Defining the mechanism of arsenic-induced degradation of PML
Arsenic trioxide is a clinically effective treatment for the disease acute promyelocytic leukaemia (APL) which is caused by the chromosomal translocation t(15;17) which fuses the promyelocytic leukaemia (PML) protein to the retinoic receptor alpha (RARa). The PML-RARa oncoprotein disrupts normal retinoic acid signalling and the function of PML nuclear bodies (PML-NBs), subnuclear protein complexes with roles in control of apoptosis and cellular senescence. Treatment with arsenic induces rapid post translational modification of PML and with the small ubiquitin like modifier (SUMO). SUMO modification of PML recruits the SUMO targeted ubiquitin E3 ligase RNF4 via four SUMO interaction motifs within the N-terminal region of RNF4. PML is then ubiquitylated and targeted for proteasomal degradation. In APL, these events trigger degradation of PML-RARa, curing the disease. To further investigate the process of arsenic induced degradation of PML, a high content siRNA screen was designed to monitor the fate of a YFP linked version of PML after siRNA mediated knockdown of components of the ubiquitin system and arsenic treatment. RNF4 depletion prior to arsenic treatment prevented PML degradation and resulted in accumulation of PML in large, bright PML-NBs. This was used as a positive control. A library of siRNAs targeting 1067 gene products were screened to identify those which perturbed the process of arsenic mediated degradation of PML, and those which affected the stability of PML in untreated cells. A number of putative hits were identified. Depletion of the cullin RING ligase scaffold CUL3, and the NEDD8 E3 ligase DCUN1D1 resulted in striking accumulation of PML, suggesting PML may be a substrate of a CUL3 RING ligase complex. Further experiments using the inhibitor of neddylation, MLN4924 support this hypothesis. PML is expressed as a various isoforms which encode a unique C-terminal region, due to alternative splicing. The second part of this study investigated the role of this variable C-terminal region in the response of the six major PML isoforms to arsenic treatment. Using a system in which only a single eYFP-linked PML isoform is expressed, differences in the localisation of PML isoforms following arsenic treatment were identified, with PML I, II and VI found to accumulate in the cytoplasm following arsenic treatment, whereas PML III, IV and V did not. A high content imaging assay identified PML V as the isoform most readily degraded following arsenic treatment, and PML IV as relatively resistant to degradation. Using siRNA it was demonstrated that arsenic induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 resulted in marked accumulation of PML V, suggesting this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.
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