Protein-tyrosine Phosphatase PTPN9 Negatively Regulates ErbB2 and Epidermal Growth Factor Receptor Signaling in Breast Cancer Cells

Taichang Yuan,Yongping Wang,Zhizhuang J Zhao,Haihua Gu

doi:10.1074/jbc.m109.099879

Abstract

ErbB family of the receptor protein-tyrosine kinase plays an important role in the progression of human cancers including breast cancer. Finding protein-tyrosine phosphatase (PTPs) that can specifically regulate the function of ErbB should help design novel therapies for treatment. By performing a small interfering RNA screen against 43 human PTPs, we find that knockdown of protein-tyrosine phosphatase PTPN9 significantly increases ErbB2 tyrosyl phosphorylation in the SKBR3 breast cancer cell line. In addition, knockdown of PTPN9 expression also enhances tyrosyl phosphorylation of the ErbB1/epidermal growth factor receptor (EGFR) in the MDA-MB-231 breast cancer cell line. Conversely, increasing expression of PTPN9 wild type (WT) inhibits tyrosyl phosphorylation of ErbB2 and EGFR. To test whether ErbB2 and EGFR are substrates of PTPN9, PTPN9 WT, and a substrate trapping mutant (PTPN9 DA) are overexpressed in SKBR3 and MDA-MB-231 cells. Compared with vector control, expression of PTPN9 WT significantly inhibits whereas expression of PTPN9 DA dramatically enhances tyrosyl phosphorylation of ErbB2 and EGFR, respectively. In contrast, expression of PTPN9 WT or DA mutant does not affect tyrosyl phosphorylation of ErbB3 and Shc. Importantly, coimmunoprecipitation and glutathione S-transferase fusion protein pulldown experiments show that tyrosol-phosphorylated ErbB2 or EGFR is preferentially associated with PTPN9 DA compared with PTPN9 WT, indicating that ErbB2 and EGFR are substrates of PTPN9. Furthermore, PTPN9 WT expression specifically impairs EGF-induced STAT3 and STAT5 activation, and inhibits the cell growth in soft agar. Last, PTPN9 WT expression also reduces invasion and MMP2 expression of MDA-MB-231 cells. Our data suggest PTPN9 as a negative regulator of breast cancer cells by targeting ErbB2 and EGFR and inhibiting STAT activation.

Highlights

Protein-tyrosine phosphatases (PTPs), which include membrane-associated receptor and cytoplasmic types, are enzymes that remove phosphates from phosphorylated tyrosine residues in proteins [6]
We found that only the siRNA against PTPN9 significantly increases (ϳ2fold) ErbB2 tyrosine phosphorylation in SKBR3 cells compared with scramble control RNA
For the first time, we provide evidence that PTPN9 is a protein-tyrosine phosphatase specific for epidermal growth factor receptor (EGFR) and ErbB2 and that negatively regulates EGFR and ErbB2 signaling

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Reagents—293T cells and human breast cancer cell lines SKBR3 and MDA-MB-231 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 1 mM sodium pyruvate, 100 units/ml of penicillin, and 100 ␮g/ml of streptomycin (Hyclone). Plasmids and Retrovirus Production—Retroviral MSCVIRES-GFP (pMIG) plasmid expressing human PTPN9 and its substrate trapping mutant D470A (DA) cDNAs were as described [17]. To knockdown PTPN9 expression, SKBR3 and MDA-MB-231 cells were transiently transfected with 100 nM non-target control siRNA oligo or PTPN9 siRNA oligo using transfection reagent 1 (Dharmacon), and lysed for biochemical analyses 3 days after transfection. Glutathione-agarose beads containing equal amounts (15 ␮g) of GST or GST-PTPN9 fusion proteins were used to incubate with equal amounts of cell lysates from MDA-MB-231 or 293T-transfected cells for 2 h at 4 °C. Gelatin Zymography—Equal numbers of MDA-MB-231 vector control and PTPN9 WT expressing cells were plated in a 6-well plate and 24 h later changed to DMEM without FBS. Statistical Analysis—Unpaired one-tailed Student’s t test was used for statistical analysis (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001)

RESULTS

Although the phosphorylated EGFR was not found associated with

DISCUSSION