Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor receptor/NF-kappaB activation through Gab1 in glioblastoma cells
Authors: Yi Zhan
Gibbes R Johnson
Gurpreet S Kapoor
Donald M O&#x27;Rourke
Journal: Molecular and Cellular Biology (2004)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: NF-kappaB; EGFR; SHP-2; Gab1; PI3-kinase/Akt
Affiliations: Department of Neurosurgery, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104, USA., Departments of Neurosurgery, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Abstract:  The transcription factor nuclear factor kappaB (NF-kappaB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-kappaB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-kappaB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-kappaB activation. We also show that EGFR-induced NF-kappaB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-kappaB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-kappaB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-kappaB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-kappaB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-kappaB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-kappaB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-kappaB activation. 

Retracted: No

PubPeer comments:
 Figure 2B. Much more similar than expected after horizontal flip. https: imgur.com W5EbjPC , 
 Figure 8C. Much more similar than expected. https: imgur.com OOQeNy0 , 
 Response to Query 1 on Figure 2 B: The experiment was conducted on parent GMB cell line, U87MG stably transfected with either mutant EGFR (delta EGFR) or truncated erbB2 receptor (T691) to assess the status of AKT phosphorylation upon EGF stimulation in presence or absence of selective MEK inhibitor (U0126). The blot in question represents total AKT levels which are not expected to be changed after treatment as shown in the figure 2B. More importantly, it does not change the key observation that EGFR-mediated phosphorylation of AKT (P-AKT) was upregulated in untreated mutant EGFR expressing cells when compared to either parental cells (U87MG) or the cells expressing truncated erbB2 receptor. The other key observation is that MEK inhibitor did not affect the phosphorylation of AKT, but p42 44MAPK, suggesting that EGFR-mediated signaling and transformation is mediated may be dependent on PI-3K AKT signaling and there is no cross-talk between PI-3K AKT and MAPK pathways. Our previous work has shown that nonfunctional heterodimer assembly EGFR and kinase-inactive erbB2 mutant (T691S) resulted in decreased transformation. Downstream analysis of the nonfunctional assembly in this manuscript showed decreased AKT phosphorylation in U87MG T691S cells upon treatment with EGF when compared to parental and mutant EGFR cells, suggesting that EGFR-mediated transformation may be dependent on activation of PI3-K AKT pathway. Therefore, we strongly believe that the blot in question will not affect the key observations emphasizing the role of EGFR erbB2 assembly and PI-3K AKT in glioma cell transformation and will not impact the overall conclusion of the study and the manuscript. Response to Query 2 on Figure 8 C : In this experiment, stable clones of U87MG (Neo3, SHP-2 WT, SHP2 R32E and SHP-2 R138E) were treated with EGF followed by immunoprecipitation using anti-SHP-2 antibody and probing with anti-phospho-tyrosine antibody (anti-PY20) to see the effect of mutations in SH2 domains of SHP-2 on Gab1 phosphorylation. The blot in question represents total Gab1 levels which are not changed upon after treatment with EGF and we do not expect them to change with treatment as indicated in the Figure 8C. The key observation of this experiment is that phosphorylation of Gab1 was upregulated in cells overexpressing SHP-2 wild type protein and was not affected by mutations in SH2 domains of SHP-2. We sincerely believe that the blot in question does not affect the key observation made in the figure and will not impact the overall message of the manuscript. , 
 #3  Response to Query 2 on Figure 8 C ....The blot in question represents total Gab1 levels which are not changed upon after treatment with EGF and we do not expect them to change with treatment as indicated in the Figure 8C.  That would imply two samples. https: imgur.com JrEjaeU ,