The phosphoinositide kinase PIKfyve mediates epidermal growth factor receptor trafficking to the nucleus - PubMed (original) (raw)

The phosphoinositide kinase PIKfyve mediates epidermal growth factor receptor trafficking to the nucleus

Jayoung Kim et al. Cancer Res. 2007.

Abstract

ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear epidermal growth factor receptor (EGFR) in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis.

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Figures

Figure 1

Figure 1

Nuclear residency of EGFR in bladder cancer. A. (I) EGFR protein expression by standard immunohistochemistry in a human bladder cancer TMA containing human tumor tissues and normal samples. Arrows indicate nuclear localization. EGFR protein expression as determined by Automated quantitative analysis (AQUA). The Cy-5 expression (red), scored on a scale of 0–250, identifies the area of EGFR expression (II), magnified in (III), where nuclear localization of EGFR is indicated by the arrow. DAPI staining (IV) identifies nuclei both in the stroma and epithelial compartment. For each core, areas of tumor are distinguished from stromal elements by an epithelial tumor mask from the keratin signal (not shown). B. Expression of EGFR overlapping to cytokeratin and to DAPI quantified by the AQUA software. Overall and subcellular AQUA score for EGFR is shown (subcellular staining graph: blue, EGFR in cytoplasmic compartment; green, EGFR in nuclear compartment) as error bars of EGFR protein expression with 95% confidence intervals(27). The intensity is corrected for area for each tissue microarray spot and measured using arbitrary units. Expression results demonstrate measurable levels of EGFR in the nuclear compartment that are significantly higher in the tumor group than in normal samples, as demonstrated by non overlapping error bars with 95% confidence intervals (green). EGFR AQUA score in the cytosolic compartment is not significantly different between tumors and normal tissues. C. Localization of EGFR to the nuclear fraction in TCCSUP bladder cancer cells. Lamin A/C and β-tubulin were used for markers of nuclear and cytoplasmic fractions, respectively. Reduced EGFR expression elicited by EGFR siRNA was verified by western blot. D. Association of EGFR with the cyclin D1 promoter, as assayed by ChIP analysis.

Figure 2

Figure 2

EGFR nuclear trafficking stimulated by HB-EGF in bladder cancer cells. A. Immunofluorescent localization of EGFR in response to 100 ng/ml HB-EGF for 30 min (green, EGFR; blue, nucleus) in TCCSUP and 253J bladder cancer cells. B. Time-dependent localization of EGFR to nuclear fractions in response to HB-EGF (100 ng/ml, 30 min). EGFR siRNA reduced EGFR levels substantially. C. Time- and dose-ChIP experiments were performed using anti-EGFR antibody. Increased association of nuclear EGFR and the cyclin D1 promoter region following HB-EGF treatment is shown. Down-regulation of EGFR by gene silencing suppressed the association of EGFR with the cyclin D1 promoter.

Figure 3

Figure 3

Signaling pathways required for EGFR nuclear trafficking in TCCSUP cells treated with HB-EGF. A. Immunofluorescence micrographs demonstrating active (phosphorylated) EGFR in the nucleus (green, phosphorylated EGFR; blue, nucleus). B. Effect of EGFR kinase inhibitors (AG, AG1478; PD, PD153035) on EGFR trafficking under conditions where cells were treated with HB-EGF for 30 min (graph). Effect of the EGFR inhibitor, AG1478, on assembly of the nuclear EGFR/cyclin D1 promoter complex (right panels). C. Western blot analysis was performed and demonstrated that HB-EGF treatment increased expression of cyclin D1 (SF=serum-free control). D. Effect of PI3-kinase (LY, LY294002) and MEK (PD, PD98059) inhibitors on nuclear trafficking of EGFR. Nuclear extracts were subjected to Western blot analysis after 30 min of HB-EGF treatment in the absence or presence of inhibitors (upper panel). ChIP analysis was performed under the same experimental conditions as above (lower panel). Both PI3-kinase and Erk/MAPK signal pathways were activated by HB-EGF (right panel).

Figure 4

Figure 4

EGFR nuclear localization induced by agents that stimulate cleavage of proHB-EGF from TCCSUP cells which stably overexpress proHB-EGF. A. Surface EGFR translocated to the nucleus in TCCSUP/proHB-EGF cells by treatment with the strong HB-EGF secretion inducer, H2O2 (green, EGFR; red, nuclei stained with propidium iodide). B. EGFR accumulated in nuclei upon treatment with HB-EGF secretion inducers H2O2 (0,01%), TPA (10 μM), and ionomycin (10 μM).

Figure 5

Figure 5

Association of PIKfyve with the EGFR trafficking complex. A. Association of EGFR and PIKfyve were verified by co-IP and western blot analysis. TCCSUP whole cell lysates were subjected to IP with anti-EGFR antibody (Ratio: PIKfyve/EGFR). B. Immunofluorescence staining shows the cytoplasmic location of PIKfyve TCCSUP cells (red, PIKfyve; blue, nucleus). C–D. Effect of PIKfyveWT or dominant-negative, kinase-dead PIKfyveK1831E on nuclear EGFR level. After transient transfection with control (V, vector only), PIKfyveWT (WT, wild-type) or PIKfyveK1831E (KD, kinase dead), cells were harvested for nuclear extraction, followed by western blot. C. EGFR level in nuclear (Nuc) and cytosolic (Cyt) fractions from PIKfyveWT transfectants after enforced expression. D. Blockade of EGFR localization to nuclei by PIKfyveK1831E.

Figure 6

Figure 6

Cellular effects of PIKfyve loss of function demonstrated by RNA interference. A. EGFR localization after transient transfection with PIKfyve siRNA or control siRNA, +/− HB-EGF. Transfectants with siRNAs were serum starved and treated with HB-EGF for 30 min. Nuclear fractions were subjected to western blot analysis using antibodies against EGFR or Lamin. Cytoplasmic fractions were used to determine the level of PIKfyve relative to control under knockdown conditions. B. PIKfyve knockdown reduces cell cycle transit. Serum-starved TCCSUP cells were stimulated by FBS for 2 d in the presence of nocodazole, a G2/M transition blocker. FACS analysis was performed using separate cell populations at different stages in the cell cycle. C. EGFR trafficking is blocked by PIKfyve knockdown. D. Reduction of complex formation between nuclear EGFR and cyclin D1 promoter demonstrated by ChIP (upper panel, log phase growth; lower panel, HB-EGF stimulated conditions).

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