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PubMed1997

Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells.

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.

Adenosine Triphosphate↗
PubMed1974

A study of 3':5'-cyclic mononucleotide-dependent protein kinase from canine prostate glands.

1. An adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1mum-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 10(8)m(-1). 5. Stimulation of the enzyme was also observed with the 3':5'-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing alpha-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent K(m) for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent K(m) for ATP (1.2x10(-5)m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn(2+) for Mg(2+) decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg(2+) was replaced by Ca(2+) and cyclic AMP produced only little stimulation of the prostatic enzyme.

Adenosine Triphosphate↗
PubMed1983

Purification and properties of cyclic nucleotide-independent phosvitin kinase from pig testis.

Cyclic nucleotide-independent and Ca2+-independent phosvitin kinase was purified from pig testis to apparent homogeneity by DEAE-cellulose, Sephadex G-200 chromatography, followed by subsequent DEAE-cellulose chromatography and phosvitin-Sepharose 4B affinity chromatography. The purified enzyme was homogeneous by analytical polyacrylamide gel electrophoresis, and it consisted of two polypeptides with molecular weights of 92000 (alpha) and 84000 (beta), which were present in the ratio of 1:2. Molecular weight of the native enzyme was estimated at 240000 by gel chromatography on Sepharose 6B, suggesting that the enzyme consists of alpha beta 2. The enzyme had maximal activity with phosvitin as the substrate. Casein was less active than phosvitin. Histone, protamine and myosin light-chains were practically ineffective. The enzyme possessed no ability to autophosphorylate. The apparent Km values were 7.4 microM for phosvitin, 65 microM for ATP and 0.6 mM for Mg2+. Vmax was 2.16 mumol/min per mg. The enzyme was inhibited by ammonium sulfate and heparin, and it was not affected by the addition of cyclic nucleotides and Ca2+ with calmodulin or phospholipid.

Animals↗
PubMed1980

Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion.

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.

Cations↗
PubMed1990

Protein kinase C in Saccharomyces cerevisiae: comparison with the mammalian enzyme.

Protein kinase C (PKC) was detected in the yeast Saccharomyces cerevisiae with bovine myelin basic protein as the phosphate acceptor. The enzyme was purified at least 500-fold by a four-step column chromatographic procedure (phenyl-Sepharose CL-4B, Mono Q, Heparin-5PW, and hydroxyapatite). The molecular mass was approximately 90 kDa, as estimated by gel-filtration analysis. Yeast PKC was activated by the simultaneous addition of Ca2+, diacylglycerol, and phosphatidylserine. Free arachidonic acid alone could activate the enzyme to some extent. However, yeast PKC did not respond significantly to tumor-promoting phorbol esters. GTP did not serve as phosphate donor. The yeast enzyme showed substrate specificity distinctly different from that of mammalian PKCs. H1 histone and protamine were poor substrates. With myelin basic protein as a model substrate, yeast PKC phosphorylated threonyl residues preferentially, whereas rat brain PKCs phosphorylated seryl residues preferentially. Further studies should elucidate the role of yeast PKC in cellular regulation and cell cycle control.

Amino Acid Sequence↗
PubMed1985

Identification, purification, and partial characterization of plasma protein kinases.

Two isomeric forms of protein kinases, FI and FII, were isolated from human plasma. These two isomeric enzymes were isolated to apparent homogeneity on NaDodSO4-PAGE by using (NH4)2SO4 fractionation, DEAE-cellulose, hydroxylapatite, Affi-Gel blue, and high-pressure liquid column chromatography. Polyclonal antibodies were obtained from immunized rabbits and both enzymes cross-reacted with each other. Furthermore, immunoaffinity-purified anti-FI and anti-FII antibodies inhibited the enzyme activity of both FI and FII. These enzymes are cyclic nucleotides, Ca2+, calmodulin and phosphatidylserine-independent enzymes which can phosphorylate exogenously added histone, casein, protamine, phosvitin, and platelet surface proteins. The phosphorylated proteins of intact platelets by these enzymes in the presence of exogenously added [gamma-32P]ATP ranged in apparent molecular weights from 13.5K to 200K, as estimated by their mobility during NaDodSO4-PAGE. Trypsin removed the label from the platelet surface phosphoproteins without affecting the intracellularly located phosphoproteins labeled endogenously by 32PO4-prelabeling of intact platelets. These observations raise the possibility that these enzymes could play a role in modulating the properties of platelets through phosphorylation of the platelet surface proteins.

