NXF-2, REF-1, and REF-2 affect the choice of nuclear export pathway for tra-2 mRNA in C. elegans

In C. elegans, tra-2 mRNA nuclear export is controlled by a 3'UTR element, the TRE. In the absence of TRA-1, the TRE retains tra-2 mRNA in the nucleus. The binding of TRA-1 to the 3'UTR overcomes this retention resulting in export of a TRA-1/tra-2 mRNA complex. Here, we find that, unlike most mRNAs, tra-2 mRNA exits the nucleus via an alternative pathway to NXF-1 that requires CRM1 activity. Inhibition of export by NXF-1 depends upon the TRE, CeNXF-2, CeREF-1, and CeREF-2. Removal of the TRE or any one of these factors results in export of tra-2 by NXF-1. NXF-2 and REF-1 specifically bind the TRE, suggesting that they directly control tra-2 mRNA export. Furthermore, choice of proper export pathway affects tra-2 translational control. Therefore, tra-2 mRNA export is highly regulated and plays an important role in development by regulating the activity of tra-2 mRNA in the cytoplasm.     

TRA-1/GLI controls development of somatic gonadal precursors in C. elegans

TRA-1/GLI is best known as a master regulator of sex determination in the nematode C. elegans, but its fly and vertebrate homologs (e.g. Ci, GLI) regulate embryonic patterning and cell proliferation. In this paper, we show that TRA-1/GLI controls development of the two somatic gonadal precursors (SGPs) in both XX and XO animals, in addition to its role in sex determination. Normally, SGPs reside at the poles of the gonadal primordium and divide according to intrinsic gonadal axes. In tra-1-null mutants, however, SGPs assume non-polar positions and the polarity of one SGP is reversed. Consistent with its SGP function, TRA-1 protein is present in SGPs during embryogenesis and early larval development. Previous studies have shown that the ehn-3 gene also affects SGP positions, and we report here that tra-1 and ehn-3 interact genetically. Whereas SGPs in tra-1 and ehn-3 single mutants are largely normal and generate many descendants, those in tra-1; ehn-3 double mutants do not mature or divide. Furthermore, tra-1 is a dominant enhancer of the ehn-3 gonadal defect, which includes the enhancement of a weak sexual transformation in the gonad. We cloned ehn-3, and found that it encodes a C2H2 zinc-finger protein. A rescuing EHN-3::GFP reporter is predominantly nuclear and expressed specifically in SGPs. The EHN-3 protein is therefore likely to regulate gene expression. We propose that TRA-1/GLI and EHN-3 have overlapping roles in regulation of multiple steps of SGP development. We speculate that regulation of SGP development may be an evolutionarily ancient role of TRA-1/GLI in nematode development.     

Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.     

Regulation of nucleo-cytoplasmic shuttling of human annexin A2—a proposed mechanism

Studies have long been focused on the functions of annexin A2 in the cytoplasm. However, the involvement of annexin A2 in DNA replication as a part of primer recognition protein complex and the presence of nuclear export signal (NES) suggest that annexin A2 is also functional in the nucleus, and its localization in the nucleus is under regulation by interaction with other nuclear factors through its N-terminus. During the study of the mechanism of annexin A2 sequestering in the nucleus and the regulation of its export from the nucleus, in this study, we show that endogenous annexin A2 is present in both the cytoplasm and the nucleus in HeLa, PC-3 and DU-145 cells. While exogenously expressed annexin A2 is excluded from nuclei of annexin A2-null LNCaP cells in a CRM1 (Chromosome Maintenance Region 1) mediated nuclear export, endogenous annexin A2 in HeLa, PC-3 and DU-145 cell lines does not undergo the CRM1 mediated nuclear export. While investigating the mechanism of the nuclear retention of annexin A2, we found that an anti-annexin A2 antibody that recognizes the C-terminus of annexin A2 (D1/274.5) cannot recognize nuclear annexin A2, suggesting that the domain recognized by this antibody may be masked in the nuclei. In order to find out the role of annexin A2 C-terminus in the nuclear retention of annexin A2, we transiently transfected green fluorescence protein (GFP)-fused N-terminal 29 amino acids of annexin A2 to LNCaP, PC-3 and DU-145 cells, and determined that the C-terminus is not required for the nuclear retention of annexin A2. Based on the finding described above, we propose a model for nuclear retention of annexin A2 where the regulation sites reside in the N-terminus and are adjacent to the NES, and upon modification, the NES is exposed and annexin A2 is exported from the nucleus. Electronic Supplementary Material The online version of this article (doi) contains supplementary material, which is available to authorized users.     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin

Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.     

Protein and RNA export from the nucleus

The presence of the nuclear envelope necessitates the movement of proteins and RNAs between the nucleus and the cytoplasm. Elaborate cellular machinery exists to promote the nuclear transport of macromolecules. Recent advances in the field have illuminated our comprehension of both nuclear import and export as powerful means of gene regulation. As our appreciation of the importance of the process has grown, its study has matured, moving beyond the single cell to the entire organism. This review discusses basic mechanisms and regulation of protein, mRNA, and ribosome export with an emphasis on developmental examples.     

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.     

Activity of the Sex-Determining Gene tra-2 Is Modulated to Allow Spermatogenesis in the C. ELEGANS Hermaphrodite

In the nematode C. elegans, there are two sexes, the self-fertilizing hermaphrodite (XX) and the male (XO). The hermaphrodite is essentially a female that makes sperm for a brief period before oogenesis. Sex determination in C. elegans is controlled by a pathway of autosomal regulatory genes, the state of which is determined by the X:A ratio. One of these genes, tra-2, is required for hermaphrodite development, but not for male development, because null mutations in tra-2 masculinize XX animals but have no effect on XO males. Dominant, gain-of-function tra-2 mutations have now been isolated that completely feminize the germline of XX animals so that they make only oocytes and no sperm and, thus, are female. Most of the tra-2(dom) mutations do not correspondingly feminize XO animals, so they do not appear to interfere with control by her-1, a gene thought to negatively regulate tra-2 in XO animals. Thus, these mutations appear to cause gain of tra-2 function in the XX animal only. Dosage studies indicate that 5 of 7 tra-2(dom) alleles are hypomorphic, so they do not simply elevate XX tra-2 activity overall. These properties suggest that in the wild type, tra-2 activity is under two types of control: (1) in males, it is inactivated by her-1 to allow male development to occur, and (2) in hermaphrodites, tra-2 is active but transiently inactivated by another, unknown, regulator to allow hermaphrodite spermatogenesis; this mode of regulation is hindered by the tra-2(dom) mutations, thereby resulting in XX females.