Regulatory Subunit 1B Research Articles

Identification of New Molecular Targets for Treatment of Endometrial Cancers

Background: Current approaches using molecularly targeted drug alone or in combination with cytotoxic agents are associated with varying degrees of response rates and adverse effects. Clearly, there is a need to identify new molecular targets with more selectivity against the endometrial cancer cells. Aim: To perform high throughput screening to identify and validate new molecular targets for treatment of endometrial cancers. Methods: Kinome-wide shRNA library screen was performed on endometrial cancer cell line. The results were validated using viability and apoptosis assay. Immunoblotting assay was conducted to identify the target protein. Results: We identified that the knock-down of endogenous cyclin-dependent kinases regulatory subunit 1B (CKS1B) induced significant cell death in a panel of endometrial cancer cell lines (AN3CA, HEC-1A, HEC-1B, RL-95 and Ishikawa). In contrast, no significant cytotoxicity was observed in the THESC nontransformed endometrial epithelial cells suggesting that CKS1B is mediating a tumor-specific survival pathway. Analysis by immunoblotting assay revealed an increased of p27 protein expression following depletion of endogenous CKS1B in the type 2 endometrial cancer cells (AN3CA and HEC-1A), while no changes was observed in the type 1 (Ishikawa and RL95-2) endometrial cancer cells. These results suggest that CKS1B might regulate the survival of type I and type II endometrial cancer cells through distinct mechanisms. Conclusion: The results demonstrated that inhibition of CKS1B induced significant tumor-specific cell death in endometrial cancer cells. We found that the response is significant in type 2 endometrial cancer cell line and is p27 dependent. This finding suggests that CKS1B could be a potential target for therapeutic intervention and warrant further investigation.

The regulatory subunit phr2AB of Dictyostelium discoideum phosphatase PP2A interacts with the centrosomal protein CEP161, a CDK5RAP2 ortholog.

phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.

Serum exosomal miR-328, miR-575, miR-134 and miR-671-5p as potential biomarkers for the diagnosis of Kawasaki disease and the prediction of therapeutic outcomes of intravenous immunoglobulin therapy.

The present study was conducted to screen serum exosomal microRNAs (miRNAs) for the early diagnosis of Kawasaki disease (KD) and to investigate their underlying mechanisms by analyzing microarray data under accession numbers GSE60965 [exosomal miRNA, including three pooled serum samples from 5 healthy children, 5 patients with KD and 5 patients with KD following intravenous immunoglobulin (IVIG) therapy] and GSE73577 (mRNA, including peripheral blood mononuclear cell samples from 19 patients with KD prior to and following IVIG treatment) from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) and genes (DEGs) were identified using the Linear Models for Microarray data method, and the mRNA targets of DE-miRNAs were predicted using the miRWalk 2.0 database. The functions of the target genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). As a result, 65 DE-miRNAs were identified with different expression patterns between the healthy children and patients with KD and between patients with KD and patients with KD following IVIG therapy. The target genes of 15 common DE-miRNAs were predicted. Following overlapping the target genes of DE-miRNAs with 355 DEGs, 28 common genes were identified and further screened to construct a network containing 30 miRNA-mRNA regulatory associations. Of these associations, only miR-328-spectrin α, erythrocytic 1, miR-575-cyclic AMP-responsive element-binding protein 5/b-1,4-galactosyltransferase 5/WD repeat and FYVE domain-containing 3/cystatin-A/C-X-C motif chemokine receptor 1/protein phosphatase 1 regulatory subunit 3B, miR-134-acyl-CoA synthetase long chain family member 1/C-type lectin domain family 1 member A and miR-671-5p-tripartite motif containing 25/leucine rich repeat kinase 2/kinesin family member 1B/leucine rich repeat neuronal 1 were involved in the negative regulation of gene expression. Functional analysis indicated that the identified target genes may be associated with inflammation. Accordingly, serum exosomal miR-328, miR-575, miR-134 and miR-671-5p may act as potential biomarkers for the diagnosis of KD and the prediction of outcomes of the IVIG therapy by influencing the expression of inflammatory genes.

