Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

In vitro ubiquitination of cyclin D1 by ROC1-CUL1 and ROC1-CUL3

Overexpression of cyclin D1 has been implicated in a variety of tumors, such as breast cancers, gastrointestinal cancers and lymphomas. Both gene amplification and protein degradation mediated by ubiquitin (Ub)-dependent proteolysis regulate the abundance of cyclin D1. Here we report that ROC1 interacted with all three D type cyclins in vivo but did not bind to other cyclins tested. The ROC1-CUL1 and ROC1-CUL3, but not ROC1-CUL2, -CUL3 and -CUL4, immunocomplexes promoted polyubiquitination of bacterially purified cyclin D1 in vitro. RING finger mutations of ROC1 eliminated the Ub ligase activity toward cyclin D1. In all cases the ubiquitination of cyclin D1 was accompanied by autoubiquitination of the cullins. The results suggest the involvement of ROC1-cullin ligases in cyclin D1 ubiquitination and a potential mechanism whereby the cullin subunit is ubiquitinated itself while ubiquitinating a substrate.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor

Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000-fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect.     

Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1

In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)- through the binding of PKC- to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and ³⁶Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 µg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC- to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC- binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; protein-protein interaction; slot blot assay; pulldown; PDZ domain; chloride efflux; immunoprecipitation     

Fibrillin-1 regulates the bioavailability of TGFbeta1

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) beta1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFbeta signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44-49 releases endogenous TGFbeta1, thereby stimulating TGFbeta receptor-mediated Smad2 signaling. This altered TGFbeta1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFbeta1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFbeta1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFbeta-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44-49 can contribute to MFS and related diseases.     

HWP1 Functions in the Morphological Development of Candida albicans Downstream of EFG1, TUP1, and RBF1

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Deltaefg1 mutant, reduced in an Deltarbf1 mutant, and derepressed in a Deltatup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.     

RACK1/TRAF2 regulation of modulator of apoptosis-1 (MOAP-1)

MOAP-1 is a pro-apoptotic tumor suppressor molecule with a growing set of known interacting partners. We have demonstrated that during death receptor-dependent apoptosis, MOAP-1 is recruited to TNF-R1 or TRAIL-R1, followed by RASSF1A and Bax association. MOAP-1/Bax association promotes Bax conformational change resulting in the translocation of Bax into the mitochondrial membrane, mitochondrial membrane insertion and dysregulation resulting in several hallmark events that execute apoptosis. Although a role in apoptosis is established, it is currently unknown how MOAP-1 is regulated and how it links to Bax to promote apoptosis. In this study, we demonstrate robust association with RACK1, a versatile scaffolding protein that responds to activation of protein kinase C. Furthermore, we can demonstrate that RACK1 functions to bring the E3 ligase, TRAF2, to MOAP-1 in order to undergo a K63-dependent ubiquitination. Furthermore, RACK1 associates with MOAP-1 via electrostatic associations similar to those observed between MOAP-1/RASSF1A and MOAP-1/TNF-R1. These events illustrate the complex nature of MOAP-1 regulation and characterizes the important role of the scaffolding protein, RACK1, in influencing MOAP-1 biology.     

Inhibition of vitamin D metabolism by ethane-1-hydroxyl-1, 1-diphosphonate

The administration of disodium-ethane-1-hydroxy-1,1-diphosphonate (20 mg/kg body weight subcutaneously) to chicks given adequate amounts of vitamin D₃ causes a hypercalcemia, inhibits bone mineralization, and inhibits intestinal calcium transport. The administration of 1,25-dihydroxyvitamin D₃, a metabolically active form of vitamin D₃, restores intestinal calcium absorption to normal but does not restore bone mineralization in disodium-ethane-1-hydroxy-1,1-diphosphonate-treated chicks. In rachitic chicks, the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment does not further reduce the low intestinal calcium transport values while it nevertheless further reduces bone ash levels and increases serum calcium concentration.These observations prompted a more detailed study of the relationship between disodium-ethane-1-hydroxy-1,1-diphosphonate treatment and vitamin D metabolism. A study of the hydroxylation of 25-hydroxyvitamin D₃ in an in vitro system employing kidney mitochondria from chicks receiving disodium-ethane-1-hydroxy-1,1-diphosphonate treatment demonstrates a marked decrease in 1,25-dihydroxyvitamin D₃ production and a marked increase in the 24,25-dihydroxyvitamin D₃ production. In addition, the in vivo metabolism of 25-hydroxy-[26,27-³H]vitamin D₃ in disodium-ethane-1-hydroxy-1,1-diphosphonate treated chicks supports the in vitro observations. In rachitic chicks the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment markedly reduces the 25-hydroxyvitamin D₃-1-hydroxylase activity of kidney, but does not increase the 25-hydroxyvitamin D₃-24-hydroxylase.These results provide strong evidence that large doses of disodium-ethane-1-hydroxy-1,1-diphosphonate produce a marked effect on calcium metabolism via alterations in the metabolism of vitamin D as well as the expected direct effect on the bone.