Receptor For Activated C-kinase Research Articles

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, β-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.

Proteomic analysis of rice embryo: An approach for investigating Gα protein‐regulated proteins

The rice dwarf1 (d1) mutant, which lacks the alpha subunit of a heterotrimeric G protein (Galpha protein), shows abnormal morphology due to shortened internodes, dark green leaves and grains that are small and round. Proteome analysis was used in this study to aid in determining the function of Galpha protein in rice embryos. Using 2-DE, seven seed embryo proteins were shown to be down-regulated in the d1 mutant as compared with its wild type. These seven proteins included a receptor for activated C-kinase (RACK) and six rice embryo globulin-2 proteins (REG2). The six REG2 have similar molecular masses with minor differences in pI. In addition to the reduced accumulation of RACK in the d1 mutant, the increase in QL/d1, in which a constitutively active form of the Galpha protein is expressed, was significantly higher as compared with wild type. The level of accumulation of these seven proteins during seed development and maturation did not change significantly until the 2nd wk after pollination. Reduced accumulation of these seven proteins started in the d1 mutant at the 3rd wk after pollination, and continued until seed maturation was complete. All seven proteins were completely absent 24 h after imbibition in both d1 mutant and its wild type. However, the phytohormone abscisic acid promoted the expression level of RACK after imbibition in the wild type as compared with d1 mutant. These results suggest that RACK is regulated by Galpha-protein and plays an important role in a basic cellular process as well as in rice embryogenesis and germination.

Protein Kinase C Isozyme Selective Peptides - A Current View of What they Tell Us About Location and Function of Isozymes in the Heart

The identification of protein kinase C isozymes in distinct localities within the cell has led to the suggestion that each isozyme mediates a unique function. This has necessitated the development of methodologies that are capable of assigning specific function to an isozyme. For many years the location of individual isozymes in a particulate fraction was used to correlate specific isozymes with cellular function. More recently over-expression of selective isozymes and genetic knockouts have provided tissue-specific and developmentally regulated information on function. It is now known that specific proteins act as isozyme selective receptors for activated C kinase (RACKs) which determine subcellular localization of specific isozymes. As a result, peptides have been designed from the interaction site between the isozyme and its RACK that prevent the binding of isozymes to their respective RACKs. This has allowed the modulation of function of individual isozymes. This review will examine the development of the peptides as isozyme selective inhibitors or activators of PKC and its impact on understanding the role of isozymes in cellular function in the healthy and diseased heart. The possible development of isozyme-specific drugs for therapeutic use will be discussed.

Farnesyl pyrophosphate promotes and is essential for the binding of RACK1 with beta-tubulin.

Receptors for activated C kinase (RACKs) are a group of protein kinase C (PKC) binding proteins that have been shown to be crucial in the translocation and subsequent functioning of PKC on activation. RACK1 isolated from BALB/3T3 cells transformed with S-ras(Q61K) exhibits receptor activity for PKCgamma as competent as that of RACK1 from BALB/3T3 cells without transformation. However, the ability of RACK1 from transformed cells to bind with beta-tubulin peptide specific for Taxol (PEPtaxol) is defective. Interestingly, when farnesyl pyrophosphate was added at the submicrogram level, the association between RACK1 and PEPtaxol was enhanced significantly in a dosage-dependent manner. A parallel finding for the enhanced effect of farnesyl pyrophosphate on tubulin binding was established with mice RACK1 expressed in vitro. On the other hand, geranylgeranyl pyrophosphate, and retinoic acid failed to modulate the binding between RACK1 and tubulin. The dissociation of RACK1 and tubulin was not effective at damaging the binding between RACK1 and membrane receptor integrin beta1 in transformed cells. These findings indicate that depletion of farnesyl pyrophosphate provides a mechanism to seal PKC signaling on the membrane with immobile RACK1 and to divert cells to aberrant growth, such as transformation.