Hepatic steatosis risk is partly driven by increased de novo lipogenesis following carbohydrate consumption

BackgroundDiet is a major contributor to metabolic disease risk, but there is controversy as to whether increased incidences of diseases such as non-alcoholic fatty liver disease arise from consumption of saturated fats or free sugars. Here, we investigate whether a sub-set of triacylglycerols (TAGs) were associated with hepatic steatosis and whether they arise from de novo lipogenesis (DNL) from the consumption of carbohydrates.ResultsWe conduct direct infusion mass spectrometry of lipids in plasma to study the association between specific TAGs and hepatic steatosis assessed by ultrasound and fatty liver index in volunteers from the UK-based Fenland Study and evaluate clustering of TAGs in the National Survey of Health and Development UK cohort. We find that TAGs containing saturated and monounsaturated fatty acids with 16–18 carbons are specifically associated with hepatic steatosis. These TAGs are additionally associated with higher consumption of carbohydrate and saturated fat, hepatic steatosis, and variations in the gene for protein phosphatase 1, regulatory subunit 3b (PPP1R3B), which in part regulates glycogen synthesis. DNL is measured in hyperphagic ob/ob mice, mice on a western diet (high in fat and free sugar) and in healthy humans using stable isotope techniques following high carbohydrate meals, demonstrating the rate of DNL correlates with increased synthesis of this cluster of TAGs. Furthermore, these TAGs are increased in plasma from patients with biopsy-confirmed steatosis.ConclusionA subset of TAGs is associated with hepatic steatosis, even when correcting for common confounding factors. We suggest that hepatic steatosis risk in western populations is in part driven by increased DNL following carbohydrate rich meals in addition to the consumption of saturated fat.

Protein phosphatase 1 regulatory subunit 3B gene variation protects against hepatic fat accumulation and fibrosis in individuals at high risk of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver damage and has a strong genetic component. The rs4841132 G>A variant, modulating the expression of protein phosphatase 1 regulatory subunit 3B (PPP1R3B), which is involved in glycogen synthesis, has been reported to reduce the risk of NAFLD but at the same time may favor liver disease by facilitating glycogen accumulation. The aim of this study was to assess the impact of rs4841132 on development of histologic steatosis and fibrosis in 1,388 European individuals in a liver biopsy cohort, on NAFLD hepatocellular carcinoma in a cross‐sectional Italian cohort (n = 132 cases), and on liver disease at the population level in the United Kingdom Biobank cohort. We investigated the underlying mechanism by examining the impact of the variant on gene expression profiles. In the liver biopsy cohort, the rs4841132 minor A allele was associated with protection against steatosis (odds ratio [OR], 0.63; 95% confidence interval [CI], 0.42‐0.95; P = 0.03) and clinically significant fibrosis (OR, 0.35; 95% CI, 0.14‐0.87; P = 0.02) and with reduced circulating cholesterol (P = 0.02). This translated into protection against hepatocellular carcinoma development (OR, 0.22; 95% CI, 0.07‐0.70; P = 0.01). At the population level, the rs4841132 variation was not associated with nonalcoholic or nonviral diseases of the liver but was associated with lower cholesterol (P = 1.7 × 10–8). In individuals with obesity, the A allele protecting against steatosis was associated with increased PPP1R3B messenger RNA expression and activation of lipid oxidation and with down‐regulation of pathways related to lipid metabolism, inflammation, and cell cycle. Conclusion: The rs4841132 A allele is associated with protection against hepatic steatosis and fibrosis in individuals at high risk of NAFLD but not in the general population and against dyslipidemia. The mechanism may be related to modulation of PPP1R3B expression and hepatic lipid metabolism. (Hepatology Communications 2018;2:666‐675)

Relationship between genetic variation at PPP1R3B and levels of liver glycogen and triglyceride.

These observations are consistent with the notion that the minor allele of rs4841132 promotes a mild form of hepatic glycogenosis that is associated with hepatic injury. (Hepatology 2018;67:2182-2195).

Association between the PPP1R3B polymorphisms and serum lipid traits, the risk of coronary artery disease and ischemic stroke in a southern Chinese Han population

