radix, and Scutellariae radix-improved IRS2 induction in rat islets, glucose-stimulated insulin secretion and beta-cell survival. R. radix, Ginseng radix and S. radix significantly enhanced glucose-stimulated insulin secretion compared to the control, i.e., by 49, 67 and 58%, respectively. These herbs induced the expression of IRS2, pancreas duodenum homeobox-1 (PDX-1), and glucokinase. The increased level of glucokinase could explain the enhancement of glucose-stimulated insulin secretion with these extracts. Increased PDX-1 expression was associated with beta-cell proliferation, which was consistent with the cell viability assay. In conclusion, R. radix, Ginseng radix and S. radix had an insulinotropic action similar to that of exendin-4.