Monoclonal and anti-idiotypic anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on glycoprotein 140, the EBV/C3dR, expressed on human B lymphocytes

Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.     

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.     

Epstein-Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.     

EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr¹⁴⁵ residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.     

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.     

Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.     

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Infection of human B lymphoma cells by Mycoplasma fermentans induces interaction of its elongation factor with the intracytoplasmic domain of Epstein-Barr virus receptor (gp140, EBV/C3dR, CR2, CD21)

Human cell lines are often infected by mycoplama strains. We have demonstrated that when infected by Mycoplasma fermentans, human B lymphoma cell proliferation increased strongly. These infected B cells expressed a p45 kDa protein which interacted with the intracellular domain of CD21, the EBV/C3d receptor. p45 analysis demonstrated that this is a new gene which encodes an elongation factor originating from Mycoplasma fermentans. p45 interaction with CD21 was specific, there being no interaction with CD19. This is the first demonstration that Mycoplasma fermentans, in infecting human B cells, generates a p45 Mycoplasma component that interacts with CD21, which is involved in B cell proliferation.     

gp 140, the C3d/EBV receptor (CR2), is phosphorylated upon in vitro activation of human peripheral B lymphocytes

gp 140, the C3d/EBV receptor (CR2), is a specific marker of human B lymphocytes. Very recent data suggest that CR2 is a membrane site involved in early B cell activation. These properties of CR2 led us to analyze the molecular events associated with gp 140. We analyzed whether in some conditions of B lymphocyte activation, CR2 could be phosphorylated. We have found that when highly enriched peripheral B cells were cultured for 48 h with anti-mu Ab and/or SAC, in order to provide an optimal activating signal, phosphorylation of the CR2 was induced.     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay     

Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.     

Decrease in the phosphorylation of the proteins associated with heterogeneous nuclear RNA after the application of 3'-methyl-4-dimethylaminoazobenzene

The effect of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) on the phosphorylation of the proteins of the nuclear ribonucleoprotein (RNP) particles was studied in liver of rats. Forty eight hours after the application of 4 mg of the hepatocarcinogen per 100 g of body wt. by stomach intubation the particle proteins contained only 7% as much phosphate per mg of protein as the proteins of the same particles isolated from liver of control animals. Determination of the protein kinase and protein phosphatase activities in the total fraction of the non-histone nuclear proteins 48 h after the application of the carcinogen have shown an increase (200% and 159%, respectively) in both enzymatic activities. These results suggest that the hepatocarcinogen could induce the observed high turnover of the phosphates on the proteins of the liver nuclear ribonucleoprotein particles and the resulting dephosphorylation of these particles by stimulation of nuclear protein kinases and phosphatases. Qualitatively the same, but quantitatively much smaller changes were also observed 48 h after the application of the non-carcinogenic p-aminoazobenzene (AB) by stomach intubation and in regenerating liver. After the application of AB phosphorylation of the proteins of rat liver nuclear ribonucleoprotein particles decreased to 70% and in regenerating liver to 61% of the phosphorylation of particle proteins in control liver. Since it is assumed that nuclear RNP particles are involved in the processing and transport of newly synthesized premessenger RNA it is possible that the drastic dephosphorylation of the particle proteins induced by the carcinogen could be connected with the distortion of RNA processing which is observed in liver of animals treated with hepatocarcinogens.     

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.     

Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV)+ B Cell Lymphomas

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.