Protein kinase C-epsilon promotes adipogenic commitment and is essential for terminal differentiation of 3T3-F442A preadipocytes.

The role of protein kinase C (PKC) isoforms in the commitment of multipotent fibroblasts to the adipocyte lineage and in their terminal differentiation into mature adipocytes was investigated. Ectopic overexpression of PKC-epsilon, but not other PKC isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers. In committed 3T3-F442A preadipocytes, PKC-epsilon protein expression increased during the course of terminal differentiation and cell-permeable PKC-epsilon inhibitory peptides, which prevent interaction with RACK (receptor for activated C-kinase) proteins, severely inhibited differentiation. PKC-epsilon accumulated in the nuclei of 3T3-F442A cells shortly after induction of differentiation and exhibited a distinctive punctate speckling immunocytochemical staining pattern. The spatiotemporal aspects of PKC-epsilon localization and expression coincided with that of C/EBP-beta, a transcription factor critically involved in promoting the early phase of adipogenesis. Collectively, these results demonstrate a role for PKC-epsilon in both adipogenic commitment and preadipocyte terminal differentiation.

Receptor for activated C kinase (RACKI) mediates protein kinase C (PKC) signaling induced by Clostridium difficile toxin A

Receptor for activated C kinase (RACKI) mediates protein kinase C (PKC) signaling induced by Clostridium difficile toxin A

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.

Interaction with factor associated with neutral sphingomyelinase activation, a WD motif-containing protein, identifies receptor for activated C-kinase 1 as a novel component of the signaling pathways of the p55 TNF receptor.

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.

Ethanol induces gene expression via nuclear compartmentalization of receptor for activated C kinase 1.

Scaffolding proteins such as receptor for activated C kinase (RACK) 1 are involved in the targeting of signaling proteins and play an important role in the regulation of signal transduction cascades. Recently, we found that in cultured cells and in vivo, acute ethanol exposure induces the nuclear compartmentalization of RACK1. To elucidate a physiological role for nuclear RACK1, the Tat protein transduction system was used to transduce RACK1 and RACK1-derived fragments into C6 glioma cells. We found that nuclear RACK1 is mediating the induction of the immediate early gene c-fos expression induced by ethanol. First, transduction of full-length RACK1 (Tat-RACK1) resulted in the induction of c-fos expression and enhancement of ethanol activities. Second, we determined that the C terminus of RACK1 (Tat-RACK1DeltaN) is mediating transcription. Third, we identified a dominant negative fragment of RACK1 that inhibited the nuclear compartmentalization of endogenous RACK1 and inhibited ethanol-induction of c-fos mRNA and protein expression. Last, acute exposure to ethanol or transduction of full-length Tat-RACK1 resulted in an increase in mRNA levels of an activator protein 1 site-containing gene, PAC1 (pituitary adenylate cyclase-activating polypeptide receptor type I), suggesting that nuclear RACK1 is involved in the regulation of the expression of genes that are altered upon acute ethanol treatment. These results may therefore have important implications for the study of alcohol addiction.

Impaired Protein Kinase C Activation/Translocation in Epstein-Barr Virus-infected Monocytes

Infection of human monocytes by Epstein-Barr virus (EBV) has been linked to a decrease in the production of proinflammatory mediators as well as an impairment of phagocytosis. Considering the key role of protein kinases C (PKCs) in many biological functions of monocytes, including phagocytosis, we investigated the effects of EBV on the PKC activity in infected monocytes. Our results indicate that infection of monocytes by EBV impairs both phorbol 12-myristate 13-acetate (PMA)-induced translocation of PKC isozymes alpha and beta from cytosol to membrane as well as the PKC enzymatic activity. Similarly, the subcellular distribution of the receptor for activated C kinase (RACK), an anchoring protein essential to PKC translocation, was also found to be reduced in EBV-infected monocytes. Transfection of 293T cells with an expression vector coding for the immediate-early protein ZEBRA of EBV resulted in impaired PMA-induced translocation and activity of PKC. Using co-immunoprecipitation assays, the ZEBRA protein was found to physically interact with the RACK1 protein. Thus interaction of ZEBRA with RACK likely results in the inhibition of PKC activity, which in turn affects functions of monocytes, such as phagocytosis.

Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC epsilon-induced heart failure: a role for RACK1.

Recent investigations have established a role for the beta II-isoform of protein kinase C (PKC beta II) in the induction of cardiac hypertrophy and failure. Although receptors for activated C kinase (RACKs) have been shown to direct PKC signal transduction, the mechanism through which RACK1, a selective PKC beta II RACK, participates in PKC beta II-mediated cardiac hypertrophy and failure remains undefined. We have previously reported that PKC epsilon activation modulates the expression of RACKs, and that altered epsilon-isoform of PKC (PKC epsilon)-RACK interactions may facilitate the genesis of cardiac phenotypes in mice. Here, we present evidence that high levels of PKC epsilon activity are commensurate with impaired left ventricular function (dP/dt = 6,074 +/- 248 mmHg/s in control vs. 3,784 +/- 269 mmHg/s in transgenic) and significant myocardial hypertrophy. More importantly, we demonstrate that high levels of PKC epsilon activation induce a significant colocalization of PKC beta II with RACK1 (154 +/- 7% of control) and a marked redistribution of PKC beta II to the particulate fraction (17 +/- 2% of total PKC beta II in control mice vs. 49 +/- 5% of total PKC beta II in hypertrophied mice), without compensatory changes of the other eight PKC isoforms present in the mouse heart. This enhanced PKC beta II activation is coupled with increased RACK1 expression and PKC beta II-RACK1 interactions, demonstrating PKC epsilon-induced PKC beta II signaling via a RACK1-dependent mechanism. Taken together with our previous findings regarding enhanced RACK1 expression and PKC epsilon-RACK1 interactions in the setting of cardiac hypertrophy and failure, these results suggest that RACK1 serves as a nexus for at least two isoforms of PKC, the epsilon-isoform and the beta II-isoform, thus coordinating PKC-mediated hypertrophic signaling.

Adaptor proteins in protein kinase C-mediated signal transduction.

Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

PKCepsilon activation induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression.

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCepsilon transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCepsilon activity (228 +/- 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCepsilon activity (452 +/- 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCepsilon activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCepsilon enhanced RACK2 expression (178 +/- 13% of control) and particulate PKCepsilon-RACK2 protein-protein interactions (178 +/- 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 +/- 33% of control) and particulate PKCepsilon-RACK2 interactions (154 +/- 14% of control) but also in RACK1 protein expression (174 +/- 10% of control). Most notably, PKCepsilon-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCepsilon, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCepsilon activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCepsilon and suggest that differential patterns of PKCepsilon-RACK interactions may be important determinants of PKCepsilon-dependent cardiac phenotypes.

Blockade of PKCϵ Activation Attenuates Phorbol Ester-Induced Increase of α-Secretase-Derived Secreted Form of Amyloid Precursor Protein

The role of PKCϵ in amyloid precursor protein (APP) processing was investigated using APP-overexpressing B103 cells. As reported previously, a PKC activator, phorbol-12,13-dibutyrate (PDBu), enhanced secretion of APPα, and this effect was blocked by a PKC inhibitor, GF109203X in this system. Selective inhibition of PKCϵ by overexpressing the PKCϵ V1 region, which binds specifically to the receptor for activated C-kinase (RACK), blocked PDBu-induced enhancement of APPα secretion as well as PDBu-induced decrease in β-secretase-derived APP C-terminal fragment production. On the other hand, the level of PKCϵ, but not that of PKCα or PKCγ, was substantially lower in the brains of Alzheimer's disease patients compared to age-matched controls. These results add to a growing body of evidence that PKCϵ plays an important role in modulating APP processing, and suggest that reduced PKCϵ activity may contribute to the development of Alzheimer's disease.

