Activation of Rac1 by Src-dependent phosphorylation of Dock180Y1811 mediates PDGFRα-stimulated glioma tumorigenesis in mice and humans
Haizhong Feng, … , Webster K. Cavenee, Shi-Yuan Cheng
Haizhong Feng, … , Webster K. Cavenee, Shi-Yuan Cheng
Published November 14, 2011
Citation Information: J Clin Invest. 2011;121(12):4670-4684. https://doi.org/10.1172/JCI58559.
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Research Article Oncology

Activation of Rac1 by Src-dependent phosphorylation of Dock180Y1811 mediates PDGFRα-stimulated glioma tumorigenesis in mice and humans

Abstract

Two hallmarks of glioblastoma multiforme, the most common malignant brain cancer in humans, are aggressive growth and the ability of single glioma cells to disperse throughout the brain. These characteristics render tumors resistant to current therapies and account for the poor prognosis of patients. Although it is known that oncogenic signaling caused by overexpression of genes such as PDGFRA is responsible for robust glioma growth and cell infiltration, the mechanisms underlying glioblastoma malignancy remain largely elusive. Here, we report that PDGFRα signaling in glioblastomas leads to Src-dependent phosphorylation of the guanine nucleotide exchange factor Dock180 at tyrosine 1811 (Dock180Y1811) that results in activation of the GTPase Rac1 and subsequent cell growth and invasion. In human glioma cells, knockdown of Dock180 and reversion with an RNAi-resistant Dock180Y1811F abrogated, whereas an RNAi-resistant Dock180WT rescued, PDGFRα-promoted glioma growth, survival, and invasion. Phosphorylation of Dock180Y1811 enhanced its association with CrkII and p130Cas, causing activation of Rac1 and consequent cell motility. Dock180 also associated with PDGFRα to promote cell migration. Finally, phosphorylated Dock180Y1811 was detected in clinical samples of gliomas and various types of human cancers, and coexpression of phosphorylated Dock180Y1811, phosphorylated SrcY418, and PDGFRα was predictive of extremely poor prognosis of patients with gliomas. Taken together, our findings provide insight into PDGFRα-stimulated gliomagenesis and suggest that phosphorylated Dock180Y1811 contributes to activation of Rac1 in human cancers with PDGFRA amplification.

Authors

Haizhong Feng, Bo Hu, Kun-Wei Liu, Yanxin Li, Xinghua Lu, Tao Cheng, Jia-Jean Yiin, Songjian Lu, Susan Keezer, Tim Fenton, Frank B. Furnari, Ronald L. Hamilton, Kristiina Vuori, Jann N. Sarkaria, Motoo Nagane, Ryo Nishikawa, Webster K. Cavenee, Shi-Yuan Cheng

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Figure 6

Src mediates PDGFRα stimulation of p-Y of Dock180Y1811, activation of Rac1, and glioma cell migration.

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Src mediates PDGFRα stimulation of p-Y of Dock180Y1811, activation of Ra...
(A) Serum-starved LN444 cells were pretreated with or without PP3 (2 μM), PP2 (2 μM), and SU6656 (2 μM) for 2 hours, and then stimulated with or without PDGF-A (50 ng/ml) for 5 minutes. (B) In vitro cell migration assays were performed and analyzed as in Figure 1C. (C) Src phosphorylates Dock180. Dock180WT or Dock180Y1811F and WT, KD, or CA Src were separately cotransfected into HEK293T cells. (D) Dock180WT or Dock180Y1811F and KD Src were separately cotransfected into HEK293T cells that were serum-starved and treated with or without PDGF-A. (E) In vitro Src kinase assay using recombinant active Src and (His)6-Dock180WT or (His)6-Dock180Y1811F proteins. Purified (His)6-Dock180WT or (His)6-Dock180Y1811F proteins were visualized by Coomassie brilliant blue staining. (F) Src phosphorylation of Dock180Y1811 enhanced the association of Dock180 to Rac1. PTP, a recombinant YOP PTP. (G) Knockdown of Src inhibited PDGF-A–induced p-Y of Dock180Y1811 and Rac1 activation. LN444/PDGF-A/shDock180 or LN444/PDGF-A/shControl cells with or without restored Dock180WT*, Dock180Y1811F*, or pLVX-Puro vector control were transfected with Src shRNA4 (#4), Src shRNA5 (#5), or a shGFP control. (H) In vitro cell migration assays were performed and analyzed as in Figure 1C. (A, C, and D) p-Y of Dock180 was detected with a pan-phosphotyrosine antibody (4G10). (E–G) p-Y1811 was detected with a specific anti–p-Dock180Y1811 antibody. Data (± SD) represent 3 independent experiments with similar results. *P < 0.05.