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Cooperation between the RING + B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival
M Fagioli1, M Alcalay, L Tomassoni
1Istituto di Medicina Intema e Scienze Oncologiche, Università degli Studi di Perugia, Policlinico Monteluce, Italy.
Abstract:
PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.
Insights
The PML protein suppresses cell growth by inducing apoptosis, and its key domains are essential for this activity. This suggests the PML part of the PML/RARalpha fusion protein drives its effects on cell survival.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Acute Promyelocytic Leukemia (APL) is characterized by the PML/RARalpha fusion protein.
- PML/RARalpha blocks hematopoietic precursor differentiation and enhances their survival.
- The specific role of PML in normal physiology and its contribution to APL pathogenesis are unclear.
Purpose of the Study:
- To investigate the biological activities of the PML protein.
- To determine the domains of PML responsible for its growth suppressive and localization functions.
- To elucidate the contribution of the PML component to the oncogenic functions of PML/RARalpha.
Main Methods:
- Transfection of various cell lines with PML constructs.
- Analysis of cell growth, apoptosis, and protein localization.
- Site-directed mutagenesis to delete specific PML domains.
Main Results:
- PML exhibits growth suppressive activity across diverse cell lines by inducing apoptosis.
- Nuclear localization of PML is crucial for its growth suppressive function.
- The Ring, B1-B2, and coiled-coil domains are essential for PML localization to nuclear bodies and its growth suppressive activity.
Conclusions:
- PML is a critical regulator of cell survival, primarily through apoptosis induction.
- The PML moiety within the PML/RARalpha fusion protein retains its intrinsic growth suppressive activity.
- The oncogenic effects of PML/RARalpha on cell survival are mediated by the functional PML component.