Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition
Pace UniversityCell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 09/2012; 11(17):3324-30. DOI: 10.4161/cc.21693
The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G 1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be that dephosphorylation of specific amino acids signals apoptosis vs. cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants of Rb to alter serine/threonine to glutamic acid to mimic the phosphorylated state. By calcium phosphate transfection, mutant plasmids were introduced into C33A Rb-null cells, and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. Our results show that only mutation of threonine-821 to glutamic acid (T821E) blocked apoptosis by 50%, whereas other sites tested had little effect. In Rb-null Saos-2 and SKUT-1 cells, the T821E mutation also blocked apoptosis induced by the cdk inhibitor, Roscovitine, by 50%. In addition, we show that endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Thus, our data indicates that dephosphorylation of threonine-821 of Rb is required for cells to undergo apoptosis.
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ABSTRACT: Multisite phosphorylation modulates the function of regulatory proteins with complex signaling properties and outputs. The retinoblastoma tumor suppressor protein (Rb) is inactivated by cyclin-dependent kinase (Cdk) phosphorylation in normal and cancer cell cycles, so understanding the molecular mechanisms and effects of Rb phosphorylation is imperative. Rb functions in diverse processes regulating proliferation, and it has been speculated that multisite phosphorylation might act as a code in which discrete phosphorylations control specific activities. The idea of an Rb phosphorylation code is evaluated here in light of recent studies of Rb structure and function. Rb inactivation is discussed with an emphasis on how multisite phosphorylation changes Rb structure and associations with protein partners.Trends in Biochemical Sciences 12/2012; 38(1). DOI:10.1016/j.tibs.2012.10.007 · 11.23 Impact Factor
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ABSTRACT: Lung cancer is a high-grade malignancy with poor 5 year-survival rates that remains incurable with current therapies. Different cellular stresses, including antitumor agents, ionizing radiation and ultraviolet (UV) light, can induce apoptosis and activate signaling pathways. UV has multiple effects on tumor cells, including DNA damage, and increases the expression of some genes involved in tumor cell apoptosis and DNA repair. It has been reported that UV can also activate casein kinase 2 (CK2). CK2, a Ser/Thr protein kinase, has been reported to be frequently overexpressed in various types of human cancer, including lung cancer, and is associated with tumor development. Thus, combination of UV and CK2 inhibitors may be a new strategy for the treatment of lung cancer. Our results demonstrated that inhibition of CK2a through CK2 siRNA or a CK2 inhibitor [(4,5,6,7-tetrabromobenzotriazole (TBB)] enhances the decrease in cell viability of lung cancer cells (A549 and H2030) induced by UV. Western blot analysis demonstrated that the combination increased the expression of apoptotic protein markers cytochrome c and the cleavage of poly ADP-ribose polymerase (PARP) and caspase-3. Furthermore, our results indicated that UV decreased the expression of the tumor suppressor protein PML through activation of CK2. Inhibition of CK2 by CK2 siRNA and TBB can recover the reduction of PML induced by UV. Collectively, these results demonstrate the significant apoptosis of lung cancer cells induced by combination treatment of the CK2 inhibitor and UV radiation. CK2 enhanced cell apoptosis by UV radiation may due, at least partly, to recover the expression of PML. These findings warrant the clinical testing of CK2 inhibitors which, when used in conjunction with DNA-damaging agents such as radiation, may be an effective cancer therapeutic strategy.Oncology Reports 04/2013; 30(1). DOI:10.3892/or.2013.2407 · 2.30 Impact Factor
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ABSTRACT: Purpose: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance. Experimental design: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts. Results: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05). Conclusions: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy.Clinical Cancer Research 08/2014; 20(21). DOI:10.1158/1078-0432.CCR-14-0326 · 8.72 Impact Factor
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