scholarly journals mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

2009 ◽  
Vol 30 (4) ◽  
pp. 908-921 ◽  
Author(s):  
Louis-Andre Julien ◽  
Audrey Carriere ◽  
Julie Moreau ◽  
Philippe P. Roux
Keyword(s):  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Glycogen Synthase ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Growth Factor Signaling ◽  
Mtorc1 Signaling ◽  
Ribosomal S6 Kinase

ABSTRACT The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.

scholarly journals BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

2006 ◽  
Vol 401 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Gopal P. Sapkota ◽  
Lorna Cummings ◽  
Felicity S. Newell ◽  
Christopher Armstrong ◽  
Jennifer Bain ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Camp Response Element Binding ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

scholarly journals The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

2002 ◽  
Vol 13 (6) ◽  
pp. 2016-2030 ◽  
Author(s):  
Mitsuru Okuwaki ◽  
Masafumi Tsujimoto ◽  
Kyosuke Nagata
Keyword(s):  
Cell Cycle ◽  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Rna Binding ◽  
Intracellular Localization ◽  
Binding Activity ◽  
Cyclin B ◽  
Cycle Dependent

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.

scholarly journals An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss)

2008 ◽  
Vol 295 (1) ◽  
pp. R329-R335 ◽  
Author(s):  
Iban Seiliez ◽  
Jean-Charles Gabillard ◽  
Sandrine Skiba-Cassy ◽  
Daniel Garcia-Serrana ◽  
Joaquim Gutiérrez ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Target Of Rapamycin ◽  
Nutritional Regulation ◽  
Tor Signaling ◽  
Meal Feeding

In mammals, feeding promotes protein accretion in skeletal muscle through a stimulation of the insulin- and amino acid- sensitive mammalian target of rapamycin (mTOR) signaling pathway, leading to the induction of mRNA translation. The purpose of the present study was to characterize both in vivo and in vitro the activation of several major kinases involved in the mTOR pathway in the muscle of the carnivorous rainbow trout. Our results showed that meal feeding enhanced the phosphorylation of the target of rapamycin (TOR), PKB, p70 S6 kinase, and eIF4E-binding protein-1, suggesting that the mechanisms involved in the regulation of mRNA translation are well conserved between lower and higher vertebrates. Our in vitro studies on primary culture of trout muscle cells indicate that insulin and amino acids regulate TOR signaling and thus may be involved in meal feeding effect in this species as in mammals. In conclusion, we report here for the first time in a fish species, the existence and the nutritional regulation of several major kinases involved in the TOR pathway, opening a new area of research on the molecular bases of amino acid utilization in teleosts.

scholarly journals LARP1 is a major phosphorylation substrate of mTORC1

2018 ◽  
Author(s):  
Bruno D. Fonseca ◽  
Jian-Jun Jia ◽  
Anne K. Hollensen ◽  
Roberta Pointet ◽  
Huy-Dung Hoang ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Cellular Functions ◽  
Multiple Substrates ◽  
Eif4f Complex ◽  
Mtorc1 Pathway ◽  
Terminal Oligopyrimidine

AbstractThe mammalian target of rapamycin complex 1 (mTORC1) controls critical cellular functions such as protein synthesis, lipid metabolism, protein turnover and ribosome biogenesis through the phosphorylation of multiple substrates. In this study, we examined the phosphorylation of a recently identified target of mTORC1: La-related protein 1 (LARP1), a member of the LARP superfamily. Previously, we and others have shown that LARP1 plays an important role in repressing TOP mRNA translation downstream of mTORC1. LARP1 binds the 7-methylguanosine triphosphate (m7Gppp) cap moiety and the adjacent 5’terminal oligopyrimidine (5’TOP) motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 plays a critical role in the control of TOP mRNA translation via LARP1 but the precise mechanism by which this occurs is incompletely understood. The data described herein help to elucidate this process. Specifically, it show that: (i) mTORC1 interacts with LARP1, but not other LARP superfamily members, via the C-terminal region that comprises the DM15 domain, (ii) mTORC1 pathway controls the phosphorylation of multiple (up to 26) serine and threonine residues on LARP1 in vivo, (iii) mTORC1 regulates the binding of LARP1 to TOP mRNAs and (iv) phosphorylation of S689 by mTORC1 is particularly important for the association of the DM15 domain of LARP1 with the 5’UTR of RPS6 TOP mRNA. These data reveal LARP1 as a major substrate of mTORC1.

