This study uncovered a novel mechanism for the phosphorylation-dependent nuclear import of NLS-containing proteins. To the best of our knowledge, this is the first study to demonstrate that 14-3-3 promotes the nuclear import of a cargo protein. In particular, we established that phosphorylated myopodin directly interacts with 14-3-3 proteins, and that this interaction is functionally significant because it promotes the binding of myopodin to importin α and, thereby, promotes the nuclear import of myopodin.
Nuclear import is regulated at several levels. The binding of NLS-containing proteins to cytosolic receptors of the importin/karyopherin superfamily is critical because the affinity of this interaction determines transport efficiency (Weis, 2003
). Therefore, targeting sequence recognition is a key control point in the regulation of nuclear import (Jans et al., 2000
). One way to regulate nuclear transport is by changing the phosphorylation status of the cargo protein. Phosphorylation of the SV40 large tumor antigen and the Drosophila melanogaster
morphogen dorsal upstream of the NLSs directly enhances the affinity between NLSs and importin α/β1 (Hubner et al., 1997
; Briggs et al., 1998
). The deletion of the phosphorylation sites in these nuclear cargoes causes a decreased nuclear import rate, which reveals the importance of serine/threonine phosphorylation for nuclear import efficiency. Phosphorylation of a cargo protein may modulate the affinity between NLS and importin α or may cause a conformational change in the cargo, thereby exposing an NLS (Harreman et al., 2004
). In addition, phosphorylation may cause the release or binding of a heterologous, NLS-masking protein.
Recent proteomic analyses of 14-3-3–binding proteins revealed that importins were putative 14-3-3 targets (Meek et al., 2004
). However, 14-3-3 proteins do not have an intrinsic NLS, and, therefore, the direct binding of importins to 14-3-3 is unlikely (Brunet et al., 2002
). Instead, these interactions are indirect and are mediated by proteins like myopodin that bind both 14-3-3 proteins and importin α ( D).
14-3-3 proteins are highly acidic, chaperone-like molecules that can induce conformational changes in their binding partners. Myopodin, on the other hand, is a very basic protein with an isolectric point of 9.34 (Weins et al., 2001
). Hence, it seems conceivable that 14-3-3 binding neutralizes the negative charge of myopodin. This, in turn, may change the structure of myopodin, thereby unmasking and exposing NLSs. In this study, we have shown that myopodin contains two functional 14-3-3–binding sites that are required for efficient 14-3-3 binding ( H). Because each monomer of a 14-3-3 dimer binds its target in opposite directions (Yaffe et al., 1997
), the binding of a 14-3-3 dimer could change the predicted linear structure of myopodin (Weins et al., 2001
) to a U-shaped conformation. This is a potential regulatory conformational change in myopodin that could alter its biochemical features, such as the capability to interact with other proteins (e.g., importin α). Clearly, structural analysis of the myopodin–14-3-3 interaction will be required to confirm or refute this hypothesis.
14-3-3 proteins can participate in protein translocation into mitochondria (Alam et al., 1994
) and chloroplasts (May and Soll, 2000
) by serving as cytoplasmic chaperones. In this study, we describe a novel role of 14-3-3 as a regulator of nuclear import by showing that 14-3-3 binding enables myopodin to interact with importin α. Protein import into mitochondria and chloroplasts is mechanistically and evolutionary very different from nuclear import. Nevertheless, the results of this study suggest that cargoes with different subcellular destinations may share 14-3-3 as a cytoplasmic chaperone in order to achieve an import-compatible structure.
14-3-3 proteins are involved in the regulation of cell proliferation, differentiation, and cell death (van Hemert et al., 2001
). For example, the epithelial 14-3-3 isoform σ serves as a tumor suppressor (Hermeking, 2003
), and the loss of 14-3-3σ expression can cause cell transformation (Dellambra et al., 2000
). Several studies have shown a decrease or loss of 14-3-3σ expression in transformed cells (Simooka et al., 2004
; Urano et al., 2004
), including transitional urinary bladder carcinomas (Ostergaard et al., 1997
; Moreira et al., 2004
). Myopodin not only binds to 14-3-3σ but also acts as a tumor suppressor in bladder carcinomas (Sanchez-Carbayo et al., 2003
). In fact, the relocalization of myopodin from the nucleus to the cytoplasm predicts the clinical outcome of these tumors (Sanchez-Carbayo et al., 2003
). Based on the results of this study, it is tempting to speculate that the loss of nuclear myopodin in invasive bladder tumors results from the loss of 14-3-3σ expression or from the loss of myopodin phosphorylation, which, in turn, abrogates 14-3-3 and importin α binding. Future studies will explore the phosphorylation stage of myopodin in normal urothelium and in bladder tumors.
To summarize, this study has helped to identify a functional role for 14-3-3 in promoting nuclear import. In particular, we have shown that importin α binding and the subsequent nuclear import of the tumor suppressor myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3 (). Altogether, these data provide a novel paradigm for the regulation of nuclear import by 14-3-3 in that 14-3-3 regulates the binding of a phosphorylated cargo protein to importin α and the nuclear import machinery.
Figure 6. A model for phosphorylation- and 14-3-3-dependent nuclear import of myopodin. (1) When phosphoacceptor sites in 14-3-3–binding motifs#1 (S225) and #2 (T272) are not phosphorylated, myopodin cannot interact with 14-3-3. Therefore, the NLSs in myopodin (more ...)