Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

Autoradiographic Imaging of [3H]Phorbol 12,13-Dibutyrate Binding to Protein Kinase C in Alzheimer''s Disease

Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being greater than 60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CA1 of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex; e.g., layers I-II control, 491 +/- 46 versus AD, 537 +/- 39 pmol/g of tissue; middle temporal cortex, e.g., layers I-II control, 565 +/- 68 versus AD, 465 +/- 72 pmol/g of tissue; and hippocampal formation, e.g., CA1 control, 511 +/- 28 versus AD, 498 +/- 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well-defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical correlates of AD.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Kinetic evidence that 1,2-diolein inhibits phorbol ester binding to protein kinase C via a competitive mechanism

Diacylglycerols inhibit binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) to protein kinase C (the phorbol ester receptor). This inhibition could reflect competitive binding by the diglyceride. Alternatively, it might simply represent perturbation of the lipid environment required for binding activity. As predicted for a competitive mechanism, we report here that inhibitory concentrations of the diglyceride 1,2-diolein do not affect the off-rate of [3H]PDBu from its receptor. This behavior contrasts with that of arachidonic acid, which appears to interact via a mixed mechanism.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

In Vitro Stimulation of Protein Kinase C by Melatonin

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca²⁺ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca²⁺ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [³H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [³H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca²⁺. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca²⁺ dependent proteins.     

An automated filtration assay for protein kinase C ligands

[3H]Phorbol dibutyrate ([3H]PDBu) binding to soluble mouse brain protein kinase C (PKC) was established in a 96-well microtiter plate assay. [3H]PDBu-PKC receptor complexes were rapidly aspirated from wells, filtered, and washed onto glass fiber filter mats using an automated cell harvester. Results were compared to a modification of a previously described assay in which components were incubated in tubes, and manually delivered and washed onto filters with a manifold filtration apparatus. Both 96-well plate and tube assays gave qualitatively and quantitatively similar results since: (i) [3H]PDBu binding to PKC was phosphatidylserine (PS) dependent and calcium stimulatable; (ii) the amounts of [3H]PDBu bound by filters with each technique at receptors excess were similar, 3.2 +/- 0.3 and 3.1 +/- 0.4 pmol respectively; and (iii) the affinities of [3H]PDBu for PKC were comparable; Kd's were 1.95 +/- 0.3 and 2.2 +/- 0.55 nM, respectively. The 96-well plate assay was more accurate and rapid than the tube assay. The microtiter plate assay was adapted for use with [N,N-dimethyl-3H]N,N-dimethylstaurosporine ([3H]DMS). With [3H]PDBu and [3H]DMS as ligands, the 96-well plate method was used for the rapid discrimination of agents which bound selectively at the regulatory and/or catalytic domains of PKC.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.     

Rapid and serial determination of protein kinase C activity and of the associated [3H]PDBu binding using a 96-well microtiter plate and a cell harvester

We propose a serial assay of both protein kinase C activity and the related [3H]phorbol 12,13-dibutyrate binding, each carried out in 96-multiwell dishes, started and stopped row by row using a multipipet. Protein kinase C activity is observed through the transfer of the gamma-phosphoryl group of radioactive ATP onto histone H1 type III-S. Enzymatic reactions are started by adding enzyme extracts and stopped by adding trichloroacetic acid. Acidic precipitates of each row are simultaneously collected on glass fiber paper using a cell harvester. The addition of bovine serum albumin and cold ATP at the end of the reaction and the addition of trichloroacetic acid in the washing fluid lead to a high recovery of protein kinase C activity and reproducible results. Measurement of [3H]phorbol 12,13-dibutyrate binding to protein kinase C was carried out in a mixed micellar solution as described elsewhere (Y. Hannun and R. M. Bell (1987) in Methods in Enzymology, Vol. 141, pp. 287-293). The quaternary complex formed from protein kinase C, phosphatidylserine, calcium, and [3H]phorbol 12,13-dibutyrate was then bound to a beaded anionic exchanger which was automatically separated from the free phorbol 12,13-dibutyrate by microfiltration using a cell harvester. The binding reaction was highly calcium- and phosphatidylserine-dependent and calcium had to be added to washing fluid for optimal recovery. Determination of protein kinase C activity and phorbol 12,13-dibutyrate binding gave results similar to those of other published methods and the signal/noise ratio was greatly increased. Using a semi-automated cell harvester, the system is partially automated and provides accurate and reproducible results.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C.

The effects of hydrophobic interaction on the activation of Ca2+-stimulated phospholipid-dependent protein kinase (protein kinase C), isolated from mouse brain, by phosphatidylserine (PS) and diacylglycerol (DAG) or phorbol 12-myristate 13-acetate were studied. To maintain bilayer structure during assay conditions, phosphatidylcholine was added to the PS vesicles. The vesicular structure of all types of PS was confirmed by freeze-fracture electron microscopy. The PS-dependent activation of purified protein kinase C from mouse brain is affected by the fatty acid composition of PS: an inverse relationship between the unsaturation index of PS (isolated from bovine heart, bovine spinal cord or bovine brain) and the ability to activate protein kinase C was demonstrated. In highly saturated PS lipid dispersions, only slight additional activation of protein kinase C by DAG was found, in contrast with highly unsaturated PS lipid dispersion, where DAG increased protein kinase C activity by 2-3-fold at optimal PS concentrations. We quantified the formation of the protein kinase C-Ca2+-PS-phorbol ester complex by using [3H]phorbol 12,13-dibutyrate [( 3H]PDBu). The efficiency of complex-formation, determined as the amount of [3H]PDBu bound, is not affected by variations in the hydrophobic part of PS. These results indicate a role of the hydrophobic part of the activating phospholipid in the activation mechanism of protein kinase C and in the action of cofactors.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

1,25-Dihydroxyvitamin D3 regulation of phorbol ester receptors in HL-60 leukemia cells

In this study the relationship between cell binding of phorbol 12,13-dibutyrate (PDBu) and induction of differentiation by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was examined. Binding of [3H]PDBu increased within 12 h of 1,25-(OH)2D3 treatment, and a 60-130% increase in [3H]PDBu receptor levels was observed within 24 h. By 48 h, however, [3H]PDBu binding was not different from control. Scatchard analysis of [3H]PDBu binding showed no statistical differences in Kd value (Kd approximately equal to 30 nM) between 1,25-(OH)2D3-treated and control cells 22 h post-treatment; however, a 2-fold increase in Bmax was observed in treated (338 +/- 24 pmol/10(9) cells) compared to control cultures (170 +/- 14 pmol/10(9) cells). Stimulation of [3H]PDBu binding was dependent on 1,25-(OH)2D3 concentrations over a range of 1-100 nM. Homogenates from 1,25-(OH)2D3-treated HL-60 cells also demonstrated an increase (70%) in [3H]PDBu binding to the Ca2+/phospholipid-dependent enzyme protein kinase C as assessed by incubation of cell homogenates with [3H]PDBu in the presence of saturating phosphatidylserine and calcium concentrations. This suggests that the increase in [3H]PDBu binding cannot be entirely explained by modulation of the latter two agents. Cycloheximide (5 microM), an inhibitor of protein synthesis, ablated the 1,25-(OH)2D3-stimulated increase in [3H]PDBu binding to intact HL-60 cells. These data demonstrate that an increase in [3H]PDBu binding occurs early in the course of 1,25-(OH)2D3-induced differentiation, results from an increased number of [3H]PDBu-binding site, and is dependent on protein synthesis.     

