Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1)

2010 ◽  
Vol 431 (2) ◽  
pp. 245-255 ◽  
Author(s):  
Laura R. Pearce ◽  
Gordon R. Alton ◽  
Daniel T. Richter ◽  
John C. Kath ◽  
Laura Lingardo ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Mammalian Target Of Rapamycin ◽  
Specific Inhibitor ◽  
Signalling Pathways ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase ◽  
Phosphoinositide 3 Kinase ◽  
20 Nm ◽  
Mtor Complex 1

S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

scholarly journals Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

2009 ◽  
Vol 421 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Juan M. García-Martínez ◽  
Jennifer Moran ◽  
Rosemary G. Clarke ◽  
Alex Gray ◽  
Sabina C. Cosulich ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Mammalian Target Of Rapamycin ◽  
Specific Inhibitor ◽  
Initiation Factor ◽  
Target Of Rapamycin ◽  
S6 Kinase ◽  
Class 1 ◽  
Hydrophobic Motif ◽  
Ribosomal S6 Kinase

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

scholarly journals Balancing Akt with S6K

2004 ◽  
Vol 167 (3) ◽  
pp. 399-403 ◽  
Author(s):  
Brendan D. Manning
Keyword(s):  
Feedback Loop ◽  
Metabolic Diseases ◽  
Mammalian Target Of Rapamycin ◽  
Akt Pathway ◽  
Negative Feedback Loop ◽  
Insulin Receptor Substrate ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase ◽  
Phosphoinositide 3 Kinase ◽  
Diabetes And Obesity

Proper regulation of the phosphoinositide 3-kinase–Akt pathway is critical for the prevention of both insulin resistance and tumorigenesis. Many recent studies have characterized a negative feedback loop in which components of one downstream branch of this pathway, composed of the mammalian target of rapamycin and ribosomal S6 kinase, block further activation of the pathway through inhibition of insulin receptor substrate function. These findings form a novel basis for improved understanding of the pathophysiology of metabolic diseases (e.g., diabetes and obesity), tumor syndromes (e.g., tuberous sclerosis complex and Peutz-Jegher's syndrome), and human cancers.

scholarly journals Mutation of the PDK1 PH Domain Inhibits Protein Kinase B/Akt, Leading to Small Size and Insulin Resistance

2008 ◽  
Vol 28 (10) ◽  
pp. 3258-3272 ◽  
Author(s):  
Jose R. Bayascas ◽  
Stephan Wullschleger ◽  
Kei Sakamoto ◽  
Juan M. García-Martínez ◽  
Carol Clacher ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Protein Kinase B ◽  
Ph Domain ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
Insulin Resistant ◽  
Ribosomal S6 Kinase ◽  
Mtor Complex 1

ABSTRACT PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

Role of PRAS40 in Akt and mTOR signaling in health and disease

2012 ◽  
Vol 302 (12) ◽  
pp. E1453-E1460 ◽  
Author(s):  
Claudia Wiza ◽  
Emmani B. M. Nascimento ◽  
D. Margriet Ouwens
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cellular Growth ◽  
S6 Kinase ◽  
Binding Partners ◽  
Health And Disease ◽  
Mtor Complex 1 ◽  
Mtor Activity

The proline-rich Akt substrate of 40 kDa (PRAS40) acts at the intersection of the Akt- and mammalian target of rapamycin (mTOR)-mediated signaling pathways. The protein kinase mTOR is the catalytic subunit of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTORC2, that link energy and nutrients to the regulation of cellular growth and energy metabolism. Activation of mTOR in response to nutrients and growth factors results in the phosphorylation of numerous substrates, including the phosphorylations of S6 kinase by mTORC1 and Akt by mTORC2. Alterations in Akt and mTOR activity have been linked to the progression of multiple diseases such as cancer and type 2 diabetes. Although PRAS40 was first reported as substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity. Adding to the complexity is that gene silencing studies indicate that PRAS40 is also necessary for the activity of the mTORC1 complex. This review summarizes the regulation and potential function(s) of PRAS40 in the complex Akt- and mTOR-signaling network in health and disease.

scholarly journals Single-gene mutations and healthy ageing in mammals

2011 ◽  
Vol 366 (1561) ◽  
pp. 28-34 ◽  
Author(s):  
Andrzej Bartke
Keyword(s):  
Insulin Resistance ◽  
Stress Resistance ◽  
Insulin Signalling ◽  
Single Gene ◽  
Mammalian Target Of Rapamycin ◽  
Gene Mutations ◽  
Signalling Pathways ◽  
Antioxidant Defences ◽  
Gh Receptor ◽  
Inflammatory Profile

Studies of the effects of single-gene mutations on longevity in Caenorhabditis elegans, Drosophila melanogaster and Mus musculus identified homologous, highly conserved signalling pathways that influence ageing. In each of these very distantly related species, single mutations which lead—directly or indirectly—to reduced insulin, insulin-like growth factor (IGF) or insulin/IGF-like signalling (IIS) can produce significant increases in both average and maximal lifespan. In mice, most of the life-extending mutations described to date reduce somatotropic (growth hormone (GH) and IGF-1) signalling. The reported extensions of longevity are most robust in GH-deficient and GH-resistant mice, while suppression of somatotropic signalling ‘downstream’ of the GH receptor produces effects that are generally smaller and often limited to female animals. This could be due to GH influencing ageing by both IGF-1-mediated and IGF-1-independent mechanisms. In mutants that have been examined in some detail, increased longevity is associated with various indices of delayed ageing and extended ‘healthspan’. The mechanisms that probably underlie the extension of both lifespan and healthspan of these animals include increased stress resistance, improved antioxidant defences, alterations in insulin signalling (e.g. hypoinsulinaemia combined with improved insulin sensitivity in some mutants and insulin resistance in others), a shift from pro- to anti-inflammatory profile of circulating adipokines, reduced mammalian target of rapamycin-mediated translation and altered mitochondrial function including greater utilization of lipids when compared with carbohydrates.

Activation of the cardiac mTOR/p70S6K pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation

2010 ◽  
Vol 298 (4) ◽  
pp. E761-E769 ◽  
Author(s):  
Cossette Sanchez Canedo ◽  
Bénédicte Demeulder ◽  
Audrey Ginion ◽  
Jose R. Bayascas ◽  
Jean-Luc Balligand ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Target Of Rapamycin ◽  
Phosphorylation Site ◽  
Phosphatidylinositol 3 Kinase ◽  
Dependent Protein Kinase ◽  
S6 Kinase ◽  
Mtor Phosphorylation ◽  
Dependent Protein ◽  
Ribosomal S6 Kinase ◽  
Mtor Activity

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70S6K activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70S6K activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70S6K activity induced by insulin and leucine correlated with changes in phosphorylation of Thr389, the mTOR phosphorylation site on p70S6K, and of Ser2448 on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70S6K, leading to the absence of p70S6K activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70S6K, suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70S6K. We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70S6K pathway requires PDK1 in a way that differs from that of insulin.

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