Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta

Conventional (alpha, betaI, betaII, gamma) and novel (delta, epsilon, eta, theta) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring, showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed 8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4 showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated PKC epsilon and eta from the cytoplasm to the plasma membrane of HeLa cells at 1 microM, whereas other PKC isozymes did not respond even at 10 microM. These results indicate that 4 could be a selective activator for PKC epsilon and eta.     

Indolactam and benzolactam compounds as new medicinal leads with binding selectivity for C1 domains of protein kinase C isozymes

Protein kinase C (PKC) isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta) are major receptors of tumor promoters and also play a crucial role in cellular signal transduction via the second messenger, 1,2-diacyl-sn-glycerol (DG). Each isozyme of PKC is involved in diverse biological events, indicating that it serves as a novel therapeutic target. Since PKC isozymes contain two possible binding sites of tumor promoters and DG (C1A and C1B domains), the design of agents with binding selectivity for individual PKC C1 domains is a pressing need. We developed a synthetic C1 peptide library of all PKC isozymes for high-throughput screening of new ligands with such binding selectivity. This peptide library enabled us to determine that indolactam and benzolactam compounds bound to the C1B domains of novel PKC isozymes (delta, epsilon, eta, theta) in some selective manner, unlike phorbol esters and DG. Simpler in structure and higher in stability than the other potent tumor promoters, a number of indolactam and benzolactam derivatives have been synthesized to develop new PKC isozyme modulators by several groups. We focused on the amide function of these compounds because recent investigations revealed that both the amide hydrogen and carbonyl oxygen of indolactam-V (ILV) are involved in hydrogen bonding with the C1B domains of PKCdelta. Synthesis of several conformationally fixed analogues of ILV led to the conclusion that the trans-amide restricted analogues with a hydrophobic chain at an appropriate position (2,7) are promising leads with a high binding selectivity for novel PKC isozyme C1B domains. We also developed a new lactone analogue of benzolactam-V8 (17) which shows significant binding selectivity for the C1B domains of PKCepsilon and PKCeta. Furthermore, our synthetic approach with the PKC C1 homology domains clarified that diacylglycerol kinase beta and gamma are new targets of phorbol esters.     

Potential role of PKC inhibitors in the treatment of hematological malignancies

The serine/threonine protein kinase C (PKC) family, the main target of tumor-promoting phorbol esters, is functionally associated to cell cycle regulation, cell survival, malignant transformation, and tumor angiogenesis. Although PKC isozymes represent an attractive target for novel anticancer therapies, our knowledge of PKC in tumorigenesis is still only partial and each PKC isoform may contribute to tumorigenesis in a distinct way. Specifically, PKC isoforms have wide and different roles, which vary depending on expression levels and tissue distribution, cell type, intracellular localization, protein-protein and lipid-protein interactions. Although PKC activation has been linked to tumor cell growth, motility, invasion and metastasis, other reports have shown that some PKC isoforms can also have opposite effects. Therefore, it will be necessary to analyze the relative contribution of each PKC isozymes in the development and progression of different tumors in order to identify therapeutic opportunities, using either PKC inhibitors or PKC activators as molecular tools of investigation. This minireview is focussed on the role of PKC signaling and on the perspective of PKC inhibition in hematological malignancies.     

Pharmacology of the receptors for the phorbol ester tumor promoters: multiple receptors with different biochemical properties

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.     

Bryostatin-1: pharmacology and therapeutic potential as a CNS drug

Bryostatin-1 is a powerful protein kinase C (PKC) agonist, activating PKC isozymes at nanomolar concentrations. Pharmacological studies of bryostatin-1 have mainly been focused on its action in preventing tumor growth. Emerging evidence suggests, however, that bryostatin-1 exhibits additional important pharmacological activities. In preclinical studies bryostatin-1 has been shown at appropriate doses to have cognitive restorative and antidepressant effects. The underlying pharmacological mechanisms may involve an activation of PKC isozymes, induction of synthesis of proteins required for long-term memory, restoration of stress-evoked inhibition of PKC activity, and reduction of neurotoxic amyloid accumulation and tau protein hyperphosphorylation. The therapeutic potential of bryostatin-1 as a CNS drug should be further explored.     

