sirolimus and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

The involvement of mammalian target of rapamycin (mTOR) in the positive regulation of oncogenesis has been well documented and thus mTOR has emerged as an attractive cancer therapeutic target. Although rapamycin and its analogues (rapalogs) are FDA-approved for the treatment of certain cancers, major success in targeting mTOR, particularly with new generation mTOR kinase inhibitors, for the effective treatment of cancers has not been achieved. Hence, a thorough understanding of the biology of the mTOR axis in cancer is still needed. It is now recognized that programmed death-ligand 1 (PD-L1) expression on cancer cells is a critical mechanism contributing to immunosuppression and immune escape via interacting with program death-1 (PD-1) on immune cells. This study has revealed a previously undiscovered role of the mTOR complex 1 (mTORC1)/p70 S6 kinase (p70S6K) in the negative regulation of PD-L1 on cancer cells and tissues. We demonstrate that disruption of this signaling pathway with mTOR inhibitors, raptor knockdown or p70S6K inhibitors elevated PD-L1 levels in some lung and other cancer cell lines. Elevation of PD-L1 by inhibition of mTORC1/p70S6K signaling is likely due to suppression of β-TrCP-mediated proteasomal degradation of PD-L1, because inhibition of either mTORC1 or p70S6K facilitated β-TrCP degradation accompanied with enhanced PD-L1 protein stabilization. Our current findings indicate the complexity of the mTOR axis in cancer, which should be considered when targeting this axis for effective cancer treatment. Our findings also suggest a strong scientific rationale for enhancing PD-1/PD-L1-targeted cancer immunotherapy through co-targeting mTORC1/p70S6K signaling.

Topics: A549 Cells; B7-H1 Antigen; Benzoxazoles; beta-Transducin Repeat-Containing Proteins; Cycloheximide; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Leupeptins; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Morpholines; Naphthyridines; Neoplasms; Protein Stability; Pyrimidines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Tumor Cells, Cultured

MG132 is a pivotal inhibitor of the ubiquitin-proteasome system (UPS), and rapamycin (RAPA) is an important inducer of autophagy. MG132 and RAPA have been shown to be effective agents that can cure multiple autoimmune diseases by reducing inflammation. Although individual MG132 and RAPA showed protective effects for atherosclerosis (AS), the combined effect of these two drugs and its molecular mechanism are still unclear. In this article we investigate the regulation of oxidative modification of low-density lipoprotein (ox-LDL) stress and foam cell formation in the presence of both proteasome inhibitor MG132 and the autophagy inducer RAPA to uncover the molecular mechanism underlying this process. We established the foam cells model by ox-LDL and an animal model. Then, we tested six experimental groups of MG132, RAPA, and 3MA drugs. As a result, RAPA-induced autophagy reduces accumulation of polyubiquitinated proteins and apoptosis of foam cells. The combination of MG132 with RAPA not only suppressed expression of the inflammatory cytokines and formation of macrophage foam cells, but also significantly affected the NF-κB signaling pathway and the polarization of RAW 264.7 cells. These data suggest that the combination of proteasome inhibitor and autophagy inducer ameliorates the inflammatory response and reduces the formation of macrophage foam cells during development of AS. Our research provides a new way to suppress vascular inflammation and stabilize plaques of late atherosclerosis.

Topics: Animals; Autophagy; Cytokines; Foam Cells; Leupeptins; Lipoproteins, LDL; Macrophages; Mice; Proteasome Inhibitors; RAW 264.7 Cells; Sirolimus; Ubiquitinated Proteins

The objective of this study is to investigate the autophagy activity of cells by the intracellular release of rapamycin (Rapa) of an autophagy inducer. Rapa was incorporated into nanospheres of poly (lactic-co-glycolic acid) (PLGA) for the controlled release of Rapa. Rapa was released from the PLGA nanospheres incorporating rapamycin (Rapa-PLGA-NS) with time while the Rapa-PLGA-NS were hydrolytically degraded. When human hepatocellular carcinoma (HepG2) cells were incubated with the Rapa-PLGA-NS, the Rapa-PLGA-NS were internalized, and the intracellular concentration was maintained over four days, indicating the intracellular Rapa release. The microtubule-associated protein 1 light chain (LC3) of an autophagy marker was significantly high for the Rapa-PLGA-NS group compared with the free Rapa group even after four days incubation. In addition, intracellular harmful ubiquitinated proteins were degraded by the intracellular release of Rapa even after four days incubation in contrast to free Rapa. It is concluded that the intracellular Rapa release is effective in modulating the autophagy activity over a longer time period.

