The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.     

Involvement of Rabring7 in EGF receptor degradation as an E3 ligase

Thermosensitive TRP channels display unique thermal responses, suggesting distinct roles mediating sensory transmission of temperature. However, whether relative expression of these channels in dorsal root ganglia (DRG) is altered in nerve injury is unknown. We developed a multiplex ribonuclease protection assay (RPA) to quantify rat TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8 RNA levels in DRG. We used the multiplex RPA to measure thermosensitive TRP channel RNA levels in DRG from RTX-treated rats (300 microg/kg) or rats with unilateral sciatic nerve chronic constriction injury (CCI). TRPV1 and TRPA1 RNA were significantly decreased in DRG from RTX-treated rats, indicating functional colocalization of TRPA1 and TRPV1 in sensory nociceptors. In DRG from CCI rats, TRPA1, TRPV2, and TRPM8 RNA showed slight but significant increases ipsilateral to peripheral nerve injury. Our findings support the hypothesis that increased TRP channel expression in sensory neurons may contribute to mechanical and cold hypersensitivity.     

TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase

Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the “USP-like-TRIM” which regulates the activity of associated TRIM proteins.     

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.     

Identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is one of the most intensively studied approaches to cancer therapy. Rational design led to the identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors. Design, synthesis and structure activity relationship are reported.     

Design,synthesis and biological evaluation of novel 3H-imidazole [4,5-b] pyridine derivatives as selective mTOR inhibitors

A series of 3H-imidazo [4,5-b] pyridines derivatives were designed and synthesized as selective mTOR inhibitors. The systematic optimization of the molecules resulted in the identification of two compounds 10d and 10n with nanomolar mTOR inhibitory activity and selectivity over PI3Kα. Besides, compounds 10d and 10n demonstrated attractive potency against human breast cancer cells (MCF-7) and human ovarian cancer cell (A2780).     

缺氧诱导因子1与PI3K/Akt/mTOR信号转导通路

缺氧诱导因子1（HIF-1）是参与缺氧调节的核心因子,可调控一系列缺氧诱导基因的表达,与机体许多生理和病理过程也密切相关。尽管一些研究显示缺氧和非缺氧性刺激可通过PI3K/Akt/mTOR信号途径诱导HIF-1的表达和活性,PI3K信号途径是否参与对HIF-1的调节仍然是个有争议的研究热点。明确HIF-1和PI3K的相互作用关系,能进一步为肿瘤等相关疾病的防治提供新的思路和方法。本文主要就HIF-1和PI3K/Akt/mTOR关系作一简要综述。     

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.     

Discovery of benzenesulfonamide derivatives as potent PI3K/mTOR dual inhibitors with in vivo efficacies against hepatocellular carcinoma

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is related to cellular activities. Abnormalities of this signaling pathway were discovered in various cancers, including hepatocellular carcinoma (HCC). The PI3K/mTOR dual inhibitors were proposed to have enhanced antitumor efficacies by targeting multiple points of the signaling pathway. We synthesized a series of propynyl-substituted benzenesulfonamide derivatives as PI3K/mTOR dual inhibitors. Compound 7k (NSC781406) was identified as a highly potent dual inhibitor, which exhibited potent tumor growth inhibition in the hepatocellular carcinoma BEL-7404 xenograft model. Compound 7k may be a potential therapeutic drug candidate for HCC.     

Synthesis and structure-activity relationships of dual PI3K/mTOR inhibitors based on a 4-amino-6-methyl-1,3,5-triazine sulfonamide scaffold

Phosphoinositide 3-kinase (PI3K) is an important target in oncology due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of 4-amino-6-methyl-1,3,5-triazine sulfonamides were synthesized and evaluated as inhibitors of PI3K. The synthesis, in vitro biological activities, pharmacokinetic and in vivo pharmacodynamic profiling of these compounds are described. The most promising compound from this investigation (compound 3j) was found to be a pan class I PI3K inhibitor with a moderate (>10-fold) selectivity over the mammalian target of rapamycin (mTOR) in the enzyme assay. In a U87 MG cellular assay measuring phosphorylation of Akt, compound 3j displayed low double digit nanomolar IC(50) and exhibited good oral bioavailability in rats (F(oral)=63%). Compound 3j also showed a dose dependent reduction in the phosphorylation of Akt in a U87 tumor pharmacodynamic model with a plasma EC(50)=193nM (91ng/mL).     

Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.     

Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.