BackgroundLittle is known about the association of the protein phosphatase 1 regulatory subunit 3B gene (PPP1R3B) single nucleotide polymorphisms (SNPs) and serum lipid levels, the risk of coronary artery disease (CAD) and ischemic stroke (IS) in the Chinese populations. This study detected such association in a Southern Chinese Han population.MethodsGenotypes of 4 novel PPP1R3B SNPs (rs12785, rs330910, rs330915 and rs9949) in 1704 Han Chinese (CAD, 556; IS, 531 and control, 617) were determined by the Snapshot technology.ResultsThe rs12785A and rs9949A allele frequency was higher in both CAD/IS patients than in controls. The rs330910T and rs330915T allele frequency was also higher in CAD patients than in controls. The rs330910T allele carriers in controls had lower serum low-density lipoprotein cholesterol (LDL-C) levels than the rs330910T allele non-carriers (P < 0.0014). The rs12785A, rs9949A and rs330910T allele carriers were associated with an increased risk of CAD (P = 0.008–0.004). There was strong linkage disequilibrium among the 4 SNPs in the controls and CAD/IS patients. The T-A-A-G haplotype was associated with a decreased risk of CAD and IS, whereas the A-A-T-A haplotype was associated with an increased risk for IS. Haplotype-environment interactions on the risk of CAD and IS were also observed.ConclusionsSeveral PPP1R3B polymorphisms were associated with serum LDL-C levels, the risk of CAD and IS in the Southern Chinese Han population. But these findings still need to be confirmed in the other populations with larger sample sizes.

CK2β regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis

AbstractPlatelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β−/−) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β−/− MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β−/− platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor–dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbβ3 activation, and aggregation were abrogated in ck2β−/− platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2β−/− mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2β−/− mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2β as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

The Ndc80 complex targets Bod1 to human mitotic kinetochores.

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores.

Hepatic protein phosphatase 1 regulatory subunit 3B (Ppp1r3b) promotes hepatic glycogen synthesis and thereby regulates fasting energy homeostasis

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (GL) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion (Ppp1r3bΔhep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3bΔhep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3bΔhep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

CKS1BP7, a Pseudogene of CKS1B, is Co-Amplified with IGF1R in Breast Cancers.

Pseudogenes have been reported to exhibit functional roles. Amplification or overexpression of CDC28 protein kinase regulatory subunit 1B (CKS1B) was found in various human cancers. But it was known little about CKS1B pseudogene 7 (CKS1BP7), a pseudogene sharing considerable sequence identity with CKS1B. The aim of this study was to evaluate copy number alterations (CNAs) of CKS1BP7 and address its potential roles in breast cancer. We detected copy numbers of CKS1BP7 and insulin-like growth factor 1 receptor (IGF1R) using quantitative multi-gene fluorescence in situ hybridization (QM-FISH) technique, compared their status in both invasive carcinoma and ductal carcinoma in situ (DCIS) components within the same tumors, and investigated the associations of CNAs with tumor features and patients outcomes. Amplification of CKS1BP7 (dot-like pattern) was found in 28.8% of all cases, while amplified IGF1R (cluster pattern) was identified in 24.2% of all patients. The two events often co-existed (p=0.01). Within the same tumors, identical CNAs of CKS1BP7 and IGF1R were found in DCIS and invasive carcinoma. Moreover, amplification of both genes was more frequent in aneuploidy tumors and the tumors with high ki67, but wasn't associated with patients' outcome. In summary, CKS1BP7 amplification is a frequent event in breast cancer and often co-occurs with amplified IGF1R, which provides evidence supporting the interactions between CKS1BP7 and IGF1R during mammary carcinogenesis. Our findings suggest that CKS1BP7 as well as IGF1R may serve as potential biomarkers for early detection and predict prognosis in breast cancer.

Genome-wide association study of iron traits and relation to diabetes in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL): potential genomic intersection of iron and glucose regulation?

Genetic variants contribute to normal variation of iron-related traits and may also cause clinical syndromes of iron deficiency or excess. Iron overload and deficiency can adversely affect human health. For example, elevated iron storage is associated with increased diabetes risk, although mechanisms are still being investigated. We conducted the first genome-wide association study of serum iron, total iron binding capacity (TIBC), transferrin saturation, and ferritin in a Hispanic/Latino cohort, the Hispanic Community Health Study/Study of Latinos (>12 000 participants) and also assessed the generalization of previously known loci to this population. We then evaluated whether iron-associated variants were associated with diabetes and glycemic traits. We found evidence for a novel association between TIBC and a variant near the gene for protein phosphatase 1, regulatory subunit 3B (PPP1R3B; rs4841132, β = -0.116, P = 7.44 × 10-8). The effect strengthened when iron deficient individuals were excluded (β = -0.121, P = 4.78 × 10-9). Ten of sixteen variants previously associated with iron traits generalized to HCHS/SOL, including variants at the transferrin (TF), hemochromatosis (HFE), fatty acid desaturase 2 (FADS2)/myelin regulatory factor (MYRF), transmembrane protease, serine 6 (TMPRSS6), transferrin receptor (TFR2), N-acetyltransferase 2 (arylamine N-acetyltransferase) (NAT2), ABO blood group (ABO), and GRB2 associated binding protein 3 (GAB3) loci. In examining iron variant associations with glucose homeostasis, an iron-raising variant of TMPRSS6 was associated with lower HbA1c levels (P = 8.66 × 10-10). This association was attenuated upon adjustment for iron measures. In contrast, the iron-raising allele of PPP1R3B was associated with higher levels of fasting glucose (P = 7.70 × 10-7) and fasting insulin (P = 4.79 × 10-6), but these associations were not attenuated upon adjustment for TIBC-so iron is not likely a mediator. These results provide new genetic information on iron traits and their connection with glucose homeostasis.