Protein kinase C isozymes as potential targets for anticancer therapy

Protein kinase C (PKC) comprises a family of isozymes (alpha, betaI, betaII, gamma, delta, epsilon, theta, eta, lambda/iota [mouse/human], and zeta) which are involved in signal transduction from membrane receptors to the nucleus. Activation of PKC by phorbol esters promotes tumor formation, and from that it was concluded that inhibitors of PKC might prevent carcinogenesis or inhibit tumor proliferation. However, the situation is more complicated because the exact function of the different PKC isozymes is not known at present. They have been shown to be involved in synaptic transmissions, the activation of ion fluxes, secretion, cell cycle control, differentiation, proliferation, tumorigenesis, metastasis and apoptosis. Modulators such as bryostatin-1, phospholipid analogues, PKC-activating adriamycin derivatives, CGP41251, UCN-01, and antisense oligonucleotides directed against PKCalpha, have shown antitumor activity in cancer patients. PKC inhibitors are not specific to PKC, but also interact with other signaling molecules, which may contribute to the antitumor effects. Modulators of PKC have also been shown to influence non-MDR1-mediated and MDR1-mediated antitumor drug resistance. This review is focussed on the role of PKC isozymes in human cell proliferation, apoptosis and antitumor drug resistance, and on the use of PKC modulators as antitumor agents.     

Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy

Recent years have seen extensive growth in the understanding of the role(s) of the various PKC isozymes and novel receptors for the phorbol ester tumor promoters. The PKC family of serine-threonine kinases is an important regulator of signaling cascades that control cell proliferation and death, and therefore represent targets for cancer therapy. While past interests have focused on PKC-selective inhibitors, more recently, intensive research has been underway for selective activators and inhibitors for each individual PKC isozyme. In the past few years a large number of PKC activators and inhibitors with potential as anticancer agents have been developed. A number of these compounds are already in Phase II clinical testing. As a new generation of cancer chemotherapeutic agents are designed, developed and put through a series of rigorous clinical trials, we can anticipate achieving exquisite control over PKC-mediated regulatory pathways, leading ultimately to a greater understanding of different cancers.     

Identification of PKC isozymes and effect of knockdown of PKC alpha by antisense oligodeoxynucleotide on iNOS expression via interleukin-1 receptor in vascular smooth muscle cells

Protein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKC alpha knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-kappa B in RASMC. Western blot analysis revealed the presence of cPKC (alpha), nPKCs (delta and epsilon) and aPKCs (tau and lambda). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKC alpha from cytosolic to particulate fraction. PKC alpha was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKC alpha mRNA depleted PKC alpha specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-kappa B, stimulated by IL-1 beta was decreased by PKC alpha knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKC alpha is involved in iNOS expression triggered by IL-1 beta, supporting our previous pharmacological conclusion.     

Divergence and complexities in DAG signaling: looking beyond PKC

For many years protein kinase C (PKC) has been the subject of extensive studies as a molecular target for the treatment of cancer and other diseases. To better define the role of PKC isozymes in the control of cell proliferation, survival and transformation, the examination of PKC-mediated signal transduction pathways by isozyme-specific intervention has become essential. However, issues related to the selectivity of activators and inhibitors of PKC isozymes, in addition to convoluted cross-talks between phorbol ester-regulated pathways, have greatly complicated our understanding of PKC-mediated responses. An additional level of complexity is provided by the fact diacylglycerol (DAG) signals can be transduced by phorbol ester receptors other than PKC. These receptors include chimaerins, RasGRPs, MUNC13s, PKD (PKC mu) and DAG kinases beta and gamma. Thus, it is conceivable that some of the effects that were originally attributed to PKC isozymes in response to phorbol esters might be mediated by PKC-independent pathways. A key issue for the design of novel therapeutic strategies that target PKC isozymes is a comprehensive analysis of isozyme-specific signal transduction pathways in different cell types and the development of pharmacological and molecular tools that can distinguish between the various PKC and 'non-PKC' phorbol ester receptors.     

