Abstract
The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Calpha (PKCalpha) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.
Highlights
To fulfill their functions in complex organisms, most proteins possess specialized domains that have been conserved throughout evolution
A Specific Domain of Ret finger protein (RFP) Is Required for Its Cytoplasmic Localization in NIH3T3 Cells—RFP is highly expressed in tumor cell lines, and its subcellular localization depends on cell type
Our study demonstrates that the coiled-coil domain of RFP containing the leucine-rich nuclear export signal (NES) controls shuttling of RFP between the nucleus and the cytoplasm
Summary
To fulfill their functions in complex organisms, most proteins possess specialized domains that have been conserved throughout evolution. Depending on their overall domain structure, proteins have been grouped into gene families, whose members often share similar functions in cells or organisms Members of one such gene family, the RING-B box-coiled-coil (RBCC) family [1], are characterized by their possession of a tripatic motif consisting of a RING finger [2, 3], one or two B boxes [4], and an ␣-helical coiled-coil domain [5]. These domains are involved in protein-protein interactions and allow RBCC family members to participate in various cellular processes depending on their subcellular localization. The regulation of RFP localization by PKC points to an important role for RFP in the control of cellular differentiation and proliferation
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