Activation of Nonreceptor Tyrosine Kinase Bmx/Etk Mediated by Phosphoinositide 3-Kinase, Epidermal Growth Factor Receptor, and ErbB3 in Prostate Cancer Cells

Xinnong Jiang,Robert A Borgesi,Nicole C Mcknight,Ramneet Kaur,Christopher L Carpenter,Steven P Balk

doi:10.1074/jbc.m703412200

Xinnong Jiang, Robert A Borgesi + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m703412200

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Abstract

Pathways activated downstream of constitutively active phosphatidylinositol (PI) 3-kinase in PTEN-deficient prostate cancer (PCa) cells are possible therapeutic targets. We found that the nonreceptor Tec family tyrosine kinase Bmx/Etk was activated by tyrosine phosphorylation downstream of Src and PI 3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and that Bmx down-regulation by short interfering RNA markedly inhibited LNCaP cell growth. Bmx also associated with ErbB3 in LNCaP cells, and heregulin-beta1 enhanced this interaction and further stimulated Bmx activity. Epidermal growth factor (EGF) similarly stimulated an interaction between Bmx and EGF receptor and rapidly increased Bmx kinase activity. Bmx stimulation in response to heregulin-beta1 and EGF was Src-dependent, and heregulin-beta1 stimulation of Bmx was also PI 3-kinase-dependent. In contrast, the rapid tyrosine phosphorylation and activation of Bmx in response to EGF was PI 3-kinase-independent. Taken together, these results demonstrate that Bmx is a critical downstream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and further show that Bmx is recruited by the EGF receptor and ErbB3 and activated in response to their respective ligands. Therefore, Bmx may be a valuable therapeutic target in PCa and other epithelial malignancies in which PI 3-kinase or EGF receptor family pathways are activated.

Highlights

Quent activation of PI 3-kinase signaling makes a major contribution to prostate cancer (PCa), with a large fraction of metastatic PCa and many high grade primary PCa being PTENdeficient [2]
Bmx-deficient mice have a defect in ischemia-meditated angiogenesis that correlates with decreased TNFR2 and VEGFR2 signaling in endothelium and bone marrow cells, indicating that Bmx functions physiologically through these receptors in endothelial cells [28]
This study shows that Bmx is activated downstream of the constitutively active PI 3-kinase pathway in PTEN-deficient LNCaP and PC3 PCa cells, and that Bmx down-regulation by RNA interference markedly inhibits cell growth, indicating that Bmx is a critical downstream effector of PI 3-kinase in these PCa cells

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Mouse anti-Bmx antibody was from BD Biosciences. Mouse anti-phosphotyrosine antibody (4G10) and 4G10-conjugated agarose beads were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse anti-FLAG antibody (M2)conjugated agarose beads, 3xFLAG peptide, IL-6, and PI 3-kinase inhibitor LY294002 were from Sigma. For anti-Tyr(P) immunoprecipitations, supernatants were transferred to new microcentrifuge tubes, and equal amounts of proteins (1–5 mg) from each sample were mixed with 20 –50 ␮l of 4G10-conjugated agarose beads and incubated at 4 °C overnight with continuous agitation. For Bmx immunoprecipitations by M2-conjugated agarose beads, cells were lysed with TBS containing 1%Triton X-100, 1 mM Na3VO4, and a mixture of protease inhibitors. The beads were transferred to MicroSpin columns, washed, and incubated with 10 ␮l of 3xFLAG peptide (100 ␮g/ml) at 4 °C overnight with continuous agitation to elute 3xFLAG-Bmx and associated proteins. 5 mg of proteins from each sample were mixed with 5 ␮g of mouse anti-Bmx antibody (BD Biosciences) and 5 ␮g of rabbit anti-Bmx antibody (Cell Signaling) and incubated overnight at 4 °C with continuous agitation. The samples were resolved by 4 –12% NuPAGE gel, and Bmx autophosphorylation was visualized by autoradiography

RESULTS

Growth Factors Enhance Bmx

DISCUSSION