Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy

The mechanistic target of rapamycin complex 1 (mTORC1) increases translation, cell size and angiogenesis, and inhibits autophagy. mTORC1 is negatively regulated by hamartin and tuberin, the protein products of the tumor suppressors TSC1 and TSC2 that are mutated in Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM). Hamartin interacts with the centrosomal and mitotic kinase polo-like kinase 1 (PLK1). Hamartin and tuberin deficient cells have abnormalities in centrosome duplication, mitotic progression, and cytokinesis, suggesting that the hamartin/tuberin heterodimer and mTORC1 signaling are involved in centrosome biology and mitosis. Here we report that PLK1 protein levels are increased in hamartin and tuberin deficient cells and LAM patient-derived specimens, and that this increase is rapamycin-sensitive. Pharmacological inhibition of PLK1 by the small-molecule inhibitor BI-2536 significantly decreased the viability and clonogenic survival of hamartin and tuberin deficient cells, which was associated with increased apoptosis. BI-2536 increased p62, LC3B-I and GFP-LC3 punctae, and inhibited HBSS-induced degradation of p62, suggesting that PLK1 inhibition attenuates autophagy. Finally, PLK1 inhibition repressed the expression and protein levels of key autophagy genes and proteins and the protein levels of Bcl^-2 family members, suggesting that PLK1 regulates both autophagic and apoptotic responses. Taken together, our data point toward a previously unrecognized role of PLK1 on the survival of cells with mTORC1 hyperactivation, and the potential use of PLK1 inhibitors as novel therapeutics for tumors with dysregulated mTORC1 signaling, including TSC and LAM.     

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.     

IFN-γ/mTORC1 decreased Rab11 in Schwann cells of diabetic peripheral neuropathy,inhibiting cell proliferation via GLUT1 downregulation

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus. Rab11 is conserved gene-regulating vesicle traffic and reported to be involved in the pathogenesis of diabetes mellitus by affecting insulin sensitivity. We aimed to investigate the role of Rab11 in the pathogenesis of DPN. In this study, Rab11 expression decreased in the sciatic nerves of diabetic mice with impaired conduction function versus those of normal mice. In vitro experiment revealed interferon-γ (IFN-γ), not high glucose and interleukin 1β was the main factor to lead to Rab11 downregulation in RSC96 cells. Again, both Rab11 knockdown and IFN-γ treatment caused cell viability inhibition and the decrease in BrdU-positive cells. In contrast, overexpression of Rab11 reversed IFN-γ-reduced cell proliferation. Furthermore, mTORC1 not mTORC2 was proven to be suppressed by IFN-γ treatment in RSC96 cells, indicated in decreased phospho-p70S6K. Inhibition of the mTORC1 pathway resulted in Rab11 expression downregulation in RSC96 cells. Activation of the mTORC1 pathway effectively prevented IFN-γ-reduced Rab11 expression in RSC96 cells. Also, glucose transporter 1 (GLUT1) was found to be downregulated in RSC96 cells with Rab11 silence and overexpression of GLUT1 reversed Rab11 blocking-caused proliferation inhibition. Taken together, our findings suggest that IFN-γ decreases Rab11 expression via the inhibition of the mTORC1 signaling pathway, causing reduced cell proliferation in Schwann cells of DPN by GLUT1 downregulation.     

The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Although TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (Mtor^A/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promotes mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 coimmunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal cross talk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.     

Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation

Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.     

Defective Autophagy and mTORC1 Signaling in Myotubularin Null Mice

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.     

TBK1 has a new Akt

TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley et al. demonstrated that TBK1-dependent activation of downstream kinase Akt is mediated via mechanistic target of rapamycin complex 2. This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.

