Regulation of phosphoinositide-specific phospholipase C isozymes.
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Animals↗
Biomedical subjects
Y S Bae
Publications and source records attributed to Y S Bae.
Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation.
Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-gamma1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested.
Epidermal Growth Factor↗
Molecular cloning and characterization of cDNAs for anionic and neutral peroxidases from suspension-cultured-cells of sweet potato and their differential expression in response to stress.
Two peroxidase (POD) cDNAs, swpal and swpn1, were isolated and characterized from suspension-cultured cells of sweet potato in order to understand the physiological function of POD isozymes. Sequence analysis showed that swpa1 encoded an anionic POD and swpn1 encoded a neutral POD. The swpa1 and swpn1 genes were both highly expressed in suspension-cultured cells in accordance with the high POD activity of these cells. Although both gene transcripts were detected in the stems of intact plants, their transcription levels were much lower than in suspension-cultured cells. During cell growth the pattern of mRNA accumulation of swpa1 differed from that of swpn1, suggesting that expression of these genes is differentially regulated by cell growth stage. In addition, the swpa1 and swpn1 genes responded differently to oxidative stress induced by chilling. The expression of swpa1 was weakly induced by 15 degrees C acclimation and strongly induced by 4 degrees C chilling, whereas the mRNA level of swpn1 was increased by 15 degrees C acclimation and reduced by 4 degrees chilling. This indicates that the two isozymes encoded by swpa1 and swpn1 might contribute to protection against cold-induced oxidative stress through different signaling pathways. In leaves, both genes were induced by wounding with broadly similar expression. patterns. Genomic analysis suggests that the two isozymes are encoded by different loci in the sweet potato genome.
Amino Acid Sequence↗
Interaction of the beta subunit of casein kinase II with the ribosomal protein L5.
Casein kinase II (CKII) usually exists as a heterotetramer with alpha 2 beta 2, alpha alpha'beta 2, or alpha'2 beta 2. The alpha or alpha' subunits catalyze protein phosphorylation, whereas the function of the beta subunit remains unclear. One of the possible functions of the beta subunit may be to mediate the interaction of the catalytic subunit with target proteins. To identify proteins capable of associating with the beta subunit in vivo, we have used a two-hybrid system. One protein identified is human ribosomal protein L5. The protein L5 does not interact with the alpha or alpha' subunits of CKII, supporting the idea that the beta subunit can determine a substrate specificity of CKII. These results furthermore suggest a novel role for CKII in ribosomal L5 phosphorylation, in ribosomal assembly, or ribosomal transport in the intact cells. The protein L5 may act as a regulator of the activity or subcellular localization of CKII.
Amino Acid Sequence↗
Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function.
Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC-gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC-delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.
Blood Platelets↗
Activation of phospholipase C-gamma by the concerted action of tau proteins and arachidonic acid.
Phospholipase C-gamma (PLC-gamma) isozymes are thought to be activated by receptor-induced tyrosine phosphorylation. Proteins that activate PLC-gamma1 have now been purified from bovine brain and identified as members of the tau family of microtubule-associated proteins. Activation of PLC-gamma by tau was enhanced in the presence of unsaturated fatty acids such as arachidonic acid, saturated fatty acids being ineffective. Maximal (15-20-fold) activation was apparent in the presence of 0.15 microM tau and 25 microM arachidonic acid (AA). The effect of tau and AA was specific to PLC-gamma isozymes in the presence of submicromolar concentrations of Ca2+ and was markedly inhibited by phosphatidylcholine. These results suggest that in cells that express tau, receptors coupled to cytosolic phospholipase A2 may activate PLC-gamma isozymes indirectly in the absence of tyrosine phosphorylation through the hydrolysis of phosphatidylcholine to generate AA.
Amino Acid Sequence↗
Multiple forms of phospholipase D inhibitor from rat brain cytosol. Purification and characterization of heat-labile form.
Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP2). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70 degrees C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP2, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP2-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.
ADP-Ribosylation Factors↗
PLC gamma 1 Src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts.
Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways.
3T3 Cells↗
Flavan and procyanidin glycosides from the bark of blackjack oak.
The bark of blackjack oak contains (+)-catechin, (-)-epicatechin, (+)-3-O-[beta-D-glucopyranosyl]-catechin, catechin-(4 alpha-->8)-catechin, epicatechin-(4 beta-->8)-catechin as well as the novel 3-O-[beta-D-glucopyranosyl]-catechin-(4 alpha-->8)-catechin and 3-O-[alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranosyl]-catechin, as major components. The latter rutinoside of catechin is especially interesting because of the exceptionally small J2,3 C-ring coupling. The glucoside of catechin-(4 alpha-->8)-catechin is only the second proanthocyanidin found in nature in which a 3-O-glycoside is present in the upper 'extender unit' of a dimer.
Biflavonoids↗
Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.
To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.
Animals↗
Development of a recombinant RNA technique for the construction of chimeric RNA with a long poly(C) tract.
The murine cardioviruses and bovine aphthoviruses are distinguished from other (+) strand RNA viruses by their long poly(C) tract in the 5'-noncoding region. The presence of this poly(C) tract has long hampered the construction of full-length cDNA with the complete poly(C) tract, because long poly(dC-dG) homopolymer-containing plasmids are difficult to amplify in bacterial systems. To overcome this problem, we constructed a chimeric RNA by joining the poly(C) region of the viral RNA to the 5'-truncated RNA transcript of the encephalomyocarditis (EMC) virus cDNA. The non-chimeric, recombinant EMC virus with a short poly(C) tract produces recombinant progeny virus, but this is not pathogenic in vivo. On the other hand, the EMC viral RNA chimera with the complete poly(C) tract produces recombinant progeny virus that is pathogenic in vivo. This method of viral RNA construction will be invaluable for functional studies of other cardioviruses and aphthoviruses, as well as for recombinant RNA manipulations.
