GSK-3 Phosphorylates ��-Catenin and Negatively Regulates Its Stability via Ubiquitination/Proteosome-mediated Proteolysis

δ-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby δ-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates δ-catenin and thus affects its stability. Initially, we found that the level of δ-catenin was greater and the half-life of δ-catenin was longer in GSK-3β^−/− fibroblasts than those in GSK-3β^+/+ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3α and -3β kinase dead constructs, consistently showed that the levels of endogenous δ-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3α and -3β interact with and phosphorylate δ-catenin. The phosphorylation of ΔC207-δ-catenin (lacking 207 C-terminal residues) and T1078A δ-catenin by GSK-3 was noticeably reduced compared with that of wild type δ-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr¹⁰⁷⁸ residue of δ-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased δ-catenin levels and caused an accumulation of ubiquitinated δ-catenin. It was also found that GSK-3 triggers the ubiquitination of δ-catenin. These results suggest that GSK-3 interacts with and phosphorylates δ-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.δ-Catenin was first identified as a molecule that interacts with presenilin-1 (PS-1)² by yeast two-hybrid assay (1) and was found to belong to the p120-catenin subfamily of armadillo proteins, which characteristically contain 10 Arm repeats (2). In addition to its interaction with PS-1 and its abundant expression in brain (3, 4), several lines of evidence indicate that δ-catenin may play a pivotal role in cognitive function. First, the hemizygous loss of δ-catenin is known to be closely correlated with Cri-du-Chat syndrome, a severe form of mental retardation in humans (5). Second, severe learning deficits and abnormal synaptic plasticity were found in δ-catenin-deficient mice (6). Moreover, in δ-catenin^−/− mice, paired pulse facilitation (a form of short term plasticity) was found to be reduced, and long term potentiation, which is related to the forming and storage mechanisms of memory, was deficient (7, 8). Third, δ-catenin interacting molecules, such as PSs (1, 9), cadherins (10), S-SCAM (2), and PSD-95 (11), have been shown to play important roles in modulating synaptic plasticity. However, even though the maintenance of an adequate δ-catenin level is known to be critical for normal brain function, few studies have been undertaken to identify the factors that regulate δ-catenin stability in cells. We have previously demonstrated that PS-1 inhibits δ-catenin-induced cellular branching and promotes δ-catenin processing and turnover (12).Because of structural similarities among β-catenin, p120-catenin, and δ-catenin and to their shared binding partners (i.e. PS-1 (1, 9) and cadherins (10)), glycogen-synthase kinase-3 (GSK-3) drew our attention as a potential candidate effector of δ-catenin stability in cells. GSK-3 is a serine/threonine kinase and has two highly homologous forms, GSK-3α and GSK-3β, in mammals (13). Although GSK-3α and GSK-3β have similar structures, they differ in mass (GSK-3α (51 kDa) and GSK-3β (47 kDa) (13)) and to some extent in function (14). GSK-3 is a well established inhibitor of Wnt signaling. Moreover, it is known to phosphorylate β-catenin, which results in its degradation via ubiquitination/proteosome-dependent proteolysis (15). GSK-3 is ubiquitously distributed in the human body, but it is particularly abundant in brain (13), and it is interesting that δ-catenin is also abundant in the nervous system (4) and that GSK-3 participates in the progression of Alzheimer disease (16). The majority of GSK-3 substrates have the consensus sequence (Ser/Thr)-Xaa-Xaa-Xaa-(Ser/Thr) (17). Interestingly, we found that δ-catenin has several putative phosphorylation sites targeted by GSK-3, which suggests that δ-catenin can be regulated by GSK-3 in the same way as β-catenin.In this report, we demonstrate that both GSK-3α and -3β interact with and phosphorylate δ-catenin and that this leads to its subsequent ubiquitination and degradation via proteosome-dependent proteolysis. Our results strongly suggest that GSK-3 is a key regulator of δ-catenin stability in cells.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Experimental translocation of juvenile water voles in a Scottish lowland metapopulation

