Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization

Abstract

EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.

Keywords: BUB1; EGFR; cancer; endocytosis; signaling.

Figure 1

BUB1 depletion stabilizes EGFR. (A) A549 cells were transfected with non-targeting control scrambled (NSS) siRNA or BUB1siRNA. 48 hours post-transfection cells were cross-linked with 100 μM DSS for 30 minutes followed by 30 ng/mL EGF for an additional 30 minutes. Resulting lysates were resolved on SDS-PAGE gels and probed with indicated antibodies. (B) Quantitation of western blots from (A) only the monomer species of EGFR is plotted. Control siRNA (NSS) transfected, EGF treated lanes were set as 1 fold and used as a baseline for estimating fold enrichment in other samples. (C) Quantitation of pEGFR and EGFR dimers from SDS and EGF treated lanes only (lanes 4 and 8 only in A). NSS transfected lane was set as 1 fold and used as a baseline for estimating fold enrichment in BUB1 siRNA transfected samples. (D) Gene expression values from non-metastatic adenocarcinoma samples (N=331) from TCGA lung dataset were log₂ transformed and median centered and correlation coefficient (r) was calculated. BUB1 and EGFR expression is expressed as log₂ transformed values. Correlation coefficient and p-value are listed.

Figure 2

BUB1 inhibition blocks EGFR signaling. MDA-MB-231-1833 (A) NCI-H358 (B) and MRC5 (C) cells were starved and pretreated with BUB1 kinase inhibitor 2OH-BNPP1 (10 μM) or EGFR inhibitor erlotinib (10 μM) for 1 hour followed by EGF (30 ng/mL) treatment for an additional 30 minutes. Resulting lysates were run on 4-12% Bis-Tris SDS-PAGE gels, transferred to PVDF membranes and probed with indicated antibodies. (D–F) western blots for phosphorylated proteins from (A–C) were quantitated (three separate biological repeats) using ImageJ and plotted.

Figure 3

BUB1 inhibition reduces EGFR activation. MDA-MB-231 (A) NCI-H358 (B) and MRC5 (C) cells were serum starved and pre-treated with 10 μM 2OH-BNPP1 for 1 hour followed by 50 ng/mL EGF. Cells were harvested at the indicated time-points (10-180 minutes) after EGF treatment. Whole cell lysates from these samples were resolved on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% de-fatted milk-TBST and probed with pEGFR (Y845), pAKT (S473), and pERK1/2 antibodies. The blots were also probed with antibodies raised against total proteins.

Figure 4

Inhibition of BUB1 kinase activity reduces EGFR active dimers without affecting inactive-EGFR dimers. A549 (A) MRC5 (B) and MDA-MB-231-1833 (C) cells were serum starved for 3-4 hours, pretreated with 2OH-BNPP1 (10 μM), erlotinib (10 μM) or cetuximab (50 μg/mL) for 1 hour followed by EGF (50 ng/mL) for 10 min. DSS (200 μM) was added for an additional 20 minutes. Total cell lysates were made, resolved on 3-8% gels and probed with pEGFR (Y845), EGFR, Her2 and c-Met antibodies.

Figure 5

Inhibition of BUB1 kinase activity by 2OH-BNPP1 prolongs EGFR half-life. (A) A549 lung adenocarcinoma cells were treated with 2OH-BNPP1 (10 μM) and Cycloheximide (50 μg/ML) for 1 hour followed by EGF (50 ng/mL). Cells were harvested at different time points after EGF treatment and resulting lysates were run on SDS-PAGE gels and probed with EGFR, Her2 and Actin antibodies. (B) Densitometric analysis of EGFR blots in A549 was performed using ImageJ. Resulting data was analyzed in MS-Excel. The protein half-life plots (t1/2) were generated using GraphPad Prism. The plots are of combined data from 2-3 biological repeats is shown. (C, D) NCI-H358 cell lines was treated similar to A549 and EGFR protein half-life was estimated by densitometric analysis.

Figure 6

BUB1 inhibitor reduces endocytosis of active EGFR in MDA-MB-231-1833 cells. (A) Cells were plated on glass coverslips, serum starved for 3-4 hours and pretreated with 2OH-BNPP1 (10 μM) or erlotinib (10 μM) for 1 hour followed by EGF treatment (50 ng/mL). Cells were fixed at different time points (5, 20, 40 and 80 minutes) post EGF treatment and processed for staining with pEGFR (Y1068) and EEA1 antibodies. Representative confocal images 20 minutes post EGF treatment are shown. (B) co-localization of pEGFR (Y1068) with EEA1 was estimated on ImageJ using JACOMP plugin. Data at 20 min post EGF treatment is plotted. Two-sided students t-test was performed on MS-Excel (p values, ** = 0.00097, ***=3.17 X 10^-5).

Figure 7

Proposed model of how BUB1 may regulate EGFR receptor endocytosis.