Estrogen potentiates the combined effects of transforming growth factor-beta and tumor necrosis factor-alpha on adult human osteoblast-like cell prostaglandin E2 biosynthesis.

Reports that estrogen treatment modulates arachidonic acid metabolism by bone and bone cells are found in the literature. However, conflicting indications of the relationship that exists between estrogen and arachidonic acid metabolism emerge from the analysis of those reports. The present studies were undertaken to determine if estrogen effected the production of prostaglandins (PG) in human osteoblast-like (hOB) cell cultures derived from adults, under basal or cytokine-stimulated conditions. A 48-hour estrogen pretreatment did not modify hOB cell PG biosynthesis on a qualitative basis, and PGE2 formation predominated under all tested conditions. Estrogen pretreatment did lead to increased PGE2 production in specimens stimulated conjointly with transforming growth factor-beta1 and tumor necrosis factor-alpha ( p < 0.001). No changes in PGE2 production were observed in estrogen pretreated specimens stimulated singly with either tested cytokine, nor in samples in which either TGFbeta or TNF was replaced by interleukin-1beta. Anti-estrogen (ICI 164,384) inclusion prevented the estrogen-dependent increase in PGE2 production in the TGFbeta plus TNF-stimulated samples. These results suggest that an estrogen effect on bone cell prostaglandin biosynthesis may be most evident and significant under conditions in which the cells are exposed to multiple osteotropic cytokines, a condition that applies during the bone remodeling process.

Drosophila melanogaster casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8 derived from the Enhancer of split complex.

Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a D. melanogaster embryo cDNA library for proteins that interact with DmCKIIalpha. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex (E(spl)C), which regulates neurogenesis. m7 interacts with DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKIIalpha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIalpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIalpha, as well as by the alpha2beta2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. We also demonstrate that m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Finally, substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKIIalpha, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis.

Identification and characterization of proteins that interact with Drosophila melanogaster protein kinase CK2.

D. melanogaster CK2 (DmCK2) is a highly conserved protein kinase that is composed of catalytic, alpha, and regulatory, beta, subunits associated as an alpha2beta2 heterotetramer. In order to analyze the functions of CK2 in this metazoan model, we have used the two hybrid approach to identify interacting proteins. One of these cDNAs, DmA24, encodes a novel polypeptide with no homologs in GenBank, and is notable in that it contains a bipartite nuclear localization signal and two sites for phosphorylation by CK2. In situ hybridization to polytene chromosomes indicates that the DmA24 gene is located at the 61 D interval of chromosome II a region that also harbors 3 additional genes with similar structure. DmA24p interacts with DmCK2alpha, but not with DmCK2beta, demonstrating that this interaction is specific for the catalytic subunit of CK2. In addition, the protein is phosphorylated by the holoenzyme purified from Drosophila embryos. These studies identify DmA24p as a potentially new physiological partner of DmCK2. In addition, we also report the results of a large-scale screen that has identified a new set of DmCK2-interacting proteins. Most notable among these are Surf6, a nucleolar protein involved in RNA processing, and Spalt, a homeotic protein.

A gene located at 72A in Drosophila melanogaster encodes a novel zinc-finger protein that interacts with protein kinase CK2.

Drosophila melanogaster protein kinase CK2 (DmCK2) is a Ser/Thr protein kinase composed of catalytic alpha and regulatory beta subunits associated as an alpha2beta2 heterotetramer. Using the two hybrid system, we have screened a Drosophila embryo cDNA library in order to identify proteins that interact with DmCK2alpha. One of these cDNAs encodes a novel previously undescribed zinc-finger protein, which we call ZFP47. ZFP47 interacts with DmCK2alpha but not with DmCK2beta, indicating that this interaction is specific for the catalytic subunit of CK2. In situ hybridization to polytene chromosomes indicates that the corresponding gene is located at the 72A interval of chromosome III. Sequence analysis indicates that ZFP47 contains a consensus site for phosphorylation by CK2, 4 C1H1-type zinc-fingers, and a bipartite nuclear localization signal. Consistent with the prediction of a site for phosphorylation by CK2, we demonstrate that ZFP47 is phosphorylated by CK2 purified from Drosophila embryos. These studies demonstrate that ZFP47 is a new physiological partner and substrate of CK2.

