A Radioimmunoassay for the Phosphoprotein B-50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.     

Phosphorylation of Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) by Akt Promotes Stability and Mitogenic Function of S-phase Kinase-associated Protein-2 (Skp2)

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21^cip1 and p27^kip1 thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.     

Identification of Members of the Family Enterobacteriaceae by the R-B System

The R-B system was evaluated in parallel with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae by using bacterial strains from a variety of clinical specimens and from stock cultures. The R-B tests found to be reliable were, in decreasing order, the reactions for phenylalanine deaminase, hydrogen sulfide, and indole, the production of gas from glucose, and the decarboxylation of ornithine. The reactions in the R-B system found to be unreliable were motility, the decarboxylation of lysine, and the fermentation of both glucose and lactose. In addition, the reactions of the R-B system were more difficult to read and interpret than those of the conventional system. On the basis of this evaluation, it was concluded that the R-B system is not an acceptable alternative to the conventional methods in the identification of the Enterobacteriaceae.     

Affinity-Purified Anti-B-50 Protein Antibody: Interference with the Function of the Phosphoprotein B-50 in Synaptic Plasma Membranes

Abstract: Affinity-purified anti-B-50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B-50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B-50 protein and its adrenocorticotropin-sensitive protein kinase, obtained from rat brain. Anti-B-50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B-50 protein isolated by an improved procedure. The purified antibodies reacted only with the B-50 and B-60 protein, a proteolysis derivative (of B-50), as assessed by the sodium dodecyl sulfate-gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B-50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4-phosphate was not altered. Inhibition by ACTH _1–24 of the endogenous phosphorylation of B-50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-diphosphate. These data support our hypothesis on the functional interaction of B-50 protein and phosphatidylinositol 4-phosphate kinase in rat brain membranes. The evidence shows that purified anti-B-50 protein antibodies can be used to probe specifically the function of B-50 protein in membranes.     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.     

Evaluation of the Enterotube System for Identification of Members of the Family Enterobacteriaceae

The Enterotube system was evaluated, in parallel with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae, by using bacterial strains from a variety of clinical specimens and from stock cultures. Excellent agreement between the two test systems was obtained with the following reactions: hydrogen sulfide, indole, Simmons' citrate, glucose, and lactose. Agreement was not as good (<85%) with the urea, phenylalanine deaminase, and dulcitol reactions. The Enterotube lysine decarboxylase test was unsatisfactory. The Enterotube method will correctly identify strains of the family Enterobacteriaceae approximately 50% of the time; if identification only as Klebsiella-Enterobacter-Serratia group is needed, the method will be correct 85% of the time. On the basis of this evaluation, the Enterotube system appears to be both simple and rapid for the presumptive identification of these bacteria. Because of the limited usefulness of the lysine decarboxylase test, the results obtained by this test system are less reliable than those obtained by conventional methods.     

ADP-Ribosylation of the Neuronal Phosphoprotein B-50/GAP-43

Abstract: The neuronal phosphoprotein B-50/GAP-43 is associated with growth and regeneration within the nervous system and its posttranslational status can be correlated with its cellular localization during growth and regeneration. Recently, B-50 has been shown to interact with certain G protein subunits. Regulation of G protein-mediated signal transduction may involve ADP-ribosylation in vivo. In the present study we have demonstrated that B-50 is a substrate for endogenous ADP-ribosyltransferases. The results are discussed with respect to the possible interaction of B-50 with G proteins, but also with regard to the posttranslational modification of B-50 by all major regulatory mechanisms that act at, or through, the neuronal membrane.     

Evaluation of Modified R-B System for Identification of Members of the Family Enterobacteriaceae

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.     

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.     

Identification of Members of the Dimocarpus Longan Flowering Locus T Gene Family with Divergent Functions in Flowering

Dimocarpus longan is a subtropical fruit crop whose year-round production relies on the application of KClO₃ to induce flowering; however, the mechanism by which this chemical causes flowering is yet unknown. To further characterize floral signaling in this species, we have isolated three longan FLOWERING LOCUS T (FT)-like genes and studied their activities by heterologous expression in Arabidopsis. Expression of two of these genes (DlFT2 and DlFT3) accelerates flowering, whereas expression of the third gene (DlFT1) causes delayed flowering and produced floral morphology defects. This anti-florigenic protein may be a member of a class of FT-like family involved in flowering time control in biennial and perennial species. Surprisingly, KClO₃ treatment also suppressed the expression of both DlFT2 and DlFT3 in a field trial.     

Identification of EBP50 as a specific biomarker for carcinogens via the analysis of mouse lymphoma cellular proteome

To identify specific biomarkers generated upon exposure of L5178Y mouse lymphoma cells to carcinogens, 2-DE and MALDI-TOF MS analysis were conducted using the cellular proteome of L5178Y cells that had been treated with the known carcinogens, 1,2-dibromoethane and Onitrotoluene and the noncarcinogens, emodin and Dmannitol. Eight protein spots that showed a greater than 1.5-fold increase or decrease in intensity following carcinogen treatment compared with treatment with noncarcinogens were selected. Of the identified proteins, we focused on the candidate biomarker ERM-binding phosphoprotein 50 (EBP50), the expression of which was specifically increased in response to treatment with the carcinogens. The expression level of EBP50 was determined by western analysis using polyclonal rabbit anti-EBP50 antibody. Further, the expression level of EBP50 was increased in cells treated with seven additional carcinogens, verifying that EBP50 could serve as a specific biomarker for carcinogens.     

Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.     

Identification and Characterization of Chalcone Synthase Gene Family Members in Nicotiana tabacum

Journal of Plant Growth Regulation - Chalcone synthase (CHS, EC 2.3.1.74) is a member of the plant polyketide synthase superfamily; it catalyzes the first committed step in the flavonoid...     

Production of Secreted Carbohydrates that Present Immunologic Similarities with the Cryptococcus Glucuronoxylomannan by Members of the Trichosporonaceae Family: A Comparative Study Among Species of Clinical Interest

Mycopathologia - Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease....     

Identification of New Members of the GS ADP-Forming Family from the De Novo Purine Biosynthesis Pathway

Most living organisms can synthesize isosinate from 5-phosphoribosyl 1-pyrophosphate in the de novo purine biosynthesis pathway, which is basically composed of 10 reaction steps. Phosphoribosylglycinamide synthetase (GARS) catalyzes the second step of the pathway. We found that the enzyme shows weak, but significant, sequence similarity to phosphoribosylglycinamide formyltransferase 2 (GART2) and the ATPase domain of phosphoribosylaminoimidazole carboxylase (AIRCA), which catalyze the third and sixth steps of the pathway, respectively. In addition, the three enzymes were similar in amino acid sequence to biotin carboxylase (BC) and carbamoylphosphate synthetase (CPS), which are the members of the GS ADP-forming family. This family has been identified through a tertiary structure comparison and includes glutathione synthetase, d-alanine:d-alanine ligase, BC, succinyl-CoA synthetase β-chain, and phosphoribosylaminoimidazole-succinocarboxamide synthase. Molecular phylogenetic analysis based on a multiple alignment of GARS, GART2, AIRCA, BC, and CPS suggests that GART2 is more closely related to AIRCA than to GARS among the three enzymes from the pathway, though the three enzymes are relatively close to each other within the GS ADP-forming family. Moreover, the analysis showed that archaeal GARS had diverged before the speciation between bacteria and eucarya. Received: 3 June 1998 / Accepted: 8 September 1998     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not

The ezrin/radixin/moesin (ERM) proteins are regulated microfilament membrane linking proteins. Previous tissue localization studies have revealed that the three related proteins show distinct tissue distributions, with ezrin being found predominantly in polarized epithelial cells, whereas moesin is enriched in endothelial cells and lymphocytes. EBP50 and E3KARP are two related scaffolding proteins that bind to the activated form of ERM proteins in vitro, and through their PDZ domains to the cytoplasmic domains of specific membrane proteins, including the Na+/H+ exchanger isoform (NHE3) present in kidney proximal tubules and the beta2-adrenergic receptor. Using specific antibodies to EBP50 and E3KARP for localization in murine tissues, we find that the cellular distribution of EBP50 and E3KARP is mutually exclusive. Epithelial cells expressing ezrin generally co-express EBP50, such as intestinal epithelial cells, gastric parietal cells, the epithelial cells of the kidney proximal tubule, the terminal bronchiole of the lung, and in mesothelia. This correlation is not absolute as cells of the mucous epithelium of the stomach and in the renal corpuscle, express ezrin but no detectable EBP50, whereas the bile canaliculi of hepatocytes express EBP50 and not ezrin. E3KARP has a restricted tissue distribution with the highest expression being found in lung. It is largely colocalized with moesin and radixin, especially in the alveoli of the lung, as well as being highly enriched in the renal corpuscle. These results document a preference for co-expression of EBP50, but not E3KARP, with ezrin in polarized epithelia. These results place constraints on the physiological roles that can be proposed for these scaffolding molecules.     

碎米荠超氧化物歧化酶基因家族成员的鉴定和分析

为了解碎米荠(Cardamine hirsuta)的SOD基因特征,对SOD家族成员的基因结构、染色体定位、系统进化关系进行了分析,对顺式作用元件和蛋白结构进行了预测,并利用qRT-PCR技术检测各家族成员的组织表达模式。结果表明,碎米荠基因组中共有10个SOD基因(ChSODs),包括6个Cu/Zn-SOD、3个Fe-SOD和1个Mn-SOD。编码的ChSODs蛋白有57~ 324个氨基酸,分子量为6 419.41~34 659.01 kDa,理论等电点为4.92~9.60;系统进化树分析表明,碎米荠的ChSOD与拟南芥的AtSOD的同源性较高;ChSODs在根、茎、叶中均有表达,且在叶中高表达,其中CARHR085500和CARHR256690在叶和茎中表达量较高;顺式作用元件预测表明,碎米荠SOD响应多种非生物胁迫,其中对ABA和低温胁迫较为敏感; ChSODs蛋白质的二级和三级结构具有差异性。这表明碎米荠SOD基因在抗氧化过程中发挥重要作用。