Signal strength dictates phosphoinositide 3-kinase contribution to Ras/extracellular signal-regulated kinase 1 and 2 activation via differential Gab1/Shp2 recruitment: consequences for resistance to epidermal growth factor receptor inhibition
Authors: Carla Sampaio
Thomas Edouard
Jean-Pierre Salles
Armelle Yart
Patrick Raynal
Bertrand Perret
Marie Dance
Alexandra Montagner
Nicole Malet
Journal: Molecular and Cellular Biology (2008)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: PI3K; ERK1-2; Gab1/Shp2; Ras; EGFR
Affiliations: INSERM U563, Dept. Lipoprot√©ines et M√©diateurs Lipidiques, Site Purpan, BP3028, Toulouse F-31024, France.

Abstract:  Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP(3), according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP(3) is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors. 

Retracted: No

PubPeer comments:
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 Figure 2E. Much more similar than you would expect.  , 
  , 
 Figure 7B. Much more similar than you would expect.  , 
 #3  , 
 It gets better:  , 
 Re-use of Actin loading controls in Fig 8.  With a vertical flip:  , 
 Unexpected horizontal symmetry in Figure 5A...  , 
 Figures 1 and 2...  Unexpected similarity within actin bands of 1A, 2B.  More similarities between 1B, 1C, 2A.  , 
 #8 Figure 5A. Gab1-Myc panel. More similar than you would expect.  , 
 #10  , 
 #1 As corresponding author of this article, I apologize for these issues since obviously I did not pay enough attention to the origin of the images shown to control gel loading or protein expression (e.g., actin, Ras, or Gab1 immunoblots) when these figures were assembled for publication. However I am quite confident in the validity and reproducibility of each main result shown in each of these figures (i.e., the result shown in the upper image of each figure), as the top image shown is representative of several experiments. Please note that none of these top images has been significantly edited in this article. Patrick Raynal , 
 #12 Dr. Raynal: Are you intending to address the image concerns raised above with the journal? This paper appears to include multiple images that may have been manipulated and your response indicates that you apparently agree with these concerns. , 
 As I tried to explain in my previous comments, these image editing did not change the main outcome of the figures shown, since all the images shown in these figures are representative of several experiments (even those showing controls of gel loading or protein expression that could have been awkwardly recycled or edited). Signed: Patrick Raynal , 
 I m slightly confused, the evidence is of recycling individual bands and full blots between separate, isolated experiments, yet you are saying there is no issue with this? They may well  only be loading controls but if you cannot show equal loading without awkwardly recycling or editing (as you call it) different experimental conditions to represent something new then it does, in fact, cast serious doubt on the reliability of the main outcome you wish figures derived from those experiments to show. , 
 The images that are displayed in these figures are all extracted from experiments performed with equal gel loading, as any result obtained from experiments with incidental unequal gel loading was systematically discarded throughout these studies. I apologize again if some of the blots showing equal gel loading in these figures have been edited. Signed as Patrick Raynal (as I cannot access to reply as an author) , 
 Figure 1. Lowest panel on right. Much more similar than expected.  , 
 #16 I believe the image modifications may run afoul of the journals standards for image manipulation, as stipulated in the link below. Having said that, the link is for the most recent version, not necessarily the version at publication. Link: https: journals.asm.org ethics-checklist?journal=mcb ,