Amino Acids↗
PubMed1999

Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

Animals↗
PubMed1991

Influence of di- and tri-phenylethylene estrogen/antiestrogen structure on the mechanisms of protein kinase C inhibition and activation as revealed by a multivariate analysis.

We have performed a systematic study of the interaction of 36 di- and tri-phenylethylene derivatives (DPEs and TPEs) with protein kinase C (PKC). The results were submitted to a multivariate analysis in order to identify the structural features that might be implicated in interference with the activity of three PKC subspecies under three enzyme activation conditions. Four groups of test-compounds, each with common chemical features, could be distinguished clearly. The first group comprised all TPEs substituted with at least one basic dialkylaminoethoxy side-chain. These inhibited type alpha, beta and gamma PKC subspecies activated by Ca2+ and phosphatidylserine (PS) with or without diolein (DO) at micromolar concentrations but did not inhibit protamine sulfate phosphorylation. The other effectors, which all possessed a 1,1-bis-(p-hydroxyphenyl) ethylene moiety, influenced PKC activity at high concentrations (30-200 microM) and could be divided into two groups. One group constituted PKC inhibitors in the TPE series and inhibited PKC activated by Ca2+, PS and DO, as well as protamine sulfate phosphorylation. The other group constituted dual-type inhibitors/activators in the DPE series and stimulated PKC in the presence of Ca2+ and low PS concentrations but inhibited the enzyme in the simultaneous presence of DO. The fourth group of compounds was inactive and had, for the most part, one or two substituents with weak steric hindrance. In agreement with previous data for six lead compounds, this study suggests that, in these chemical series, a basic amino side-chain leads to interaction with phospholipid and the regulatory domain of PKC, whereas a 1,1-bis-(p-hydroxyphenyl) ethylene moiety leads to interaction with the catalytic domain of the enzyme.

Animals↗
PubMed1989

Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure-activity relationships.

Protein kinase C (PKC) from rat brain was inhibited by plant flavonoids in a concentration-dependent manner depending on flavonoid structure. Of the fifteen flavonoids studied, fisetin, quercetin and luteolin were the most potent, while hesperetin, taxifolin and rutin were among the least potent. The flavonol fisetin was almost 100% inhibitory at a concentration of 100 microM. The extent of inhibition was the same whether diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate was used as enzyme activator. Inhibition was independent of Ca2+, phospholipid, and enzyme activator, as shown by inhibition of protamine phosphorylation in the absence of the regulatory components. Fisetin was a competitive inhibitor with respect to ATP binding and noncompetitive with respect to protein substrate. The X-ray crystal structure analysis of hesperetin monohydrate showed that the molecule is essentially planar despite the sofa conformation of the gamma-pyran ring and the 27 degrees twist of the 2-phenyl ring. Comparison of this inactive flavanone with those of the active flavones showed that, although hesperetin can adopt a planar profile similar to those of fisetin and quercetin, the 4'-methoxy substituent blocks an essential structural feature required for inhibitory activity. Analysis of these structure-activity data revealed a model of the minimal essential features required for PKC inhibition by flavonoids: a coplanar flavone structure with free hydroxyl substituents at the 3', 4' and 7-positions.

Animals↗
PubMedDate not supplied

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7.

Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.

Animals↗
PubMed1993

Reversible palmitoylation of the protein-tyrosine kinase p56lck.

The myristoylated protein-tyrosine kinase, p56lck, is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56-kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [3H]myristate or [3H]palmitate. This protein was identified as p56lck based on its specific immunoprecipitation with polyclonal antisera to p56lck, by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and by co-chromatography with p56lck on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56lck-associated radioactivity from [3H]myristate-labeled cells, the p56lck-associated radioactivity from [3H]palmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse-chase analyses revealed that the labeling of p56lck with [3H]palmitate, but not [3H]myristate, was reversible. The presence of covalently attached palmitate on p56lck from [3H]palmitate-labeled cells was verified by thin-layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of p56lck with [3H]myristate without affecting its labeling with [3H]palmitate. These studies indicate that p56lck is both a cotranslationally myristoylated and post-translationally palmitoylated protein.

Animals↗
PubMed1984

Developmental studies of phospholipid-sensitive Ca2+-dependent protein kinase and its substrates and of phosphoprotein phosphatases in rat brain.

