Epidermal growth factor receptor potentiates MCM7-mediated DNA replication through tyrosine phosphorylation of Lyn kinase in human cancers

Abstract

Epidermal growth factor receptor (EGFR) initiates a signaling cascade that leads to DNA synthesis and cell proliferation, but its role in regulating DNA replication licensing is unclear. Here, we show that activated EGFR phosphorylates the p56 isoform of Lyn, p56(Lyn), at Y32, which then phosphorylates MCM7, a licensing factor critical for DNA replication, at Y600 to increase its association with other minichromosome maintenance complex proteins, thereby promoting DNA synthesis complex assembly and cell proliferation. Both p56(Lyn) Y32 and MCM7 Y600 phosphorylation are enhanced in proliferating cells and correlated with poor survival of breast cancer patients. These results establish a signaling cascade in which EGFR enhances MCM7 phosphorylation and DNA replication through Lyn phosphorylation in human cancer cells.

Figure 1

MCM7 is Tyr phosphorylated by Lyn kinase. (A) Immunoprecipitates of endogenous MCM7 were analyzed by Western blotting using 4G10 antibody for detecting Tyr phosphorylation in various cancer cell lines stimulated with EGF for 1 hr. (B) Western blot of endogenous MCM2, MCM3, and MCM4 in anti-MCM7 immunoprecipitates from A431 cells stimulated with EGF at various time points. The cell lysate from A431 cells treated with EGF for 2 hr was used for both the anti-MCM7 and control IgG immunoprecipitation. The right graph shows quantified densities of bands at different time points, and the band’s density in control lane (i.e. EGF 0 hr) was set as 1. (C) Cell lysates from 293T cells expressing exogenous Myc-MCM7 and Flag-p56^Lyn were subjected to immunoprecipitation and immunoblotting with indicated antibodies. (D) A431 cell lysates with different treatments were subjected to immunoprecipitation and immunoblotting with indicated antibodies to detect the association of endogenous MCM7 with Lyn and MCM7 Tyr phosphorylation. (E) Western blot of Tyr phosphorylation of GST-MCM7 by recombinant p56^Lyn (top) and phosphorylation of p56^Lyn itself (middle) using 4G10 antibody. Bottom, Coomassie Blue staining of the GST-MCM7 input. (F) Western blot of endogenous MCM7 Tyr phosphorylation in A431 cells with or without EGF stimulation (lanes 3 and 2). Endogenous Lyn was knocked down by siRNA (targeting 3′-UTR; lane 4) and rescued with a siRNA-resistant Flag-Lyn (lane 5). See also Figure S1 and Table S1.

Figure 2

Lyn phosphorylates MCM7 at Y600. (A) Mass spectrometry analysis of endogenous MCM7 immunopurified from A431 cells. (B) Western blot of Tyr phosphorylation of MCM7 or MCM7 Y600F from 293T cells transfected with EGFR, Flag-p56^Lyn and Myc-MCM7, as indicated. The number under each band is quantified relative density with lane 3 being set as 1. (C) Western blot analysis of Tyr phosphorylation of GST-MCM7 and GST-MCM7 Y600F by recombinant p56^Lyn kinase (top) and phosphorylation of p56^Lyn using 4G10 antibody (middle). Bottom, Coomassie Blue staining of the GST fusion protein input. (D) Western blot of phosphorylation of the endogenous MCM7 Y600 in A431 cells with or without EGF stimulation (lanes 3 and 2). Endogenous Lyn was knocked down by siRNA (targeting 3′-UTR; lane 4) and rescued with a siRNA-resistant Flag-p56^Lyn (lane 5). The number under each band is quantified relative density with lane 3 being set as 1. (E) Cell lysates from A431 cells transfected with the indicated siRNA with or without EGF stimulation were subjected to Western blot analysis of endogenous MCM2, MCM3, and MCM7 in the soluble fraction or chromatin fraction. (F) ChIP analysis of endogenous MCM2, MCM3, and MCM7 binding to the DNA replication origins in LMNB2 (Lamin B2), MCM4, and control locus (2 kb downstream of LMNB2 locus) in A431 cells transfected with the indicated siRNA and stimulated with or without EGF for 7 hr. Results are normalized to levels in cells without EGF stimulation (n = 3). Error bars, ± SD. See also Figure S2.

Figure 3

MCM7 Y600 phosphorylation enhances MCM complex assembly and cancer cell proliferation. (A) MDA-MB-468 (1 × 10⁴) cells carrying the indicated constructs were seeded in the presence or absence of 1 μM PP2, and cell number was determined at day 7 (n = 3). (B) MDA-MB-468 cells were treated as (A) and DNA synthesis rate was assayed at day 5 by detection of BrdU incorporation (n = 3). (C) In vivo tumor growth of orthotopically transplanted MDA-MB-468 cells (n = 5). (D) Western blot of endogenous MCM2, MCM3, and MCM5 in the anti-Myc-MCM7 immunoprecipitates from the soluble fraction of A431 cells with or without EGF stimulation. (E) Same as (D) except binding of MCM7 with other MCM members in chromatin fraction was assessed. (F) ChIP analysis of Myc-MCM7 or Myc-MCM7 Y600F binding to the DNA replication origins in LMNB2 (Lamin B2), MCM4, and control locus (2 kb downstream of LMNB2 locus) in A431 cells stimulated with EGF for 7 hr. Results are normalized to levels in cells without EGF stimulation (n = 5). (G) Top, HeLa cells expressing siRNA-resistant Myc-MCM7 or Myc-MCM7 Y600F were transfected with a control siRNA or a siRNA targeting 3′UTR of MCM7 as indicated. After 3 days, cells were lysed and subjected to western blot analysis using the indicated antibodies. Bottom, after siRNA transfection, 1 × 10⁴ cells were seeded in the presence or absence of 10 ng/ml EGF and DNA synthesis rate was assayed at day 3 by detection of BrdU incorporation (n = 3). (H) HeLa cells expressing siRNA-resistant Myc-MCM7 or Myc-MCM7 Y600F were transected with siRNA targeting MCM7 3′UTR. Cells were synchronized by 48 hr of serum starvation and then stimulated with EGF for 12 hr. Cell cycle progression was analyzed by PI staining using FACS. Error bars, ± SD. See also Figure S3.

