PMID- 16709153
OWN - NLM
STAT- MEDLINE
DCOM- 20060906
LR  - 20191210
IS  - 1470-8728 (Electronic)
IS  - 0264-6021 (Print)
IS  - 0264-6021 (Linking)
VI  - 398
IP  - 2
DP  - 2006 Sep 1
TI  - Ras activation in response to phorbol ester proceeds independently of the EGFR 
      via an unconventional nucleotide-exchange factor system in COS-7 cells.
PG  - 243-56
AB  - Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various 
      mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, 
      Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and 
      Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and 
      Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Hohn and 
      Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target 
      GAPs (GTPase-activating proteins) for inactivation and those that result in 
      activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless 
      homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link 
      between PE and GAP inactivation is missing and GEF stimulation is hard to 
      reconcile with the observation that dominant-negative S17N-Ras does not 
      compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation 
      by PE. We have addressed this controversy and carried out an in-depth biochemical 
      study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization 
      approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced 
      Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange 
      stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by 
      EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that 
      EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen 
      homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) 
      association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in 
      growth-arrested COS-7 cells. These data disclose the existence of two separate 
      GEF systems that operate independently from each other to accomplish PE-dependent 
      formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We 
      document that COS-7 cells do not express RasGRP and present evidence that the 
      PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More 
      fundamentally, these observations shed new light on enigmatic issues such as the 
      inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in 
      heterologous agonist activation of the Ras/ERK pathway.
FAU - Rubio, Ignacio
AU  - Rubio I
AD  - Institute of Molecular Cell Biology, Medical Faculty, 
      Friedrich-Schiller-University Jena, Drackendorfer Str. 1, 07747 Jena, Germany. 
      b5igru@rz.uni-jena.de
FAU - Rennert, Knut
AU  - Rennert K
FAU - Wittig, Ute
AU  - Wittig U
FAU - Beer, Katrin
AU  - Beer K
FAU - Durst, Matthias
AU  - Durst M
FAU - Stang, Stacey L
AU  - Stang SL
FAU - Stone, Jim
AU  - Stone J
FAU - Wetzker, Reinhard
AU  - Wetzker R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (Culture Media, Serum-Free)
RN  - 0 (Guanine Nucleotide Exchange Factors)
RN  - 0 (Phorbol Esters)
RN  - 0 (Reactive Oxygen Species)
RN  - 0 (Shc Signaling Adaptor Proteins)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - EC 2.7.10.1 (ErbB Receptors)
RN  - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt)
RN  - EC 2.7.11.13 (Protein Kinase C)
RN  - EC 3.6.5.2 (ras Proteins)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing/metabolism
MH  - Animals
MH  - COS Cells
MH  - Chlorocebus aethiops
MH  - Culture Media, Serum-Free
MH  - Enzyme Activation/drug effects
MH  - ErbB Receptors/metabolism
MH  - Guanine Nucleotide Exchange Factors/*metabolism
MH  - Guanosine Triphosphate/metabolism
MH  - MAP Kinase Signaling System
MH  - Phorbol Esters/*pharmacology
MH  - Phosphatidylinositol 3-Kinases/metabolism
MH  - Phosphorylation
MH  - Protein Binding
MH  - Protein Kinase C/metabolism
MH  - Proto-Oncogene Proteins c-akt/metabolism
MH  - Reactive Oxygen Species/metabolism
MH  - Shc Signaling Adaptor Proteins
MH  - Transcriptional Activation
MH  - ras Proteins/genetics/*metabolism
PMC - PMC1550314
EDAT- 2006/05/20 09:00
MHDA- 2006/09/07 09:00
PMCR- 2007/03/01
CRDT- 2006/05/20 09:00
PHST- 2006/05/20 09:00 [pubmed]
PHST- 2006/09/07 09:00 [medline]
PHST- 2006/05/20 09:00 [entrez]
PHST- 2007/03/01 00:00 [pmc-release]
AID - BJ20060160 [pii]
AID - bj3980243 [pii]
AID - 10.1042/BJ20060160 [doi]
PST - ppublish
SO  - Biochem J. 2006 Sep 1;398(2):243-56. doi: 10.1042/BJ20060160.