Ontogenetic changes in the protein phosphorylation/dephosphorylation systems in rat brain were investigated. It was found that the activity level of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) in the particulate fraction of grey and white matter and the soluble fraction of grey matter increased rapidly and markedly after birth, reached the highest level at day 30, and declined slightly or remained unchanged thereafter. The enzyme level in the soluble fraction of white matter, in contrast, remained constant throughout the development and maturation of brain. Various ontogenetic changes in the substrate proteins for PL-Ca-PK were also noted. The levels of myelin basic protein and other substrates (notably the Mr 87,000, 58,000, 54,000, and 50,000 protein in grey matter) progressively increased during development, reaching the highest level at adulthood. The level of the Mr 66,000 protein from the particulate fraction of white and grey matter, on the other hand, increased rapidly after birth, reached a peak at day 18, and then declined to the initial neonatal level at the adult stage. The time scale for the increases in the levels of PL-Ca-PK and its many substrates paralleled that of brain development and maturation (synaptogenesis and myelinogenesis). The activity levels of phosphoprotein phosphatases (assayed using 32P-labeled myelin basic protein, histone, and protamine sulfate) were found to only slightly (up to 60%) increase or decrease in certain fractions from different brain regions during development, suggesting that phosphorylation, compared to dephosphorylation, may be more important in determining the phosphorylation state of cellular proteins.

Aging↗
PubMed1990

Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets.

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.

Animals↗
PubMed1984

Subunit structure and autophosphorylation mechanism of casein kinase-TS (type-2) from rat liver cytosol.

Type-2 casein kinase-TS (Ck-TS) purified to homogeneity from rat liver cytosol exhibits a molecular mass of 130000 daltons in non-denaturating media and a subunit composition consistent with an alpha 2 beta 2 heterotetramer. The quaternary structure of Ck-TS is not compromised by limited proteolysis with trypsin which converts the 38-kDa alpha subunit into 36-kDa (alpha') and 34-kDa (alpha") derivatives, inducing a parallel decrease of enzymatic activity. Since the 25-kDa beta subunit is unaffected under comparable conditions, the catalytic activity seemingly resides in the alpha subunits. The beta subunit, on the other hand, undergoes a very rapid phosphorylation upon incubation of Ck-TS with ATP/Mg2+: 0.8-1.5 mol P/mol Ck-TS are incorporated within 30 s. Such a fast autophosphorylation is neither prevented nor slowed down by the addition of a large excess of phosphorylatable substrates and takes place through an intra-molecular rather than inter-molecular process. This conclusion is supported by the following data. (a) The autophosphorylation rate is linearly proportional to the concentration of Ck-TS. (b) Thermally inactivated Ck-TS is not phosphorylated by catalytic amounts of active enzyme. (c) Basic polypeptides like protamine and polylysine stimulate the activity of Ck-TS toward phosphorylatable substrates while preventing the autophosphorylation reaction. Since the effectors that inhibit autophosphorylation also induce a remarkable decrease of the Km values for the protein substrates, the possibility is discussed that autophosphorylation might represent a regulatory device by which Ck-TS could be converted into a partially inactivated form exhibiting reduced affinity toward its endogenous targets.

Animals↗
PubMed2001

Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase.

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.

Darkness↗
PubMed1998

A baculovirus mutant defective in PKIP, a protein which interacts with a virus-encoded protein kinase.

We have found that a temperature-sensitive mutant of the baculovirus AcMNPV, tsB97, is defective in PKIP, the product of ORF24 which was previously found to interact with and stimulate the activity of a virus-encoded protein kinase, PK-1. The mutant lacks the ability to form plaques and occlusion bodies at the nonpermissive temperature. The mutant displays several properties which suggest a defect in the latter half of the late phase of infection; these properties include a delay in the shutoff of host protein synthesis, the presence of aberrant electron-dense bodies associated with the virogenic stroma, and the production of few, if any, progeny budded virus. A study of the expression of selected late genes showed no difference in the timing or level of transcription or translation of most late genes. However, elevated levels of the late 6.9K protein, a protamine-like protein, were observed in mutant-infected cells at 24 h postinfection, suggesting a defect in the regulation of this protein. Two polypeptides, 40 and 6 kDa, exhibited considerably higher levels of steady-state phosphorylation in wt-infected cells versus tsB97-infected cells at 24 h p.i. and could be candidates for PK-1/PKIP-mediated phosphorylation. The tsB97 mutant also displayed a severe defect in very late gene transcription which accounts for its inability to form occlusion bodies. The effect of PKIP on very late gene transcription may be a secondary effect of the block in the late phase of infection. PKIP showed no ability to transactivate expression from a very late promoter in transient expression assays.

Animals↗
PubMed1991

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets.

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.

Alkaline Phosphatase↗