Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.
The members of the atypical subfamily of protein kinase C (PKC) show dramatic structural and functional differences from other PKC isotypes. Thus, in contrast to the classical or novel PKCs, they are not activated by diacylglycerol or phorbol esters. However, the atypical PKCs are the target of important lipid second messengers such as ceramide, phosphatidic acid, and 3'-phosphoinositides. The catalytic and pseudosubstrate sequences in the two atypical PKCs (lambda/iota PKC and zeta PKC) are identical but are significantly different from those of conventional or novel PKCs. It has been shown that microinjection of a peptide with the sequence of the pseudosubstrate of the atypical PKC isotypes but not of alpha PKC or epsilon PKC dramatically inhibited maturation and NF-kappa B activation in Xenopus oocytes, as well as reinitiation of DNA synthesis in quiescent mouse fibroblasts. This indicates that either or both atypical isoforms are important in cell signalling. Besides the pseudosubstrate, the major differences in the sequence between lambda/iota PKC and zeta PKC are located in the regulatory domain. Therefore, any functional divergence between the two types of atypical PKCs will presumably reside in that region. We report here the molecular characterization of lambda-interacting protein (LIP), a novel protein that specifically interacts with the zinc finger of lambda/iota PKC but not zeta PKC. We show in this paper that this interaction is detected not only in vitro but also in vivo, that LIP activates lambda/iota PKC but not zeta PKC in vitro and in vivo, and that this interaction is functionally relevant. Thus, expression of LIP leads to the transactivation of a kappa B-dependent promoter in a manner that is dependent on lambda/iota PKC. To our knowledge, this is the first report on the cloning and characterization of a protein activator of a PKC that binds to the zinc finger domain, which has so far been considered a site for binding of lipid modulators. The fact that LIP binds to lambda/iota PKC but not to the highly related zeta PKC isoform suggests that the specificity of the activation of the members of the different PKC subfamilies will most probably be accounted for by proteins like LIP rather than by lipid activators.
An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of λ/ιPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both λ/ιPKC and ζPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56lck SH2-interacting protein, as a molecule that interacts potently with the V1 domain of λ/ιPKC and, albeit with lower affinity, with ζPKC. We also show in this study that ectopically expressed p62 colocalizes perfectly with both λ/ιPKC and ζPKC. Interestingly, the endogenous p62, like the ectopically expressed protein, displays a punctate vesicular pattern and clearly colocalizes with endogenous λ/ιPKC and endogenous ζPKC. P62 colocalizes with Rab7 and partially with lamp-1 and limp-II as well as with the epidermal growth factor (EGF) receptor in activated cells, but not with Rab5 or the transferrin receptor. Of functional relevance, expression of dominant negative λ/ιPKC, but not of the wild-type enzyme, severely impairs the endocytic membrane transport of the EGF receptor with no effect on the transferrin receptor. These findings strongly suggest that the aPKCs are anchored by p62 in the lysosome-targeted endosomal compartment, which seems critical for the control of the growth factor receptor trafficking. This is particularly relevant in light of the role played by the aPKCs in mitogenic cell signaling events.
Increased protein kinase C (PKC) activity in malignant breast tissue and positive correlations between PKC activity and expression of a more aggressive phenotype in breast cancer cell lines suggest a role for this signal transduction pathway in the pathogenesis and/or progression of breast cancer. To examine the role of PKC in the progression of breast cancer, human MCF-7 breast cancer cells were transfected with PKC-alpha, and a group of heterogenous cells stably overexpressing PKC-alpha were isolated (MCF-7-PKC-alpha). MCF-7-PKC-alpha cells expressed fivefold higher levels of PKC-alpha as compared to parental or vector-transfected MCF-7 cells. MCF-7-PKC-alpha cells also displayed a substantial increase in endogenous expression of PKC-beta and decreases in expression of the novel delta- and eta-PKC isoforms. MCF-7-PKC-alpha cells displayed an enhanced proliferative rate, anchorage-independent growth, dramatic morphologic alterations including loss of an epithelioid appearance, and increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells exhibited a significant reduction in estrogen receptor expression and decreases in estrogen-dependent gene expression. These findings suggest that the PKC pathway may modulate progression of breast cancer to a more aggressive neoplastic process.
The lipid second messenger diacylglycerol (DAG) acutely controls the rate, amplitude, duration, and location of protein kinase C (PKC) activity in the cell. There are three classes of PKC isozymes and, of these, the conventional and novel isozymes are acutely controlled by DAG. The kinetics of DAG production at various intracellular membranes, the intrinsic affinity of specific isoforms for DAG-containing membranes, the coordinated use of additional membrane-binding modules, the intramolecular regulation of DAG sensitivity, and the competition from other DAG-responsive proteins together result in a unique, context-dependent activation signature for each isoform. This review focuses on the spatiotemporal dynamics of PKC activation and how it is controlled by lipid second messengers.
