Signaling to Nuclear Transport

In a recent issue of Molecular Cell, Yoon et al. provide evidence for the control of nucleocytoplasmic transport by protein kinase signaling pathways through phosphorylation of RanBP3, an accessory factor in the Ran GTPase system. This mechanism may coordinate nucleocytoplasmic transport with other mitogenic effects of these pathways. In a recent issue of Molecular Cell, Yoon et al. provide evidence for the control of nucleocytoplasmic transport by protein kinase signaling pathways through phosphorylation of RanBP3, an accessory factor in the Ran GTPase system. This mechanism may coordinate nucleocytoplasmic transport with other mitogenic effects of these pathways. The nucleocytoplasmic transport of many macromolecules depends upon Ran, a small GTPase of the Ras superfamily. Ran is concentrated in the nucleus where it is maintained in a GTP-bound form by RCC1, its chromatin-bound guanine nucleotide exchange factor. Ran-GTP levels in the cytoplasm are kept low by RanGAP1, which stimulates the GTPase activity of Ran. Proteins carrying a classical lysine-rich nuclear localization signal (NLS) are recognized in the cytoplasm by importin-β via the adaptor importin-α. This cargo complex translocates through the central hydrophobic channel of the nuclear pore complex (NPC), the large multiprotein structure that fenestrates the double membrane of the nuclear envelope. Within the nucleus, Ran-GTP binds to importin-β and causes dissociation of the cargo complex. Ran-GTP also promotes the export of proteins to the cytoplasm through its interaction with Crm1, another member of the importin/karyopherin family that recognizes leucine-rich nuclear export signals. Nucleocytoplasmic transport is modulated at multiple levels (Terry et al., 2007Terry L.J. Shows E.B. Wente S.R. Science. 2007; 318: 1412-1416Crossref PubMed Scopus (394) Google Scholar), including the expression of specific transport factors, modification of NPCs and substrate-specific effects, such as the posttranslational modification of cargo. Furthermore, because nucleocytoplasmic transport is important for general protein synthesis—ribosomal components are imported and assembled into ribosomes, mRNA and tRNA are exported through the NPC—one would perhaps expect the rate of transport to be enhanced when cells receive mitogenic signals to elevate protein synthesis. Now, Yoon et al., 2008Yoon S.-O. Shin S. Liu Y. Ballif B.A. Woo M.S. Gygi S.P. Blenis J. Mol. Cell. 2008; 29: 362-375Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar identify a mechanism by which extracellular signals transduced through kinase signaling pathways can modulate Ran-dependent nucleocytoplasmic transport. The authors set out to identify targets of RSK, the 90 kDa ribosomal S6 kinase that is activated by a wide variety of growth factors and other extracellular signals via the Ras-ERK MAP kinase pathway (Figure 1). Using a yeast two-hybrid assay to screen a mouse embryonic library for RSK binding partners, they identified RanBP3, a nuclear protein that binds to Ran-GTP and associates with RCC1 in complex with Ran (Mueller et al., 1998Mueller L. Cordes V.C. Bischoff F.R. Ponstingl H. FEBS Lett. 1998; 427: 330-336Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Nemergut et al., 2002Nemergut M.E. Lindsay M.E. Brownawell A.M. Macara I.G. J. Biol. Chem. 2002; 277: 17385-17388Crossref PubMed Scopus (40) Google Scholar). RanBP3 also interacts directly with Crm1, promoting Crm1-dependent nuclear export (Englmeier et al., 2001Englmeier L. Fornerod M. Bischoff F.R. Petosa C. Mattaj I.W. Kutay U. EMBO Rep. 2001; 2: 926-932Crossref PubMed Scopus (93) Google Scholar, Lindsay et al., 2001Lindsay M.E. Holaska J.M. Welch K. Paschal B.M. Macara I.G. J. Cell Biol. 2001; 153: 1391-1402Crossref PubMed Scopus (114) Google Scholar, Nemergut et al., 2002Nemergut M.E. Lindsay M.E. Brownawell A.M. Macara I.G. J. Biol. Chem. 2002; 277: 17385-17388Crossref PubMed Scopus (40) Google Scholar, Sabri et al., 2007Sabri N. Roth P. Xylourgidis N. Sadeghifar F. Adler J. Samakovlis C. J. Cell Biol. 2007; 178: 557-565Crossref PubMed Scopus (44) Google Scholar). Yoon et al. showed that RanBP3 interacts with RSK in human cells, and although the interaction was weak, this might be expected for a substrate of the kinase. Indeed, they found that RSK phosphorylates RanBP3 at serine 58, which lies within a kinase consensus motif RxRxxS. This motif is also recognized by other members of the “AGC” kinase family, and the authors confirmed that serine 58 is also phosphorylated by Akt/protein kinase B. Inhibitors of the MEK/ERK/RSK and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways indicated that both