scholarly journals mTORC1 Inhibition Is Required for Sensitivity to PI3K p110α Inhibitors inPIK3CA-Mutant Breast Cancer

2013 ◽  
Vol 5 (196) ◽  
pp. 196ra99-196ra99 ◽  
Author(s):  
Moshe Elkabets ◽  
Sadhna Vora ◽  
Dejan Juric ◽  
Natasha Morse ◽  
Mari Mino-Kenudson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Mammalian Target Of Rapamycin ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Clinical Activity ◽  
Mtorc1 Signaling

Activating mutations of thePIK3CAgene occur frequently in breast cancer, and inhibitors that are specific for phosphatidylinositol 3-kinase (PI3K) p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition amongPIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly, in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. InPIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as insulin-like growth factor 1 and neuregulin 1 can activate mammalian target of rapamycin (mTOR) and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α-targeted drugs and delay the appearance of resistance.

scholarly journals Identification and targeting of novel CDK9 complexes in acute myeloid leukemia

Blood ◽  
2019 ◽  
Vol 133 (11) ◽  
pp. 1171-1185 ◽  
Author(s):  
Elspeth M. Beauchamp ◽  
Sameem M. Abedin ◽  
Sara G. Radecki ◽  
Mariafausta Fischietti ◽  
Ahmet Dirim Arslan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Mtor Signaling ◽  
Signaling Activation ◽  
Acute Myeloid

Abstract Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

scholarly journals Basement Membrane Laminin α2 Regulation of BTB Dynamics via Its Effects on F-Actin and Microtubule Cytoskeletons Is Mediated Through mTORC1 Signaling

Endocrinology ◽  
2017 ◽  
Vol 158 (4) ◽  
pp. 963-978 ◽  
Author(s):  
Ying Gao ◽  
Haiqi Chen ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
C. Yan Cheng
Keyword(s):  
Basement Membrane ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Mammalian Target Of Rapamycin ◽  
Growth Factor Receptor ◽  
Negative Control ◽  
Mtorc1 Signaling ◽  
Laminin Α2

Abstract A local axis connects the apical ectoplasmic specialization (ES) at the Sertoli-spermatid interface, the basal ES at the blood–testis barrier (BTB), and the basement membrane across the seminiferous epithelium functionally in rat testes. As such, cellular events that take place simultaneously across the epithelium such as spermiation and BTB remodeling that occur at the apical ES and the basal ES, respectively, at stage VIII of the cycle are coordinated. Herein, laminin α2, a structural component of the basement membrane, was found to regulate BTB dynamics. Sertoli cells were cultured in vitro to allow the establishment of a tight junction (TJ) barrier that mimicked the BTB in vivo. Knockdown of laminin α2 by transfecting Sertoli cells with laminin α2-specific short hairpin RNA vs the nontargeting negative control was shown to perturb the Sertoli cell TJ barrier, illustrating laminin α2 was involved in regulating BTB dynamics. This regulatory effect was mediated through mammalian target of rapamycin complex 1 (mTORC1) signaling because the two mTORC1 downstream signaling molecules ribosomal protein S6 and Akt1/2 were activated and inactivated, respectively, consistent with earlier findings that mTORC1 is involved in promoting BTB remodeling. Also, laminin α2 knockdown induced F-actin and microtubule (MT) disorganization through changes in the spatial expression of F-actin regulators actin-related protein 3 and epidermal growth factor receptor pathway substrate 8 vs end-binding protein 1 (a MT plus-end tracking protein, +TIP). These laminin α2 knockdown-mediated effects on F-actin and MT organization was blocked by exposing Sertoli cells to rapamycin, an inhibitor of mTORC1 signaling, and also SC79, an activator of Akt. In summary, laminin α2-mediated regulation on Sertoli cell BTB dynamics is through mTORC1 signaling.