TANK-binding kinase 1 (TBK1), a serine/threonine kinase that belongs to the noncanonical IκB kinase family, plays an essential role in the innate immune response to viral and bacterial pathogens by regulating the type I interferon–mediated T cell response (1). Although TBK1 has been most widely studied in this context, more recent investigations using tissue-specific KO mice and drugs that inhibit kinase activity have revealed novel roles for this kinase in nonimmune cells, particularly at the intersection of immunity and metabolism. For example, TBK1 expression and activity are induced in adipose tissue in obesity by elevated expression of proinflammatory cytokines such as tumor necrosis factor α (2). TBK1 contributes to obesity by repressing energy expenditure and increasing anabolic functions as determined from analysis of mice with conditional adipose cell KO of TBK1 (3). TBK1 has also been reported to promote activation of Akt, a central kinase involved in metabolic regulation (4). However, the mechanism by which TBK1 regulates Akt has remained unclear.Akt is an essential regulator of glucose metabolism and plays an important role in controlling cellular glucose uptake and utilization through both positive and negative regulatory actions (4). Phosphorylation of Akt on T308 in its activation loop stimulates kinase activity, and phosphorylation on S473 further enhances activity and determines substrate specificity (4). Although it had been previously reported that TBK1 can directly phosphorylate Akt at S473 and T308 in in vitro kinase assays, the ability of TBK1 to mediate these phosphorylation events under physiological conditions was not known (5). In a recent study, Tooley et al. (6) contributed to the mechanistic understanding of TBK1 function in metabolic regulation by demonstrating a role for TBK1 in mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) activation and subsequent phosphorylation of Akt.To investigate how TBK1 regulates Akt activation, mouse embryonic fibroblasts (MEFs) were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and Akt-T308 phosphorylation (6). The intensity and duration of Akt phosphorylation at both sites was diminished significantly, both in the absence of TBK1 and in the presence of the TBK1 inhibitor amlexanox. Restoration of endogenous levels of TBK1, but not kinase-dead TBK1, rescued EGF-stimulated Akt-S473 phosphorylation. The stimulation of Akt-S473 phosphorylation by EGF, as well as by other growth factors and the hormone insulin, was found to be dependent upon mTOR activity. Together, these results validate the ability of TBK1 to regulate Akt-S473 phosphorylation and show that in response to normal growth regulatory signaling, this regulation is mediated through mTOR kinase.The kinase mTOR is the core catalytic kinase of two multisubunit complexes, mTOR complex 1 (mTORC1) and mTORC2, which are distinguished by the scaffolding proteins Raptor and Rictor, respectively (7). mTORC1 is regulated by the combination of growth factor/hormone signaling and nutrient availability to drive anabolic metabolism. mTORC2, on the other hand, is regulated by growth factor/hormone signaling to activate Akt. Together, mTORC1 and mTORC2 are key signaling nodes in the regulation of cell growth and proliferation, and dysregulation of these signaling pathways contributes to metabolic disease and cancer. In previous investigations, the authors had demonstrated that phosphorylation of mTOR on S2159 by TBK1 enhanced mTORC1 activation and downstream signaling to promote cell growth and proliferation (8). To investigate if TBK1 acts upstream of mTORC2 to regulate Akt-S473 phosphorylation through a similar mechanism, MEFs derived from mice with an alanine knock-in at S2159 (Mtor^A/A) were stimulated with EGF. A marked reduction of Akt-S473 phosphorylation was observed in Mtor^A/A MEFs compared with WT MEFs (Mtor^+/+). Using immunoprecipitation of Rictor to isolate the mTORC2 complex, TBK1 was observed to interact with mTORC2 and directly phosphorylate mTOR-S2159 to activate mTORC2 intrinsic kinase activity toward Akt-S473. TBK1 activity is increased by phosphorylation of S172 in its activation loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al. (6) found that EGF stimulation did not enhance S172 phosphorylation, supporting that it is the basal activity of TBK1 that is important for mTORC2 signaling downstream of growth factors. However, when RAW264.7 macrophages and primary bone marrow–derived macrophages were stimulated with the dsRNA mimetic poly(I:C), which induces TBK1-S172 phosphorylation, TBK1 and mTOR-S2159 were also found to be required for mTORC2-dependent phosphorylation of Akt-S473. Finally, the physiological regulation of mTORC2 activity by TBK1 was assessed by injection of Mtor^A/A and Mtor^+/+ mice with poly(I:C). Spleen tissue isolated from Mtor^A/A mice showed diminished Akt-S473 phosphorylation. Therefore, the authors conclude that under both basal and activated states, the activation of Akt by TBK1 is mediated through mTORC2 (Fig. 1) (6).Open in a separate windowFigure 1TBK1 promotes AKT activation through mTORC2. TBK1 interacts with and phosphorylates mTORC2 on S2159 of mTOR in response to either growth factor stimulation or innate immune agonists to promote AKT activation. Created using BioRender.com. mTORC2, mTOR complex 2; SGK, serum/glucocorticoid-regulated kinase; TBK1, TANK-binding kinase 1.TBK1 regulation of mTORC2-dependent phosphorylation of Akt shown in this study adds to the growing role of TBK1 as a signaling node in the regulation of cellular metabolism and growth by diverse environmental inputs. In response to foreign pathogens or inflammatory cytokines that stimulate TBK1 activation, or growth factor/hormone signaling that requires basal TBK1 activity, mTORC2 is activated to promote Akt-S473 phosphorylation and its downstream functions. Given that TBK1 expression and activity are enhanced in metabolic diseases and cancer, and the important role that Akt plays in these pathological conditions, identifying TBK1 as an upstream regulator of Akt reveals a potential novel approach to disrupt this signaling axis for therapeutic benefit (4, 9). In this regard, drugs such as amlexanox and other compounds are under investigation for their potential clinical use (10). Of note, the study by Tooley et al. (6) only examined the TBK1-dependent phosphorylation of Akt-S473 by mTORC2; mTORC2 also has additional substrates, including serum/glucocorticoid-regulated kinase and members of the PKC family (Fig. 1) (4). These kinases regulate unique cellular functions, such as regulation of the actin cytoskeleton. It will be important to determine if TBK1 regulates the activation of these kinases through mTORC2 as well, to understand the full impact of inhibiting TBK1 function therapeutically.The mechanism by which TBK1 regulates mTORC2 function has not been established. Although the kinase activity of TBK1 is required for Akt-S473 phosphorylation, neither phosphorylation of S172 in the activation loop of TBK1 nor phosphorylation of mTOR-S2159 was increased by growth factor stimulation in this study. Phosphorylation of S172 stabilizes the active confirmation of TBK1 and it is possible that additional uncharacterized phosphorylation sites could serve a similar function. Alternatively, the interaction of TBK1 with mTORC2 could impact TBK1 conformation, or multimerization, to enhance activity. Intracellular localization of mTORC2 could also be determined by TBK1 interaction, which could affect substrate availability. As little is known about the upstream regulation of mTORC2, the next acts should be elucidating further the mechanism of its activation by TBK1 to reveal novel approaches for targeting the mTORC2-Akt signaling pathway.     