Basolateral to Central Amygdala Neural Circuits for Appetitive Behaviors

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia. VIDEO ABSTRACT.

Generation and quantitative proteomics analysis of CK2\u03b1/\u03b1\u2019(\u2212/\u2212) cells

CK2 is a ubiquitous, constitutively active, highly pleiotropic, acidophilic Ser/Thr protein kinase whose holoenzyme is composed of two catalytic (α and/or α’) subunits and a dimer of a non-catalytic β subunit. Abnormally high CK2 level/activity is often associated with malignancy and a variety of cancer cells have been shown to rely on it to escape apoptosis. To gain information about the actual “druggability” of CK2 and to dissect CK2 dependent cellular processes that are instrumental to the establishment and progression of neoplasia we have exploited the CRISPR/Cas9 genome editing technology to generate viable clones of C2C12 myoblasts devoid of either both the CK2 catalytic subunits or its regulatory β-subunit. Suppression of both CK2 catalytic subunits promotes the disappearance of the β-subunit as well, through its accelerated proteasomal degradation. A quantitative proteomics analysis of CK2α/α’(−/−) versus wild type cells shows that knocking out both CK2 catalytic subunits causes a rearrangement of the proteomics profile, with substantially altered level ( > 50%) of 240 proteins, 126 of which are up-regulated, while the other are down-regulated. A functional analysis reveals that up- and down-regulated proteins tend to be segregated into distinct sub-cellular compartments and play different biological roles, consistent with a global rewiring underwent by the cell to cope with the lack of CK2.

Role of vernalization-mediated demethylation in the floral transition of Brassica rapa.

Vernalization-mediated demethylation of BrCKA2 (casein kinase II α-subunit) and BrCKB4 (casein kinase II β-subunit) shorten the period of the clock gene BrCCA1 (circadian clock associated 1) in Brassica rapa. Photoperiod and vernalization are two environmental cues involved in the regulation of floral transition, but the ways in which they interact remain unclear. DNA methylation is one of the main mechanisms involved in controlling the functional state of chromatin and gene expression in response to environmental signals. To study the interaction between photoperiod and vernalization in floral transition, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in normal (CK) and vernalized (CA) leaves from Brassica rapa using methylated-DNA immunoprecipitation sequencing (MeDIP-seq). Two subunits of casein kinase II (CK2), BrCKA2 (catalytic α-subunit of CK2) and BrCKB4 (regulatory β-subunit of CK2), exhibited gradual DNA demethylation and increased expression in vernalized B. rapa. DNA methylation-defective plants demonstrated the causal link between DNA demethylation changes and changes in gene expression. Virus-induced gene silencing (VIGS) of BrCKA2 and BrCKB4 in B. rapa resulted in no change to the period of BrCCA1 (circadian clock associated 1) and a 1-week late flowering time. Finally, we demonstrated that increased levels of BrCKA2 and BrCKB4 in vernalized B. rapa confer elevated CK2 activity, resulting in a shortened period of the clock gene BrCCA1, which plays an important role in perceiving photoperiod in plants. Thus, our results suggest that there is a direct interaction between photoperiod and vernalization through DNA methylation mechanisms.

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.

The Intracellular Signaling Molecule Darpp-32 Is a Marker for Principal Neurons in the Cerebellum and Cerebellum-Like Circuits of Zebrafish.

