AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Randependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKBmediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.Protein kinase B (PKB), also known as Akt, promotes cell survival in many different cell types (24,38,40,66). Following its initial cloning (17, 34), PKB was isolated as the transforming gene of v-Akt, hence the name c-Akt and its classification as a proto-oncogene (6, 67). Activation of PKB requires the lipid phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) (15) and phosphorylation by an upstream kinase, PDK1 (2, 69, 72). PIP 3 is produced by phosphatidylinositol 3-kinase in response to signals from extracellular growth factors (for a review see reference 60). PKB has been shown to phosphorylate and regulate the activity of transcription factors in response to survival factors. Genetic studies of Caenorhabditis elegans have demonstrated that the PKB signal transduction pathway inhibits the activity of the Forkhead transcription factor, daf-16, a gene that regulates longevity (55). There are three human orthologues of daf-16, AFX (13), FKHR (27), and FKHRL1 (3), that were first identified as chromosomal breakpoints in human tumors.AFX is phosphorylated by PKB in response to insulin and serum at three sites: T28, S193, and S258 (42). Phosphorylation of these residues by PKB leads to both inhibition of the transcriptional activity of AFX and cytoplasmic retention and/or nuclear exclusion of the protein. Withdrawal of serum or insulin results in AFX dephosphorylation, nuclear localization, and target gene activation. In the absence of survival f...

A concentration gradient of the GTP-bound form of the GTPase Ran across nuclear pores is essential for the transport of many proteins and nucleic acids between the nuclear and cytoplasmic compartments of eukaryotic cells [1] [2] [3] [4]. The mechanisms responsible for the dynamics and maintenance of this Ran gradient have been unclear. We now show that Ran shuttles between the nucleosol and cytosol, and that cytosolic Ran accumulates rapidly in the nucleus in a saturable manner that is dependent on temperature and on the guanine-nucleotide exchange factor RCC1. Nuclear import in digitonin-permeabilized cells in the absence of added factors was minimal. The addition of energy and nuclear transport factor 2 (NTF2) [5] was sufficient for the accumulation of Ran in the nucleus. An NTF2 mutant that cannot bind Ran [6] was unable to facilitate Ran import. A GTP-bound form of a Ran mutant that cannot bind NTF2 was not a substrate for import. A dominant-negative importin-beta mutant inhibited nuclear import of Ran, whereas addition of transportin, which accumulates in the nucleus, enhanced NTF2-dependent Ran import. We conclude that NTF2 functions as a transport receptor for Ran, permitting rapid entry into the nucleus where GTP-GDP exchange mediated by RCC1 [7] converts Ran into its GTP-bound state. The Ran-GTP can associate with nuclear Ran-binding proteins, thereby creating a Ran gradient across nuclear pores.

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5 end and a 3-8-nt protruding 3 end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.Nucleo-cytoplasmic transport of most RNAs and proteins is dependent on soluble receptors called karyopherins that can dock at and translocate through the nuclear pore complex. Interaction between cargo and karyopherin ␤ is governed by the GTPase Ran. The asymmetric distribution of the Ran regulatory proteins provides a steep gradient of RanGDP (cytoplasmic)/RanGTP (nuclear) across the nuclear envelope that ensures the directionality of nuclear transport (1, 2). Nuclear import receptors unload their cargo upon binding to RanGTP in the nucleus, whereas RanGTP is used to assemble export complexes which are in turn destabilized by dissociation of RanGTP in the cytoplasm (3, 4).Our understanding of the nuclear export of RNAs has been greatly facilitated by the study of viral RNAs. For this reason, we focused our attention on the adenovirus VA1 RNA (VA1), a 160-nt 1 -long RNA transcribed by RNA polymerase III that massively accumulates in the cytoplasm of infected cells. It serves to antagonize the interferon-induced cellular antiviral defense system. Indeed, VA1 binds and inhibits the doublestranded RNA-dependent protein kinase R (PKR), which otherwise phosphorylates eIF2␣ and leads to the inhibition of protein synthesis (5, 6). Adenovirus VA1 RNA contains a new cis-acting RNA export motif that comprises a double-stranded stem (Ͼ14 nt) with a base-paired 5Ј end and a 3-8-nt protruding 3Ј end and that can tolerate some mismatches and bends (7). This export signal, called minihelix, is present not only in VA1 but in a large family of small viral and cellular RNAs transcribed by polymerase III. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin). This exportin is distinct from Crm1 and exportin-t (7, 8). Therefore, we sought to identify cellular factors that bind to and mediate the export of miniheli...

First defined in the mid-1990s, prebiotics, which alter the composition and activity of gastrointestinal (GI) microbiota to improve health and well-being, have generated scientific and consumer interest and regulatory debate. The Life Sciences Research Organization, Inc. (LSRO) held a workshop, Prebiotics and the Health Benefits of Fiber: Future Research and Goals, in February 2011 to assess the current state of the science and the international regulatory environment for prebiotics, identify research gaps, and create a strategy for future research. A developing body of evidence supports a role for prebiotics in reducing the risk and severity of GI infection and inflammation, including diarrhea, inflammatory bowel disease, and ulcerative colitis as well as bowel function disorders, including irritable bowel syndrome. Prebiotics also increase the bioavailability and uptake of minerals and data suggest that they reduce the risk of obesity by promoting satiety and weight loss. Additional research is needed to define the relationship between the consumption of different prebiotics and improvement of human health. New information derived from the characterization of the composition and function of different prebiotics as well as the interactions among and between gut microbiota and the human host would improve our understanding of the effects of prebiotics on health and disease and could assist in surmounting regulatory issues related to prebiotic use.