Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb 21;109(8):3018-23.
doi: 10.1073/pnas.1121457109. Epub 2012 Feb 7.

Phosphorylation of Dedicator of Cytokinesis 1 (Dock180) at Tyrosine Residue Y722 by Src Family Kinases Mediates EGFRvIII-driven Glioblastoma Tumorigenesis

Affiliations
Free PMC article

Phosphorylation of Dedicator of Cytokinesis 1 (Dock180) at Tyrosine Residue Y722 by Src Family Kinases Mediates EGFRvIII-driven Glioblastoma Tumorigenesis

Haizhong Feng et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Dock180 is required for EGFRvIII-induced Rac1 activity, glioblastoma cell migration, and survival in vitro. (A) IB analyses. (B and D) In vitro cell migration assays. Data are presented as percentage of control cells. (C) IB analyses. C, control siRNA; D1, Dock180 siRNA pool. In A and C, β-actin, Akt, Erk1/2, and Rac1 were used as loading controls. (E) Cell apoptosis. Data are presented as percentage of apoptotic cells. (F and G) Cell proliferation; data were calculated by dividing the total cell number by 50,000 and converting it to a log2 value. Data in B and DG were from six replicates per pair per cell line. Data are representative from three independent experiments with similar results. *P < 0.05. (Scale bars, ± SD.)
Fig. 2.
Fig. 2.
EGFRvIII induces p-Y of Dock180 at Y722. (A) IP and IB analyses. (B) Schematic of deletion mutants of Dock180. (C) EGFRvIII induces p-Y of Dock180 in HEK293T cells. (D) IB analyses. Red arrows, IgG; blue arrows, p-Y of Del mutant. (E) Mutation of Y722F in Del-5 decreased p-Y of Dock180. (F) Y722 is a major EGFRvIII-induced p-Y site in Dock180. (Scale bars, ± SD.) Bar graph underneath: relative amount of p-Y of Dock180 was determined from three separate in IP-IB blots by ImageJ and normalized to the amount of Dock180. *P < 0.05. (G) Y722 is conserved in Dock180 of various species and in Dock protein family. Black, conserved amino acids; blue, nonconserved amino acids. In A and CF, a pan-phospho-tyrosine antibody was used to detect p-Y-Dock180. Data are representative of three independent experiments with similar results.
Fig. 3.
Fig. 3.
Phosphorylation of Dock180Y722 is critical for EGFRvIII-driven glioblastoma growth and invasion. (A) EGF, but not PDGF-A or HGF, induces p-Y of endogenous Dock180Y722 (detected with a specific anti–p-Dock180Y722 antibody) in glioblastoma cells. (B) Effect of Dock180WT, Dock180Y722F, or a vector control on p-Dock180Y722, p-Akt, p-Erk1/2, and Rac1 activity in EGFRvIII-expressing cells. Dock180, Akt, Erk1/2, Rac1, and β-actin were used as loading controls. (C) Dock180Y722F inhibits EGFRvIII-promoted glioblastoma growth and invasion in the brain. Representative H&E and IHC images of brain sections of mice receiving various SNB19 cells (8 wk postinjection, five mice per group). (ac) H&E staining. (Scale bars, 1 mm.) (df) Enlarged areas in a to c marked with squares. (Scale bars, 200 μm.) (gi) GFP images of the same areas in d to f. (Scale bars, 200 μm.) Arrows indicate invasive tumor cells (di). (D and E) Quantification of Ki-67 and TUNEL staining, respectively. *P < 0.05. (Scale bars, ± SD.) Data represent three independent experiments with similar results.
Fig. 4.
Fig. 4.
EGFRvIII-induced p-Dock180Y722 is Src-dependent. (A) Inhibition of Src by PP2 (2 μM) or SU6656 (2 μM) attenuates EGFRvIII-stimulated p-Y of Dock180 (detected with a pan-phospho-tyrosine antibody, 4G10) and Rac1 activity. (B) In vitro cell migration. (C) Src phosphorylates Dock180 at Y722 by Src. Dock180WT or Dock180Y722F and WT, a KD or a CA Src were separately coexpressed in HEK293T cells. (D) Src-KD inhibits EGFRvIII-induced p-Dock180Y722. (E) In vitro Src kinase assay. Various proteins were visualized by Coomassie brilliant blue staining. (F) Src-dependent p-Y of Dock180 at Y722 enhances association of Dock180 with Rac1. (G) Knockdown of Src inhibits EGFRvIII-induced p-Dock180Y722 and Rac1 activation. (H) In vitro cell migration. In A and CG, Dock180, Src, Rac1, and β-actin were used as loading controls. In A, C, and D, a pan anti–p-Y antibody (4G10) was used to detect p-Y of Dock180. (EG) A specific anti–p-Dock180Y722 antibody was used to detect p-Y722 of Dock180. In B and H, data are presented as percentage of the control from six replicates per pair per cell line. *P < 0.05 and **, P < 0.01. (Scale bars, ± SD.) Data represent three independent experiments with similar results.
Fig. 5.
Fig. 5.
EGFRvIII-induced p-Dock180Y722 is also dependent on SFKs, Fyn and Lyn. (A and C) Knockdown of Lyn or Fyn inhibits EGFRvIII-induced p-Dock180Y722 and Rac1 activation. Dock180, Rac1, and β-actin were used as loading controls. (B and D) In vitro cell migration assays; data are presented as percentage of the control from six replicates per pair per cell line. *P < 0.05 and **, P < 0.01. (Scale bars, ± SD.) Data represent three independent experiments with similar results.
Fig. 6.
Fig. 6.
Coexpression of p-Dock180Y722, EGFRvIII and p-SrcY418 correlates with an extremely poor prognosis in patients with glioblastomas. (A) IHC analysis. A total of 57 specimens that express EGFRvIII and/or p-Dock180Y722 and p-SrcY418 are listed in Table S1. Representative images of GBM (grade IV) tissue stained by anti-EGFRvIII (a and d), anti–p-SrcY418 (b and e), and anti–p-Dock180Y722 (c and f) antibodies. Arrows, positive staining for EGFRvIII, p-SrcY418, and p-Dock180Y722. (Scale bars, 50 μm.) (B) IB analysis of a separate and independent cohort of 38 snap-frozen GBM specimens. Dock180 and β-actin were used as loading controls. (C) Kaplan-Meier curves with long-rank analyses for patients with high EGFRvIII/p-Dock180Y722–expressing tumors (red line) versus low-expression tumors (blue line) of two separate cohorts of glioblastomas examined in A and B. P values were determined by using the log-rank test. Black bars, censored data. Data represent three independent experiments with similar results.

Similar articles

See all similar articles

Cited by 31 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback