Vitamin E inhibits protein kinase C activity

Vitamin E (dl-alpha-tocopherol) has been found to inhibit in vitro brain protein kinase c with a half inhibitory concentration of 450 microM. The known plasma concentrations of vitamin E are one order of magnitude lower than the protein kinase c half-inhibitory concentration but it is also known that, at the membrane level where the active protein kinase c is located, the lipophilic vitamin E is more concentrated (Burton, G.W., Joyce, A. and Ingold, K.U. and Locke, S. (1983) Arch. Biochem. Biophys. 221, 281-290). It appears that vitamin E, in addition to its antioxidant function, may play a role in regulating the activity of protein kinase c.     

Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.     

Lidocaine inhibits stimulation-coupled responses of neutrophils and protein kinase C activity

The effects of lidocaine, a local anesthetic, on various stimulation-coupled responses of neutrophils were studied. Superoxide generation, generation of chemiluminescence, depolarization of membrane potential and transitional increase in intracellular Ca2+ were inhibited by lidocaine in a concentration dependent manner. Lidocaine also inhibited Ca(2+)-activated phospholipid-dependent protein kinase (PKC) in the presence of various concentrations of Ca2+, phosphatidylserine and dioleoylglycerol. For the inhibition of all these stimulation-coupled responses, a similar order of the lidocaine concentration was needed. As in the case of dibucaine (Mori, T., Takai, Y., Minakuchi, R., Yu, B. and Nishizuka, Y., J. Biol. Chem. 255:8378-8380, 1980), lidocaine inhibited PKC activity in a manner competitive with phosphatidylserine. Lidocaine also inhibited the phosphorylation of 47 kDa neutrophil cytosplasmic protein, a phosphorylated protein required for NADPH oxidase activation. Thus, the cellular membrane phospholipid may be one of the target sites of lidocaine for the inhibitory action on the various stimulation-coupled responses of neutrophils, and these effects of lidocaine may correlate with its inhibitory action on PKC activity.     

Diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C) activity in bovine thyroid

Bovine thyroid 100,000 X g supernatant contained diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C). The protein kinase C was partially purified using ion-exchange chromatography and characterized. Substrate specificity studies revealed that the enzyme was most active when histone F1 was used as substrate. The thyroid protein kinase C was not stimulated by Ca2+ or phosphatidylserine (PS), but was stimulated by the combination of the two by 570%. Diolein stimulated the kinase by increasing its sensitivity to Ca2+. Other phospholipids could not substitute for PS and were ineffective in stimulating the protein kinase C in the absence of diolein. However, in the presence of diolein some of the other phospholipids were stimulatory albeit not to the extent of PS. Quercitin, a protein kinase C inhibitor in other systems, inhibited the thyroid enzyme in a dose-related manner. Protein kinase C could also be demonstrated using endogenous thyroid proteins as substrate. Separation of these 32P-labelled proteins by electrophoresis and subsequent autoradiography revealed that three proteins were phosphorylated by the protein kinase C of approximate molecular weights 60,000, 45,000, and less than 29,000. These results offer a possible mechanism by which Ca2+ and/or diacylglycerol effects may be mediated in thyroid.     

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.     

Diabetes-related changes in cAMP-dependent protein kinase activity and decrease in relaxation response in rat mesenteric artery

Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283-H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6,O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-alpha were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-beta were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an imbalance between PKA catalytic and regulatory subunit expressions.     

Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.     

Arginine vasopressin inhibits Kir6.1/SUR2B channel and constricts the mesenteric artery via V1a receptor and protein kinase C

Kir6.1/SUR2B channel is the major isoform of K(ATP) channels in the vascular smooth muscle. Genetic disruption of either subunit leads to dysregulation of vascular tone and regional blood flows. To test the hypothesis that the Kir6.1/SUR2B channel is a target molecule of arginine vasopressin (AVP), we performed studies on the cloned Kir6.1/SUR2B channel and cell-endogenous K(ATP) channel in rat mesenteric arteries. The Kir6.1/SUR2B channel was expressed together with V1a receptor in the HEK-293 cell line. Whole cell currents of the transfected HEK cells were activated by K(ATP) channel opener pinacidil and inhibited by K(ATP) channel inhibitor glibenclamide. AVP produced a concentration-dependent inhibition of the pinacidil-activated currents with IC(50) 2.0 nM. The current inhibition was mediated by a suppression of the open-state probability without effect on single-channel conductance. An exposure to 100 nM PMA, a potent PKC activator, inhibited the pinacidil-activated currents, and abolished the channel inhibition by AVP. Such an effect was not seen with inactive phorbol ester. A pretreatment of the cells with selective PKC blocker significantly diminished the inhibitory effect of AVP. In acutely dissociated vascular smooth myocytes, AVP strongly inhibited the cell-endogenous K(ATP) channel. In isolated mesenteric artery rings, AVP produced concentration-dependent vasoconstrictions with EC(50) 6.5 nM. At the maximum effect, pinacidil completely relaxed vasoconstriction in the continuing exposure to AVP. The magnitude of the AVP-induced vasoconstriction was significantly reduced by calphostin-C. These results therefore indicate that the Kir6.1/SUR2B channel is a target molecule of AVP, and the channel inhibition involves G(q)-coupled V1a receptor and PKC.     

The activation of protein kinase C by the polyphosphoinositides phosphatidylinositol 4,5-diphosphate and phosphatidylinositol 4-monophosphate

Protein kinase C(PKC) is a Ca2+- and phospholipid-dependent protein kinase which can be activated by diacylglycerol, a product of polyphosphoinositide hydrolysis. In this report, we show that the polyphosphoinositides L-alpha-phosphatidylinositol 4-monophosphate (PI 4P) and L-alpha-phosphatidylinositol 4,5-diphosphate (PI 4.5DP) can serve as phospholipid cofactors of isolated rat brain PKC. The order of potency of the phosphoinositides in the activation of PKC, PI greater than PI 4P greater than PI 4,5DP, shows a negative correlation with the degree of acidity of the phospholipid head group, whether 1 mM Ca2+ or 200 nM TPA is present in the reaction assay mixture. Although the polyphosphoinositides are by themselves weaker activators of PKC than PI, small amounts of PI 4,5DP cause a two-fold enhancement of PKC in the presence of Ca2+ and PI. While the endogenous phospholipid cofactors of PKC remain to be identified, these results suggest that the small amounts of polyphosphoinositides which are present in cell membranes may play a direct role in the activation of PKC in vivo, by serving as phospholipid cofactors of the enzyme.     

Isotypes of protein kinase C in bovine sperm.

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.     

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.     

Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate.

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of glyceryl trinitrate biotransformation and glyceryl trinitrate induced relaxation in bovine pulmonary artery and vein

Recent evidence supports the hypothesis that the mechanism by which glyceryl trinitrate induces relaxation of vascular smooth muscle involves the biotransformation of glyceryl trinitrate. This study was conducted to determine if there was a direct correlation between the capacity of vascular smooth muscle preparations to biotransform glyceryl trinitrate and their sensitivity to the relaxant effect of this organic nitrate. Isolated bovine pulmonary arteries and veins were contracted submaximally and cumulative dose-response relationships to glyceryl trinitrate were obtained; the vein was approximately 10 times more sensitive than the artery to glyceryl trinitrate induced relaxation. In a separate series of experiments, these vascular tissues were contracted submaximally and incubated with 0.5 microM [14C]glyceryl trinitrate for 2 min, during which glyceryl trinitrate induced relaxation was monitored. At 2 min, tissue samples were taken for determination of glyceryl trinitrate and glyceryl-1,2- and 1,3-dinitrate content by thin-layer chromatography and liquid scintillation spectrometry. Biotransformation of glyceryl trinitrate to glyceryl dinitrate occurred concomitantly with relaxation of these blood vessels. The concentration of glyceryl dinitrate in the vein was significantly less than that in the artery (p less than or equal to 0.05), even though significantly greater relaxation of the vein than the artery was observed (p less than or equal to 0.05). From these data, a simple linear relationship between glyceryl trinitrate biotransformation and relaxation is not apparent.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA-sensitive but not in RA-resistant cells

Treatment with 10(-5) M retinoic acid causes loss of anchorage-independent growth in src-transformed RR1022 cells but not in ras-transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10(-5) M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10(-5) M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage-independent growth in these two cell lines.     

Activation of protein kinase C inhibits human keratinocyte migration

The involvement of protein kinase C (PKC) in epidermal growth factor (EGF)-induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF-induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H-7 did not inhibit EGF-induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol-12-myristate-13-acetate (PMA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoly-sn-glycerol inhibited migration, but biologically inactive 4α-PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC-depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC-depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post-receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF-induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin-like growth factor-1, and keratinocyte growth factor. © 1993 Wiley-Liss, Inc.     

Phosphorylation of bovine platelet myosin by protein kinase C

Bovine platelet myosin is phosphorylated by protein kinase C at multiple sites. Most of the phosphate is incorporated in the 20,000-dalton light chain although some phosphate is incorporated in the heavy chain. Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10 times faster than the phosphorylation of smooth muscle myosin. Platelet myosin light chain is first phosphorylated at a threonine residue followed by a serine residue. Dominant phosphorylation sites of the 20,000-dalton light chain are estimated as serine-1, serine-2, and threonine-9. Prolonged phosphorylation by protein kinase C resulted in an additional phosphorylation site which, on the basis of limited proteolysis, appears to be either serine-19 or threonine-18. Phosphorylation by protein kinase C causes an inhibition of actin-activated ATPase activity of platelet myosin prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. It is shown that platelet myosin also exhibits the 6S to 10S conformation transition as judged by viscosity and gel filtration methods. Mg2(+)-ATPase activity of platelet myosin is paralleled with the 10S-6S transition. Phosphorylation by protein kinase C affects neither the 10S-6S transition nor the myosin filament formation. Therefore, the inhibition of actin-activated ATPase activity of platelet myosin is not due to the change in the myosin conformation.     

Heat-resistant inhibitors of protein kinase C from bovine brain

Bovine brain cytosol is shown to contain two heat-resistant inhibitors of protein kinase C, with the following characteristics: 1. One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19,000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 microM and the inhibition is Ca2+-independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition. 2. Another protein kinase C inhibitory activity has been partially purified. Its Mr is low (Mr 600-700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid- and alkali-resistant, acts Ca2+-independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin-dependent protein kinase II is inhibited to a similar extent. cAMP-dependent protein kinase is not affected.     

Wild-type p53 inhibits protein kinase CK2 activity

The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory beta-subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy-terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild-type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose-dependent and was seen for various CK2 substrates. Experiments with heat-denatured p53 and the conformational mutant p53(R175H) revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild-type and of mutant p53 into p53-/- cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild-type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down-regulation of protein kinase CK2.