The PI3K/Akt and Ras/ERK pathways are two major signaling systems that regulate cell fate decisions in a variety of cell types in both normal and disease settings. Although the basic backbone of these pathways has been defined, subtle but critical differences in the strength, location and duration of signaling by these pathways is now known to occur. Exactly how these different aspects of PI3K/Akt and Ras/ERK signaling are regulated and how these differences result in the differential regulation of downstream effectors and cell fates are not well understood. It is clear that signaling in the cytoplasm versus the nucleus will yield very different outcomes; therefore regulating nucleo-cytoplasmic transport may provide a means for modifying the cellular response to these “generic” signaling pathways. The data reported here highlight a previously unreported role for the PI3K/Akt and Ras/ERK pathways on nuclear transport. We demonstrate that two basophilic protein serine/threonine kinases, RSK and Akt, associate with and phosphorylate a relatively uncharacterized Ran binding protein, RanBP3, at the same site. This observation immediately raised the possibility that RanBP3 is a site of signal convergence downstream of the Ras and PI3K pathways, respectively. Furthermore, these results suggested the possibility that both pathways might modulate the function of the small GTPase, Ran, in response to growth factor signaling. Indeed, we have found that RanBP3 is involved in the formation of the Ran gradient, and phosphorylation of RanBP3 by RSK and Akt regulates Ran gradient formation and nuclear transport.
The Ran gradient plays a key role in the proper spatial activation of important regulatory proteins by regulating their nuclear transport (Li et al., 2003
; Sazer, 2005
), thus the finding that RanBP3 contributes to establishing the Ran gradient in interphase cells is an important discovery. The establishment of the Ran-GTP gradient across the nuclear envelope imposes directionality on nucleocytoplasmic transport by insuring that both import and export cargo bind to and subsequently dissociate from their respective transport receptors in the appropriate cellular compartment. Ran-GTP is thus perfectly positioned to provide the spatial cues essential for insuring that many proteins, particularly those that participate in or regulate the cell cycle, are present and activated in the right place at the right time (Sazer, 2005
). Despite the important function of the Ran gradient, the precise mechanisms by which this gradient is established and prevented from collapsing remain largely unknown. The most acceptable theory is that the compartmentalization of RCC1 (nucleus) and RanGAP (cytosol) is responsible for this gradient. In addition, nuclear transport factor 2 (NTF2) appears to be involved in maintenance of the Ran gradient by regulating Ran-GDP transport (Smith et al., 1998
). NTF2 binds to Ran-GDP in the cytosol and facilitates import of Ran-GDP into the nucleus. Here, we show that RanBP3 regulates the Ran gradient without affecting RCC1 protein levels or localization. Our data suggest that RanBP3 may regulate the Ran gradient in part by a direct interaction with Ran and in part by regulating RCC1 activity, a well-known contributing factor for establishment of the Ran gradient. RanBP3 has a nuclear localization signal (Welch et al., 1999
) and in interphase cells most RanBP3 is found in the nucleus under a variety of growth conditions such as during serum-deprivation, after stimulation of cells with serum, defined growth factors or tumor promoting PMA, in the absence or presence of a variety of signaling inhibitors (unpublished data). Although the interaction is relatively weak compared to RanBP1 (Noguchi et al., 1997
), RanBP3 has a Ran-binding domain and prefers Ran-GTP to Ran-GDP for its binding partner (Macara, 2001
; Mueller et al., 1998
). Through the interaction with Crm1 and cargo molecules, RanBP3 facilitates the export of the Ran-GTP-cargo complex through the nuclear pore (Englmeier et al., 2001
; Lindsay et al., 2001
). In this regard, the RanBP3 function uncovered in our study is closely related to the function of NTF2. Whereas NTF2 binds to and transports Ran-GDP into the nucleus, which becomes Ran-GTP by RCC1, RanBP3 interacts with Ran-GTP and contributes to its transport into the cytosol where Ran-GTP is hydrolyzed to Ran-GDP by RanGAP. Therefore, RanBP3 and NTF2 regulate the formation of the Ran gradient by modulating the cycling of Ran-GDP and Ran-GTP. At this point however, we cannot exclude the possibility that RanBP3 may sequester free Ran-GTP in the nucleus for later usage.