LDL receptor related protein 2 to promote colorectal cancer metastasis via enhancing GSK3β/β-catenin signaling.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15507-e15507
Author(s):  
Xiaobin Zheng ◽  
Huashan Liu ◽  
Xiaoli Zhong ◽  
Xuanhui Liu ◽  
Zerong Cai ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Cellular Localization ◽  
Glycogen Synthase ◽  
Ldl Receptor ◽  
Gene Set Enrichment Analysis ◽  
Related Protein ◽  
Rt Pcr

e15507 Background: LDL receptor related protein 2 (LRP2), a multi-ligand endocytic receptor, has been implicated in the tumourigenesis of various human cancers. However, the biological roles and underlying regulatory mechanism of LRP2 in CRC remain unclear. Methods: Real-time PCR (RT-PCR), western blot, and immunohistochemistry were used to examine LRP2 expression. Gene silencing and overexpressing efficiencies of LRP2 were confirmed by RT-PCR and western blot. Then wound-healing, migration assay and invasion assay were used to investigate the function of LRP2 in CRC cells. The protein expression and cellular localization of LRP2 and β-catenin were characterized by immunofluorescence staining. The nude mice tail vein metastasis model was established to observe the effect of LRP2 on the lung metastasis of CRC cells in vivo. In addition, gene set enrichment analysis was carried out to explore the potential mechanism of LRP2 in CRC. Results: LRP2 expression was highly upregulated in CRC compared with matched adjacent normal tissue. LRP2 overexpression was positively correlated with shorter overall survival in CRC patients. The biological function observation indicated that LRP2 promoted CRC metastasis in vitro and in vivo. Further molecular mechanism investigation demonstrated that LRP2 protein interacted with glycogen synthase kinase-3β (GSK-3β) and led to decreased expression of GSK3β at ser9, followed by enhanced β-catenin signaling pathway in CRC cells. Conclusions: Our study demonstrated that LRP2 could enhance the metastatic abilities of CRC cells in vitro and in vivo by inhibiting the expression of GSK3β and enhancing the activity of β-catenin signaling pathway, providing that LRP2 might be a novel therapeutic target for metastasis in CRC.

Targeting Tau Hyperphosphorylation via Kinase Inhibition: Strategy to Address Alzheimer's Disease

2020 ◽  
Vol 20 (12) ◽  
pp. 1059-1073 ◽  
Author(s):  
Ahmad Abu Turab Naqvi ◽  
Gulam Mustafa Hasan ◽  
Md. Imtaiyaz Hassan
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Tau Protein ◽  
Glycogen Synthase ◽  
Paired Helical Filaments ◽  
Tau Hyperphosphorylation ◽  
Microtubule Stabilization ◽  
Extracellular Signal

Microtubule-associated protein tau is involved in the tubulin binding leading to microtubule stabilization in neuronal cells which is essential for stabilization of neuron cytoskeleton. The regulation of tau activity is accommodated by several kinases which phosphorylate tau protein on specific sites. In pathological conditions, abnormal activity of tau kinases such as glycogen synthase kinase-3 β (GSK3β), cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and microtubule affinity regulating kinase (MARK) lead to tau hyperphosphorylation. Hyperphosphorylation of tau protein leads to aggregation of tau into paired helical filaments like structures which are major constituents of neurofibrillary tangles, a hallmark of Alzheimer’s disease. In this review, we discuss various tau protein kinases and their association with tau hyperphosphorylation. We also discuss various strategies and the advancements made in the area of Alzheimer's disease drug development by designing effective and specific inhibitors for such kinases using traditional in vitro/in vivo methods and state of the art in silico techniques.

scholarly journals GSK-3β in Pancreatic Cancer: Spotlight on 9-ING-41, Its Therapeutic Potential and Immune Modulatory Properties

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 610
Author(s):  
Robin Park ◽  
Andrew L. Coveler ◽  
Ludimila Cavalcante ◽  
Anwaar Saeed
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Potential ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
In Vivo Models ◽  
Gsk 3Β ◽  
Early Phase Clinical Trials

Glycogen synthase kinase-3 beta is a ubiquitously and constitutively expressed molecule with pleiotropic function. It acts as a protooncogene in the development of several solid tumors including pancreatic cancer through its involvement in various cellular processes including cell proliferation, survival, invasion and metastasis, as well as autophagy. Furthermore, the level of aberrant glycogen synthase kinase-3 beta expression in the nucleus is inversely correlated with tumor differentiation and survival in both in vitro and in vivo models of pancreatic cancer. Small molecule inhibitors of glycogen synthase kinase-3 beta have demonstrated therapeutic potential in pre-clinical models and are currently being evaluated in early phase clinical trials involving pancreatic cancer patients with interim results showing favorable results. Moreover, recent studies support a rationale for the combination of glycogen synthase kinase-3 beta inhibitors with chemotherapy and immunotherapy, warranting the evaluation of novel combination regimens in the future.

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