Raf-1 activates MAP kinase-kinase

Kyriakis, John; App, Harald; Zhang, Xianfeng; Banerjee, Papia; Brautigan, David L.; Rapp, Ulf R.; Avruch, Joseph

doi:10.1038/358417a0

Cited by 1,255 publications

(723 citation statements)

References 23 publications

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“…Once activated by autophosphorylation or upstream kinases (Gomez and Cohen, 1991;Dent et al, 1992;Howe et al, 1992;Kyriakis et al, 1992;Ahn et al, 1993;Itoh et al, 1993;Matsuda et al, 1993;Posada et al, 1993;Gardner et al, 1994;Haystead et al, 1994;Papin et al, 1998), MEK activity is the main upstream mechanism leading to phosphorylation of both tyrosine and serine/ threonine residues and subsequent activation of ERK (Seger and Krebs, 1995). This, in turn, results in the regulation of a large number of proteins both in the cytoplasm and, following ERK translocation, in the nucleus (Khokhlatchev et al, 1998).…”

Section: Treatment With Ifn-α Reduces the Levels Of Phosphorylation Amentioning

confidence: 99%

Interferon-α 2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism

2000

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Summary Interferon (IFN)-α affects the growth, differentiation and function of various cell types by transducing regulatory signals through the Janus tyrosine kinase/signal transducers of activation and transcription (Jak/STAT) pathway. The signalling pathways employing the mitogenactivated ERK-activating kinase (MEK) and the extracellular-regulated kinase (ERK) are critical in growth factors signalling. Engagement of the receptors, and subsequent stimulation of Ras and Raf, initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation and proliferation. We demonstrate here that 24-48 h following treatment of transformed T-and monocytoid cell lines with recombinant human IFN-α2b both the phosphorylation and activity of MEK1 and its substrates ERK1/2 were reduced. In contrast, the activities of the upstream molecules Ras and Raf-1 were not affected. No effect on MEK/ERK activity was observed upon short-term exposure (1-30 min) to IFN. The anti-proliferative effect of IFN-α was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-α regulates the activity of the MEK/ERK pathway and consequently modulates cellular proliferation through a Ras/Raf-independent mechanism. Targeting the MEK/ERK pathway may strengthen the IFN-mediated anti-cancer effect.

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Section: Treatment With Ifn-α Reduces the Levels Of Phosphorylation Amentioning

confidence: 99%

Interferon-α 2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism

2000

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“…The expression levels of Raf proteins in transfected COS-7 were determined by lysing cells in bu er A (20 mM Tris-Cl, pH 8.0, 2 mM EDTA, 50 mM b-glycerophosphate, 1 mM Na 3 VO 4 , 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM PMSF, 1 mg/ml pepstatin A, 1 mg/ml leupeptin and 2.2 mg/ ml aprotinin) (Kyriakis et al, 1992). The lysate was cleared by centrifugation at 10 000 g for 10 min at 48C.…”

Section: Immunoblottingmentioning

confidence: 99%

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

et al. 1998

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We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Speci®-cally, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491 ± 508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCa phosphorylation sites failed to signi®cantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no e ect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of di erent stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a signi®cant mechanism of Raf-1 kinase regulation in COS-7 cells.

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“…The proteins (p21Ras) encoded by the ras genes (Harvey-ras/Ha-ras, Kirsten-ras/Ki-ras and N-ras) bind guanine nucleotides, possess an intrinsic GTPase activity and cycle between an active (GTP bound) form and an inactive (GDP bound) state (Barbacid, 1987). Activated p21Ras proteins stimulate a cascade of cytoplasmic protein kinases including Raf, MEK, MAPK (Mitogen Activated Protein Kinase) which in turn culminates in the phosphorylation of nuclear transcription factors resulting in transcriptional activation of growth-mediating genes (Bollag and McCormick, 1991;Boulton et al, 1991;de Vries-Smit et al, 1992;Dent et al, 1992;DeÂ rijard et al, 1994;Hibi et al, 1993;Howe et al, 1992;Kyriakis et al, 1992;Lowenstein et al, 1992;Wood et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Transformation by ras modifies AP1 composition and activity

et al. 1997

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The Ras proteins play a central role in regulating cell growth and their mutation can lead to abnormal proliferation. To analyse the potential link betwen AP1 activity, encoded by members of the jun and fos gene families, and Ras-mediated cellular transformation, we have studied several NIH3T3 clones which overexpress the Ha-Ras or Ki-Ras oncogenes. These transformed ®broblasts accumulated higher levels of cJun, JunB, Fra1 and Fra2 proteins relative to their normal counterparts. They also displayed increased AP1 DNA binding activity which was predominantly composed of cJun and Fra1 containing dimers. Following serum stimulation of Ras clones, the elevated levels of cJun and Fra1 remained steady, while the induction of JunB and Fra2 was partially attenuated. Moreover, deregulated Ras signaling resulted in a complete loss of the serum inducibility of cFos and FosB. Ectopic co-expression of cJun and Fra1 in NIH3T3 ®broblasts led to a transformed phenotype, attenuation of cFos serum inducibility, increased AP1 activity and Cyclin D1 accumulation, all characteristics of oncogenic Ras expressing cells. These results demonstrate that cJun and Fra1 are crucial mediators of the Ras-transformation process.

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Raf-1 activates MAP kinase-kinase

Cited by 1,255 publications

References 23 publications

Interferon-α 2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism

Interferon-α 2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

Transformation by ras modifies AP1 composition and activity

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