Multiple, closely spaced alternative 5' exons in the DmCKIIbeta gene of Drosophila melanogaster.

Drosophila melanogaster casein kinase II (CKII) is composed of catalytic alpha and regulatory beta subunits. Using the two-hybrid system, we have isolated a number of cDNAs that are related to a previously published cDNA encoding the beta subunit, but exhibit divergent 5' sequences. To determine the source of this sequence variation, we have isolated the gene encoding the beta subunit of CKII. The beta gene contains five exons encompassing the complete open reading frame, as well as five alternative exons in the 5' untranslated region (UTR). Only one 5' UTR exon is contained in each cDNA, implying five distinct classes of transcript. In addition, the beta gene contains at least two poly(A) addition signals which generate additional complexity at the 3' end. The complex pattern of transcription may serve a role in the spatial and/or temporal expression of the beta subunit since, with one exception, all transcripts encode the full-length beta polypeptide. Phylogenetic comparison of the beta genes of Drosophila, C. elegans, and mammals reveals three invariant introns as well as evidence of recent intron gain/loss.

A gene located at 56F1-2 in Drosophila melanogaster encodes a novel metazoan beta-like subunit of casein kinase II.

Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic alpha and regulatory beta subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a Drosophila embryo cDNA library for proteins that interact with DmCKII alpha. One of the cDNAs encodes a novel beta-like polypeptide, which we designate beta'. In situ hybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the beta gene and the Stellate family of beta-like sequences. The predicted sequence of beta' is more closely related to the beta subunit of Drosophila and other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit. In vitro reconstitution studies show that a GST-beta' fusion protein associates with the alpha subunit to generate a tetrameric complex with regulatory properties similar to those of the native alpha2beta2 holoenzyme. The data are consistent with the proposed role of the beta' subunit as an integral component of the holoenzyme.

Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.

Cloning and disruption of CKB1, the gene encoding the 38-kDa beta subunit of Saccharomyces cerevisiae casein kinase II (CKII). Deletion of CKII regulatory subunits elicits a salt-sensitive phenotype.

Saccharomyces cerevisiae casein kinase II (CKII) contains two distinct catalytic (alpha and alpha') and regulatory (beta and beta') subunits. We report here the isolation and disruption of the gene, CKB1, encoding the 38-kDa beta subunit. The predicted Ckb1 sequence includes the N-terminal autophosphorylation site, internal acidic domain, and potential metal binding motif (CPX3C-X22-CPXC) present in other beta subunits but is unique in that it contains two additional autophosphorylation sites as well as a 30-amino-acid acidic insert. CKB1 is located on the left arm of chromosome VII, approximately 33 kilobases from the centromere and does not correspond to any previously characterized genetic locus. Haploid and diploid strains lacking either or both beta subunit genes are viable, demonstrating that the regulatory subunit of CKII is dispensable in S. cerevisiae. Such strains exhibit wild type behavior with regard to growth on both fermentable and nonfermentable carbon sources, mating, sporulation, spore germination, and resistance to heatshock and nitrogen starvation, but are salt-sensitive. Salt sensitivity is specific for NaCl and LiCl and is not observed with KCl or agents which increase osmotic pressure alone. These data suggest a role for CKII in ion homeostasis in S. cerevisiae.

Cloning and disruption of CKB2, the gene encoding the 32-kDa regulatory beta'-subunit of Saccharomyces cerevisiae casein kinase II.

