Functional effects of PTPN11 (SHP2) mutations causing LEOPARD syndrome on epidermal growth factor-induced phosphoinositide 3-kinase/AKT/glycogen synthase kinase 3beta signaling
Authors: Francoise Conte-Auriol
Audrey Nédélec
Mélanie Métrich
Stanislas Lyonnet
Frank Lezoualc&#x27;h
Thomas Edouard
Armelle Yart
Jean-Pierre Salles
Patrick Raynal
Sophie Bel-Vialar
Béatrice Parfait
Maithé Tauber
Jean-Philippe Combier
Journal: Molecular and cellular biology (2010)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: LEOPARD syndrome; SHP2 mutations; EGF-induced phosphorylation; AKT; GSK-3beta
Affiliations: INSERM U563, Centre de Physiopathologie de Toulouse-Purpan, BP3028, Toulouse, F-31024, France., INSERM U563, Centre de Physiopathologie de Toulouse-Purpan, F-31024 Toulouse, France, Centre de Biologie du Développement, CNRS UMR5547, F-31062 Toulouse, France, INSERM UMRS 769, Signalisation et Physiopathologie Cardiaque, Université Paris Sud 11, F-92296 Châtenay-Malabry, France, CHRU and Université Toulouse III, Hôpital Purpan, F-31059 Toulouse, France, Université Paris Descartes and INSERM U781, Hôpital Necker, F-75743 Paris, France, Université Paris Descartes and INSERM U745, UFR Pharmacie, F-75270 Paris, France

Abstract:  LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology. 

Retracted: No

PubPeer comments:
 The same strip was used to illustrate AKT controls in two different experiments. With different exposures, and horizontal mirror. ![file] , 
 Figure 1E and 1G. Much more similar than you would expect. ![file] , 
 Figure 4A. Much more similar than you would expect. ![file] , 
 Figure 4. Tubulin band in A contains four repeated lanes. It also overlaps with the tubulin in B, after a 180-deg rotation. ![file] , 
 Figure 3A. Unexpected symmetry. ![file] , 
 #1 I was not aware of this particular issue when we published this article. I apologize for it. However this does not change the outcome of the experiments depicted in Fig. 1 and 2, as the experiments shown were representative of several experiments showing similar results in terms of Akt phosphorylation. , 
 #2 Same comment as above. I was not aware of this issue when we submitted the manuscript. I apologize again. Obviously I did not pay enough attention to the images that were used as control for gel loading in this manuscript, but on a daily basis, controlling gel loading was a priority for me. And thus the main outcome of these experiments cannot be reconsidered. , 
 #3 I do not understand where is the issue in this figure. , 
 #4 Same comment as above. I was not aware of this issue when we submitted the manuscript. My apologies again. Obviously I did not pay enough attention to the images that were used as control for gel loading in this manuscript, but on a daily basis, controlling gel loading was a priority for me. And thus the main outcome of these experiments cannot be reconsidered. , 
 #5 Indeed the symetry of these two lanes is questioning. , 
 #6 Dr. Raynal: Thank you for your replies. However, I am not sure I fully understand your reply here (and in other threads). It appears that you agree with specific concerns raised, e.g., u201cI was not aware of this particular issue when we published this article. I apologize for it.u201d However, as you stand by the conclusions of the experiments, your remarks suggest that the matter is closed, e.g., u201cAnd thus the main outcome of these experiments cannot be reconsidered.u201d Does this accurately summarize your replies? , 
 #5 More specificially: ![file] The horizontally-flipped, 2-deg rotated version of Lane 7, when it is black white reversed and superimposed on lane 8, cancels out impressively well. ![file] How does this happen? , 
 #11 I guess you are right. Obviously I did not pay enough attention to the images controlling gel loading or protein expression when these figures were assembled. My priority was that the top strip of each figure (which is usually the most meaningful blot of any figure) was absolutely representative of the  average experiment (the ideal situation would have been to display data representing the average of each experiment, as they were performed at least 3 or 4 times. However it was not possible when showing images, because images cannot be aggregated from different experiements). , 
 #12 Indeed, if the scheme described is correct, this is questioning. As a hypothetical explanation, since this is a pretty wide strip (wider than the gel loading apparatus that we used to have in the lab), maybe this blot is putting together two different blots, for presentation reasons. I mean that the left part and the right part of the image could come from two different blots. Or maybe there were bubbles in these particular lanes. In any case, I apologize about that. However, even if there is an issue with certains lanes in this strip, it seems to be a minor issue and does not change the main outcome of the whole figure. , 
 #14 Dr. Raynal, I believe the modifications described in this thread violate the terms of MCB s publishing terms regarding image manipulation (described [here] ), if you have access to the original blots please post them here, or consider a corrigendum. ,