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Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors
Published on: July 17, 2020
[SHP2 and MKP5 in P2Y purinergic receptor-mediated prostate cancer invasion]
Hui-ying He1, Jie Zheng, Yan Li
1Department of Pathology, Health Science Center, Peking University, Beijing 100083, China.
Objective:
To investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.
Methods:
The wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.
Results:
ATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).
Conclusions:
Both SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.
Insights
Protein tyrosine phosphatase-SHP2 (SHP2) promotes prostate cancer cell invasion by activating ERK signaling, while dual-specificity MAPK phosphatase-MKP5 inhibits invasion by suppressing p38 activation. Both phosphatases influence P2Y receptor-mediated pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Prostate cancer cell invasion is a critical determinant of metastasis.
- P2Y purinergic receptors modulate cellular processes, including cancer cell behavior.
- Mitogen-activated protein kinases (MAPKs) are key regulators of cell proliferation and invasion.
Purpose of the Study:
- To elucidate the roles of SHP2 and MKP5 in P2Y receptor-induced MAPK activation and prostate cancer cell invasion.
- To compare the effects of SHP2 and MKP5 in prostate cancer cell lines with varying metastatic potentials.
Main Methods:
- Stable transfection of wild-type and mutant SHP2 and MKP5 expression vectors into human prostate cancer cell lines (1E8 and 2B4).
- Analysis of SHP2 tyrosine phosphorylation via immunoprecipitation.
- Western blot analysis of ERK1/2 and p38 activation using phospho-specific antibodies.
- Assessment of in-vitro cell invasion using Boyden-chamber assays.
Main Results:
- ATP stimulation induced stronger and prolonged SHP2 phosphorylation in highly metastatic 1E8 cells compared to non-metastatic 2B4 cells.
- SHP2 positively modulated ATP-induced ERK1/2 activation and cell invasion, with SHP2-wt enhancing invasion in 2B4 cells and SHP2-cs reducing it in 1E8 cells.
- MKP5-wt suppressed ATP-induced p38 phosphorylation and significantly reduced cell invasion in both cell lines.
Conclusions:
- SHP2 positively regulates ERK activation and prostate cancer cell invasion.
- MKP5 inhibits prostate cancer cell invasion by suppressing p38 activation.
- Both SHP2 and MKP5 are key mediators in P2Y receptor-driven prostate cancer progression.
