Phosphorylation of initiation factor 4E is resistant to SB203580 in cells expressing a drug-resistant mutant of stress-activated protein kinase 2a/p38

Morley, Simon; Naegele, Susanne

doi:10.1016/s0898-6568(03)00008-1

Cited by 10 publications

(11 citation statements)

References 35 publications

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“…Antisera specific to eIF4GI and PABP were generated inhouse. [28][29][30] Immobilon PVDF was from GE Healthcare and unless otherwise stated, all other chemicals were from Sigma.…”

Section: Chemicals and Biochemicalsmentioning

confidence: 99%

“…Pellets were resuspended in 100 ml ice-cold Buffer A (20 mM MOPS(KOH), pH 7.2, 20 mM sodium fluoride, 1 mM microcystin, 75 mM KCl, 2 mM MgCl 2 , 2 mM benzamidine, 2 mM Na 3 VO 4 , complete protease inhibitor mix (-EDTA; Roche) and lysed by vortexing following the addition of 0.5% (v/v) Igepal and 0.5% (v/v) deoxycholate. Cell debris was removed by centrifugation in a microfuge for 5 min at 4 C [28][29][30] ; a Bradford assay was used to determine protein content of extracts which were then frozen in liquid N 2 .…”

Section: Preparation Of Cell Extractsmentioning

confidence: 99%

“…26,[28][29][30] Briefly, membranes were blocked using Tris-Buffered Saline (TBS)/0.5% (v/v) Tween containing 3% (w/v) BSA for 1 hour and incubated with antisera diluted in the same overnight at 4 C. Following washing in TBS-Tween, membranes were incubated with horseradish peroxidase-conjugated secondary antibody and signals developed using ECL. [28][29][30] Lambda phosphatase treatment of cell extracts Aliquots of total cell extract (90 mg) were incubated with 200 units of lambda phosphatase (NEB) using the manufacturer's conditions and buffers, in a final volume of 100 ml, in the presence of MnCl 2 . Incubations were carried out for the times indicated, in the absence or presence of 50 mM NaF; aliquots containing 10 mg extract protein were then removed into SDS-PAGE sample buffer and the phosphorylation of 4E-BP1 visualized by Western blotting.…”

Section: Polyacrylamide Gel Electrophoresis (Sds-page) and Immunoblotmentioning

confidence: 99%

“…12 Protein synthesis is carried out in 3 stages (initiation, elongation and termination), with the initiation stage of translation generally accepted as a major site of regulation of gene expression in mammalian cells. 5,7,[22][23][24][25][26][27][28][29][30] This step in protein synthesis is regulated by a family of proteins, the initiation factors 5,23,24 which interact with each other and the mRNA. These proteins modulate the binding of mRNA to the ribosome, a process facilitated by the assembly of the cap binding protein, eIF4E, a helicase, eIF4A, and a scaffold protein, eIF4G, into the eIF4F complex (eIF4E/eIF4A/eIF4G).…”

Section: Introductionmentioning

confidence: 99%

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mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

2014

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Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptorindependent regulation of downstream signaling is important for myogenic differentiation.

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“…Antisera specific to eIF4GI and PABP were generated inhouse. [28][29][30] Immobilon PVDF was from GE Healthcare and unless otherwise stated, all other chemicals were from Sigma.…”

Section: Chemicals and Biochemicalsmentioning

confidence: 99%

Section: Preparation Of Cell Extractsmentioning

confidence: 99%

Section: Polyacrylamide Gel Electrophoresis (Sds-page) and Immunoblotmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

2014

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“…Using SB203580, these investigators demonstrated that p38 regulates the stability of PGHS-2 mRNA, suggesting that IFN-dependent activation of the pathway also exerts effects at the post-transcriptional level (32). Other studies have also shown that a downstream effector of p38, the kinase Mnk1 (60,61), regulates phosphorylation of the eukaryotic initiation factor-4E (eIF4E) on serine 209 (62,63,64), to initiate mRNA translation. Thus, it is likely that activation of the p38 pathway regulates both gene transcription and mRNA translation using distinct downstream effectors, to mediate optimal induction of interferon responses.…”

Section: P38 Map Kinase and Interferon Signalingmentioning

confidence: 99%

Role of p38α Map Kinase in Type I Interferon Signaling

Sassano

Majchrzak

et al. 2004

Journal of Biological Chemistry

113

111

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Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38␣ isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38␣ gene. Our data demonstrate that p38␣ activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38␣ is not required for IFN-dependent

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Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver

et al. 2004

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Following acute injuries that diminish functional liver mass, the remaining hepatocytes substantially increase overall protein synthesis to meet increased metabolic demands and to allow for compensatory liver growth. Previous studies have not clearly defined the mechanisms that promote protein synthesis in the regenerating liver. In the current study, we examined the regulation of key proteins involved in translation initiation following 70% partial hepatectomy (PH) in mice. PH promoted the assembly of eukaryotic initiation factor (eIF) 4F complexes consisting of eIF4E, eIF4G, eIF4A1, and poly-A binding protein. eIF4F complex formation after PH occurred without detectable changes in eIF4E-binding protein 1 (4E-BP1) phosphorylation or its binding eIF4E. The amount of serine 1108-phosphorylated eIF4G (but not Ser209-phosphorylated eIF4E) was induced following PH. These effects were antagonized by treatment with rapamycin, indicating that target of rapamycin (TOR) activity is required for eIF4F assembly in the regenerating liver. Rapamycin inhibited the induction of cyclin D1, a known eIF4F-sensitive gene, at the level of protein expression but not messenger RNA (mRNA) expression. In conclusion, increased translation initiation mediated by the mRNA cap-binding complex eIF4F contributes to the induction of protein synthesis during compensatory liver growth. Further study of factors that regulate translation initiation may provide insight into mechanisms that govern metabolic homeostasis and regeneration in response to liver injury. (HEPATOLOGY 2004;40:537-544.)

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Phosphorylation of initiation factor 4E is resistant to SB203580 in cells expressing a drug-resistant mutant of stress-activated protein kinase 2a/p38

Cited by 10 publications

References 35 publications

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

Role of p38α Map Kinase in Type I Interferon Signaling

Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver

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