Casein kinase II of Saccharomyces cerevisiae is composed of two distinct catalytic subunits, alpha and alpha', and two distinct regulatory subunits, beta and beta' (Padmanabha, R. and Glover, C. V. C. (1987) J. Biol. Chem. 262, 1829-1835; Bidwai, A. P., Reed, J. C., and Glover, C. V. C. (1994) Arch. Biochem. Biophys. 309, 348-355). We report here the cloning, sequencing, and disruption of the CKB2 gene encoding the beta'-subunit. The deduced amino acid sequence of Ckb2 displays only 40-45% identity to other beta-subunit sequences reported to date, allowing a better definition of conserved features of this protein. Most notable is the conservation of a cysteine-containing sequence, CPX3C-X22-CPXC, which may constitute a novel metal-binding motif. The degree of sequence divergence of Ckb2 is comparable to that of the Drosophila Stellate protein, a testis-specific protein of unknown function, suggesting that the latter may function as a second beta-subunit in Drosophila. CKB2 is located on the right arm of chromosome XV between the HIR2 and WHI2 loci and has not been previously identified genetically. Haploid and homozygous diploid cells harboring a ckb2 null allele are viable, demonstrating that the beta'-subunit does not have an essential function distinct from that of beta. Strains lacking a functional CKB2 gene appear to grow normally on both fermentable and non-fermentable carbon sources, mate and sporulate normally, and display normal resistance to nitrogen starvation and heat shock. However, haploid strains harboring disruptions of both the beta' gene and either of the catalytic subunit genes exhibit a synthetic phenotype consisting of slow growth and flocculation in rich glucose medium. The occurrence of this synthetic phenotype implies that the beta'-subunit interacts physically and/or functionally with both the alpha- and alpha'-subunits in vivo.

Casein kinase II of Saccharomyces cerevisiae contains two distinct regulatory subunits, beta and beta'.

The subunit composition of casein kinase II (CKII) from S. cerevisiae has been difficult to define, particularly with respect to the existence and number of regulatory (beta) subunits. A single, integral beta subunit, a loosely associated beta subunit, two distinct beta subunits, and a complete absence of beta subunits have all been proposed. Our laboratory reported yeast CKII to be composed of four polypeptides of 42, 41, 35, and 32 kDa (R. Padmanabha and C. V. C. Glover, 1987, J. Biol. Chem. 262, 1829-1835). The 42- and 35-kDa polypeptides were identified as distinct catalytic subunits, alpha and alpha', on the basis of N-terminal sequencing and subsequent molecular cloning. The 41- and 32-kDa polypeptides were found to undergo autophosphorylation, a characteristic of the beta subunit in other species, but antibodies raised against the beta subunit of Drosophila CKII crossreacted only with the 41-kDa polypeptide. In order to clarify the subunit composition of yeast CKII, particularly with regard to the 32-kDa polypeptide, we have purified the enzyme to homogeneity using a modified procedure. Based on the results of autophosphorylation studies, Western blotting, peptide mapping of the 41- and 32-kDa peptides, and sequencing of subunit-specific peptides, we demonstrate that the 32-kDa polypeptide is an additional beta subunit (beta') distinct from the 41-kDa beta subunit. This represents the first demonstration of beta subunit heterogeneity in purified CKII from any species.

Structure and function of Saccharomyces cerevisiae casein kinase II.

Analysis of casein kinase II in organisms amenable to genetic manipulation is essential to elucidating the physiological function(s) of this ubiquitous protein kinase. This paper summarizes work from our laboratory on the enzyme from Saccharomyces cerevisiae. The biochemistry, molecular biology, and genetics of S. cerevisiae casein kinase II are reviewed and discussed.

Phosphorylation of calmodulin by the catalytic subunit of casein kinase II is inhibited by the regulatory subunit.

Casein kinase II (CKII) is composed of a catalytic subunit (alpha) and a regulatory subunit (beta) that combine to form an alpha 2 beta 2 holoenzyme. The alpha-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified beta subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983, J. Biol. Chem. 258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the alpha subunit and the holoenzyme of Drosophila melanogaster CKII. We demonstrate that the alpha subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (RII) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as alpha-casein and the synthetic peptide RRREEETEEE. The Km of the alpha subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the alpha subunit alone. While the Km values of the alpha subunit and the holoenzyme for calmodulin are similar, the Vmax of the alpha subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the alpha subunit contains the necessary determinants for CKII substrate specificity, the beta subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the alpha subunit as well.

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.

Studies on the active site of the Neurospora crassa plasma membrane H+-ATPase with periodate-oxidized nucleotides.

