Covalent Modification of PML by the Sentrin Family of Ubiquitin-like Proteins

Abstract

PML, a RING finger protein with tumor suppressor activity, has been implicated in the pathogenesis of acute promyelocytic leukemia that arises following a reciprocal chromosomal translocation that fuses the PML gene with the retinoic acid receptor ␣ (RAR␣) gene. Immunocytochemical analysis has demonstrated that PML is co-localized with a novel ubiquitin-like protein in the nuclear bodies, which could be disrupted by the PML-RAR␣ fusion protein. The physical nature of this co-localization is unknown. Using a COS cell expression system, we show that PML is covalently modified by all three members of the sentrin family of ubiquitin-like proteins. Covalent modification of PML requires the conserved Gly residue near the C termini of sentrin proteins. Sentrinization of PML is highly specific because neither NEDD8 nor ubiquitin could modify PML. Similar specificity is also observed for the covalent modification of RanGAP1 by the sentrin member of ubiquitin-like proteins. These observations highlight the fine substrate specificity of the sentrinization pathway. In acute promyelocytic leukemia, two forms of PML-RAR␣ fusion proteins have been reported. Remarkably, both forms of PML-RAR␣ fusion proteins could not be sentrinized. Thus differential sentrinization of PML and PML-RAR␣ could play an important role in regulating the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.Chromosomal translocation t(15;17), detected in the majority of patients with acute promyelocytic leukemia, generates a fusion protein composed of portions of the retinoic acid receptor ␣ (RAR␣) 1 and RING finger protein called PML (1-4). In normal myeloid cells, PML is localized to a nuclear multiprotein complex called nuclear bodies or PML oncogenic domains (5, 6). In cell lines derived from patients with acute promyelocytic leukemia, the nuclear bodies are disrupted into a microparticulate pattern, which is reversible by treatment with retinoic acid (5, 6). PML has also been shown to suppress the transformation of NIH3T3 cells by the activated neu oncogene (7). Using fulllength PML as bait in a yeast two-hybrid interaction screening, Boddy et al. have isolated a novel ubiquitin-like protein called PIC1 that interacted specifically with PML in the yeast interaction assay (8). They have further shown that PIC1 was colocalized with PML to the nuclear bodies. In NB4 cells, which are derived from a patient with acute promyelocytic leukemia, there was no significant co-localization of PIC1 with PML. However, following retinoic acid treatment, a significant relocalization of PIC1 with PML was observed. These observations suggest that the association of PIC1 with PML may play an important role in the pathogenesis of acute promyelocytic leukemia.Our laboratory has recently reported the cloning of a novel ubiquitin-like molecule, called sentrin, which interacts specifically with the death domains of both Fas and tumor necrosis factor receptor 1 (9). Overexpression of sentrin protects cells against anti-Fas or tumor necrosis f...

Covalent Attachment of the SUMO-1 Protein to the Negative Regulatory Domain of the c-Myb Transcription Factor Modifies Its Stability and Transactivation Capacity

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Regulation of Heat Shock Transcription Factor 1 by Stress-induced SUMO-1 Modification

“…A number of SUMO-1-modified proteins localize in discrete structures in the nucleus including PML, HIPK2, and Sp100 (15)(16)(17)(18)(19). Therefore, to determine whether there is a relationship between HSF1 and SUMO-1 in stress-induced nuclear granules, we performed double labeled immunofluorescence analysis on normal and heat-treated HeLa cells using antibodies that recognize each of these proteins.…”

Identification and Characterization of a SUMO-1 Conjugation System That Modifies Neuronal Calcium/Calmodulin-dependent Protein Kinase II in Drosophila melanogaster

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