Bioflavonoids as poisons of human topoisomerase II alpha and II beta

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.     

Membrane binding of human phospholipid scramblase 1 cytoplasmic domain

Human phospholipid scramblase 1 (SCR) consists of a large cytoplasmic domain and a small presumed transmembrane domain near the C-terminal end of the protein. Previous studies with the SCRΔ mutant lacking the C-terminal portion (last 28 aa) revealed the importance of this C-terminal moiety for protein function and calcium-binding affinity. The present contribution is intended to elucidate the effect of the transmembrane domain suppression on SCRΔ binding to model membranes (lipid monolayers and bilayers) and on SCRΔ reconstitution in proteoliposomes. In all cases the protein cytoplasmic domain showed a great affinity for lipid membranes, and behaved in most aspects as an intrinsic membrane protein. Assays have been performed in the presence of phosphatidylserine, presumably important for the SCR cytoplasmic domain to be electrostatically anchored to the plasma membrane inner surface. The fusion protein maltose binding protein-SCR has also been studied as an intermediate case of a molecule that can insert into the bilayer hydrophobic core, yet it is stable in detergent-free buffers. Although the intracellular location of SCR has been the object of debate, the present data support the view of SCR as an integral membrane protein, in which not only the transmembrane domain but also the cytoplasmic moiety play a role in membrane docking of the protein.     

c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1

Phospholipid scramblase 1 (PLSCR1) is a plasma membrane protein that has been proposed to play a role in the transbilayer movement of plasma membrane phospholipids. PLSCR1 contains multiple proline-rich motifs resembling Src homology 3 (SH3) domain-binding sites. An initial screen against 13 different SH3 domains revealed a marked specificity of PLSCR1 for binding to the Abl SH3 domain. Binding between intracellular PLSCR1 and c-Abl was demonstrated by co-immunoprecipitation of both proteins from several cell lines. Deletion of the proline-rich segment in PLSCR1 (residues 1--118) abolished its binding to the Abl SH3 domain. PLSCR1 was Tyr-phosphorylated by c-Abl in vitro. Phosphorylation was abolished by mutation of Tyr residues Tyr(69)/Tyr(74) within the tandem repeat sequence (68)VYNQPVYNQP(77) of PLSCR1, implying that these residues are the likely sites of phosphorylation. Cellular PLSCR1 was found to be constitutively Tyr-phosphorylated in several cell lines. The Tyr phosphorylation of PLSCR1 was increased upon overexpression of c-Abl and significantly reduced either upon cell treatment with the Abl kinase inhibitor STI571, or in Abl-/- mouse fibroblasts, suggesting that cellular PLSCR1 is a normal substrate of c-Abl. Cell treatment with the DNA-damaging agent cisplatin activated c-Abl kinase and increased Tyr phosphorylation of PLSCR1. The cisplatin-induced phosphorylation of PLSCR1 was inhibited by STI571 and was not observed in Abl-/- fibroblasts. These findings indicate that c-Abl binds and phosphorylates PLSCR1, and raise the possibility that an interaction between c-Abl and plasma membrane PLSCR1 might contribute to the cellular response to genotoxic stress.     

Both alpha and beta isoforms of mammalian DNA topoisomerase II associate with chromosomes in mitosis.

Two isoforms of DNA topoisomerase II, alpha and beta, coded by separate genes, are expressed in actively cycling vertebrate cells. Some previous studies have suggested that only topoisomerase II alpha remains associated with chromosomes at mitosis. Here, the distributions of topoisomerase II alpha and beta in mitosis were studied by subcellular fractionation and by immunolocalization. Both isoforms of topoisomerase II were found to remain associated with mitotic chromatin. Topoisomerase II alpha was distributed along chromosome arms throughout mitosis and was highly concentrated at centromeres until mid-anaphase, particularly in some cell types. Topoisomerase II beta showed weak concentration at centromeres in early mitosis in some cell types and was distributed along chromosome arms at every stage of mitosis through telophase. These studies suggest that in most cells both the major topoisomerase II isoforms may play roles in chromatin remodeling during M phase.     

A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a -/- mutants

Background

Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima.

Results

We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes.

Conclusions

Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.     

Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II.

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.     

Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane

Phospholipid scramblase 1 (PLSCR1) is a Ca(2+)-binding, endofacial plasma membrane protein thought to contribute to the transbilayer movement of phosphatidylserine and other membrane phospholipids that is observed upon influx of calcium into the cytosol. Expression of PLSCR1 is markedly induced by interferon and other cytokines, and PLSCR1-/- bone marrow cells exhibit defective myeloid proliferation and differentiation in response to stimulation by select growth factors, implying that PLSCR1 also functions in cytokine signaling or response pathways. PLSCR1 is multiply palmitoylated and partitions into membrane lipid raft domains. We have now identified the Cys-rich sequence (184)CCCPCC(189) in PLSCR1 as required for palmitoylation of the polypeptide. Mutation of these five cysteines abrogates PLSCR1 trafficking to the plasma membrane and results in virtually all of the expressed protein localizing to the nucleus. Consistent with this observation, cell treatment with the palmitoylation inhibitor, 2-bromo-palmitate, results in a marked redistribution of endogenous PLSCR1 from plasma membrane to nucleus. In a small percentage of untreated cells, predominantly nuclear localization of PLSCR1 is also observed. Furthermore, PLSCR1 is also found in the nucleus following its cytokine-induced expression. These data suggest that under the circumstance of rapid biosynthesis in response to gene induction by cytokines, PLSCR1 traffics into the nucleus, implying a potential nuclear function for this protein.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE))

