Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.

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Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5670 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5670-5677 ◽

Cited By ~ 82

Author(s):

Ossama Abu Hatoum ◽

Shlomit Gross-Mesilaty ◽

Kristin Breitschopf ◽

Aviad Hoffman ◽

Hedva Gonen ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Specific Inhibitor ◽

Inhibitory Effect ◽

26S Proteasome ◽

Intact Cells ◽

Free System ◽

Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

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Ganciclovir Suppresses Human T Lymphocyte Proliferation In Vitro.

Blood ◽

10.1182/blood.v106.11.5378.5378 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5378-5378

Author(s):

Minoo Battiwalla ◽

Yiyuan Wu ◽

Ryotaro Nakamura ◽

Marija Radovic ◽

Rajinder P.S. Bajwa ◽

...

Keyword(s):

Dna Synthesis ◽

T Lymphocyte ◽

Lymphocyte Proliferation ◽

Brdu Incorporation ◽

Human Pbmcs ◽

Post Transplant ◽

T Lymphocyte Proliferation ◽

Proliferation In Vitro ◽

Cmv Reactivation

Abstract Clinically significant cytomegalovirus (CMV) infection in allogeneic blood or marrow transplant recipients has dramatically declined in recent years by the strategy of early detection of reactivation and pre-emptive therapy with Ganciclovir (GCV). We have previously shown that even in the absence of overt CMV disease, persisting post-transplant antigenemia predicts for increased late relapse and treatment failure. (Nakamura, et al BBMT 2004) In other words, frequent CMV reactivation serves as a surrogate for impaired post-transplant immune reconstitution. To explain the observed association between CMV reactivation and relapse we also raised the possibility that several weeks of GCV therapy could exert a deleterious effect on a fragile immune system. Clinical association between GCV administration and impaired lymphocyte function has not received attention previously; perhaps because of confounding effects from the underlying conditions (HIV or post-transplant) that induce CMV reactivation. We examined the effect of GCV in vitro on normal human PBMCs. Human PBMCs were extracted from normal volunteers and subjected to mitogenic stimulation (PHA) in the absence or presence of varying concentrations of GCV. PHA-induced proliferation, measured by uptake of 3[H]-thymidine after 5 days incubation in RPMI-10% AB serum, was reduced by 35% at peak therapeutic concentrations (10mg/ml) of GCV as opposed to 73% by Tacrolimus (10ng/ml). GCV did not induce lymphocyte apoptosis in the presence or absence of stimulation. Flow cytometry-based BrdU incorporation assays show that GCV exerts a time-dependent impairment of DNA synthesis in lymphocytes. Collectively, these results show that GCV suppresses T-lymphocyte proliferation in vitro at therapeutic concentrations and the likely mechanism of action is inhibition of DNA synthesis. Further work is ongoing to evaluate the effect of GCV on proliferative responses to specific antigens and to confirm these effects in comparison to other drugs used in the transplant setting. Figure Figure

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Insulin Stimulates Goose Liver Cell Growth by Activating PI3K-AKT-mTOR Signal Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000438650 ◽

2016 ◽

Vol 38 (2) ◽

pp. 558-570 ◽

Cited By ~ 10

Author(s):

Chunchun Han ◽

Shouhai Wei ◽

Qi Song ◽

Fang He ◽

Xiangping Xiong ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Dna Synthesis ◽

Protein Content ◽

Mrna Level ◽

Signal Pathway ◽

Mtor Pathway ◽

Brdu Incorporation ◽

Primary Hepatocytes

Background/Aims: Recent studies have suggested a crucial role for PI3K-Akt-mTOR pathway in regulating cell proliferation, so we hypothesize that insulin acts goose hepatocellular growth by PI3K-Akt-mTOR signal pathway. Because the physiological status of liver cells in vitro is different from that in vivo, a simplified cell model in vitro was established. Methods: Goose primary hepatocytes were isolated and incubated in either no addition as a control or insulin or PI3K-Akt-mTOR pathway inhibitors or co-treatment with glucose and PI3K-Akt-mTOR pathway inhibitors; Then, cell DNA synthesis and cell cycle analysis were detected by BrdU-incorporation Assay and Flow cytometric analysis; the mRNA expression and protein expression of factors involved in the cell cycle were determined by Real-Time RT-PCR, ELISA, and western blot. Results: Here we first showed that insulin evidently increased the cell DNA synthesis, the mRNA level and protein content of factors involved in the cell proliferation of goose primary hepatocytes. Meanwhile, insulin evidently increased the mRNA level and protein content of factors involved in PI3K-Akt-mTOR pathway. However, the up-regulation of insulin on cell proliferation was decreased significantly by the inhibitors of PBK-Akt-mTOR pathway, LY294002, rapamycin or NVP-BEZ235. Conclusion: These findings suggest that PI3K-Akt-mTOR pathway plays an essential role in insulin-regulated cell proliferation of goose hepatocyte.

