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14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

The retinoblastoma-related pocket proteins pRb, p107, and p130 are implicated in the control of cell proliferation, differentiation, and transformation. The function of pocket proteins is in part mediated by their ability to inhibit specific E2F transcription factors. The transcriptional activity of E2Fs is controlled by alteration of their nucleocytoplasmic localization during the cell cycle. p130 was observed to shuttle between the nucleus and cytoplasm in a heterokaryon fusion assay, suggesting the presence of nuclear and cytoplasmic localization signals. Two independent nuclear localization signals (NLS) that could target reporter proteins to the nucleus in transient transfection and microinjection experiments were identified in the C terminus of p130. In addition to the C-terminal NLS, the intact pocket domain of p130 itself was sufficient for nuclear translocation. Moreover, an additional functional NLS was mapped within the unique Loop region of p130. An N-terminal domain that conferred cytoplasmic localization was identified. Removal of the entire N terminus did not affect the ability of p130 to interact with E2F and to induce growth arrest. A model suggesting that the activity of pRb family members can be regulated by intracellular trafficking of the proteins is proposed.