129/SvJ mice have mutated CD23 and hyper IgE

CD23, the low affinity IgE receptor, is hypothesized to function as a negative regulator of IgE production. Upon discovering reduced CD23 surface levels in 129/SvJ inbred mice, we sought to further investigate 129/SvJ CD23 and to examine its influence on IgE levels. Five amino acid substitutions were found in 129/SvJ CD23. Identical mutations were also observed in CD23 from New Zealand Black and 129P1/ReJ mice. 129/SvJ B cells proliferated more rapidly than those from BALB/c after stimulation with IL-4 and CD40 ligand trimer. However, in vitro IgE levels in supernatants from stimulated 129/SvJ B cells were significantly reduced. Contrary to the in vitro findings, the 129/SvJ CD23 mutations correlated with a hyper IgE phenotype in vivo and 129/SvJ were able to clear Nippostrongylus brasiliensis infection more rapidly than either BALB/c or C57BL/6. Overall, this study further suggests that CD23 is an important regulatory factor for IgE production.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

Mutation of cysteine 21 inhibits nucleophosmin/B23 oligomerization and chaperone activity

Nucleophosmin (NPM/B23) is a multifunctional nucleolar protein to which both tumor-suppressor and oncogenic functions have been attributed. NPM/B23 has a variety of binding partners including ribosomes, nucleic acids, the centrosome and tumor suppressors such as p53 and p19ARF. These disparate functions are likely due to its ability to oligomerize and display molecular chaperone activity. In this report we identify a single amino acid residue, Cys²¹, of nucleophosmin as important for the oligomerization and chaperone activity. Mutation of Cys²¹ to aromatic hydrophobic residues (e.g., Phe or Try), but not to a conserved polar residue (e.g., Ser) inhibited the pentameric oligomerization of NPM/B23. However, only Phe substitution of Cys²¹ drastically inhibited NPM/B23 chaperone activity. Interestingly, expression of Cys21Phe mutant in MCF7 cells demonstrated that this mutant protein does not co-polymerize with endogenous wild-type NPM/B23 and acts as negative dominant by destabilizing the endogenous dimer, trimer oligomerization. Taken together, the results in this study identify Cys²¹ as critical residue for NPM/B23 oligomerization and chaperone functions. In addition, Cys²¹ mutant provide a strong link between the oligomerization and chaperone functions of NPM/B23.     

Nucleophosmin/B23 Inhibits Eg5-mediated Microtubule Depolymerization by Inactivating Its ATPase Activity

Nucleophosmin/B23, an abundant nucleolar protein, plays multiple roles in cell growth and proliferation, and yet, little has been studied about its function in regulating dynamics of microtubules. Here, we report that B23 directly interacts with Eg5, a member of the kinesin family, in the cytosol. The DNA/RNA binding domain of B23 and the motor domain of Eg5 were found to be involved in their interaction. Both in vivo and in vitro evidences showed that B23 acts as an upstream regulator of Eg5 in promoting microtubule polymerization. Moreover, we further demonstrated that B23 regulates microtubule dynamics by directly inhibiting Eg5 ATPase activity.     

Sensitivity of tumor cells towards CIGB‐300 anticancer peptide relies on its nucleolar localization

CIGB‐300 is a novel anticancer peptide that impairs the casein kinase 2‐mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB‐300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB‐300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB‐300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB‐300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB‐300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull‐down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB‐300‐treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB‐300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide‐based drug should entail its more efficient intracellular delivery at such subcellular localization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.     

Phosphorylation regulates interaction of 210-kDa myosin light chain kinase <Emphasis Type="Italic">N</Emphasis>-terminal domain with actin cytoskeleton

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303–314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.     

Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

The Non-Coding RNA Llme23 Drives the Malignant Property of Human Melanoma Cells

Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) protein-associated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors. To identify the PSF-binding lncRNA involved in human oncogenesis, we screened a nuclear RNA repertoire of human melanoma cell line, YUSAC, through RNA-SELEX affinity chromatography. A previously unreported lncRNA, termed as Llme23, was found to bind immobilized PSF resin. The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo. The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines. Knocking down Llme23 remarkably suppressed the malignant property of YUSAC cells, accompanied by the repressed expression of proto-oncogene Rab23. These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding lncRNA with human melanoma.     

Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus

Severe acute respiratory syndrome-coronavirus (SARS-CoV) causes an infectious disease through respiratory route. Diagnosing the disease effectively and accurately at early stage is essential for preventing the disease transmission and performing antiviral treatment. In this study, we raised monoclonal antibodies (mAbs) against the nucleocapsid (N) protein of SARS-CoV and mapped epitopes by using different truncated N protein fragments. The mapping of those epitopes was valuable for constructing pair-Abs used in serological diagnosis. The results showed that all of the six raised mAbs were divided into two groups recognizing the region of amino acids 249-317 (A group) or 317-395 (B group). This region spanning amino acids 249-395 contains predominant B cell epitopes located at the C-terminus of N protein. One pair-Abs, consisting of N protein-specific rabbit polyclonal antibody and SARS-CoV N protein-specific mAb, was selected to construct a sandwich ELISA-kit. The kit was able to specifically detect SARS-CoV N proteins in serum samples.     

B23 is a downstream target of polyamine- modulated CK2

Our previous studies have shown that the overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, increases the enzymatic activity of the polyamine-responsive enzyme casein kinase 2 (CK2). Because CK2 is known to preferentially associate with the nuclear matrix in response to other trophic stimuli, we investigated the effects of ODC overexpression on CK2 localisation and on the CK2-mediated phosphorylation of a known CK2 substrate, the nucleolar phosphoprotein B23. Immunofluorescence analysis of CK2 and B23 in primary keratinocytes revealed that ODC overexpression resulted in the colocalisation of CK2 with B23 at the nucleolar borders. ODC overexpression also increased CK2 kinase activity 2-fold at the nuclear matrix, a response which could be abrogated by treatment of K6/ODC transgenic keratinocytes with the ODC inhibitor α-difluoromethylornithine (DFMO). Levels of B23 protein were also elevated in ODC-overexpressing cells compared to normal cells or transgenic cells treated with DFMO. This increase in protein level was neither due to an increase in steady-state mRNA levels, nor was it due to increased stability of B23 protein. Phosphorylation of B23 was also increased in ODC-overexpressing cells, and this increased phosphorylation could be blocked by treatment of the cells with the CK2 kinase inhibitors apigenin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). These data suggest that B23 may be a downstream effector of polyamines via phosphorylation by the protein kinase CK2.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

Skeletal FGFR1 signaling is necessary for regulation of serum phosphate level by FGF23 and normal life span

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1^fl/fl; Ocn^Cre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.     

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.     

The design,synthesis and evaluation of 2-aminobenzoxazole analogues as potent and orally efficacious ChemR23 inhibitors

We previously reported 2-aminobenzoxazole analogue 1 as a potent ChemR23 inhibitor. The compound showed inhibitory activity against chemerin-induced calcium signaling through ChemR23 internalization in CAL-1 cells, which are cell lines of plasmacytoid dendric cells (pDCs). Furthermore, compound 2 inhibited chemotaxis of CAL-1 triggered by chemerin in vitro. However, we noted a difference in the ChemR23 response to our inhibitor between rodents and non-rodents in a previous study. To address this issue, we performed optimization of ChemR23 inhibitors using CAL-1 cells endogenously expressing human ChemR23 and conducted a pharmacokinetics study in cynomolgus monkeys. Various substituents at the 4-position of the benzoxazole ring exhibited potent in vitro bioactivity, while those at the 6-position were not tolerated. Among substituents, a carboxyl group was identified as key for improving the oral bioavailability in cynomolgus monkeys. Compound 38a with the acidic part changed from a tetrazole group to a 1,2,4-oxadiazol-5-one group to improve bioactivity and pharmacokinetic parameters exhibited inhibitory activity against chemerin-induced chemotaxis in vitro. In addition, we confirmed the ChemR23 internalization of pDCs by compound 38a orally administered to cynomolgus monkeys. These 2-aminobenzoxazole-based ChemR23 inhibitors may be useful as novel immunotherapeutic agents capable of suppressing the migration of pDCs, which are known to be major producers of type I interferons in the lesion area of certain autoimmune diseases, such as systemic lupus erythematosus and psoriasis.