Topics: Antineoplastic Agents; Autophagy; Drug Carriers; Drug Liberation; Hep G2 Cells; Humans; Lactic Acid; Leupeptins; Microtubule-Associated Proteins; Nanospheres; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Proteasome Inhibitors; Sirolimus; Surface Properties

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Hypoxia; Cell Line; Cell Survival; Doxorubicin; Endoplasmic Reticulum Stress; Histone Deacetylase 6; Histone Deacetylases; Leupeptins; Lung; Male; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Rats; Reperfusion Injury; Sirolimus; Ubiquitin

MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment.

Topics: Amino Acid Sequence; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Biomarkers; Bombyx; Cell Line; Green Fluorescent Proteins; Insect Proteins; Leupeptins; Lysosomes; Macrolides; Proteasome Inhibitors; Proteolysis; Sirolimus

Biologists have long been concerned about what constrains variation in cell size, but progress in this field has been slow and stymied by experimental limitations. Here we describe a new method, ergodic rate analysis (ERA), that uses single-cell measurements of fixed steady-state populations to accurately infer the rates of molecular events, including rates of cell growth. ERA exploits the fact that the number of cells in a particular state is related to the average transit time through that state. With this method, it is possible to calculate full time trajectories of any feature that can be labelled in fixed cells, for example levels of phosphoproteins or total cellular mass. Using ERA we find evidence for a size-discriminatory process at the G1/S transition that acts to decrease cell-to-cell size variation.

Topics: Cell Count; Cell Cycle; Cell Line; Cell Proliferation; Cell Size; Cycloheximide; Dimethyl Sulfoxide; Feedback, Physiological; G1 Phase; HeLa Cells; Humans; Leupeptins; Phosphoproteins; S Phase; Single-Cell Analysis; Sirolimus

Tuberous sclerosis complex (TSC), an inherited tumor predisposition syndrome associated with mutations in TSC1 or TSC2, affects ∼1 in 6,000 individuals. Eighty percent of TSC patients develop renal angiomyolipomas, and renal involvement is a major contributor to patient morbidity and mortality. Recent work has shown that mammalian target of rapamycin complex 1 (mTORC1) inhibition caused angiomyolipoma shrinkage but that this treatment may cause cytostatic not a cytotoxic effect. Endoplasmic reticulum (ER) stress can develop in TSC-associated cells due to mTORC1-driven protein translation. We hypothesized that renal angiomyolipoma cells experience ER stress that can be leveraged to result in targeted cytotoxicity. We used immortalized human angiomyolipoma cells stably transfected with empty vector or TSC2 (encoding tuberin). Using cell number quantification and cell death assays, we found that mTORC1 inhibition with RAD001 suppressed angiomyolipoma cell proliferation in a cytostatic manner. Angiomyolipoma cells exhibited enhanced sensitivity to proteasome inhibitor-induced ER stress compared with TSC2-rescued cells. After proteasome inhibition with MG-132, Western blot analyses showed greater induction of C/EBP-homologous protein (CHOP) and more poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage, supporting ER stress-induced apoptosis. Live cell numbers also were decreased and cell death increased by MG-132 in angiomyolipoma cells compared with TSC2 rescued. Intriguingly, while pretreatment of angiomyolipoma cells with RAD001 attenuated CHOP and BiP induction, apoptotic markers cleaved PARP and caspase-3 and eukaryotic translation initiation factor 2α phosphorylation were increased, along with evidence of increased autophagy. These results suggest that human angiomyolipoma cells are uniquely susceptible to agents that exacerbate ER stress and that additional synergy may be achievable with targeted combination therapy.

