Forced expression of RNF36 induces cell apoptosis.
RNF36 (ring finger protein 36; alias Trif), a member of the RING zinc finger protein family, is expressed in germ cells at round spermatid stages during spermatogenesis. RING finger proteins have been implicated in a variety of functions including oncogenesis, viral replication, and developmental processes. Since no germ cell line is presently available to study the function of RNF36, in this research, we expressed RNF36 truncated and full-length proteins in COS-7 and HEK-293 cell lines to study the effect of RNF36 in somatic cells. The full-length RNF36 protein in both cell lines showed a speckled pattern in the nucleus. Truncated RNF36-1 protein with its putative nuclear localization signal (NLS) remained within the nucleus but lost the speckled pattern. The promyelocytic leukemia (PML) protein, another RING finger protein, was previously identified as present in the nucleus with a speckled pattern. Double-staining and coimmunoprecipitation analyses suggested that RNF36 colocalizes and interacts with PML. In vitro phosphorylation analysis further suggested that RNF36 nuclear localization is under the control of phosphorylation, which might be mediated by p38. Treatment with the p38 inhibitor SB203580 resulted in the cytoplasmic translocation of RNF36. Overexpression of full-length RNF36 in cells induced about half of the transfected cells to undergo cell death. The results of DNA fragmentation assays, flow cytometry assay, and TUNEL staining suggest that the death of RNF36-transfected cells was caused by apoptosis. Following further characterization of the molecular mechanism of RNF36-induced apoptosis, we found that the expression of Bax, caspase-2, and receptor-interacting protein were elevated upon RNF36 induction in test cells. These results suggest that RNF36 may interact with PML and induce cell apoptosis. We suspect that RNF36 may play a role in germ cell homeostasis during spermatogenesis.
Shyu
Huey-Wen
HW
Graduate Institute of Life Science, National Defense Medical Center, National Defense University, and Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Hsu
Shih-Hsien
SH
Hsieh-Li
Hsiu-Mei
HM
Li
Hung
H
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D019556:COS Cells; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D002479:Inclusion Bodies / Q000378:metabolism; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000502:physiology*; D016335:Zinc Fingers / Q000235:genetics*; D051028:bcl-2-Associated X Protein; D048051:p38 Mitogen-Activated Protein Kinases
C494826:BAX protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D000070584:Tripartite Motif Proteins; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins; C579345:TRIM69 protein, human; C476388:Trim69 protein, mouse; D044767:Ubiquitin-Protein Ligases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases; C093642:SB 203580