No abstract

The oncogenic protein ␤-catenin is overexpressed in many cancers, frequently accumulating in nuclei where it forms active complexes with lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factors, inducing genes such as c-myc and cyclin D1. In normal cells, nuclear ␤-catenin levels are controlled by the adenomatous polyposis coli (APC) protein through nuclear export and cytoplasmic degradation. Transient expression of LEF-1 is known to increase nuclear ␤-catenin levels by an unknown mechanism. Here, we show that APC and LEF-1 compete for nuclear ␤-catenin with opposing consequences. APC can export nuclear ␤-catenin to the cytoplasm for degradation. In contrast, LEF-1 anchors ␤-catenin in the nucleus by blocking APC-mediated nuclear export. LEF-1 also prevented the APC/CRM1-independent nuclear export of ␤-catenin as revealed by in vitro assays. Importantly, LEF-1-bound ␤-catenin was protected from degradation by APC and axin in SW480 colon cancer cells. The ability of LEF-1 to trap ␤-catenin in the nucleus was down-regulated by histone deacetylase 1, and this correlated with a decrease in LEF1 transcription activity. Our findings identify LEF-1 as key regulator of ␤-catenin nuclear localization and stability and suggest that overexpression of LEF-1 in colon cancer and melanoma cells may contribute to the accumulation of oncogenic ␤-catenin in the nucleus.␤-Catenin accumulates to excessive levels in different cancers, including melanomas, colon cancer, breast cancer, and hepatocarcinomas (1-3). Whether overexpressed in cancer (2, 3), in transfected cells (4), or in transgenic mice (5), the induced ␤-catenin accumulates throughout the cell with frequent concentration in the nucleus. ␤-Catenin is thought to be the key mediator of the Wnt signaling pathway (3), and when overexpressed it can cause cell transformation (6). The oncogenic potential of ␤-catenin is mediated by its association with a class of related (but not identical) transcription factors that include lymphoid enhancer factor-1 (LEF-1), 1 and the T cell factors including TCF-1, -3, and -4 (3). Of these, LEF-1 is of particular interest as it is overexpressed in colon cancer cell lines (7) and colon tumors (8), and in metastatic melanoma cells (9), and is known to form nuclear ␤-catenin-LEF-1 complexes in vivo, which activate transcription of various transforming genes, including cyclin D1 (10) and c-myc (11). ␤-Catenin overexpression results from stabilizing cancer mutations within the ␤-catenin gene (see Ref. 2) or in genes that regulate its degradation such as APC and axin (see Ref.3). Recently, we and others showed that the nuclear build-up of ␤-catenin is normally prevented by a combination of nuclear export and cytoplasmic degradation (12-14). ␤-Catenin can exit the nucleus by two distinct pathways: the CRM1 export pathway, which requires its association with the shuttling protein APC (12)(13)(14), and an alternative CRM1-independent export route (15, 16). Despite the ability of ␤-catenin to efficiently exit the nucleus of SW480 colon cancer c...

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56␣, -␤, and -⑀ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56␣ subcellular localization. We detected B56␣ in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56␣. Conversely, CRM1 overexpression shifted B56␣ to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451-469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56␣. Active NESs were identified at similar positions in the cytoplasmic B56-␤ and ⑀ isoforms, but not in the nuclear-localized B56-␦ or ␥ isoforms. The transient expression of B56␣ induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56␣ co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56␣-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56␣ can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.Reversible protein phosphorylation is a key mechanism regulating a myriad of cellular processes. A delicate balance between the opposing effects of protein kinases and phosphatases determines the functional state of many proteins. Protein phosphatase 2A (PP2A) 3 refers to a major family of heterotrimeric serine-threonine phosphatase enzymes in the cell (1-3). The core enzyme is a dimer consisting of a 36-kDa catalytic C-subunit and a 65-kDa structural regulatory A-subunit, which acts as a scaffold to bring into proximity the C-subunit and protein substrates bound by the diverse regulatory B-subunits. There are four B-subunit gene families each with multiple genes that encode a range of splice variant peptides. The diverse nature of PP2A is inherent in its composition, which is potentially comprised of over 200 distinct protein complexes each containing different combinations of the A-, B-, and C-subunits, and hence allowing for variability and subtle regulation in phosphatase action (3).The B-subunits are postulated to regulate PP2A activity in different ways: (a) by targeting the holoenzyme to specific subcellular locations (e.g. B55␣ directs PP2A to microtubules (4)), (b) determining substrate specificity (e.g. PP2A complexes containing B55 or B72 B-subunits cause the activation or inhibition of SV40 DNA replication, respectively, because of differences in substrate recognition sites on...

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism.