PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system.

Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.

Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole.

Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.

Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1.

Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ domain-containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are thought to be important in the targeting and anchoring of receptors to specific synapses. Here, we show that PICK1 (protein interacting with C kinase), a PDZ domain-containing protein, interacts with the C termini of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in vitro and in vivo. In neurons, PICK1 specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors.

PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands.

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1.

PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.

A critical compilation of Henry's law constant temperature dependence relations for organic compounds in dilute aqueous solutions.

A comprehensive compilation of published studies reporting directly measured experimental determinations of Henry's law constant (HLC) temperature dependence relationships for organic compounds in dilute, non-saline aqueous solutions under ambient conditions was conducted. From this effort, 55 such studies (covering 204 organic compounds) were identified, critically reviewed, summarized and discussed. Of the 204 compounds, 57 were studied in more than one investigation. For the 57 'multi-studied' compounds, relatively good agreement (averaging within 20-30%) was found between the results from different investigations. Given such results, a 'consensus' relationship (i.e., an average temperature dependence relation) was generated for each of the multi-studied compounds. Overall, considering relations established for 197 of the 204 compounds investigated (the results for the other 7 compounds being excluded due to poor correlation coefficients), the average slope of the temperature dependence line was found to correspond to an increase in HLC values by a factor of 1.88 (i.e., an 88% increase) per 10 degrees C rise in temperature (thermodynamically equivalent to an average enthalpy of volatilization of 47 kJ/mole). The associated range found for the temperature dependence slope corresponds to an increase in HLC values by a factor ranging from 1.12 to 3.55 (i.e., a 12-255% increase) per 10 degrees C rise (equivalent enthalpy of volatilization range: 8-93 kJ/mole). The wide range of slope values found indicates that serious errors may result if one applies the commonly cited 'rule of thumb' that HLC values double per 10 degrees C rise in temperature to a specific compound. In light of this finding, when faced with a lack of data, a prudent course for practitioners to take appears to be conducting a laboratory study to determine the exact temperature dependence for the compound(s) of interest.

Mutant allele frequencies in domestic cat populations in Arkansas and Tennessee.

We conducted surveys of mutant allele frequencies of four cat populations in Arkansas and Tennessee during 2002. Our calculations and analyses support that Southwestern cat populations were relatively more genetically similar to each other than compared to cat populations in other areas of North America. However, the cat population of Fort Smith is slightly different from the other cat populations studied in the Southwestern United States. Although there is a clear significant spatial geographic pattern for many mutant coat allele frequencies in the United States and Canada cat populations (d, l, S, and W), our results revealed that there is not a significant isolation-by-distance model affecting these cat populations. Our data also support the historical migration hypothesis because our calculated allele frequencies were genetically similar to cat populations located in ancestral areas of Europe. Different phenograms, including new European cat genetic profiles, showed that the Southwestern cat populations studied are of a clear British origin. Therefore, migration routes of early Arkansas and Tennessee settlers help explain the similarities of allele frequencies among domestic cat populations.

Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha.

PICK1 is a protein kinase C (PKC) alpha-binding protein initially identified using the yeast two-hybrid system. Here we report that PICK1 contains a PDZ domain that interacts specifically with a previously unidentified PDZ-binding domain (QSAV) at the extreme COOH terminus of PKCalpha and that mutation of a putative carboxylate-binding loop within the PICK1 PDZ domain abolishes this interaction. The PDZ-binding domain in PKCalpha is absent from other PKC isoforms that do not interact with PICK1. We also demonstrate that PICK1 can homooligomerize through sequences that are distinct from the carboxylate-binding loop, suggesting that self-association and PKCalpha binding are not mutually exclusive. A Caenorhabditis elegans PICK1-like protein is also able to bind to PKCalpha, suggesting a conservation of function through evolution. Association of PKCalpha with PICK1 provides a potential mechanism for the selective targeting of PKCalpha to unique subcellular sites.

Comparison of molecular typing methods for Candida albicans.

