title: Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor receptor/NF-kappaB activation through Gab1 in glioblastoma cells.
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.
authors.affiliation: Department of Neurosurgery, University of Pennsylvania School of Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104, USA.
authors.forename: Gurpreet S
authors.initials: GS
authors.lastname: Kapoor
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhan
authors.forename: Gibbes R
authors.initials: GR
authors.lastname: Johnson
authors.forename: Donald M
authors.initials: DM
authors.lastname: O'Rourke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004273:DNA, Neoplasm; D066246:ErbB Receptors; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; C098587:GAB1 protein, human; C494918:AKT1 protein, human; C516724:PTPN11 protein, human