Animals↗
Determination of diabetogenicity attributable to a single amino acid, Ala776, on the polyprotein of encephalomyocarditis virus.
The best experimental evidence indicating that viruses have an etiological role in the pathogenesis of diabetes comes from studies of mice infected with EMC virus. For this study we generated mutant viruses from stocks of diabetogenic EMC-D and nondiabetogenic EMC-B viruses by serial passages of the viruses in L-cell cultures at high MOI. The genomic sequence information and the biological activities of three different plaque-purified diabetogenic variants of EMC virus (EMC-D, EMC-D1/6A, EMC-D2/4) and six different plaque-purified nondiabetogenic variants (EMC-B, EMC-BS, EMC-B1/G, EMC-DV1, EMC-D4/1B, EMC-D3/1) revealed that only one amino acid, Ala (776th amino acid on the polyprotein), is critical for the diabetogenicity of EMC virus. The G base at the nucleotide position 3155 (Ala[GCC]776 in the polyprotein) is unique to all diabetogenic variants, whereas the A base at the same position (Thr[ACC]776 in the polyprotein) is identical to all nondiabetogenic variants. A single-point mutation (G to A; Ala to Thr) results in the conversion of the diabetogenic variant into a nondiabetogenic variant of EMC virus. On the basis of these observations, we conclude that a single amino acid, Ala776, on the polyprotein of EMC virus appears responsible for the inducement of diabetes in susceptible mice. Conversion of Ala776 into Thr776 on the polyprotein by a point mutation, G to A at the nucleotide position 3155, results in the loss of diabetogenicity.
Alanine↗
Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle.
The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.
Amino Acid Sequence↗
A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency.
To study the mechanism of illegitimate recombination in mammalian cells, we have developed a shuttle vector, pNK1, that contains three bacterial markers, amp (ApR), galK, and neo (KmR). The frequency of deletions occurring in autonomously replicating pNK1 DNA during the growth of monkey COS1 cells was measured by transfecting the plasmid into Escherichia coli cells and counting the number of galK- ApS double mutants among total KmR cells. This method allowed us to test the effects of topoisomerase inhibitors on deletion formation in mammalian cells. The DNA topoisomerase II (TopII) inhibitor, 4'-dimethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM26), stimulated deletions in pNK1 DNA in monkey cells. Since VM26 does not inhibit the strand-break activity of TopII, but rather stabilizes an enzyme-DNA complex in which DNA is cleaved upon treatment with sodium dodecyl sulfate, it is implicated that TopII participates in the deletion process in mammalian cells.
Amsacrine↗
Characterization of synthetic peptide substrates for p34cdc2 protein kinase.
Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.
Amino Acid Sequence↗
A new type of insertion mutation in monkey cells: insertion accompanied by long target site duplication.
We have developed a system for the detection of a new type of insertion mutation in mammalian cells. We have used a shuttle vector, plasmid pNK1, which contains the SV40 and pBR322 replication origins, and ApR, galK, and neoR genes. This plasmid was introduced into monkey COS1 cells, allowed to replicate, and then recovered plasmids were reintroduced into Escherichia coli HB101 to detect insertion mutations in the galK gene. We selected galK- KmR ApR mutants in order to eliminate galK- KmS deletion mutants. Insertion mutations in the plasmids recovered were then screened by agarose gel electrophoresis. Finally, insertion mutants that had the following characteristics were selected. First, they had the ability to produce gal+ revertants caused by the precise excision of inserted DNA in E. coli, implying that they had a target site duplication on both sides of the insertion. Second, they contained some repetitive sequence(s) as judged by hybridization with a bulk monkey DNA probe. Nucleotide sequence analysis of one of the mutants, 15K-1, showed that it contained alpha-satellite sequences within the coding region of the galK gene. It contained 13 1/2 tandem repeat units of alpha-satellite sequence and was flanked by a 64 bp target site duplication, indicating that the alpha-satellite sequence had been translocated from the monkey genome into the plasmid by illegitimate recombination. Another insertion mutant, N11-1, contained an 11 kb insert which included an unknown repetitive sequence that was also flanked by a target site duplication of 353 bp.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Immunocytochemical localization of casein kinase II during interphase and mitosis.
We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.
Amino Acid Sequence↗
Two amino acids, Phe 16 and Ala 776, on the polyprotein are most likely to be responsible for the diabetogenicity of encephalomyocarditis virus.
The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to differ from the non-diabetogenic B variant (EMC-B) by 14 nucleotides out of 7829 bases. Similar approaches with a new nondiabetogenic variant, EMC-DV1, obtained by plaque purification of the EMC-D variant stock pool, enabled us to narrow down further the possible genomic area responsible for the diabetogenicity of EMC virus. EMC-DV1 does not induce interferon in vitro, differing from the highly interferon-inducing EMC-B. The complete nucleotide sequence of EMC-DV1 was determined by RNA-dependent DNA sequencing and cDNA sequencing. The genomic size and organization of EMC-DV1 are similar to those of EMC-D and EMC-B, with a long open reading frame encoding a polyprotein of 2292 amino acids. Comparative analyses of sequence information as well as biological activities of EMC-DV1 with EMC-D and EMC-B suggest that (i) the diabetogenicity is apparently distinct from the ability to induce interferon, which is probably due to the single U base insertion at position 765 in EMC-B, and (ii) the diabetogenicity of EMC virus is most probably controlled by one or both of two amino acids, Phe 16 (on the leader peptide) and Ala 776 (152nd amino acid on the VP1) on the polyprotein.
Alanine↗