Effects of resveratrol on the autophosphorylation of phorbol ester-responsive protein kinases: inhibition of protein kinase D but not protein kinase C isozyme autophosphorylation

The natural product resveratrol is a potent antagonist of phorbol ester-mediated tumor promotion and in vitro cellular responses to phorbol-ester tumor promoters, but it is only weakly inhibitory against the phosphorylation of conventional exogenous substrates by phorbol ester-responsive protein kinase C (PKC) isozymes. In this report, we compare the effects of resveratrol against the autophosphorylation reactions of PKC isozymes versus the novel phorbol ester-responsive kinase, protein kinase D (PKD). We found that resveratrol inhibits PKD autophosphorylation in a concentration-dependent manner, but has only negligible effects against the autophosphorylation reactions of representative members of each PKC isozyme subfamily (cPKC-alpha, -beta(1), and -gamma, nPKC-delta and -epsilon, and aPKC-zeta). Resveratrol was comparably effective against PKD autophosphorylation (IC(50) = 52 microM) and PKD phosphorylation of the exogenous substrate syntide-2 (IC(50) = 36 microM). The inhibitory potency of resveratrol against PKD is in line with the potency of resveratrol observed in cellular systems and with its potency against other purified enzymes and binding proteins that are implicated in the cancer chemopreventive activity of the polyphenol. Thus, PKD inhibition may contribute to the cancer chemopreventive action of resveratrol.     

The therapeutic role of targeting protein kinase C in solid and hematologic malignancies

The protein kinase C (PKC) family, the most prominent target of tumor-promoting phorbol esters, is functionally linked to cell differentiation, growth, survival, migration and tumorigenesis and so mediates tumor cell proliferation, survival, multidrug resistance, invasion, metastasis and tumor angiogenesis. Therefore, targeting PKC isozymes may represent an attractive target for novel anticancer therapies. Recent preclinical and clinical studies using the macrocyclic bisindolylmaleimide enzastaurin or the N-benzylstaurosporine midostaurin demonstrate promising activity of PKC inhibitors in a variety of tumors, including diffuse large B-cell lymphoma, multiple myeloma and Waldenstroem's macroglobulinemia. However, our knowledge of PKCs in tumorigenesis is still only partial and each PKC isoform may contribute to tumorigenesis in a distinct way. Specifically, PKC isoforms have vastly different roles, which vary depending on expression levels of organ and tissue distribution, cell type, intracellular localization, protein-protein and lipid-protein interactions and the biologic environment. Although PKC activation generally positively affects tumor cell growth, motility, invasion and metastasis, recent reports show that many PKCs can also have negative effects. Therefore, it is necessary to further dissect the relative contribution of PKC isozymes in the development and progression of specific tumors in order to identify therapeutic opportunities, using either PKC inhibitors or PKC activators.     

The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKC? specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKC? in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKC? kinase activity in vitro. VHHs A10, C1 and D1 increased PKC? kinase activity in a concentration-dependent manner (EC₅₀ values: 212–310 nM), whereas E6 and G8 inhibited PKC? activity (IC₅₀ values: 103–233 nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKC? specific modulators is an important contribution to PKC research.     

Differential phosphorylation of sites in the linker region of P-glycoprotein by protein kinase C isozymes alpha, betaI, betaII, gamma, delta, epsilon, eta, and zeta.

To determine whether individual protein kinase C (PKC) isozymes differentially phosphorylate sites in the linker region of human P-glycoprotein (P-gp), we used a synthetic peptide substrate, PG-2, exactly corresponding to amino acid residues spanning the region 656-689 of the multidrug resistance gene (MDRI). All tested PKC isozymes phosphorylated PG-2. The maximum phosphate incorporation by calcium-dependent PKC isozymes alpha, betaI, betaII, and gamma was 3, 2, 2, and 3 mol phosphate/mol PG-2, respectively. The maximum phosphate incorporation by calcium-independent isozymes delta, epsilon, eta, and zeta was 1.5, 0.5, 1.5, and 1.5 mol phosphate/mol PG-2, respectively. Two-dimensional tryptic phosphopeptide mapping indicated differential phosphorylation of the PKC consensus sites Ser-661, Ser-667, and Ser-671 by individual isozymes, which may be functionally significant. These data suggest that differential phosphorylation by PKC isoenzymes of PKC sites within the P-gp linker region may play a role in modulating P-gp activity.     