Four molecular approaches to determining the types of Candida albicans strains were compared. The strains used were those whose repeated DNA (ribosomal and mitochondrial) EcoRI restriction fragment length polymorphisms (RFLP) were determined by Stevens et al. (D. A. Stevens, F. C. Odds, and S. Scherer, Rev. Infect. Dis. 12:258-266, 1990). Scherer and Stevens (S. Scherer and D. A. Stevens, Proc. Natl. Acad. Sci. USA 85:1452-1456, 1988) used the same strains to examine the Southern blots of genomic EcoRI digests probed with the repeated sequence 27A. The results of these investigators were compared with determinations of RFLPs generated from repeated DNA by the enzyme HinfI and examination of the karyotypes of strains under two sets of conditions, one for the smaller chromosomes and one for the larger ones. Analysis of RFLPs of repeated DNA is most convenient but shows the lowest degree of resolution. Use of the repeated sequence and use of karyotype have very high resolution, but the former method is more convenient than the latter. HinfI digestion is more sensitive than EcoRI digestion but equally convenient. By using all four methods, separate types were identified for 18 of the 20 strains examined.

Structure, expression, and properties of an atypical protein kinase C (PKC3) from Caenorhabditis elegans. PKC3 is required for the normal progression of embryogenesis and viability of the organism.

Little is known about differential expression, functions, regulation, and targeting of "atypical" protein kinase C (aPKC) isoenzymes in vivo. We have cloned and characterized a novel cDNA that encodes a Caenorhabditis elegans aPKC (PKC3) composed of 597 amino acids. In post-embryonic animals, a 647-base pair segment of promoter/enhancer DNA directs transcription of the 3.6-kilobase pair pkc-3 gene and coordinates accumulation of PKC3 protein in approximately 85 muscle, epithelial, and hypodermal cells. These cells are incorporated into tissues involved in feeding, digestion, excretion, and reproduction. Mammalian aPKCs promote mitogenesis and survival of cultured cells. In contrast, C. elegans PKC3 accumulates in non-dividing, terminally differentiated cells that will not undergo apoptosis. Thus, aPKCs may control cell functions that are independent of cell cycle progression and programmed cell death. PKC3 is also expressed during embryogenesis. Ablation of PKC3 function by microinjection of antisense RNA into oocytes yields disorganized, developmentally arrested embryos. Thus, PKC3 is essential for viability. PKC3 is enriched in particulate fractions of disrupted embryos and larvae. Immunofluorescence microscopy revealed that PKC3 accumulates near cortical actin cytoskeleton/plasma membrane at the apical surface of intestinal cells and in embryonic cells. A candidate anchoring/targeting protein, which binds PKC3 in vitro, has been identified.

Coordinate regulation of xenobiotic and bile acid homeostasis by pregnane X receptor.

Identification and characterization of the pregnane X receptor (PXR) as a key regulator of cytochrome P450 3A (CYP3A) gene expression has led to an increased understanding of the molecular basis of many drug-drug interactions. Mice lacking PXR (PXR-KO) were used in the present study to delineate the role of PXR in regulating hepatomegaly and regulating the activity of CYP3A, organic anion transporting polypeptide-2 (Oatp2), and Cyp7a1 (cholesterol 7alpha-hydroxylase) gene products in vivo. Pregnenolone-16alpha-carbonitrile (PCN) produced hepatomegaly in the wild-type mice but not in the PXR-KO mice. PCN increased both the number of proliferating cell nuclear antigen immuno-positive nuclei and apparent cell size in the wild-type mice but not in the PXR-KO mice. To determine the role PXR plays in regulating CYP3A activity, 6beta-hydroxylation of testosterone and the duration of the loss of righting reflex following administration of the muscle-relaxant zoxazolamine were measured. PCN increased the level of testosterone 6beta-hydroxylation and decreased the duration of the loss of righting-reflex time following zoxazolamine administration in wild-type mice, but did not effect either of these parameters in PXR-KO mice. PCN increased the hepatic uptake of [(3)H]digoxin, an Oatp2 substrate, in wild-type mice but not in the PXR-KO mice. Similarly, PCN decreased bile acid excretion in wild-type mice but not in the PXR-KO mice. Taken together, these data demonstrate a pivotal role for PXR in the regulation of drug-induced hepatomegaly and in the metabolism (CYP3A), transport (Oatp2), biosynthesis (Cyp7a1), and excretion of xenobiotics and bile acids in vivo.

Interactions among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system.

The helix-loop-helix (HLH) motif is contained in a rapidly growing family of transcription factors and has been shown to mediate dimerization among heterologous HLH-containing proteins. E12 is a widely expressed HLH protein that preferentially forms heterodimers with cell type-specific HLH proteins such as MyoD, myogenin, and the achaete-scute gene products. As a first step toward screening for novel cell type-specific partners of E12, we used a modification of the two-hybrid assay for detection of protein-protein interactions in vivo to determine whether dimerization of HLH proteins with E12 can occur in yeast. Using the GAL4 DNA-binding domain fused to the E12 HLH motif and the GAL4 transcription activation domain fused to MyoD, we show that E12 and MyoD can efficiently dimerize in yeast and reconstruct a hybrid transcription factor that activates reporter genes linked to the GAL4 DNA-binding site. The GAL4 DNA-binding domain fused to E12 was used to screen a mouse T-cell cDNA library in which the cDNA was fused to the GAL4 activation domain. Several cDNA clones encoding proteins that interact with E12 were isolated, one of which corresponded to the HLH protein Id-2. Given the ability of E12 to dimerize preferentially with cell type-specific HLH proteins, this strategy should be useful for cloning novel partners for E12 from a variety of cell types.

Regulation of peroxisome proliferator-activated receptor alpha-induced transactivation by the nuclear orphan receptor TAK1/TR4.

Recently, we reported the cloning of the nuclear orphan receptor TAK1. In this study, we characterized the sequence requirements for optimal TAK1 binding and analyzed the repression of the peroxisome proliferator-activated receptor alpha (PPARalpha) signaling pathway by TAK1. Site selection analysis showed that TAK1 has the greatest affinity for direct repeat-1 response elements (RE) containing AGGTCAAAGGTCA (TAK1-RE) to which it binds as a homodimer. TAK1 is a very weak inducer of TAK1-RE-dependent transcriptional activation. We observed that TAK1, as PPARalpha, is expressed within rat hepatocytes and is able to bind the peroxisome proliferator response elements (PPREs) present in the promoter of the PPARalpha target genes rat enoyl-CoA hydratase (HD) and peroxisomal fatty acyl-CoA oxidase (ACOX). TAK1 is unable to induce PPRE-dependent transcriptional activation and represses PPARalpha-mediated transactivation through these elements in a dose-dependent manner. Two-hybrid analysis showed that TAK1 does not form heterodimers with either PPARalpha or retinoid X receptor (RXRalpha), indicating that this repression does not involve a mechanism by which TAK1 titrates out PPARalpha or RXRalpha from PPAR.RXR complexes. Further studies demonstrated that the PPARalpha ligand 8(S)-hydroxyeicosatetraenoic acid strongly promotes the interaction of PPARalpha with the co-activator RIP-140 but decreases the interaction of PPARalpha with the co-repressor SMRT. In contrast, TAK1 interacts with RIP-140 but not with SMRT and competes with PPARalpha for RIP-140 binding. These observations indicated that the antagonistic effects of TAK1 on PPARalpha.RXRalpha transactivation act at least at two levels in the PPARalpha signaling pathway: competition of TAK1 with PPARalpha.RXR for binding to PPREs as well as to common co-activators, such as RIP-140. Our results suggest an important role for TAK1 in modulating PPARalpha-controlled gene expression in hepatocytes.

Physical and genetic mapping of Candida albicans: several genes previously assigned to chromosome 1 map to chromosome R, the rDNA-containing linkage group.

Analysis of the karyotypes of multiple Candida albicans isolates by pulsed-field electrophoresis confirms the observation by Lasker et al. of eight chromosomes. The genes previously assigned to chromosome 1 in fact fall into two groups, one (including ADE1, SOR9, and CDC10) is linked to the ribosomal DNA genes on a chromosome called R, whereas the others are found on chromosome 1. Chromosome R varies in electrophoretic mobility among strains, usually running equal to or faster than chromosome 1 but in rare cases running slower than chromosome 1. In strain 1012A, the decreased mobility of one homolog is associated with the very large majority of the rDNA genes being on that homolog; the second homolog, with only a few copies, migrates with chromosome 2. Linkage analysis by using spheroplast fusion confirms the gene assignments made by hybridization to blots of the electrophoretic karyotype. A newly cloned gene, LYS2, hybridizes to chromosome 1.

CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11.

This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.

The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16alpha-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7alpha-hydroxylase (Cyp7a1) and the Na(+)-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.