The Neurospora crassa plasma membrane H+-ATPase is inactivated by the periodate-oxidized nucleotides, oATP, oADP, and oAMP, with oAMP the most effective. Inhibition of the ATPase is essentially irreversible, because Sephadex G-50 column chromatography of the oAMP-treated ATPase does not result in a reversal of the inhibition. Inhibition of the ATPase by oAMP is protected against by the H+-ATPase substrate ATP, the product ADP, and the competitive inhibitors TNP (2',3'-O-(2,4,6-trinitrocyclohexadienylidine)-ATP and TNP-ADP, suggesting that oAMP inhibition occurs at the nucleotide binding site of the enzyme. The rate of inactivation of the ATPase by oAMP is only slightly affected by EDTA, indicating that the oAMP interaction with the nucleotide binding site of the H+-ATPase occurs in the absence of a divalent cation. The protection against oAMP inhibition by ADP is likewise unaffected by EDTA. The inhibition of the ATPase by oAMP is absolutely dependent on the presence of acidic phospholipids or acidic lysophospholipids known to be required for H+-ATPase activity, suggesting that these lipids either aid in the formation of the nucleotide binding site or render it accessible. Incubation of the ATPase with Mg2+ plus vanadate, which locks the enzyme in a conformation resembling the transition state of the enzyme dephosphorylation reaction, completely protects against inhibition by oAMP, suggesting that in this transition state conformation the nucleotide site either does not exist, or is inaccessible to oAMP. Labeling studies with [14C] oAMP indicate that the incorporation of 1 mol of oAMP is sufficient to cause complete inactivation of the ATPase.

Bacterial phytotoxin, syringomycin, induces a protein kinase-mediated phosphorylation of red beet plasma membrane polypeptides.

Syringomycin, a peptide toxin and a virulence factor produced by the bacterial phytopathogen Pseudomonas syringae pv. syringae, stimulated the phosphorylation of several plasma membrane polypeptides of red beet storage tissue. Among these was a 100-kDa polypeptide, which corresponds in size to the proton pump ATPase. The phosphorylations were insensitive to hydroxylamine, indicating that the polypeptide phosphorylated intermediates involved phosphate ester bonds characteristic of protein kinase-mediated phosphorylation. Phosphorylation of the 100-kDa polypeptide and of most of the other polypeptides was reduced or eliminated by extraction of the membranes with 0.1% (wt/vol) sodium deoxycholate, a treatment that also eliminated the ability of the toxin to stimulate ATPase activity. Phosphorylation of the 100-kDa polypeptide was highest with 10-20 mug of syringomycin; the same amounts gave the highest degree of ATPase activity stimulation. Phosphorylation of some of the polypeptides was eliminated or decreased by the Ca(2+) chelator EGTA. Addition of excess Ca(2+) restored the phosphorylation of most of these polypeptides. We conclude that syringomycin acts by stimulating an endogenous membrane protein kinase activity, which results in the phosphorylation of several membrane polypeptides. One of the phosphorylated polypeptides corresponds in size to the proton pump ATPase.

Mechanism of Action of Pseudomonas syringae Phytotoxin, Syringomycin : Stimulation of Red Beet Plasma Membrane ATPase Activity.

Syringomycin, a peptide toxin produced by the phytopathogen Pseudomonas syringae pv syringae preferentially stimulated (2-fold) the vanadate-sensitive ATPase activity associated with the plasma membrane of red beet storage tissue. The toxin had a very slight effect on the tonoplast ATPase and had no detectable effect on the mitochondrial ATPase. Optimal stimulation was achieved with 10 to 50 micrograms of syringomycin per 25 micrograms of membrane protein. Treatment of membranes with 0.1% (weight/volume) deoxycholate eliminated the activation effect, and enzyme solubilized with Zwittergent 3-14 was not affected by syringomycin. ATPase activity was activated to the same extent at KCl concentrations ranging from 0 to 50 millimolar. Valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenylhydrazone, and gramicidin did not increase the plasma membrane ATPase activity. However, these ionophores did not hinder the ability of syringomycin to stimulate the activity. We suggest that syringomycin does not increase ATPase activity by altering membrane ion gradients nor directly interacting with the enzyme, but possibly through regulatory effectors or covalent modification of the enzyme.