PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

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.

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

Biochemical Journal ◽

10.1042/bj20061088 ◽

2006 ◽

Vol 401 (1) ◽

pp. 29-38 ◽

Cited By ~ 200

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.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5714957.5695055 ◽

2010 ◽

Author(s):

Alex Toker

Keyword(s):

Specific Inhibitor ◽

S6 Kinase ◽

Ribosomal S6 Kinase

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Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1

Biochemical Journal ◽

10.1042/bj20101732 ◽

2010 ◽

Vol 433 (2) ◽

pp. 357-369 ◽

Cited By ~ 87

Author(s):

Ayaz Najafov ◽

Eeva M. Sommer ◽

Jeffrey M. Axten ◽

M. Phillip Deyoung ◽

Dario R. Alessi

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Embryonic Stem ◽

Specific Inhibitor ◽

Prolonged Treatment ◽

Pleckstrin Homology Domain ◽

Biological Processes ◽

S6 Kinase ◽

Ribosomal S6 Kinase ◽

Kinase Pathway

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC50 of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.

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

Biochemical Journal ◽

10.1042/bj20101024 ◽

2010 ◽

Vol 431 (2) ◽

pp. 245-255 ◽

Cited By ~ 174

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.

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Aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.00870.2004 ◽

2005 ◽

Vol 98 (4) ◽

pp. 1562-1566 ◽

Cited By ~ 34

Author(s):

Troy A. Hornberger ◽

R. D. Mateja ◽

E. R. Chin ◽

J. L. Andrews ◽

K. A. Esser

Keyword(s):

Skeletal Muscle ◽

Signaling Pathways ◽

Ex Vivo ◽

Mechanical Stimuli ◽

S6 Kinase ◽

Diminished Capacity ◽

Ribosomal S6 Kinase ◽

Passive Stretch ◽

Old Mice ◽

Dependent Mechanism

The capacity for skeletal muscle to recover its mass following periods of unloading (regrowth) has been reported to decline with age. Although the mechanisms responsible for the impaired regrowth are not known, it has been suggested that aged muscles have a diminished capacity to sense and subsequently respond to a given amount of mechanical stimuli (mechanosensitivity). To test this hypothesis, extensor digitorum longus muscles from young (2–3 mo) and old (26–27 mo) mice were subjected to intermittent 15% passive stretch (ex vivo) as a source of mechanical stimulation and analyzed for alterations in the phosphorylation of stress-activated protein kinase (p38), ribosomal S6 kinase (p70S6k), and the p54 jun N-terminal kinase (JNK2). The results indicated that the average magnitude of specific tension (mechanical stimuli) induced by 15% stretch was similar in muscles from young and old mice. Young and old muscles also revealed similar increases in the magnitude of mechanically induced p38, p70S6k (threonine/serine 421/424 and threonine 389), and JNK2 phosphorylation. In addition, coincubation experiments demonstrated that the release of locally acting growth factors was not sufficient for the induction of JNK2 phosphorylation, suggesting that JNK2 was activated by a mechanical rather than a mechanical/growth factor-dependent mechanism. Taken together, the results of this study demonstrate that aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

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p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in α-tropomyosin Glu180Gly transgenic mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00237.2013 ◽

2013 ◽

Vol 305 (7) ◽

pp. H1010-H1019 ◽

Cited By ~ 10

Author(s):

Catherine L. Passariello ◽

Marjorie Gayanilo ◽

Michael D. Kritzer ◽

Hrishikesh Thakur ◽

Zoharit Cozacov ◽

...

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Cardiac Function ◽

Interstitial Fibrosis ◽

Cardiac Insufficiency ◽

S6 Kinase ◽

Transgenic Expression ◽

P90 Ribosomal S6 Kinase ◽

Ribosomal S6 Kinase

Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of α-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout ( RSK3−/−) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180; RSK3−/− mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure.

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