Nuclear protein kinase C isoforms: key players in multiple cell functions?

Protein kinase C (PKC) isozymes are a family of serine/threonine protein kinases categorized into three subfamilies: classical, novel, and atypical. PKC isozymes, whose expression is cell type-specific and developmentally regulated, are key transducers in many agonist-induced signaling cascades. To date at least 10 different PKC isotypes have been identified and are believed to play distinct regulatory roles. PKC isoforms are catalytically activated by several lipid cofactors, including diacylglycerol. PKC is thought to reside in the cytoplasm in an inactive conformation and to translocate to the plasma membrane or cytoplasmic organelles upon cell activation by different stimuli. However, a sizable body of evidence collected over the last 15 years has shown PKC to be capable of translocating to the nucleus. Furthermore, PKC isoforms can reside within the nucleus. Studies from independent laboratories have to led to the identification of several nuclear proteins which act as PKC substrates as well as to the characterization of some nuclear PKC-binding proteins which may be of fundamental importance for finely tuning PKC function in this peculiar cell microenvironment. Most likely, nuclear PKC isozymes are involved in the regulation of several important biological processes such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we shall summarize the most intriguing evidence about the roles played by nuclear PKC isozymes.     

Protein kinase C isoforms and lipid second messengers: a critical nuclear partnership?

A growing body of evidence, accumulated over the past 15 years, has highlighted that the protein kinase C family of isozymes is capable of translocating to the nucleus or is resident within the nucleus. The comprehension of protein kinase C isoform regulation within this organelle is under development. At present, it is emerging that lipid second messengers may play at least two roles in the control of nuclear protein kinase C: on one side they serve as chemical attractants, on the other they directly modulate the activity of specific isoforms. One of the best characterized lipid second messenger that could be involved in the regulation of nuclear PKC activity is DAG. The existence of two separate pools of nuclear DAG suggests that this lipid second messenger might be involved in distinct pathways that lead to different cell responses. Nuclear phosphatidylglycerol, D-3 phosphorylated inositol lipids and nuclear fatty acids are involved in a striking variety of critical biological functions which may act by specific PKC activation. The fine tuning of PKC regulation in cells subjected to proliferating or differentiating stimuli, might prove to be of great interest also for cancer therapy, given the fact that PKC-dependent signaling pathways are increasingly being seen as possible pharmacological target in some forms of neoplastic diseases. In this article, we review the current knowledge about lipid second messengers that are involved in regulating the translocation and/or the activity of different protein kinase C isoforms identified at the nuclear level.     

Multiple biological responses activated by nuclear protein kinase C.

Protein kinase C is a family of serine-threonine kinases that are physiologically activated by a number of lipid cofactors and are important transducers in many agonist-induced signaling cascades. To date, 12 different isozymes of this kinase have been identified and are believed to play distinct regulatory roles. Protein kinase C was thought to reside in the cytosol in an inactive conformation and translocate to the plasma membrane upon cell activation by different stimuli. Nevertheless, a growing body of evidence has illustrated that this family of isozymes is capable of translocating to other cellular sites, including the nucleus. Moreover, it seems that some protein kinase C isoforms are resident within the nucleus. A wealth of data is being accumulated, demonstrating that nuclear protein kinase C isoforms are involved in the regulation of several critical biological functions such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we will discuss the most significant findings concerning nuclear protein kinase C which have been published during the past 5 years.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Specific subcellular targeting of PKCα and PKCε in normal and tumoral lactotroph cells by PMA-mitogenic stimulus

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCα and PKCε in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCα to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCε in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCα could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.     

Nuclear protein kinase C isoforms and apoptosis

The process of apoptosis is regulated at multiple levels through phosphorylation by several different protein kinases. The protein kinase C (PKC) family of isozymes have been shown to exert both inhibitory and stimulatory influences on apoptosis. During the apoptotic process phosphorylative events are known to occur also at the nuclear level. Evidence suggests that PKC isoforms play a key role in some steps that lead to nuclear disassembly during the execution phase of apoptosis. This review highlights the recent progress made in determining the roles played by individual PKC nuclear isoforms in the control of apoptosis.     

Nuclear expression of diacylglycerol kinases: possible involvement in DNA replication

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.     

Oncogenic and activated wild-type ras-p21 proteins induce different isoforms of protein kinase C in mitogenic signal transduction

We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.     

Proliferating or differentiating stimuli act on different lipid-dependent signaling pathways in nuclei of human leukemia cells

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-beta II migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the alpha isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-beta II occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-alpha to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Adenosine triggers the nuclear translocation of protein kinase C epsilon in H9c2 cardiomyoblasts with the loss of phosphorylation at Ser729

Adenosine is a major mediator of ischaemic preconditioning (IPC) and cardioprotection. The translocation and activation of protein kinase C epsilon, triggered by adenosine, are essential for these processes. We report here that H9c2 cardiomyoblasts express five PKC isoforms (α, β_I, δ, ε and ζ). PKCε is predominantly associated with F‐actin fibres in unstimulated H9c2 cells but translocates to the nucleus on stimulation with adenosine. Cytosolic PKCε associated with F‐actin fibres is phosphorylated at Ser729 but nuclear PKCε lacks phosphorylation at this site. Adenosine triggers the nuclear translocation after 5 min stimulation. PKCε Ser729Ala and Ser729Glu mutants showed no translocation on adenosine stimulation suggesting both phosphorylation and serine at 729 are critical for this translocation. Among five PKC isoforms (α, β_I, δ, ε and ζ) detected, PKCε is the only isoform translocating to the nucleus upon adenosine stimulation. Disruption of microtubules (MTs), but not F‐actin‐rich fibres, blocked translocation of both endogenous PKCε and overexpressed GFP‐PKCε to the nucleus. Ten proteins interacted with cytosolic PKCε; five of which are components of myofibrils. Matrin 3 and vimentin interacted with nuclear PKCε. These findings suggest that adenosine stimulates PKCε translocation to the nucleus in H9c2 cells in a mechanism involving dephosphorylation at Ser729 and MT, which should advance our understanding of the signalling pathways stimulated by adenosine in IPC and cardioprotection. J. Cell. Biochem. 106: 633–642, 2009. © 2009 Wiley‐Liss, Inc.     

Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a β-like molecule during encystment

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.     

Characterization of protein kinase C in rat and human prostates

The properties of protein kinase C (PKC) activity have been studied in cytosolic and membrane fractions from rat and human prostate. Ion exchange chromatography indicated the existence of different PKC isoforms, PKC from rat ventral prostate behaved as a classical Ca²⁺- and phospholipid-dependent enzyme and was activated by 1,2-diacylglycerol as well as by high concentrations of arachidonic acid. PKC activity in the cytosolic fraction was higher and presented different cofactor requirements than that in the membrane fraction. PKC from human benign hyperplastic prostate was also phospholipid dependent, activated by tumor-promotong phorbol esters, and appeared to belong to the group of PKC isozymes which lack Ca²⁺ sensitivity. Human prostatic PKC activity appeared to be of similar nature in both membrane and cytosolic fractions but the specific activity was higher in the particulate preparation which could be related to the stage of endogenous activation of the enzyme. These results extend previous observations in rat ventral prostate and present evidences on the human counterpart. Forthcoming experiments are needed to establish the exact nature of PKC isozymes and their physiological and pathophysiological role in this gland.     

Nuclear import and export signals enable rapid nucleocytoplasmic shuttling of the atypical protein kinase C lambda

The atypical protein kinase C (PKC) isoenzymes, lambda/iota- and zetaPKC, play important roles in cellular signaling pathways regulating proliferation, differentiation, and cell survival. By using green fluorescent protein (GFP) fusion proteins, we found that wild-type lambdaPKC localized predominantly to the cytoplasm, whereas both a kinase-defective mutant and an activation loop mutant accumulated in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal part of the zinc finger domain of lambdaPKC. Leptomycin B treatment induced rapid nuclear accumulation of GFP-lambda as well as endogenous lambdaPKC suggesting the existence of a CRM1-dependent nuclear export signal (NES). Consequently, we identified a functional leucine-rich NES in the linker region between the zinc finger and the catalytic domain of lambdaPKC. The presence of both the NLS and NES enables a continuous shuttling of lambdaPKC between the cytoplasm and nucleus. Our results suggest that the exposure of the NLS in both lambda- and zetaPKC is regulated by intramolecular interactions between the N-terminal part, including the pseudosubstrate sequence, and the catalytic domain. Thus, either deletion of the N-terminal region, including the pseudosubstrate sequence, or a point mutation in this sequence leads to nuclear accumulation of lambdaPKC. The ability of the two atypical PKC isoforms to enter the nucleus in HeLa cells upon leptomycin B treatment differs substantially. Although lambdaPKC is able to enter the nucleus very rapidly, zetaPKC is much less efficiently imported into the nucleus. This difference can be explained by the different relative strengths of the NLS and NES in lambdaPKC compared with zetaPKC.     

Associations of PKC isoforms with the cytoskeleton of B16F10 melanoma cells.

Although PKC plays a major role in regulating the morphology and function of the cytoskeleton, little is known about in situ associations of specific isoforms with the cytoskeleton. We demonstrate that seven PKC isoforms are expressed in B16F10 melanoma cells and show different levels of induction by serum. Using cell cytoskeleton preparations (CSKs), confocal microscopy, and immunocytochemistry, all isoforms show specific patterns of localization to focal contact-like structures (alpha, delta), very small cytoplasmic granules/vesicles (all isoforms), dense ordered arrays of small granules in the perinuclear region (alpha, delta), granules/vesicles associated with a homogeneous framework in the cytoplasm adjacent to the nucleus (gamma), or irregular-shaped patches of granules at or near the nuclear perimeter (eta, theta). In addition, several isoforms are present as cytoplasmic granules/ vesicles in linear or curvilinear arrays (alpha, delta, epsilon, theta). When isoform localization is examined using 3.7% formaldehyde or methanol:acetone, the patterns of localization in CSKs are often difficult or impossible to detect, and many are described here for the first time. Double-labeling experiments with CSK demonstrate that PKC actin co-localizes with punctate alpha-rich particles above the nucleus, granules of epsilon throughout the cytoplasm, and with theta in irregular-shaped aggregates associated with the nucleus. Vimentin co-localizes with perinuclear granules of delta and beta(2), and alpha-tubulin co-localizes with theta in structures at or near the nuclear surface and in microtubules associated with the microtubule organizing center (MTOC). In summary, the present study demonstrates that seven PKC isoforms are endogenously expressed in B16F10 melanoma cells. These isoforms show various levels of induction by serum and specific patterns of association with various components of the detergent-resistant cell cytoskeleton.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.     

Nuclear diacylglycerol kinases: emerging downstream regulators in cell signaling networks

There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.     

Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.

Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.