Uncoupling of betaIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.

Identification and characterization of PRKCBP1, a candidate RACK-like protein.

The receptors for activated C-kinase (RACK) family of proteins function as anchors for activated protein kinase C (PKC) isoenzymes. Using a monoclonal antibody to RACK1 in the screening of a human hippocampus cDNA library, we identified a novel member of the RACK family, designated PRKCBP1. Immunoprecipitation assays performed with a GST-fused PRKCBP1 protein suggest the carboxy terminus of PRKCBP1 interacts specifically with PKCbetaI. Northern analysis detected a 3.1-kb PRKCBP1 transcript in all tissues examined including brain, heart, liver, lung, pancreas, skeletal muscle, kidney, and spleen. The PRKCBP1 gene has been localized to human Chromosome (Chr) 20q12-13.1. Several groups have reported evidence for genetic linkage of Type II diabetes to this region in Caucasian families. This localization, combined with the observation that abnormalities in the activation, translocation, and inhibition of PKC occur in the development of Type II diabetes, suggested that PRKCBP1 was a candidate for contributing to Type II diabetes. We determined the PRKCBP1 coding sequence is 1845 bp in length, dispersed over 9 exons, spanning approximately 34 kb of genomic DNA. SSCP analysis was used to screen PRKCBP1 coding regions for mutations or polymorphisms in 100 Caucasian Type II diabetics and 100 Caucasian unaffected individuals. A silent C/T transition (bp1413, Thr137) was present in 23% of both diabetic and control individuals. A C/T transition (bp198) was also identified in a single diabetic individual, which resulted in no coding change (Ser66). The results of this analysis suggest that PRKCBP1 coding variations are uncommon and do not contribute to Type II diabetes in the general population.

Cardiotrophic Effects of Protein Kinase C ε

Abstract —Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [ r eceptors for a ctivated C k inase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.

