Identification of p122RhoGAP (Deleted in Liver Cancer-1) Serine 322 as a Substrate for Protein Kinase B and Ribosomal S6 Kinase in Insulin-stimulated Cells

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.

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

Biochemical Journal ◽

10.1042/bj20090489 ◽

2009 ◽

Vol 421 (1) ◽

pp. 29-42 ◽

Cited By ~ 318

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.

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Arsenite-induced Phosphorylation of Histone H3 at Serine 10 Is Mediated by Akt1, Extracellular Signal-regulated Kinase 2, and p90 Ribosomal S6 Kinase 2 but Not Mitogen- and Stress-activated Protein Kinase 1

Journal of Biological Chemistry ◽

10.1074/jbc.m208581200 ◽

2003 ◽

Vol 278 (12) ◽

pp. 10588-10593 ◽

Cited By ~ 40

Author(s):

Zhiwei He ◽

Wei-Ya Ma ◽

Guangming Liu ◽

Yiguo Zhang ◽

Ann M. Bode ◽

...

Keyword(s):

Protein Kinase ◽

Histone H3 ◽

S6 Kinase ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Ribosomal S6 Kinase 2 ◽

P90 Ribosomal S6 Kinase ◽

Ribosomal S6 Kinase

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p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1α

Journal of Biological Chemistry ◽

10.1074/jbc.m109.083642 ◽

2010 ◽

Vol 285 (10) ◽

pp. 6970-6979 ◽

Cited By ~ 10

Author(s):

Xianlong Gao ◽

Deepti Chaturvedi ◽

Tarun B. Patel

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Catalytic Subunit ◽

S6 Kinase ◽

P90 Ribosomal S6 Kinase ◽

Ribosomal S6 Kinase ◽

Kinase A

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Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase

Biochemical Journal ◽

10.1042/bj3490059 ◽

2000 ◽

Vol 349 (1) ◽

pp. 59-65 ◽

Cited By ~ 5

Author(s):

Silvia FERNÁNDEZ DE MATTOS ◽

Elisabet DE LOS PINOS ◽

Manel JOAQUIN ◽

Albert TAULER

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Transcriptional Activity ◽

Protein Kinase B ◽

Dominant Negative ◽

Phosphatidylinositol 3 Kinase ◽

S6 Kinase ◽

Dominant Negative Form ◽

Negative Form ◽

Phosphatidylinositol 3

Previous studies have demonstrated that the F isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00421.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. E761-E769 ◽

Cited By ~ 33

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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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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