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The α-isoform of glycogen synthase kinase-3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase-activated protein kinase-1 in vitro

FEBS Letters ◽

10.1016/0014-5793(94)80112-6 ◽

1994 ◽

Vol 338 (1) ◽

pp. 37-42 ◽

Cited By ~ 157

Author(s):

Calum Sutherland ◽

Philip Cohen

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Map Kinase ◽

Glycogen Synthase ◽

Rabbit Skeletal Muscle ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

S6 Kinase ◽

P70 S6 Kinase

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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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The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells

Biochemical Journal ◽

10.1042/bj3030027 ◽

1994 ◽

Vol 303 (1) ◽

pp. 27-31 ◽

Cited By ~ 94

Author(s):

Y Saito ◽

J R Vandenheede ◽

P Cohen

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

A431 Cells ◽

S6 Kinase ◽

P70 S6 Kinase ◽

Epidermal Growth

Glycogen synthase kinase 3 (GSK3) was inhibited by 50% within 5 min when A431 cells were stimulated with epidermal growth factor (EGF). The inhibition was unaffected by rapamycin at concentrations which blocked the activation of p70 S6 kinase, and reversed by incubation with protein phosphatase-1. EGF stimulation of A431 cells inhibited GSK3 alpha and GSK3 beta to a similar extent, and inhibition was accompanied by phosphorylation of the tryptic peptides containing the serine residues phosphorylated in vitro by p70 S6 kinase or MAP kinase-activated protein (MAPKAP) kinase-1 beta (also termed Rsk-2). These results demonstrate that EGF inhibits GSK3 by inducing phosphorylation of a serine residue and that GSK3 is not phosphorylated in vivo by either p70 S6 kinase or protein kinase C.

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Compensatory renal hypertrophy. I. Evidence for a factor of renal origin inhibiting DNA synthesis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y77-113 ◽

1977 ◽

Vol 55 (4) ◽

pp. 839-847 ◽

Cited By ~ 4

Author(s):

J. Martel-Pelletier ◽

M. Bergeron

Keyword(s):

Molecular Weight ◽

Dna Synthesis ◽

Specific Inhibitor ◽

Inhibitory Effect ◽

Compensatory Hypertrophy ◽

Partial Purification ◽

Dna Uptake ◽

Renal Hypertrophy ◽

Compensatory Renal Hypertrophy

This study describes a method for the measurement and partial purification of a factor seemingly involved in the regulation of the renal mass.After homogenization at 4 °C, rabbit kidneys were centrifuged for 100 min at 105 000 g. The resulting supernatant (S-105) was lyophilized and tested on kidney slices obtained from rats mononephrectomized 48 h previously. We have developed a method based on the inhibition of DNA synthesis to measure the activity of the S-105. Slices of renal cortex, undergoing compensatory hypertrophy, were incubated in vitro in Hanks' medium at 37 °C, pH 7.4, in an O2–CO2 atmosphere in the presence of 0.144 μg (20 μCi (1 Ci = 37 GBq)) [3H]thymidine.An inhibition of DNA uptake of [3H]thymidine was noted in the presence of S-105. When other media (Hanks', sucrose, water) were used to extract S-105, the same type of inhibition was noted even though the sucrose buffer seemed ideal for the preservation of the inhibitory factor. The inhibitory effect was still observed after dialysis of S-105 against membranes permitting exclusion of molecules with molecular weight smaller than about 4000 (such as electrolytes and tissue thymidine). This inhibition seems to be specific, since other tissues such as liver in regeneration and rat intestine were not influenced by the dialyzed renal S-105. The dialyzable fraction did contain some inhibitors, but they were not specific for the kidney since they also acted on the liver and the jejunum.Our results suggest the existence, in the normal nephron, of a specific inhibitor of thymidine incorporation into DNA of kidneys undergoing a compensatory hypertrophy. This renal factor has a molecular weight of over 5000.

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Mitogen inactivation of glycogen synthase kinase-3β in intact cells via serine 9 phosphorylation

Biochemical Journal ◽

10.1042/bj3030701 ◽

1994 ◽

Vol 303 (3) ◽

pp. 701-704 ◽

Cited By ~ 395

Author(s):

V Stambolic ◽

J R Woodgett

Keyword(s):

Cell Fate ◽

Glycogen Synthase ◽

Regulatory Proteins ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

Serine Kinase ◽

Intact Cells ◽

S6 Kinase ◽

Mitogen Activated Protein

Glycogen synthase kinase-3 (GSK-3), a protein-serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function.

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Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

The Journal of Lipid Research ◽

10.1194/jlr.ra120000682 ◽

2020 ◽

Vol 61 (6) ◽

pp. 896-910 ◽

Cited By ~ 5

Author(s):

Eyad Naser ◽

Stephanie Kadow ◽

Fabian Schumacher ◽

Zainelabdeen H. Mohamed ◽

Christian Kappe ◽

...

Keyword(s):

Cultured Cells ◽

Specific Inhibitor ◽

Acid Sphingomyelinase ◽

Intact Cells ◽

Protein Levels ◽

High Doses ◽

Hydrolysis Of

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

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