Novel "nonkinase" phorbol ester receptors: the C1 domain connection

In recent years, there have been great advances in our understanding of the pharmacology and biology of the receptors for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG). The traditional view of protein kinase C (PKC) as the sole receptor for the phorbol esters has been challenged with the discovery of proteins unrelated to PKC that bind phorbol esters with high affinity, suggesting a high degree of complexity in the signaling pathways activated by DAG. These novel "nonkinase" phorbol ester receptors include chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), and Munc13 isoforms (scaffolding proteins involved in exocytosis). In all cases, phorbol ester binding occurs at the single C1 domain present in these proteins and, as in PKC isozymes, ligand binding is a phospholipid-dependent event. Moreover, the novel phorbol ester receptors are also subject to subcellular redistribution or "translocation" by phorbol esters, leading to their association to different effector and/or regulatory molecules. Clearly, the use of phorbol esters as specific activators of PKC in cellular models is questionable. Alternative pharmacological and molecular approaches are therefore needed to dissect the involvement of each receptor class as a mediator of phorbol ester/DAG responses.     

2,2',3,3',4,4'-Hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE)-induced neuronal apoptosis independent of classical protein kinase C alpha or gamma inhibition

Protein kinase C (PKC) isozymes constitute a family of at least 12 structurally related serine-threonine kinases that are differentially regulated and localized, and are presumed to mediate distinct intracellular functions. To explore their roles in intact cells, investigators are developing cell-permeable, isoform-selective inhibitors. 2,2',3,3',4,4'-Hexahydroxy-1, 1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE) is reported to be a selective inhibitor of PKC alpha and gamma with IC(50) values of 43 and 50 microM, respectively, using an in vitro assay. However, data examining the potency and selectivity of HBDDE in intact cells are lacking. Employing rodent cerebellar granule neurons as a model system, we investigated the effects of HBDDE using cell survival as a functional end-point. HBDDE induced an apoptotic form of cell death that was dependent upon protein synthesis and included activation of a terminal executioner of apoptosis, caspase 3. The concentration of HBDDE required for half-maximal cell death was less than 10 microM ( approximately 5-fold less than the reported IC(50) values for PKC alpha and gamma in vitro). Furthermore, HBDDE induced apoptosis even after phorbol-ester-mediated down-regulation of PKC alpha and gamma, indicating that this effect is independent of these isoforms. Consistent with this, 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl)-maleimide (GF 109203X), a general inhibitor of all classical and some novel PKCs, did not interfere with survival. Thus, HBDDE should not be used as an isoform-selective inhibitor of PKC alpha or gamma in intact cells. Nevertheless, identification of its target in granule neurons will provide valuable information about survival pathways.     

Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development.

Protein kinase C (PKC) is a family of enzymes that play a ubiquitous role in intracellular signal transduction. Our understanding of the precise role of PKC has evolved considerably as a result of improved methodology and a better understanding of the signal transduction pathways. A number of primary pathways previously attributed to PKC have been re-examined and found to involve other kinases as our understanding of the PKC isozymes has evolved. PKC isozymes appear to play distinct, and in some cases opposing roles in the transduction of intracellular signals. The development of potent and selective PKC inhibitors, including isozyme-selective inhibitors, has opened new avenues for biochemical and pharmaceutical studies. The role of PKC in some of the pathways relevant to cardiovascular, peripheral microvascular, CNS, oncology, immune and infectious disease states are surveyed. A survey of the current generation of potent and selective ATP-competitive inhibitors is provided. The progress of PKC inhibitors currently in clinical development, including LY333531, ISIS 3521 (CGP 64128A), bryostatin 1, GF109203x, Ro 32-0432 and Ro 31-8220, Go 6976 and Go 7611, CPR 1006, and balanol (SPC 100840) are discussed.     

