Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution

The ret finger protein (rfp) is a member of the B box zinc finger gene family which possesses a tripartite motif consisting of a RING finger, B box finger, and a coiled-coil domain. Rfp is expressed at specific stages of spermatogenesis and in various adult mouse and human tissues. It becomes oncogenic when the tripartite domain is recombined with the tyrosine kinase domain of the ret protooncogene. Many of the B box family proteins function as homodimers, although the role of the individual components of the tripartite motif in this process remains unclear. We demonstrate that rfp homomultimerization occurs through the coiled-coil domains; however, while the B box is not an interacting interface itself, its structural integrity is necessary for this interaction to occur. This is the first evidence that the B box zinc finger domain is involved in regulating protein-protein interactions. Interestingly, we find that mutations of the RING finger and B box affect the subcellular compartmentalization of rfp in various cell lines. These results demonstrate that the interactions of rfp with itself and its association with specific subcellular compartments is dependent upon the function of all of the components of the tripartite motif. It is likely that these domains play a crucial role in the function of the rfp protein in normal cell differentiation and in its transformation potential in the recombined state

Ret finger protein is a normal component of PML nuclear bodies and interacts directly with PML

The ret finger protein (rfp) is a member of the B-box zinc finger gene family many of which may function in growth regulation and in the appropriate context become oncogenic. Members of this family are nuclear proteins that possess a characteristic tripartite motif consisting of the RING and B-box zinc binding domains and a coiled-coil domain. The promyelocytic leukemia gene (PML), another B-box family member, produces a protein product that is detected within punctate nuclear structures called PML nuclear bodies (NBs) or PML oncogenic domains (PODs). These NBs are complex structures that consist of a number of different proteins many of which have yet to be identified. In the disease acute promyelocytic leukemia (APL) a fusion protein, PML-RARA, is produced through the t(15:17) translocation. In APL the morphology of the NBs is altered. We report that rfp co-localizes with PML in a subset of the PML NBs and that it interacts directly with PML. This interaction is mediated through the rfp B-box and the distal two coils. In contrast, homomultimerization of rfp preferentially involves the B-box and the proximal coil. The association of rfp with the PML NBs is altered by mutations that affect rfp/PML interaction and in NB4 cells that are derived from APL patients. When treated with retinoic acid, rfp reassociates with the NBs in a pattern similar to non APL cells. Additionally, we found that rfp colocalizes with PML-RARA protein produced in APL patients. These results suggest that rfp, along with the other known/unknown components of PML NBs, have an important role in regulating cellular growth and differentiation