PICK1 is a protein which was initially identified as a protein kinase Cα (αPKC) binding protein using the yeast two-hybrid system. In addition to αPKC, the PICK1 complex binds to and regulates various transmembrane proteins including receptors and transporters. However, it has not been clarified when and where PICK1 binds to αPKC. We examined the spatiotemporal interaction of PICK1 and PKC using live imaging techniques and showed that the activated αPKC binds to PICK1 and transports it to the plasma membrane. Although the membrane translocation of PICK1 requires the activation of αPKC, PICK1 is retained on the membrane even after PKC moves back to the cytosol. These results suggest that the interaction between αPKC and PICK1 is transient and may not be necessary for the regulation of receptors/transporters by PICK1 or by αPKC on the membrane.
protein kinase C; PICK1; PDZ; translocation; phosphorylation
To characterize protein kinase C (PKC) gamma, an isozyme found exclusively in brain and spinal cord, its cDNA was introduced into basophilic RBL-2H3 cells that lack this isozyme. The expression of PKC gamma significantly attenuated antigen-induced responses including hydrolysis of inositol phospholipids, increase in cytosolic calcium, and secretion of granules but enhanced antigen-induced release of arachidonic acid. Instead of a sustained increase in cytosolic calcium, antigen now induced calcium oscillations; possibly as a consequence of suppression of the phospholipase C activity and incomplete emptying of internal calcium stores. In addition, PKC gamma appeared to inhibit activation of other PKC isozymes because phorbol 12-myristate 13-acetate failed to act synergistically with the Ca(2+)-ionophore on secretion. This was confirmed in other studies where PKC gamma was shown to suppress the transduction of stimulatory signals by other isozymes of PKC on provision of these isozymes to PKC-depleted permeabilized cells. The studies in total indicated that only PKC gamma was capable of inhibiting both early and distal signals for secretion including those signals transduced by endogenous isozymes of PKC.
Protein Kinase C (PKC) is a family of serine/threonine kinases that play a central role in cellular signal transduction. The second messenger diacylglycerol having two long carbon chains acts as the endogenous ligand for the PKCs. Polyphenol curcumin, the active constituent of Curcuma longa is an anticancer agent and modulates PKC activity. To develop curcumin derivatives as effective PKC activators, we synthesized several long chain derivatives of curcumin, characterized their absorption and fluorescence properties and studied their interaction with the activator-binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. Curcumin (1) and its C16 long chain analog (4) quenched the intrinsic fluorescence of PKCδC1B, PKCεC1B and PKCθC1B in a manner similar to that of PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). The EC50s of the curcumin derivatives for fluorescence quenching varied in the range of 4-11 μM, whereas, EC50s for TPA varied in the range of 3-6 μM. Fluorescence emission maxima of 1 and 4 were blue shifted and the fluorescence anisotropy values were increased in the presence of the C1B domains similar to that shown by the fluorescent analog of TPA, sapintoxin–D, confirming that they were bound to the proteins. Molecular docking of 1 and 4 with novel PKC C1B revealed that both the molecules form hydrogen bonds with the protein residues. The present result shows that curcumin and its long chain derivatives bind to the C1B subdomain of novel PKCs and can be further modified structurally to improve its binding and activity.
Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α–pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R2 position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively-charged DAG lactone derivatives play a central role in determining biological activity and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.
diacylglycerol (DAG)-lactones; PKC; plasma membrane; membrane anchoring; vesicles
The activity of calcium-, phospholipid-dependent protein kinase (PKc) was measured in (a) total extracts, (b) crude membrane, and (c) cytosolic fractions of chick embryo myogenic cells differentiating in culture. Total PKc activity slowly declines during the course of terminal myogenesis in contrast to the activity of cAMP-dependent protein kinase, which was also measured in the same cells. Myogenic cells at day 1 of culture possess high particulate and low soluble PKc activity. A dramatic decline of particulate PKc activity occurs during myogenic cell differentiation and is accompanied, through day 4, by a striking rise of the soluble activity. The difference in the subcellular distribution of PKc between replicating myoblasts and myotubes is confirmed by phosphorylation studies conducted in intact cells. These studies demonstrate that four polypeptides whose phosphorylation is stimulated by the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in myotubes, are spontaneously phosphorylated in control myoblasts. Phosphoinositide turnover under basal conditions in [3H]inositol-labeled cells is faster in myoblasts than in myotubes, a finding that may in part explain the different distribution of PKc observed during the course of myogenic differentiation.
Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.