The cytosolic phorboid receptor correlates with hormone dependency in six mammary carcinoma cell lines

Potent, structurally different tumor promoters inhibited growth of 6 human mammary carcinoma cell lines (ROOS et al, PNAS in press). This growth inhibition was investigated by measuring the phorboid receptor binding using [3H] PDBu (4 beta-phorbol 12, 13 dibutyrate). Specific, high affinity receptors were found in all six cell lines. [3H] PDBu binding affinities were higher in the cytosolic fractions than in the corresponding intact cells (K alpha = app. 1nM vs K alpha = app. 15nM). The hormone-independent cell lines (BT-20, HBL-100 and MDA-MB-231) exhibited significantly higher levels of cytosolic [3H] PDBu receptors than the hormone-dependent cells (MCF-7, T-47-D and ZR-75-1). The subcellular distribution of the [3H] PDBu binding correlated well with the distribution of the protein kinase C activity (r = 0.95).     

Aminoacridines, potent inhibitors of protein kinase C

Acridine orange, acridine yellow G, and related compounds potently inhibited protein kinase C (Ca2+/phospholipid-dependent enzyme) activity and phorbol dibutyrate binding. Inhibition was investigated in vitro using Triton X-100 mixed micellar assays (Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 and Hannun, Y. A., and Bell, R. M. (1986) J. Biol. Chem. 261, 9341-9347). Inhibition by the acridine derivatives was subject to surface dilution; therefore, the relevant concentration unit is mol % rather than the bulk molar concentration. Fifty percent inhibition of protein kinase C activity occurred at concentrations of these compounds comparable to concentrations of sn-1,2-diacylglycerol (DAG) and phosphatidylserine (PS) required for enzyme activation (i.e. 1-6 mol %). The mechanism of inhibition appeared to be complex: both the catalytic and regulatory sites of protein kinase C were affected. Acridine orange was a competitive inhibitor with respect to MgATP when the catalytic fragment of protein kinase C was employed. Inhibition at the active site was overcome by the addition of Triton X-100 micelles or phospholipid vesicles. When the activity of intact protein kinase C was measured, inhibition was noncompetitive with respect to MgATP. Further kinetic analysis suggested a competitive type of inhibition with respect to PS and DAG implying an interaction of acridine compounds with the regulatory lipid cofactors or with the regulatory domain of protein kinase C. This was further supported by demonstrating inhibition of phorbol dibutyrate binding to both protein kinase C and the lipid-binding domain generated by trypsin hydrolysis. Acridine orange and acridine yellow G also inhibited thrombin-induced 40-kDa phosphorylation in human platelets and phorbol dibutyrate binding to platelets. These effects were also subject to surface dilution. These results suggest that acridine derivatives have multiple interactions with protein kinase C with the predominant effect being inhibition of activation within the regulatory domain of the enzyme. Some of the biologic effects of acridine derivatives including anti-tumor action may occur as a consequence of protein kinase C inhibition.     

Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion dependent conformational change

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unknown function [Coluccio, L. M., & Bretscher, A. (1988) J. Cell Biol. 106, 367-374]. By analysis of limited alpha-chymotryptic cleavage products, we now show that the molecular organization is very similar to that described for the S1 fragment of myosin. The catalytic site was identified by photoaffinity labeling with [5,6-3H]UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by [5,6-3H]UTP. Physical characterization revealed that the 45- and 32-kDa fragments exist as a complex that can bind F-actin, whereas the 40-kDa/32-kDa complex cannot bind actin. We conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles

The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Involvement of sulfatide in activation of protein kinase C by tumor promoters

Sulfatide (cerebroside sulfate) activated protein kinase C to the same extent as phosphatidylserine did with the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and debromoaplysiatoxin. Sulfatide and phosphatidylserine both induced specific binding of [3H]TPA to protein kinase C, although the ratios of specific to non-specific [3H]TPA binding to protein kinase C with the two were not the same. It is concluded that sulfatide is involved in activation of protein kinase C by tumor promoters in a slightly different way from phosphatidylserine.