Figure 4

EGFR phosphorylates p56^Lyn Y32. (A) Anti-Lyn immunoprecipitates from A431 cells with or without EGF stimulation were analyzed by western blotting using 4G10 antibody. The asterisk (*) indicates the protein phosphorylated by EGF stimulation in the anti-Lyn immuniprecipates. (B) Western blot of endogenous EGFR and phospho-EGFR Y1173 (EGFR p-Y1173) in the anti-Lyn immunoprecipitates from A431 cells with indicated treatments. (C) Western blot of endogenous Lyn in the anti-EGFR immunoprecipitates from A431 cells with different treatments. The number above (for p56^Lyn) or under (for p53^Lyn and EGFR) each band is quantified relative density with lane 2 being set as 1. (D) Western blot detection of Tyr phosphorylation of GST fused various domains of p56^Lyn by recombinant EGFR in vitro. Coomassie blue staining is shown below each blot. A schematic representation of Lyn isoforms with the 21-amino acid presented in p56^Lyn but not in p53^Lyn indicated is shown at the top. U, the unique domain; U/Y32F, the U domain with Y32F mutation. (E) Western blot of Tyr phosphorylation of GST-p56^Lyn KD and GST-p56^Lyn KD/Y32F by recombinant EGFR (middle). and phosphorylation of EGFR itself (top) using 4G10 antibody. Bottom, GST-p56^Lyn KD and GST-p56^Lyn KD/Y32F detected by western blot using an anti-GST antibody. (F) Cell lysates of A431 cells with different treatments were subjected to anti-Lyn immunoprecipitation then immunoblotted with the phospho-p56^Lyn Y32 (p56^Lyn p-Y32) antibody. See also Figure S4.

Figure 5

Phosphorylation of Y32 enhances p56^Lyn mediated MCM7 Tyr phosphorylation and cancer cell proliferation. (A) MDA-MB-468 cells (1 × 10⁴) carrying indicated constructs were seeded in the presence or absence of 500 nM inhibitors. Cell number was determined at day 7 (n = 3). (B) DNA synthesis rate of MDA-MB-468 cells treated as (A) was assayed at day 5 by detection of BrdU incorporation (n = 3). (C) In vivo tumor growth of orthotopically transplanted MDA-MB-468 cells (n = 5). (D) Western blot of p56^Lyn Y397 and MCM7 Y600 phosphorylation in Lyn knockdown A431 cells expressing Flag-p56^Lyn Y32F or Flag-p56^Lyn stimulated with EGF at indicated time point. Error bars, ± SD. See also Figure S5.

Figure 6

Phosphorylation of p56^Lyn Y32 and MCM7 Y600 correlate physiological proliferation. (A) Tyr phosphorylation of p56^Lyn Y32 and MCM7 Y600 in regenerating liver. N, normal liver; R, regenerating liver 24 hr after partial hepatectomy. (B) MCF-10A cells were serum starved for 48 hr and then stimulated with EGF at the indicated time points. Lysates were immunoblotted with indicated antibodies. The number under each band of anti-phospho MCM7 Y600 (MCM7 p-Y600) blot is quantified relative density with the lane 1 (0 hr) being set as 1. (C) Lysates of MCF-10A cell treated with EGF for 8 hr were subjected to Lyn immunoprecipitation and immunoblotting with indicated antibodies. (D) MCF-10A cells were serum starving for 48 hr and then stimulated with EGF in the presence of indicated inhibitors. Cell number was determined 48 hr after EGF stimulation (n = 3). (E) In the top panel, 1 × 10⁴ NCI-H226 cells were seeded in the presence or absence of 50 ng/ml EGF in medium with 0.5% calf serum, and cell number was determined at day 5 (n = 3). In the bottom panel, NCI-H226 cells were untreated or treated with 50 ng/ml EGF for 4 hr, and the lysates were immunoblotted with the indicated antibodies. Error bars, ± SD; ** indicates p < 0.01. See also Figure S6.

Figure 7

Phosphorylation of p56^Lyn Y32 and MCM7 Y600 correlate with the EGFR protein level of human tumor samples and poor survival of breast cancer patients. (A) IHC staining of EGFR level and Tyr phosphorylation of p56^Lyn Y32 and MCM7 Y600 of representative human breast tumor samples with high and low EGFR. Scale bar, 25 μm (B) Relationship between expression of EGFR, phospho-p56^Lyn Y32 (p56^Lyn p-Y32), phospho-MCM7 Y600 (MCM7 p-Y600), Lyn and MCM7 in human breast cancer tissues. (C) IHC staining of phospho-MCM7 Y600 and Ki-67 of representative human breast tumor samples. Scale bar, 25 μm. (D) Relationship between expression of phospho-MCM7 Y600 and Ki-67 in human breast cancer samples. (E) The Kaplan-Meier analysis of overall survival of breast cancer patients according to the phospho-Lyn Y32 level in their breast cancer. (F) The Kaplan-Meier analysis of overall survival of breast cancer patients according to the phospho-MCM7 Y600 level in their breast cancer. See also Figure S7, Table S2, S3, S4 and S5.