The ER/Golgi protein p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β-chimaerin. C1 domains from PKC isozymes can also interact with p23/Tmp21. Our study highlights the relevance of C1 domains in protein-protein interactions in addition to their well-established lipid-binding properties.
The C1 domains in protein kinase C (PKC) isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and for mediating translocation to membranes. Previous studies revealed that the C1 domain in α- and β-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the endoplasmic reticulum/Golgi protein p23/Tmp21. Here, we found that p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β2-chimaerin. Glu227 and Leu248 in the β2-chimaerin C1 domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 domains from individual PKC isozymes differentially interact with p23/Tmp21. For PKCε, it interacts with p23/Tmp21 specifically via its C1b domain; however, this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 domain-containing proteins, and it plays an essential role in β2-chimaerin relocalization. Our study also highlights the relevance of C1 domains in protein–protein interactions in addition to their well-established lipid-binding properties.
The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.
We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.
PKC-theta; microRNA; chromatin; T cells; signaling kinase; immune response gene; NF-κB; nuclear PKC-theta
Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.
N-methyl substituted diacylglycerol-indololactones (DAG-indololactones) are newly-synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKC alpha. We present a comprehensive analysis of membrane interactions and biological activities of several DAG-indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we find that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG-indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands
protein kinase C; phorbol ester; diacylglycerol-lactone; vesicles; FRET; tryptophane fluorescence
Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100–300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.
The MEK5–extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that ζPKC and λ/ιPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.
Protein Kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that εPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of εPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of εPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that εPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of εPKC activation during ischemia on cerebral blood flow (CBF). We found that ψε-Receptors for activated C kinase (RACK), a εPKC-selective peptide activator, injected intravenously 30 minutes before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψεRACK compared to Tat peptide (vehicle). Our results suggest that εPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, εPKC may be one of the treatment modalities against ischemic injury.
Ischemia; epsilon Protein Kinase C; Cerebral Blood Flow; Neuroprotection
Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3–Par-6–aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs–Stardust (Sdt)–Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3–Par-6–aPKC and Crumbs–Sdt–Patj complexes based in the posttranslational modification of Crb by DaPKC.
DaPKC; Crumbs; Drosophila; epithelial cell polarity; signal transduction
Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKC epsilon and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKC epsilon within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC- binding protein. The binding of PKC epsilon to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKC epsilon. Once established, this protein-protein interaction securely anchored PKC epsilon to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKC epsilon isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.
Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.
Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70S6 kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCζ and -λ in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKCλ from the nucleus into the cytosol, and Cdc42 and PKCλ colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKCλ. Kinase-dead PKCλ and -ζ constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKCλ and -ζ and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKCλ block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.
We previously reported that some ATP competitive protein kinase C (PKC) inhibitors are either competitive or uncompetitive inhibitors with respect to substrate peptides. In this report, we demonstrate how the interactions between PKC and inhibitors change PKC activation kinetics. A substrate competitive inhibitor, bisindolylmaleimide I, targets activated PKC and stabilizes PKC in the activated conformation. This leads to transient activation and prolonged deactivation of PKC in the presence of bisindolylmaleimide I. In contrast, an uncompetitive substrate inhibitor, bisindolylmaleimide IV, targets quiescent PKC and stabilizes PKC in the quiescent conformation, which generates slower activation and suppressed translocation upon activation of PKC.
Newly synthesized protein kinase C (PKC) undergoes a series of phosphorylation to render a mature form of the enzyme. It is this mature PKC that possesses the catalytic competence to respond to second messengers for activation and downstream signaling. The first and rate-limiting phosphorylation occurs at a threonine residue in the activation loop (AL), which triggers the rest maturation processing of PKC and regulates PKC activity in response to cellular stimulation. Given the fact that PKC is enriched in striatal neurons, we investigated the regulation of PKC phosphorylation at the AL site in the rat striatum by the psychostimulant cocaine in vivo. We found that PKC was phosphorylated at the AL site at a moderate level in the normal rat brain. Acute systemic injection of cocaine increased the PKC-AL phosphorylation in the two striatal structures (caudate putamen and nucleus accumbens). Cocaine also elevated the PKC-AL phosphorylation in the medial prefrontal cortex. The cocaine-stimulated PKC phosphorylation in the striatum is rapid and transient. A reliable increase in PKC phosphorylation was seen 7 min after drug injection, which declined to the normal level by 1 h. This kinetics corresponds to that seen for another striatum-enriched protein kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase, in response to cocaine. This study suggests a new model for exploring the impact of cocaine on protein kinases in striatal neurons. By modifying PKC phosphorylation at the AL site, cocaine is believed to possess the ability to alter the maturation processing of the kinase in striatal neurons in vivo.
protein kinase C; ERK; dopamine; stimulant; caudate; nucleus accumbens; prefrontal cortex; addiction