EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis
Haizhong Feng, … , Hai Yan, Shi-Yuan Cheng
Haizhong Feng, … , Hai Yan, Shi-Yuan Cheng
Published July 25, 2014
Citation Information: J Clin Invest. 2014;124(9):3741-3756. https://doi.org/10.1172/JCI73093.
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EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis

Abstract

Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.

Authors

Haizhong Feng, Giselle Y. Lopez, Chung Kwon Kim, Angel Alvarez, Christopher G. Duncan, Ryo Nishikawa, Motoo Nagane, An-Jey A. Su, Philip E. Auron, Matthew L. Hedberg, Lin Wang, Jeffery J. Raizer, John A. Kessler, Andrew T. Parsa, Wei-Qiang Gao, Sung-Hak Kim, Mutsuko Minata, Ichiro Nakano, Jennifer R. Grandis, Roger E. McLendon, Darell D. Bigner, Hui-Kuan Lin, Frank B. Furnari, Webster K. Cavenee, Bo Hu, Hai Yan, Shi-Yuan Cheng

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Figure 3

EGFR phosphorylation of DCBLD2 at Y750, but not at Y621, is critical for EGFR-promoted tumorigenesis.

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EGFR phosphorylation of DCBLD2 at Y750, but not at Y621, is critical for...
(A) EGFRvIII activates p-Y of DCBLD2 in glioma U87 and SNB19 cells. vIII or vIII-DK, U87 or SNB19 cells that express vIII or a mutant of vIII that lacks of cytoplasmic domain of vIII, respectively. p-Y of DCBLD2 was detected with a pan anti-tyrosine antibody, 4G10. p-Y of EGFR was detected with an anti–p-EGFRY1045 antibody. (B) EGF (50 ng/ml) stimulates p-Y of DCBLD2 in U87/EGFR cells. (C) Schematic of the DCBLD2WT TIM P-X-E-X-X (Ar/Ac). CUB, C1r/C1s, Uegf, Bmp1 domain; LCCL, Limulus factor C, Coch-5b2, and Lgl1 domain; F5/8 type C, coagulation factor V/factor VIII homology (FV/VIII) domain. (D) DCBLD2F621/F750 mutant abolishes, and DCBLD2F621 or DCBLD2F750 mutant attenuates, EGFRvIII-stimulated p-Y of DCBLD2 in U87/P or U87/EGFRvIII cells. (E) Reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2WT or DCBLD2F621, but not DCBLD2F750, DCBLD2F621/F750 mutant or a vector control (C), rescues EGFRvIII-stimulated p-AKTT308 and p-AKTS473 in U87/EGFRvIII/shD2 cells. (F) Reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2WT and DCBLD2F621, but not DCBLD2F750, DCBLD2F621/F750 mutant, or a vector control (C), rescues EGFRvIII-promoted U87 glioma growth in the brain of animals. Images represent results of 5 mice per group of 3 independent experiments. Scale bars: 1 mm. (G) Effect of reexpression of Flag-DCBLD2 shRNA-resistant DCBLD2WT or indicated mutants on tumor size, cell proliferation, and cell apoptosis in U87/EGFRvIII/shD2 tumors. Error bars ± SD. **P < 0.01, compared with shControl + C tumors or shD2 tumors expressing DCBLD2WT or DCBLD2F621 mutant. In A, B, D–G, data are representative of 3 independent experiments. β-Actin or AKT was used as a loading control.