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.     

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.     

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.     

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     

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.     

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.     

Development of a Simple and Rapid Fluorogenic Procedure for Identification of Vibrionaceae Family Members

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.     

B-50 (GAP-43): Biochemistry and Functional Neurochemistry of a Neuron-Specific Phosphoprotein

The biochemistry and functional neurochemistry of the synaptosomal plasma membrane phosphoprotein B-50 (GAP-43) are reviewed. The protein is putatively involved in seemingly diverse functions within the nervous system, including neuronal development and regeneration, synaptic plasticity, and formation of memory and other higher cognitive behaviors. There is a considerable amount of information concerning the spatial and temporal localization of B-50 (GAP-43) in adult, fetal, and regenerating nervous tissue but far less is known about the physical chemistry and biochemistry of the protein. Still less information is available about posttranslational modifications of B-50 (GAP-43) that may be the basis of neurochemical mechanisms that could subsequently permit a variety of physiological functions. Hence, consideration is given to several plausible roles for B-50 (GAP-43) in vivo, which are discussed in the context of the cellular localization of the protein, significant posttranslational enzymes, and regulatory proteins, including protein kinases, phosphoinositides, calmodulin, and proteases.     

PDZ interactions regulate rapid turnover of the scaffolding protein EBP50 in microvilli

Scaffolding proteins containing PDZ (postsynaptic density 95/discs large/zonula occludens-1) domains are believed to provide relatively stable linkages between components of macromolecular complexes and in some cases to bridge to the actin cytoskeleton. The microvillar scaffolding protein EBP50 (ERM-binding phosphoprotein of 50 kD), consisting of two PDZ domains and an ezrin-binding site, retains specific proteins in microvilli and is necessary for microvillar biogenesis. Our analysis of the dynamics of microvillar proteins in vivo indicated that ezrin and microvillar membrane proteins had dynamics consistent with actin treadmilling and microvillar lifetimes. However, EBP50 was highly dynamic, turning over within seconds. EBP50 turnover was reduced by mutations that inactivate its PDZ domains and was enhanced by protein kinase C phosphorylation. Using a novel in vitro photoactivation fluorescence assay, the EBP50-ezrin interaction was shown to have a slow off-rate that was dramatically enhanced in a PDZ-regulated manner by addition of cell extract to near in vivo levels. Thus, the linking of relatively stable microvillar components can be mediated by surprisingly dynamic EBP50, a finding that may have important ramifications for other scaffolding proteins.     

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.