The dopamine and cAMP regulated phosphoprotein of apparent molecular weight 32 kDa (Darpp-32) is an inhibitory subunit of protein phosphatase-1 (PP-1). Darpp-32 activity is regulated by multiple ligand-activated G-protein coupled receptors (GPCRs). This protein is coded for by the protein phosphatase-1 regulatory subunit 1b (ppp1r1b) gene. Here, we provide experimental evidence for the presence of multiple isoforms of ppp1r1b in zebrafish. We show that these isoforms are differentially expressed during development with the full-length isoform being maternally deposited. Next, with a custom polyclonal antibody generated against the full-length protein, we show that in the adult, Darpp-32 is strongly expressed in principal neurons of the cerebellum and cerebellum-like circuits. These include Purkinje neurons in the cerebellum, Type-I neurons in the optic tectum, and crest cells in the medial octavolateralis nucleus (MON). We confirmed the identity of these neurons through their colocalization with Parvalbumin 7 immunoreactivity. Darpp-32 is seen in the somata and dendrites of these neurons with faint staining in the axons. In all of these regions, Darpp-32-immunoreactive cells were in close proximity to tyrosine hydroxylase (TH) immunoreactive puncta indicating the presence of direct catecholaminergic input to these neurons. Darpp-32 immunoreactivity was seen in Purkinje neurons as early as 3 days post-fertilization (dpf) when Purkinje neurons are first specified. In sum, we show that Darpp-32, a signaling integrator, is a specific marker of principal neurons in the cerebellum and cerebellum-like circuits in zebrafish.

Loss of miR-1258 contributes to carcinogenesis and progression of liver cancer through targeting CDC28 protein kinase regulatory subunit 1B.

Hepatocellular carcinoma (HCC) is the leading cause of cancer related death worldwide. The number of deaths is proportional to the global incidence, which highlights the aggressive tumor biology and lack of effective therapies. Dysregulation of microRNAs has been implicated in carcinogenesis and progression of liver cancer. Here, we identified that miR-1258 was significantly downregulated in HCC and associated with poor patients' survival. Overexpression of miR-1258 significantly inhibits liver cancer cell growth, proliferation and tumorigenicity through increasing cell cycle arrest in G0/G1 phase and promotes cell apoptosis. Interestingly, stable overexpression of miR-1258 suppresses cell migration, stemness and increases sensitivity of HCC cells to chemotherapy drug like doxorubicin. The CDC28 protein kinase regulatory subunit 1B (CKS1B) was identified as a functional downstream target of miR-1258. Re-expression of CKS1B overcomes miR-1258 induced apoptosis and increases stemness of HCC cells, suggesting that loss of miR-1258 contributes to carcinogenesis and progression of liver cancer through targeting CKS1B. Therefore, loss of miR-1258 may be a potential diagnostic and prognostic biomarker and blocking miR-1258-CKS1B axis is a potential therapeutic strategy in HCC.

DARPP-32: from neurotransmission to cancer.

Dopamine and cAMP-regulated phosphoprotein Mr 32,000 (DARPP-32), also known as phosphoprotein phosphatase-1 regulatory subunit 1B (PPP1R1B), was initially discovered as a substrate of dopamine-activated protein kinase A (PKA) in the neostriatum in the brain. While phosphorylation at Thr-34 by PKA converts DARPP-32 into a potent inhibitor of protein phosphatase 1 (PP1), phosphorylation at Thr-75 transforms DARPP-32 into an inhibitor of PKA. Through regulation of DARPP-32 phosphorylation and modulation of protein phosphatase and kinase activities, DARPP-32 plays a critical role in mediating the biochemical, electrophysiological, and behavioral effects controlled by dopamine and other neurotransmitters in response to drugs of abuse and psychostimulants. Altered expression of DARPP-32 and its truncated isoform (t-DARPP), specifically in the prefrontal cortex, has been associated with schizophrenia and bipolar disorder. Moreover, cleavage of DARPP-32 by calpain has been implicated in Alzheimer's disease. Amplification of the genomic locus of DARPP-32 at 17q12 has been described in several cancers. DARPP-32 and t-DARPP are frequently overexpressed at the mRNA and protein levels in adenocarcinomas of the breast, prostate, colon, and stomach. Several studies demonstrated the pro-survival, pro-invasion, and pro-angiogenic functions of DARPP-32 in cancer. Overexpression of DARPP-32 and t-DARPP also promotes chemotherapeutic drug resistance and cell proliferation in gastric and breast cancers through regulation of pro-oncogenic signal transduction pathways. The expansion of DARPP-32 research from neurotransmission to cancer underscores the broad scope and implication of this protein in disparate human diseases.