pathways are involved in the phosphorylation of RanBP3 in human HEK293 cells. Although serine 58 lies adjacent to the NLS of RanBP3 (Welch et al., 1999Welch K. Franke J. Köhler M. Macara I.G. Mol. Cell. Biol. 1999; 19: 8400-8411Crossref PubMed Google Scholar), mutation of this residue to alanine affected neither its nuclear localization nor its interaction with Crm1. However, the rather weak interaction in cells between RanBP3 and a mutant of Ran insensitive to RanGAP1 (RanE46G) was stimulated by serum and inhibited by the S58A mutant of RanBP3, suggesting that phosphorylation of RanBP3 at serine 58 stabilizes its interaction with Ran-GTP. Short hairpin RNA-mediated ablation of RanBP3 expression in HeLa cells did not alter Crm1 localization but did cause partial redistribution of Ran to the cytoplasm. The normal accumulation of Ran in the nucleus was restored by RanBP3 expression to endogenous levels, but not by the S58A mutant. Furthermore, RanBP3 S58A did not support the efficient nuclear import of ribosomal protein L12 or an artificial construct containing an NLS. Although it is not clear if RanBP3 is required for HeLa cell proliferation, cells expressing the S58A mutant of RanBP3 failed to proliferate as well as those with the wild-type protein. Together, these results indicate that phosphorylation of RanBP3 at serine 58 by RSK and Akt could provide a mechanism to couple nucleocytoplasmic transport and ribosome biogenesis with other effects of these pathways on transcription and translation, progression through the cell cycle, and cell survival. It is not clear exactly how inhibition of RanBP3 phosphorylation affects Ran localization and nuclear protein import, but it could be through an effect on Ran-GTP production by RCC1 or possibly another step in the cycling of Ran or other transport factors between the nucleus and the cytoplasm. The apparent lack of effect on Crm1 suggests that RanBP3 phosphorylation does not specifically control Crm1-mediated nuclear export. Interestingly, Hendriksen et al., 2005Hendriksen J. Fagotto F. van der Velde H. van Schie M. Noordermeer J. Fornerod M. J. Cell Biol. 2005; 171: 785-797Crossref PubMed Scopus (69) Google Scholar identified a Crm1-independent role for RanBP3 in the nuclear export of β-catenin to which it binds directly. Overexpression of RanBP3 inhibits Wnt signaling and disrupts β-catenin-dependent dorsoventral axis formation during Xenopus embryonic development, whereas ablation of RanBP3 causes overactivation of Wnt signaling in cultured cells and in Drosophila embryos (Hendriksen et al., 2005Hendriksen J. Fagotto F. van der Velde H. van Schie M. Noordermeer J. Fornerod M. J. Cell Biol. 2005; 171: 785-797Crossref PubMed Scopus (69) Google Scholar). Furthermore, RanBP3 has a negative role in JAK/STAT signaling in Drosophila through control of STAT92E transport (Baeg et al., 2005Baeg G.H. Zhou R. Perrimon N. Genes Dev. 2005; 19: 1861-1870Crossref PubMed Scopus (174) Google Scholar). So phosphorylation of RanBP3 by RSK and Akt has the potential to regulate other signaling mechanisms through effects on the nucleocytoplasmic transport of specific components of those pathways. It is possible that RanBP3 normally has a role in restraining cell growth, proliferation, and/or differentiation in vivo, and this suppression might be relieved by its phosphorylation. Interestingly, the human RanBP3 gene is located in a chromosome region (19p13.3) that is commonly deleted in various cancers and may contain multiple tumor suppressor genes (Hendriksen et al., 2005Hendriksen J. Fagotto F. van der Velde H. van Schie M. Noordermeer J. Fornerod M. J. Cell Biol. 2005; 171: 785-797Crossref PubMed Scopus (69) Google Scholar). So, it may be interesting to determine if loss of RanBP3 or its increased phosphorylation plays a role in cancer. Although many questions remain, this study provides new food for thought on how extracellular signals can coordinate cellular processes through regulation of nucleocytoplasmic transport. Ran-Binding Protein 3 Phosphorylation Links the Ras and PI3-Kinase Pathways to Nucleocytoplasmic TransportYoon et al.Molecular CellFebruary 15, 2008In BriefThe major participants of the Ras/ERK and PI3-kinase (PI3K) pathways are well characterized. The cellular response to activation of these pathways, however, can vary dramatically. How differences in signal strength, timing, spatial location, and cellular context promote specific cell-fate decisions remains unclear. Nuclear transport processes can have a major impact on the determination of cell fate; however, little is known regarding how nuclear transport is regulated by or regulates these pathways. Full-Text PDF Open Archive