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) is an integral membrane aspartic proteinase responsible for beta-site processing of APP, and its cytoplasmic region composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE. With the yeast two-hybrid screening, we found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the cytoplasmic region of BACE. In cultured cells, BACE and PLSCR1 were colocalized in the Golgi area and in endosomal compartments, whereas they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a common trafficking pathway in cells. Co-immunoprecipitation analysis showed that both proteins form a protein complex at an endogenous expression level in the human neuroblastoma SH-SY5Ycells, and the dileucine residue of the BACE tail is also revealed to be essential for the physical interaction with PLSCR1 in vitro and in vivo. Moreover, both BACE and PLSCR1 were localized in a low buoyant lipid microdomain in SH-SY5Y cells. The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much less stably than wild-type BACE. Taken together, our current study suggests the functional involvement of PLSCR1 in the intracellular distribution of BACE and/or recruitment of BACE into the detergent-insoluble lipid raft.     

Determinants of the specificity of rotavirus interactions with the alpha2beta1 integrin

The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

beta 2-Glycoprotein-I (apolipoprotein H) interactions with phospholipid vesicles

The binding characteristics of the human serum protein beta 2-glycoprotein-I, also called apolipoprotein H, with multilamellar phospholipid vesicles has been studied. It was found that beta 2-G-I is not or almost not bound to the "neutral" phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM). The negatively charged compounds phosphatidylserine (PS) and phosphatidylinositol (PI) interact strongly with beta 2-G-I. In terms of phospholipid concentration the binding to PS is about one order of magnitude greater than to PI. The binding capacity is influenced by several parameters such as the molarity of buffer, presence of mono- or divalent cations as well as ethylenediaminotetraacetic acid (EDTA). Proteins like bovine serum albumin (BSA), human serum albumin (HSA) or horse gamma-globulin (HGG) influence the binding also in a concentration dependent manner.     

DNA ligation catalyzed by human topoisomerase II alpha

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.     

IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase

To identify new effectors of IgE receptor (FcepsilonRI) signaling, we purified proteins from FcepsilonRI-stimulated RBL-2H3 rat mast cells on anti-phosphotyrosine beads and generated mouse monoclonal antibodies (mAb) against these proteins. Two mAbs bound to a protein that was identified as a new isoform of phospholipid scramblase (PLSCR) after screening an RBL-2H3 cDNA expression library. This isoform differed from PLSCR1 by the absence of an exon 3-encoded sequence and by an insert coding six QGPY(P/A)GP repeats. The PLSCR family of proteins is responsible for a redistribution of phospholipids across the plasma membrane. Although rat PLSCR is a 37-kDa protein, anti-phosphotyrosine immunoblots revealed the presence of 37-49 kDa phosphoproteins in the material immunoprecipitated with either anti-PLSCR mAb but not with unrelated monoclonal or polyclonal antibodies. Depletion of PLSCR resulted in the absence of these phosphoproteins. Additional experiments led to the identification of these phosphoproteins as phospho-PLSCR itself. Stimulation of RBL-2H3 cells upon FcepsilonRI engagement resulted in a dramatic increase in PLSCR tyrosine phosphorylation. A comparison of the relative amounts of phospho-PLSCR and nonphosphorylated PLSCR demonstrated that only a tiny fraction was thus modified, indicating a finely targeted involvement of PLSCR in FcepsilonRI signaling. Thus, this study reports the cloning of a new isoform of PLSCR, as well as the first observation that a member of the PLSCR family is a target for tyrosine kinases and is involved in signaling by an immune receptor. These findings open new perspectives on the role of phospholipid scramblases and to the mechanisms involved in their regulation.     

The negative c-Myc target onzin affects proliferation and apoptosis via its obligate interaction with phospholipid scramblase 1

Onzin, the product of a negatively c-Myc-regulated target gene, is highly expressed in myeloid cells. As a result of its interaction with and activation of Akt1 and Mdm2, onzin down-regulates p53. The apoptotic sensitivity of several cell lines is thus directly related to onzin levels. We have conducted a search for additional onzin-interacting proteins and identified phospholipid scramblase 1 (PLSCR1), an endofacial membrane protein, which is proposed to mediate the bidirectional movement of plasma membrane phospholipids during proliferation and apoptosis. PLSCR1 interacts with the same cysteine-rich domain of onzin as do Akt1 and Mdm2, whereas the onzin-interacting domain of PLSCR1 centers around, but does not require, a previously identified palmitoylation signal. Depletion of endogenous PLSCR1 in myeloid cells leads to a phenotype that mimics that of onzin overexpression, providing evidence that PLSCR1 is a physiologic regulator of onzin. In contrast, PLSCR1 overexpression in fibroblasts, which normally do not express onzin, affects neither growth nor apoptosis unless onzin is coexpressed, in which case PLSCR1 completely abrogates onzin's positive effects on proliferation and survival. These findings demonstrate a functional interdependence between onzin and PLSCR1. They further suggest a contiguous link between the earliest events mediated by c-Myc and the latest ones, which culminate at the cell surface and lead to phospholipid reshuffling and cell death.     

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.