Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the Ba regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation-(L309D) and phosphorylation-(T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B¢, and B¢¢-type regulatory subunits in NIH 3T3 fibroblasts and neuro2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309D, and T304D mutants, which are impaired in their ability to associate with the Ba subunit, induces their loss. Silencing of Ba subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal Ba-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.

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The biophysical and molecular mechanisms that enable animals to detect magnetic fields are unknown. It has been proposed that birds have a light-dependent magnetic compass that relies on the formation of radical pairs within cryptochrome molecules. Using spectroscopic methods, we show that pigeon cryptochrome clCRY4 is photoreduced efficiently and forms long-lived spin-correlated radical pairs via a tetrad of tryptophan residues. We report that clCRY4 is broadly and stably expressed within the retina but enriched at synapses in the outer plexiform layer in a repetitive manner. A proteomic survey for retinal-specific clCRY4 interactors identified molecules that are involved in receptor signaling, including glutamate receptor–interacting protein 2, which colocalizes with clCRY4. Our data support a model whereby clCRY4 acts as an ultraviolet-blue photoreceptor and/or a light-dependent magnetosensor by modulating glutamatergic synapses between horizontal cells and cones.

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...

Cancer cells increase glucose metabolism to support aerobic glycolysis. However, only some cancer cells are acutely sensitive to glucose withdrawal, and the underlying mechanism of this selective sensitivity is unclear. We showed that glucose deprivation initiates a cell death pathway in cancer cells that is dependent on the kinase RIPK1. Glucose withdrawal triggered rapid plasma membrane depolarization and an influx of extracellular calcium into the cell through the L-type calcium channel Ca1.3 (CACNA1D), followed by activation of the kinase CAMK1. CAMK1 and the demethylase PPME1 were required for the subsequent demethylation and inactivation of the catalytic subunit of the phosphatase PP2A (PP2Ac) and the phosphorylation of RIPK1. Plasma membrane depolarization, PP2Ac demethylation, and cell death were prevented by glucose and, unexpectedly, by its nonmetabolizable analog 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor. These findings reveal a previously unknown function of glucose as a signaling molecule that protects cells from death induced by plasma membrane depolarization, independently of its role in glycolysis. Components of this cancer cell death pathway represent potential therapeutic targets against cancer.

the transcription factor ZenK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. it has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. to address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZenK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain. Over the past century Aves have served as powerful models to study vertebrate cognitive and sensory neurobiology 1. It has been shown that crows and cockatoos can develop tools to solve complex tasks 2-4 , pigeons can memorize abstract visual patterns 5,6 , songbirds are able to learn conspecific and novel vocalizations 7,8 , and robins rely on magnetic fields for long-range navigation 9,10. It is apparent from these studies that the avian brain processes information from a myriad of sensory cues allowing it to drive an array of complex behaviours. The quantitation of immediate early gene (IEG) expression is a well established methodology to map the neuronal networks that integrate this information. IEGs represent a class of genes characterized by a rapid rise in expression levels upon sustained cell stimulation and are therefore linked to synaptic activity in the brain 11,12. One commonly used IEG is the transcription factor ZENK (an acronym for zif-268, egr-1, ngfi-a, krox-24) 13-16. Upon expression, this zinc finger protein regulates the transcription of downstream targets, thereby modulating synaptic long-term adaptations to incoming information 17. ZENK mRNA is induced rapidly in response to stimulation 18 , followed by peak protein expression approximately 1-2 hours after stimulus onset 19,20. It should be noted, however, that not all neuronal populations in the bird brain express ZENK 19,21-23 , and that ZENK induction is dependent on stimulus context 24,25 or neuromodulatory factors such as the activity of the noradrenergic system 26. To date avian studies relying on ZENK expression have employed in situ hybridization, or a commercial polyclonal antibody raised against the rabbit ZENK homologue EGR-1 (C-19) 19,27-30. While C-19 has been widely used, it displays high batch-to-batch variation, requires laborious amplification methods in some species 27,28 , and its production has now been discontinued by the provider. In order to establish a reliable alternative to C-19 we set out to gen...

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.