Topics: Angiomyolipoma; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Everolimus; Humans; Immunosuppressive Agents; Kidney Neoplasms; Leupeptins; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphorylation; Poly(ADP-ribose) Polymerases; Proteasome Inhibitors; Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factor CHOP; Transfection; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell. Here the effects of biologically-relevant concentrations and short incubations (1-16 h) of perillyl alcohol or the mTOR inhibitor, rapamycin, on telomerase activity were examined in prostate cancer cell lines. A rapid suppression of telomerase activity was observed (from ∼65% to >95%) determined by real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel-analysis. Using real-time reverse transcriptase-PCR, we demonstrated that human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered. Western blot analysis revealed that hTERT protein levels decreased in response to perillyl alcohol or rapamycin. This decrease was partially blocked by pretreatment with a proteasome inhibitor MG-132, indicating that proteasomal degradation contributed to the loss of hTERT protein. No change in hTERT phosphorylation at Ser824 was observed, indicating the absence of cellular hTERT protein redistribution. These findings provide evidence for a unique link between nutrient- and macrolide-mediated regulation of mTOR and hTERT, a key enzyme that regulates DNA structure and stability.

Topics: Blotting, Western; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Male; Monoterpenes; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Telomerase; TOR Serine-Threonine Kinases

Despite incremental improvements in outcomes for patients with acute lymphoblastic leukemia, significant numbers of patients still die from this disease. Mammalian target of rapamycin inhibitors have shown potential in vitro and in vivo as therapeutic agents against a range of tumors including acute lymphoblastic leukemia.. Flow cytometry was used to evaluate drug-induced cell death in acute lymphoblastic leukemia cell lines and patients' samples. Human xenografts in immunocompromised mice were used to assess the in vivo effects of selected combinations. Pharmacological inhibitors and lentiviral small interfering ribonucleic acid knock-down of p53 were used to investigate the mechanism of cell killing involved.. Synergistic interactions between RAD001 and cytotoxic agents were demonstrated in vitro and in vivo, with increased caspase-dependent killing. RAD001 suppressed p53 and p21 responses, while suppression of p53 did not prevent killing, indicating p53 independence. RAD001 and cytotoxic agents activated the JUN N-terminal kinase pathway and the combination further increased JUN N-terminal kinase activation. JUN N-terminal kinase inhibition reduced synergistic cell killing by cytotoxic agents and RAD001 in pre-B acute lymphoblastic leukemia cell lines and patients' samples. Bortezomib and MG132, which activate the JUN N-terminal kinase pathway, also synergized with RAD001 in killing pre-B acute lymphoblastic leukemia cells. Killing was greater when RAD001 was combined with proteasome inhibitors than with cytotoxic drugs.. These observations suggest that combining mammalian target of rapamycin inhibitors with conventional chemotherapy or selected novel agents has the potential to improve clinical responses in patients with pre-B acute lymphoblastic leukemia.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Cell Line, Tumor; Combined Modality Therapy; Everolimus; Humans; Immunosuppressive Agents; Leupeptins; Mice; Mice, Inbred NOD; Mice, SCID; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Protease Inhibitors; Pyrazines; Radiation, Ionizing; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Whole-Body Irradiation

We previously identified a novel interaction between tuberous sclerosis-2 (TSC2) and death-associated protein kinase-1 (DAPK), the consequence being that DAPK catalyses the inactivating phosphorylation of TSC2 to stimulate mammalian target of rapamycin complex 1 (mTORC1) activity. We now report that TSC2 binding to DAPK promotes the degradation of DAPK. We show that DAPK protein levels, but not gene expression, inversely correlate with TSC2 expression. Furthermore, altering mTORC1 activity does not affect DAPK levels, excluding indirect effects of TSC2 on DAPK protein levels through changes in mTORC1 translational control. We provide evidence that the C-terminus regulates TSC2 stability and is required for TSC2 to reduce DAPK protein levels. Importantly, using a GTPase-activating protein-dead missense mutation of TSC2, we demonstrate that the effect of TSC2 on DAPK is independent of GTPase-activating protein activity. TSC2 binds to the death domain of DAPK and we show that this interaction is required for TSC2 to reduce DAPK protein levels and half-life. Finally, we show that DAPK is regulated by the lysosome pathway and that lysosome inhibition blocks TSC2-mediated degradation of DAPK. Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK stability, which taken together with our previous findings, reveal a regulatory loop between DAPK and TSC2 whose balance can either promote: (a) TSC2 inactivation resulting in mTORC1 stimulation, or (b) DAPK degradation via TSC2 signalling under steady-state conditions. The fine balance between DAPK and TSC2 in this regulatory loop may have subtle but important effects on mTORC1 steady-state function.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis Regulatory Proteins; Autophagy; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Cell Line; Cell Line, Tumor; Chloroquine; Death-Associated Protein Kinases; Fibroblasts; Gene Expression; HEK293 Cells; Humans; Leupeptins; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred Strains; Mice, Knockout; Models, Biological; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Mutation, Missense; Neuropeptides; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Interaction Domains and Motifs; Proteins; Ras Homolog Enriched in Brain Protein; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases; RNA, Small Interfering; Sequence Deletion; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis. Incubation of C2C12 myotubes with 0.2 x physiological amino acids concentration (0.2 x PC AA), relative to 1.0 x PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 x PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0 x PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 x PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 x PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucine. In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

Topics: Amino Acids; Animals; Cell Line; Insulin; Leucine; Leupeptins; Mice; Muscle Fibers, Skeletal; Myoblasts; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Signal Transduction; Sirolimus; Ubiquitin-Conjugating Enzymes

Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.

Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Anisomycin; CHO Cells; Cloning, Molecular; Cricetinae; Cricetulus; Cysteine Proteinase Inhibitors; Diagnostic Imaging; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Leupeptins; Long-Term Potentiation; Male; Membrane Glycoproteins; Microtubule-Associated Proteins; Neurons; Nuclear Localization Signals; Patch-Clamp Techniques; Perforant Pathway; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Rats; Rats, Wistar; Sirolimus; Synapses; Time Factors; Transfection; Viral Envelope Proteins

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Topics: Acetylcysteine; Adenine; Animals; Autophagy; Cell Line; Chromones; Epidermal Growth Factor; Glyceraldehyde-3-Phosphate Dehydrogenases; Kidney Tubules; Leupeptins; Lysosomes; Methylamines; Morpholines; PAX2 Transcription Factor; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proteasome Endopeptidase Complex; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3.. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level.. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

Topics: Animals; beta Catenin; Cell Cycle; Cell Division; Cell Line; Chromones; Cyclin D; Cyclins; Fibroblasts; Genes, bcl-1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; Lithium Chloride; Mice; Morpholines; NIH 3T3 Cells; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphothreonine; Protein Biosynthesis; Protein Kinases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Fusion Proteins; S Phase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Tyrosine dephosphorylation, serine phosphorylation, and proteasomal degradation of insulin receptor substrates (IRSs) are implicated in the negative regulation of insulin action. Here we show that simultaneous inhibition of IRS-1 tyrosine dephosphorylation and proteasomal degradation synergistically augments insulin-responsive glucose uptake. L6 skeletal muscle cells (L6 cells) were treated with inhibitors of protein-tyrosine phosphatases, proteasomal degradation, and mammalian target of rapamycin (mTOR), and the effects of insulin on glucose uptake, IRS-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity, and IRS-1 mass were examined. Pretreatment of L6 cells with sodium orthovanadate (Na(3)VO(4)) plus the mTOR inhibitor rapamycin caused a 5-fold increase in insulin-responsive glucose uptake at 2 hours when compared to insulin alone. Evaluation of IRS-1 associated PI 3-kinase activity, IRS-1-associated p85 mass, and IRS-1 tyrosine phosphorylation showed that 2 hours after insulin addition they were reduced by 70% from maximal activity. Likewise, IRS-1 mass was reduced by 50%. When L6 cells were pretreated with Na(3)VO(4) plus the proteasome inhibitor MG-132 or the mTOR inhibitor rapamycin prior to insulin addition, IRS-1 mass loss as well as IRS-1/PI-3 kinase complex decay was blocked at 2 hours and PI 3-kinase activity was increased 2.5-fold and 4-fold, respectively, over insulin alone. Finally, treatment of L6 cells with subtherapeutic amounts of vanadyl sulfate and rapamycin induced a synergistic 3-fold increase in insulin-induced glucose uptake at 2 hours. These findings indicate that vanadium and rapamycin synergize to enhance glucose uptake by preventing IRS-1 mass loss and IRS-1/PI 3-kinase complex decay and may offer a new approach to enhance glucose transport in diabetes.

Topics: Animals; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Synergism; Glucose; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Leupeptins; Multienzyme Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proteasome Endopeptidase Complex; Sirolimus; Tyrosine; Vanadates