Differential nucleocytoplasmic shuttling of beta-arrestins. Characterization of a leucine-rich nuclear export signal in beta-arrestin2

beta-arrestins (betaarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two betaarrs (betaarr1 and betaarr2) is that betaarr1 is evenly distributed throughout the cell, whereas betaarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. betaarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in betaarr1. In addition, using a nuclear import assay in yeast we showed that betaarr2 is actively imported into the nucleus, suggesting that betaarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing betaarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in betaarr2, which was sufficient to redistribute betaarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of betaarr2 controls the subcellular distribution of JNK3.     

A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport.

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Delta cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-Delta cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.     

RBEL1 is a novel gene that encodes a nucleocytoplasmic Ras superfamily GTP-binding protein and is overexpressed in breast cancer

Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.     

BAG-1, an anti-apoptotic tumour marker

BAG-1 is a multifunctional and anti-apoptotic or anti-cell death protein that interacts with a variety of cellular proteins and affects their functions. On the cell surface, it binds to the cytosolic domain of the growth factor receptors and enhances the protection from cell death triggered by growth factor receptors. In the cytosol, it binds to Bcl-2 and heat shock protein, and modulates their functions. In the nucleus, it binds to a variety of nuclear hormone receptors and inhibits hormone-induced apoptosis. BAG-1 is widely overexpressed in a variety of tumour cell lines and cancer tissues. In addition, differential expression of BAG-1 isoforms has been observed. Preclinical studies indicate that overexpression of BAG-1, especially its nuclear and cytoplasmic isoforms, may be useful as a prognostic and/or predictive biomarker. Pilot clinical studies have demonstrated that overexpression of nuclear BAG-1 may be associated with a shorter survival in breast and laryngeal carcinomas. Conversely, overexpression of cytoplasmic BAG-1 may be associated with a better clinical outcome in early stage breast cancer and in non-small cell lung cancer. Further large-scale clinical studies are warranted to establish the role of BAG-1 as a novel prognostic and/or predictive biomarker in the clinical management of these common malignancies.     

Cytosolic and Nuclear Mitogen-Activated Protein Kinases Are Regulated by Distinct Mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPKK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

Subcellular differences in post-translational modification of barley 14-3-3 proteins

Expression and post-translational modification of barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, were investigated using isoform-specific antibodies. Although all three isoforms were shown to be present in the cytosolic, the nuclear and the microsomal cell fractions, differences in post-translational modification were identified for the different cell fractions. Germination-related modifications of 14-3-3 proteins were observed in the cytosol and the microsomal fraction, but not in the nucleus. In vitro proteolytic cleavage of 14-3-3 proteins using trypsin suggests that for 14-3-3A this change was caused by proteolytic cleavage of the unconserved C-terminal region.     

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.