Population density affects dispersal success because residents can hinder or facilitate immigration into a new site, via a “social fence effect” or “social attraction” (or “conspecific attraction”), respectively. These mechanisms can affect the dynamics of fragmented populations and the success of translocations. However, information on the settlement behaviour of dispersers is rare. We conducted a manipulative field experiment using wild water voles, which exist in metapopulations along waterways in Scotland. We translocated 17 young of dispersal age into either an occupied site or a vacant site containing good habitat, which had recently become extinct due to a feral predator (American mink) moving through. We monitored the movements of translocated voles using radio telemetry. Translocated voles were less likely to settle in occupied sites with higher densities of residents, suggesting a possible social fence effect at high density. There was evidence of a social attraction mechanism, because voles never remained at new sites unless another individual arrived soon after translocation, and they were more likely to settle in occupied or colonised sites than vacant ones. Voles remained in the transient phase of dispersal for many days, and often followed a “stepping stone” trajectory, stopping for several days at successive sites. We suggest that trajectories followed by dispersing water voles, the time scale and long dispersal distances found in this species are conducive to locating conspecifics at low density and colonising vacant habitat. These results are encouraging for prospects of metapopulation persistence and future translocation success.     

Relevance of Global Forest Change Data Set to Local Conservation: Case Study of Forest Degradation in Masoala National Park,Madagascar

A global data set on forest cover change was recently published and made freely available for use (Hansen et al. 2013. Science 342: 850–853). Although this data set has been criticized for inaccuracies in distinguishing vegetation types at the local scale, it remains a valuable source of forest cover information for areas where local data is severely lacking. Masoala National Park, in northeastern Madagascar, is an example of a region for which very little spatially explicit forest cover information is available. Yet, this extremely diverse tropical humid forest is undergoing a dramatic rate of forest degradation and deforestation through illegal selective logging of rosewood and ebony, slash‐and‐burn agriculture, and damage due to cyclones. All of these processes result in relatively diffuse and small‐scale changes in forest cover. In this paper, we examine to what extent Hansen et al.'s global forest change data set captures forest loss within Masoala National Park by comparing its performance to a locally calibrated, object‐oriented classification approach. We verify both types of classification with substantial ground truthing. We find that both the global and local classifications perform reasonably well in detecting small‐scale slash‐and‐burn agriculture, but neither performs adequately in detecting selective logging. We conclude that since the use of the global forest change data set requires very little technical and financial investment, and performs almost as well as the more resource‐demanding, locally calibrated classification, it may be advantageous to use the global forest change data set even for local conservation purposes.     

Infant Growth after Preterm Birth and Mental Health in Young Adulthood

Objectives

Faster growth after preterm birth benefits long-term cognitive functioning. Whether these benefits extend to mental health remains largely unknown. We examined if faster growth in infancy is associated with better self-reported mental health in young adults born preterm at very low birth weight (VLBW) (<1500g).

Study Design

As young adults, participants of the Helsinki Study of Very Low Birth Weight Adults self-reported symptoms of depression and attention deficit/hyperactivity disorder (ADHD) (n = 157) and other psychiatric problems (n = 104). As main predictors of mental health outcomes in linear regression models, we used infant weight, length, and head circumference at birth, term, and 12 months of corrected age, and growth between these time points. Growth data were collected from records and measures at term and at 12 months of corrected age were interpolated. Additionally, we examined the moderating effects of intrauterine growth restriction.

Results

Size at birth, term, or 12 months of corrected age, or growth between these time points were not associated with mental health outcomes (p-values >0.05). Intrauterine growth restriction did not systematically moderate any associations.

Conclusions

Despite the high variability in early growth of VLBW infants, the previously described association between slow growth in infancy and poorer cognitive functioning in later life is not reflected in symptoms of depression, ADHD, and other psychiatric problems. This suggests that the development of cognitive and psychiatric problems may have dissimilar critical periods in VLBW infants.     

CYP2W1 Is Highly Expressed in Adrenal Glands and Is Positively Associated with the Response to Mitotane in Adrenocortical Carcinoma

Background

Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins.

Objective

To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC.

Material and Methods

CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples).

Results

CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0–1) in 72% of non-adrenal normal tissues, but high (H-score 2–3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01).

Conclusion

CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.     

Laying eyes on headlights: eye movements suggest facial features in cars

Humans' proneness to see faces even in inanimate structures such as cars has long been noticed, yet empirical evidence is scarce. To examine this tendency of anthropomorphism, participants were asked to compare specific features (such as the eyes) of a face and a car front presented next to each other. Eye movement patterns indicated on which visual information participants relied to solve the task and clearly revealed the perception of facial features in cars, such as headlights as eyes or grille as nose. Most importantly, a predominance of headlights was found in attracting and guiding people's gaze irrespective of the feature they were asked to compare--equivalent to the role of the eyes during face perception. This response to abstract configurations is interpreted as an adaptive bias of the respective inherent mechanism for face perception and is evolutionarily reasonable with regard to a "better safe than sorry" strategy.