Silencing of type II phosphatidylinositol 4-kinase β stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells
| Autor: | Naveen Bojjireddy, Santosh K. Sandur, Gosukonda Subrahmanyam, Sonica Chaudhry, Maiko Thoh, Vibhor Joshi |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Small interfering RNA
Kinase HEK 293 cells Amino Acid Motifs Apoptosis Cell Biology Transfection Caspase 8 Biochemistry Cell biology Neoplasm Proteins Small hairpin RNA chemistry.chemical_compound HEK293 Cells chemistry Neoplasms Cancer cell MCF-7 Cells Humans Phosphatidylinositol Amino Acid Sequence Apoptosis Regulatory Proteins Molecular Biology 1-Phosphatidylinositol 4-Kinase Sequence Deletion |
| Zdroj: | The Biochemical journal. 476(2) |
| ISSN: | 1470-8728 |
| Popis: | Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II β from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II β did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II β-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II β shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II β may be a novel regulator of Par-4 through protein–protein interactions. These studies have potential implications in cancer therapy. |
| Databáze: | OpenAIRE |
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