In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Abstract

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor a. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (Bi and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both Bi and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the Bi and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-B1/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.The human PML gene is associated with APL (acute promyelocytic leukemia), which arises due to a block in normal differentiation of promyelocytes (for review, see ref. 1). In APL cells, PML is fused with the retinoic acid receptor a (RARa) following a reciprocal chromosomal translocation t(15;17)(q22q21) (2-5). The PML protein has been shown to be part of a nuclear multiprotein complex distinct from small nuclear ribonucleoproteins and nucleoli (6-10). These PML nuclear bodies become disrupted in leukemic cells; however, treatment with retinoic acid reverses these changes (8-10). It has been shown that the PML-RARa fusion product can bind to both normal PML (8, 10) as well as RXR, another retinoic acid receptor family member (retinoid X receptor), forming heterodimers (11). This leads to speculation that there is a dominant-negative effect of PML-RARa over both the PML protein and the RXR pathways. PML, but not PML-RARa, has been shown to be a growth suppressor both in vitro and in vivo (12), suggesting that sequestration of normal PML by PML-RARa would affect the growth suppressor activity of PML and sequestration of RXR, the induction of differentiation (12). It is suggested that both events may be necessary for leukemogenesis (12,13).The PML coding sequence contains three novel cysteinerich metal binding regions and a predicted a-helical coiled-coilThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1601(see refs. 2 and 14). The first region, the RING motif, defines a family of proteins expressed in organisms ranging from plants to viruses, several of which have been implicated in oncogenesis (for revie...

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis

“…It belongs to a large family of proteins with common structural features, including a RING motif, two additional Cys/His-rich regions, named B-boxes and a coiled coil region, through which PML is able to form homodimers and, in APL cells, heterodimers with PML/RAR (Kastner et al, 1992;Goddard et al, 1991;Reddy et al, 1992;Borden et al, 1995Borden et al, , 1996. Within the nucleus, the PML protein is mainly localized in speci®c subdomains, the PML nuclear bodies (PMLNBs) (Dyck et al, 1994).…”

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein

“…Because, PML does not bind DNA, it is postulated that its e ects on transcription are through protein-protein interactions with other factors involved in transcription regulation (Borden et al, 1995). Supporting the above hypothesis, recent analysis of the solution structure of the PML RING®nger suggests that this region may be involved in protein-porotein interactions (Borden et al, 1996). Accordingly, the PML-interacting clone 1 (PIC1), a new ubiquitin-like protein, was recently cloned by twohybrid screening.…”

Finding a role for PML in APL pathogenesis: a critical assessment of potential PML activities

“…8,10,12,27,28 If W73 of eIF4E is mutated to alanine (W73A), the resulting W73A mutant is still able to bind the m 7 G cap structure with wild-type affinity, but is unable to form either an active translation initiation complex with eIF4G, or inactive complexes with negative regulators including the 4E-BPs.…”

“…1,57 The RING and B-box domains were shown to mediate protein-protein interactions instead. 24,27,28 PML may only exhibit direct transcriptional activity when artificially fused to a Gal4-DNA binding domain. 1,66 If PML has a role in transcription, then it would not be unreasonable to find PML-NBs surrounding transcriptionally active genomic loci.…”

“…24 These domains are actually used for interacting with partner proteins that preserve both the structural integrity and subsequent physiological activities of the PML-NB. 8,10,12,27,28 Upon first assessment of current PML literature there appears to be a seemingly endless list of potential PML partner proteins implicating PML in a variety of functions at every level of gene expression. 1,29 Indeed, the sheer number of potential partners has led some researchers to propose that the PML-NB is a storage site or nuclear depot.…”