Smooth muscle: PKC‐induced Ca2+ sensitisation by myosin phosphatase inhibition

When arterial smooth muscles are stimulated with certain agonists such as phenylephrine contractile force increases while the intracellular free Ca2+ concentration may increase very little or even not at all (cf. Bradley & Morgan, 1987). In this case, force may be enhanced by raising the responsiveness of the contractile machinery or - for that matter - the sensitivity of the myofilaments to intracellular free Ca2+ (some 100 nM under resting conditions). The nature of mechanisms underlying Ca2+ sensitisation in pharmacomechanical coupling has been shown to involve various quite different intracellular signalling pathways and signalling molecules, such as small G-proteins (e.g. Rho A) and enzymes such as Rho-associated kinase, MAP kinase and various isoforms of protein kinase C (PKC) (cf. Somlyo & Somlyo, 1994; for review see Arner & Pfitzer, 1999). Based on the observation that phorbol esters, which are well known activators of PKC, cause a sustained contraction of arterial smooth muscle (Rasmussen et al. 1984) and also increase the sensitivity of the contractile apparatus to added Ca2+ in permeabilised smooth muscle preparations (Chatterjee & Tejeda, 1986), a role for PKC-induced Ca2+ sensitisation in tonic smooth muscle contraction was proposed long ago. During smooth muscle activation, PKC has been shown to be translocated from the cytosol to the cell membrane (Khalil et al. 1992), where it may be activated by diacylglycerol (DAG). It then initiates a signal sequence leading to the phosphorylation of specific intracellular receptors called receptors for activated C-kinase (RACKs). Thus the question arises as to the nature of RACKs involved in Ca2+ sensitisation of the myofilamets by PKC. The primary mechanism of smooth muscle contractile activation is the phosphorylation of myosin light chain (MLC) though, admittedly, the thin filament proteins caldesmon and calponin may also play a role (Arner & Pfitzer, 1999). It is now well established that the physiological increase in the phosphorylation of MLC is caused not only via Ca2+/calmodulin activation of myosin light chain kinase but also by a decrease in the dephosphorylating enzyme activity (e.g. Kitazawa et al. 1991). In a paper appearing in this issue of The The Journal of Physiology, Kitazawa et al. (1999) now show that in permeabilised (skinned) preparations of arterial smooth muscle, PKC enhances contractile force at a given submaximal concentration of free Ca2+ by increasing the level of MLC phosphorylation via inhibition of myosin phosphatase. This inhibition is mediated by phosphorylation of a novel RACK called CPI-17. This smooth muscle specific myosin phosphatase inhibitor exerts its phosphatase inhibitory and hence Ca2+ sensitising action in permeabilised preparations only when phosphorylated by PKC. These discoveries were made by using a differential skinning method: while intact arterial smooth muscle or preparations permeabilised by Staphylococcus aureusα-toxin or with the saponin ester β-escin became sensitised to Ca2+ by phorbol esters and other activators of PKC, fibres demembranated with the detergent Triton X-100 did not. The reason was that Triton-skinned fibres had lost the smooth muscle specific phosphatase-1 inhibitor protein CPI-17 and most of PKC. However, reconstitution of these fibres with PKC and CPI-17 increased MLC phosphorylation and the response to Ca2+ suggesting that, indeed, this kinase and the phosphatase inhibitor were the key players, causing increased myosin phosphorylation and contraction at submaximal Ca2+ concentration. These important new findings challenge current ideas (cf. Singer, 1996) suggesting that the physiological activation of PKC in vascular smooth muscle and its sensitisation to Ca2+ are mediated by mechanisms involving mitogen activated protein kinase-dependent caldesmon phosphorylation rather than phosphorylation of MLC by PKC. Now, the question arises as to what extent the proposed mechanisms may be operating in smooth muscle in vivo. It seems quite puzzling how in intact cells PKC can phosphorylate CPI-17 which, apparently, is located in the cytosol, while PKC activation requires its translocation to the cell membrane. Perhaps, CPI-17 shuttles between membrane-bound active PKC and thick filament-bound myosin phosphatase. Alternatively, membrane-bound (activated) PKC may be in rapid equilibrium with cytosolic PKC. In the cytosolic compartment the quantity of activated PKC, though low, may then be still sufficient to cause the Ca2+ sensitising downstream effect mediated by the phosphorylation of the smooth muscle specific phosphatase-1 inhibitor CPI-17 targeted to the thick filaments.

Protein Kinase C Anchoring Deficit in Postmortem Brains of Alzheimer's Disease Patients

Protein kinase C (PKC) has been implicated in the pathophysiology of Alzheimer's disease (AD). The levels of particular isoforms and the activation of PKC are reduced in postmortem brain cortex of AD subjects. Receptors for activated C kinase (RACK) are a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments. Recent evidence has indicated that the impaired activation (translocation) of PKC in the aging brain is associated with a deficit in RACK1, the most well-characterized member of this family. The present study was conducted to determine whether alterations in RACK1 occurred in cortical areas where an impaired translocation of PKC has been demonstrated in AD. Here we report the presence of RACK1 immunoreactivity in human brain frontal cortex for the first time and demonstrate a decrease in RACK1 content in cytosol and membrane extracts in AD when compared with non-AD controls. By comparison, the levels of the RACK1-related PKCβII were not modified in the same membrane extracts. These observations add a new perspective in understanding the disease-associated defective PKC signal transduction and indicate that a decrease in an anchoring protein for PKC is an additional determinant of this deficit.

Coordinated Movement of RACK1 with Activated βIIPKC

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated betaIIPKC, determines the localization and functional activity of betaIIPKC. However, the mechanism by which RACK1 localizes activated betaIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and betaIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and betaIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves betaIIPKC from one intracellular site to another.