Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.     

Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.     

Influence of liposome composition and membrane binding on protein kinase activity of PI3Kγ

Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in a variety of cellular signaling processes. It is a multifunctional enzyme with lipid and protein kinase activity that also acts as a scaffold protein. Although it is well known that membrane recruitment is essential for the phosphorylation of phosphoinositides, the cellular localization of PI3Kγ as a protein kinase remains unclear. It has merely been described that PI3Kγ protein kinase activity leading to MAPK activation seems to be restricted to a cytosolic localization. Here, we demonstrate that a hybrid-PI3Kγ having protein kinase, but not lipid kinase activity shows a similar cellular distribution with a high membrane association and comparable liposome binding behavior to wild-type PI3Kγ. Binding of PI3Kγ to liposomes mimicking the natural plasma membrane slightly stimulates autophosphorylation of PI3Kγ. However, liposomes containing an unphysiologically high amount of PI inhibit autophosphorylation of PI3Kγ. Finally, PI3Kγ bound to membrane fragments does not show autophosphorylation which is possibly due to protein–protein-interactions at the plasma membrane. This indicates that not only MAPK activation, but PI3Kγ protein kinase activity in general is localized in the cytosol.     

Functional role of the charge at the T538 residue in the control of protein kinase Cθ

We show that protein kinase C (PKC) θ localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCθ, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCθ does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCθ molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCθ.     

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.     

Are calcium-dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes? Studies with Ca2+/calmodulin-dependent protein kinase and protein kinase C

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.     

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

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

The localization of the β-subtype of protein kinase C (PKC-β) in rat sympathetic neurons

Summary The localization of PKC- was studied in rat sympathetic neurons using a polyclonal antibody specific for the ₁- and ₂-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC--LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC--LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC--immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC--LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC- in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC- in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.     

Characterization of the α-cyanocinnamate binding site in rat heart mitochondria and in submitochondrial particles

The effect of pH and substrates on the binding of radiolabelled α-cyanocinnamate to mitochondria and submitochondrial particles has been investigated. It has been found that the binding is strongly influenced by the pH of the medium (it decreases on increasing the pH of the medium). The inhibition of pyruvate oxidation by this inhibitor follows the same pH dependence. The pH affects only the affinity of the α-cyanocinnamate binding site without changing their total number. A similar pH dependence has been found in inside-out submitochondrial particles where the binding sites are directly accessible. The quantitative parameters of the binding of α-cyanocinnamate in submitochondrial particles have been determined. The binding can be prevented or displaced by pyruvate and other substrates of the carrier. The turnover number for pyruvate transport in rat-heart mitochondria has been determined.     

3′,5′-Cyclic-nucleotide-dependent protein kinase of squamous cell carcinoma of the prostate

An adenosine 3'5'-cyclic-monophosphate (Cyclic AMP)-dependent protein kinase has been identified and partially purified from the rat prostate tumor induced by 20-methylcholanthrene. This enzyme is stimulated 2- to 3-fold by the nucleotide. Equilibrium studies at pH 5.0 suggest the presence of a major class of binding site for cyclic AMP with an association constant of approximately 10(8) M-1. The concentration of binding site is about 1 pmol/mg of protein of the enzyme preparation. The enzyme is stimulated by other cyclic nucleotides as well, but only by higher concentrations. In comparing the ability of different histone subfractions, casein and protamine, to serve as substrate for this particular protein kinase, maximal cyclic-AMP-dependent enzyme activity was observed with histones. The results suggest that factors contributing to the malignant growth of the prostatic tissue do not directly involve changes in the characteristics of a cyclic-AMP-dependent protein kinase.     

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

Protein kinase C (PKC) is a family of serine/threonine protein kinases, and alterations have been found in PKC isoform expression and localization in the failing heart. These alterations in PKC activation levels influence the PKC-mediated phosphorylation status of cellular target proteins involved in Ca²⁺-handling and sarcomeric contraction. The differences observed in the effects due to PKC-mediated phosphorylation may underlie part of the contractile dysfunction observed in the failing heart. It is therefore important to establish the beneficial and detrimental effects of this kinase in the healthy and failing heart. The function of PKC has been studied intensively; however, the complexity of the regulation of this kinase makes the interpretation of the different effects difficult. The main focus of this review is the (patho)physiological impact of phosphorylation of sarcomeric proteins, myosin light chain-2, troponin I and T, desmin, myosin binding protein-C, and titin by PKC.     

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.     

Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart?

The activity of the isoform of protein kinase C (PKC) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC activity in the membrane. Associated with the improvement in PKC activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

Analysis of the red cell membrane in a family with hereditary elliptocytosis — total or partial of protein 4.1

Summary In a 12-year-old boy carrying a clinically silent elliptocytosis, we observed a total lack of red cell membrane band 4.1. Band 4.1 was partially absent in the father who also displayed a clinically silent elliptocytosis and, remarkably, in the mother although she presented normal discocytes. Band (2 and 2.1) phosphorylation was sharply reduced in the three persons examined. In the propositus and his mother, but not in his father, a clearly phosphorylated band appeared at the level of band 4.2. We suggest that the father and the mother carry two distinct alleles affecting differently the interactions, within the spectrin-actin protein 4.1 complex. The father's allele is elliptocytogenic in the heterozygous state and, among other molecular alterations, prevents the attachment of protein 4.1. The mother's allele is morphologically silent in the heterozygous state, yet it also affects the binding of protein 4.1, possibly because the latter is shortened. The propositus, being doubly heterozygous, has the same morphological phenotype as his father, but his protein 4.1 electrophoretic phenotype is the addition of both parental phenotypes. The distinct phosphorylation patterns in the region of bands 4.1 and 4.2 are also consistent with the two-allele hypothesis.     

Allosterism in membrane binding: a common motif of the annexins?

Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.     

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233–1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.