Rab35 GTPase recruits NDP52 to autophagy targets

Autophagy targets intracellular molecules, damaged organelles, and invading pathogens for degradation in lysosomes. Recent studies have identified autophagy receptors that facilitate this process by binding to ubiquitinated targets, including NDP52. Here, we demonstrate that the small guanosine triphosphatase Rab35 directs NDP52 to the corresponding targets of multiple forms of autophagy. The active GTP‐bound form of Rab35 accumulates on bacteria‐containing endosomes, and Rab35 directly binds and recruits NDP52 to internalized bacteria. Additionally, Rab35 promotes interaction of NDP52 with ubiquitin. This process is inhibited by TBC1D10A, a GAP that inactivates Rab35, but stimulated by autophagic activation via TBK1 kinase, which associates with NDP52. Rab35, TBC1D10A, and TBK1 regulate NDP52 recruitment to damaged mitochondria and to autophagosomes to promote mitophagy and maturation of autophagosomes, respectively. We propose that Rab35‐GTP is a critical regulator of autophagy through recruiting autophagy receptor NDP52.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

TBC1D7 Is a Third Subunit of the TSC1-TSC2 Complex Upstream of mTORC1

The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits?cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit?of the?TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is?the functional complex that senses specific cellular?growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.     

Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy

The mechanistic target of rapamycin complex 1 (mTORC1) increases translation, cell size and angiogenesis, and inhibits autophagy. mTORC1 is negatively regulated by hamartin and tuberin, the protein products of the tumor suppressors TSC1 and TSC2 that are mutated in Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM). Hamartin interacts with the centrosomal and mitotic kinase polo-like kinase 1 (PLK1). Hamartin and tuberin deficient cells have abnormalities in centrosome duplication, mitotic progression, and cytokinesis, suggesting that the hamartin/tuberin heterodimer and mTORC1 signaling are involved in centrosome biology and mitosis. Here we report that PLK1 protein levels are increased in hamartin and tuberin deficient cells and LAM patient-derived specimens, and that this increase is rapamycin-sensitive. Pharmacological inhibition of PLK1 by the small-molecule inhibitor BI-2536 significantly decreased the viability and clonogenic survival of hamartin and tuberin deficient cells, which was associated with increased apoptosis. BI-2536 increased p62, LC3B-I and GFP-LC3 punctae, and inhibited HBSS-induced degradation of p62, suggesting that PLK1 inhibition attenuates autophagy. Finally, PLK1 inhibition repressed the expression and protein levels of key autophagy genes and proteins and the protein levels of Bcl^-2 family members, suggesting that PLK1 regulates both autophagic and apoptotic responses. Taken together, our data point toward a previously unrecognized role of PLK1 on the survival of cells with mTORC1 hyperactivation, and the potential use of PLK1 inhibitors as novel therapeutics for tumors with dysregulated mTORC1 signaling, including TSC and LAM.     

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.     

The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene‐induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site‐specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF‐receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus‐selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock‐in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN‐β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1‐mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation.     

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation.     

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.     

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.     

Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino‐acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton‐coupled amino‐acid transporter 4), whose accumulation in Rab12‐knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.     

A Highlights from MBoC Selection: Insulin-promoted mobilization of GLUT4 from a perinuclear storage site requires RAB10

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.