In addition to nucleocytoplasmic transport in interphase cells, the Ran gradient is also essential for kinetochore function, spindle assembly, microtubule dynamics, nuclear envelope reformation, and other mitotic events during mitosis (Clarke, 2005
; Sazer, 2005
). As observed during interphase, the mitotic Ran gradient is similarly formed by RCC1 (Clarke, 2005
; Moore et al., 2002
). Moreover, Crm1, a nuclear exporter in interphase cells, was recently shown to be an essential factor for the formation of kinetochore fibres and for faithful chromosome segregation, thereby, regulating mitotic spindle assembly during mitosis (Arnaoutov et al., 2005
). Considering our data and the previously known function of RanBP3 as a binding partner of Ran, RCC1, and Crm1, it will be of interest to determine if RanBP3 also regulates the Ran gradient during mitosis.
It is intriguing that Ras/ERK/RSK and PI3K/Akt pathways can contribute to the regulation of nuclear transport through RanBP3 phosphorylation. There are several ways that nuclear transport can be regulated (Kau et al., 2004
). The first is by post-translational modification of the cargo molecules themselves or their binding partners that mask or increase their transport, which in turn affects their ability to interact with their cognate transporter. A second way that nucleocytoplasmic transport is modulated is through the regulation of the level of transporters. Some importins are differentially expressed in specific tissues and therefore might transport cargoes only during specific stages of development, or function in a particular cell type. Finally, the nuclear pore itself can offer an added level of regulation. The number of functional pores varies depending on the growth state of the cell, which in turn affects the overall permeability of the nucleus. Here we identify a previously unreported mechanism for regulating nuclear transport – through growth factor regulated post-translational modification of a transporter cofactor. Considering the growing evidence linking subtle differences in the spatial and temporal regulation of signaling pathways to specific cell fate decisions, the finding that Ras/ERK/RSK and PI3K/Akt signaling can modulate the Ran gradient expands our understanding and views of how nucleocytoplasmic transport can be modulated by mitogenic inputs. Since RanBP3 phosphorylation by RSK and Akt enhances nuclear transport rate, the strength of activation of these enzymes, the duration of activation, their cellular localization, as well as the differential regulation of these pathways will all contribute to their ability to differentially modulate RanBP3 function and the Ran gradient throughout the cell cycle. This level of regulation will be layered upon the other mechanisms of regulating nucleocytoplasmic transport as discussed above, thus providing another way to fine tune this critical process at a general or very specific level.
Many signaling molecules, transcription factors, cell cycle regulators, growth factor receptors, viral proteins, and disease-related proteins are transported in and out of the nucleus (Xu and Massague, 2004
). Some proteins shuttle between the nucleus and cytoplasm continuously, some do so only once. Other proteins do not shuttle at all but localize permanently in or out of the nucleus following translation (Tartakoff et al., 2000
). For nucleocytoplasmic transport, many proteins have a nuclear localization signal (NLS) and/or nuclear export signal (NES). Importins (nuclear importers such as importin α and β) and exportins (such as the Crm1 nuclear exporter) recognize a cargo's NLS and NES sequence, respectively, and as a result properly transport them (Macara, 2001
; Weis, 2003
). However, nuclear import and/or export of some proteins are importin-independent and/or exportin-independent even when cargos have NLS and/or NES signals. In addition, although most nucleocytoplasmic protein transport is dependent on the Ran gradient, some proteins move into and/or out of the nucleus independent of Ran. Moreover, transport rates vary for different cargos. These observations imply that there are likely many ways to contribute to the general as well as specific regulation of nucleocytoplasmic transport. It is not surprising that the regulation of nuclear transport is very complicated and involves the intricate interplay of multiple protein components since nucleocytoplasmic transport contributes to the proper regulation of transcription, translation, cell cycle progression, and other cellular processes that determine cell fate; survival, apoptosis, differentiation, etc (Weis, 2003
). For example, we have found that nuclear import of the ribosomal protein L12 is affected by RanBP3. Ribosome biogenesis is a multi-step process: ribosomal protein synthesis in the cytoplasm, import into the nucleus, ribosome assembly in the nucleolus, and export to the cytoplasm. For proper functioning of ribosomes, all these steps should be efficiently controlled. These preliminary data indicate that RanBP3 may be involved in a step of ribosome biogenesis, a possibility that will require more experimentation. Further studies are also needed to identify and characterize other targets of RanBP3-mediated transport.