The effect of plant phenols on the expression and activity of phorbol ester-induced PKC in mouse epidermis

Protein kinase C (PKC) is thought to be a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The diversity of PKC isoforms, and their central role in many signaling pathways, makes them important targets for potential chemopreventive agents. Our earlier studies showed that the plant phenols protocatechuic acid, chlorogenic acid and tannic acid alter the activity of enzymes involved in carcinogen activation, inhibit the formation of polycyclic aromatic hydrocarbon (PAH)–DNA adducts in mouse epidermis and decrease the level of lipid peroxidation in the epidermal microsomes. In the present study the effects of protocatechuic acid, chlorogenic acid and tannic acid on TPA-stimulated PKC isozymes α, β₁, β₂, γ and ζ activity, and their distribution in mouse epidermis, was examined. The application of these phenolics 15 min before a single dose (3.4 nmol) of TPA resulted in significant inhibition of PKC translocation and a subsequent decrease in classical and novel/atypical PKC isoforms in comparison to a group of mice treated with TPA alone. The most potent inhibitor of PKC translocation and activity was tannic acid. This compound increased the levels of PKCα, β₁, β₂ in the cytosolic fraction by between 127% and 492% in comparison with TPA treated group of mice. Tannic acid decreased the activities of all three PKC classes by ∼94% in the membrane fraction in comparison with the TPA treated group of animals. The effect of protocatechuic and chlorogenic acids on the distribution and activity of PKC isozymes was moderate. These compounds mostly affected translocation of PKCα and subsequently the activity of classical PKC. The enzyme activity in the particulate fraction was reduced by 59% and 43% in comparison with the TPA group, respectively. Thus, the results of these studies suggest that the subcellular distribution of PKC isoforms, and the activity of PKCs, can be modulated by plant phenolic acids, particularly tannic acid, and that such actions represent a part of the anti-promotional activity of these substances in mouse epidermis.     

Protein kinase C and prostate carcinogenesis: targeting the cell cycle and apoptotic mechanisms

A series of both genetic and epigenetic factors have been implicated in the genesis and progression of prostate cancer. Recent evidence revealed that protein kinase C (PKC) isozymes play a crucial role in the control of cell proliferation and apoptosis in prostate cancer models, as well as in the transition from an androgen-dependent to an androgen-independent status. Indeed, PKCalpha and PKCdelta promote apoptosis in androgen-dependent prostate cancer cells. Due to the relevance of PKC isozymes in the control of cell cycle, both in G1/S and G2/M, the elucidation of such complex intracellular networks using cellular and animal models has become of outmost importance. In this review, we present the current knowledge on the regulation of apoptosis and tumorigenicity by PKC isozymes and the functional roles of cell cycle regulators in prostate carcinogenesis. The development of animal models where overexpression of discrete PKCs or cell cycle regulators is targeted to the prostate will greatly contribute to the understanding of the molecular basis of the disease, and more importantly, it will have profound implications for the development of novel strategies for prostate cancer therapy.     

Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems.     

Parallel modulation of receptor for activated C kinase 1 and protein kinase C-alpha and beta isoforms in brains of morphine-treated rats.

1. Receptor for activated C kinase 1 (RACK1) is an intracellular receptor for protein kinase C (PKC) that regulates the cellular enzyme localization. Because opiate drugs modulate the levels of brain PKC (Ventayol et al., 1997), the aim of this study was to assess in parallel the effects of morphine on RACK1 and PKC-alpha and beta isozymes densities in rat brain frontal cortex by immunoblot assays. 2. Acute morphine (30 mg kg(-1), i.p., 2 h) induced significant increases in the densities of RACK1 (33%), PKC-alpha (35%) and PKC-beta (23%). In contrast, chronic morphine (10-100 mg kg(-1), i.p., 5 days) induced a decrease in RACK1 levels (22%), paralleled by decreases in the levels of PKC-alpha (16%) and PKC-beta (16%). 3. Spontaneous (48 h) and naloxone (2 mg kg(-1), i.p., 2 h)-precipitated morphine withdrawal after chronic morphine induced marked up-regulations in the levels of RACK1 (38-41%), PKC-alpha (51-52%) and PKC-beta (48-62%). 4. In the same brains and for all combined treatments, there were significant positive correlations between the density of RACK1 and those of PKC-alpha (r=0.85, n = 35) and PKC-beta (r=0.75, n=32). 5. These data indicate that RACK1 is involved in the short- and long-term effects of morphine and in opiate withdrawal, and that RACK1 modulation by morphine or its withdrawal is parallel to those of PKC-alpha and beta isozymes. Since RACK1 facilitates the PKC substrate accessibility, driving its cellular localization, the coordinate regulation of the PKC/RACK system by morphine could be a relevant molecular mechanism in opiate addiction.