Despite the important function of Ran and its effectors, little is known about how these effectors are regulated in cells. Recently, it was shown that RCC1 is phosphorylated in mitosis by Cdc2 kinase (Li and Zheng, 2004
). This phosphorylation is essential for positioning a high Ran-GTP concentration on mitotic chromosomes and for spindle assembly in mammalian cells. Another Ran modulator that may be regulated is a Ran-GDP dissociation factor for the NTF2-RanGDP complex. As described above, NTF2 binds to Ran-GDP in the cytosol with high affinity and promotes translocation of this complex into the nucleus. In the nucleus, Ran-GDP is released from NTF2 and converted into Ran-GTP by RCC1. Ran-binding proteins within the nucleus sequester free Ran-GTP and further nucleotide exchange by mass action (Smith et al., 1998
). However, how Ran-GDP is dissociated from NTF2 is unknown. Recently, it was proposed that there is an unidentified protein that stimulates dissociation of Ran-GDP from NTF2 (Yamada et al., 2004
). From the data obtained from their experiments, the authors proposed that this unidentified protein might interact with RCC1 and enhance RCC1 catalytic activity. Interestingly, it was also suggested that the function of this unknown protein could be regulated by phosphorylation. From our data, the localization and function of RanBP3 reflect the properties of this unidentified protein. Since NTF2 and RanBP3 play important roles in Ran gradient formation, it will be of interest to investigate the relationship between these proteins.
It is clear that the regulation of nucleocytoplasmic transport is integrated with subtle differences in temporal and spatial regulation of signaling proteins to ensure a proper biological response to extracellular signals. Deregulation of nuclear transport on the other hand has been linked to several human diseases (Davis et al., 2007
; Fabbro and Henderson, 2003
; Kau et al, 2004
), likely the result of failed signaling at the right place and/or right time. The diseases linked to improper nuclear transport include Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), familial hypercholesterolemia, cystic fibrosis, schizophrenia, retinitis pigmentosa, nephrogenic diabetes insipidus, and others (Davis et al., 2007
). Failure to properly localize certain proteins can also render cells resistant to drugs. In the case of cancers, altered localization of tumor suppressors (p53, INI1/hSNF5, BRCA1), cell cycle regulators (p21WAF-1, p27Kip1), and transcription factors (FOXO, NF-κB) are often found to be closely linked to tumor progression (Kau et al., 2004
). The contribution of inappropriate localization of important regulatory proteins to the development or progression of various diseases has made this process an excellent target for therapeutic intervention. Therefore, further examination of nuclear transport mechanisms may reveal different ways to treat a growing number of diseases ranging from metabolic disorders to cancer that have been linked to improper regulation of nucleocytoplasmic transport (Davis et al., 2007
). In this regard, our studies provide important new insights into how the Ran gradient is established and how the protein kinases RSK and Akt, enzymes linked to many critical biological processes such as cell growth, proliferation, survival and migration, can modulate nucleocytoplasmic protein transport. Future work should now be focused toward understanding how an improperly regulated RanBP3 contributes to various human disorders and diseases.