Binding of isoxazole and pyrazole derivatives of curcumin with the activator binding domain of novel protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target because of its involvement in the regulation of various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. The endogenous PKC activator diacylglycerol contains two long carbon chains, which are attached to the glycerol moiety via ester linkage. Natural product curcumin (1), the active constituent of Curcuma L., contains two carbonyl and two hydroxyl groups. It modulates PKC activity and binds to the activator binding site (Majhi et al., Bioorg. Med. Chem.2010, 18, 1591). To investigate the role of the carbonyl and hydroxyl groups of curcumin in PKC binding and to develop curcumin derivatives as effective PKC modulators, we synthesized several isoxazole and pyrazole derivatives of curcumin (2-6), characterized their absorption and fluorescence properties, and studied their interaction with the activator-binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. The EC(50)s of the curcumin derivatives for protein fluorescence quenching varied in the range of 3-25 μM. All the derivatives showed higher binding with the PKCθC1B compared with PKCδC1B and PKCεC1B. Fluorescence emission maxima of 2-5 were blue shifted in the presence of the C1B domains, confirming their binding to the protein. Molecular docking revealed that hydroxyl, carbonyl and pyrazole ring of curcumin (1), pyrazole (2), and isoxazole (4) derivatives form hydrogen bonds with the protein residues. The present result shows that isoxazole and pyrazole derivatives bind to the activator binding site of novel PKCs and both carbonyl and hydroxy groups of curcumin play roles in the binding process, depending on the nature of curcumin derivative and the PKC isotype used.     

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection

We investigated the regulation of Hsp27 phosphorylation by protein kinase C δ (PKCδ) during etoposide-induced apoptosis. The phosphorylation of Hsp27 at Ser78 was temporally correlated with the proteolytic activation of PKCδ during apoptosis. Hsp27 phosphorylation was dependent on the activity of PKCδ since treatment with rottlerin, a chemical inhibitor of PKCδ, or overexpression of a PKCδ dominant negative mutant abolished the phosphorylation. In addition, recombinant PKCδ phosphorylated Hsp27 at Ser78 in vitro. Moreover, caspase-3 was specifically activated following Hsp27 phosphorylation at Ser78. Pull-down assays using a phosphomimetic Hsp27 mutant revealed that binding between Hsp27 and cytochrome c was abolished by the phosphorylation. These results suggest that Hsp27 dissociates from cytochrome c following PKCδ-mediated phosphorylation at Ser78, which allows formation of the apoptosome and stimulates apoptotic progression.     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδ in vivo.     

Protein kinase cθ c2 domain is a phosphotyrosine binding module that plays a key role in its activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.     

Regulated binding of importin-α to protein kinase Cδ in response to apoptotic signals facilitates nuclear import

PKCδ translocates into the nucleus in response to apoptotic agents and functions as a potent cell death signal. Cytoplasmic retention of PKCδ and its transport into the nucleus are essential for cell homeostasis, but how these processes are regulated is poorly understood. We show that PKCδ resides in the cytoplasm in a conformation that precludes binding of importin-α. A structural model of PKCδ in the inactive state suggests that the nuclear localization sequence (NLS) is prevented from binding to importin-α through intramolecular contacts between the C2 and catalytic domains. We have previously shown that PKCδ is phosphorylated on specific tyrosine residues in response to apoptotic agents. Here, we show that phosphorylation of PKCδ at Tyr-64 and Tyr-155 results in a conformational change that allows exposure of the NLS and binding of importin-α. In addition, Hsp90 binds to PKCδ with similar kinetics as importin-α and is required for the interaction of importin-α with the NLS. Finally, we elucidate a role for a conserved PPxxP motif, which overlaps the NLS, in nuclear exclusion of PKCδ. Mutagenesis of the conserved prolines to alanines enhanced importin-α binding to PKCδ and induced its nuclear import in resting cells. Thus, the PPxxP motif is important for maintaining a conformation that facilitates cytosplasmic retention of PKCδ. Taken together, this study establishes a novel mechanism that retains PKCδ in the cytoplasm of resting cells and regulates its nuclear import in response to apoptotic stimuli.     

Insulin promotes neuronal survival via the alternatively spliced protein kinase CδII isoform

Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.     

p23/Tmp21 associates with protein kinase Cdelta (PKCdelta) and modulates its apoptotic function

There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKCδ, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKCδ C1b domain associates with p23 and identified two key residues (Asp(245) and Met(266)) implicated in this interaction. Interestingly, silencing p23 from LNCaP prostate cancer cells using RNAi markedly enhanced PKCδ-dependent apoptosis and activation of PKCδ downstream effectors ROCK and JNK by phorbol 12-myristate 13-acetate. Moreover, translocation of PKCδ to the plasma membrane by phorbol 12-myristate 13-acetate was enhanced in p23-depleted LNCaP cells. Notably, a PKCδ mutant that failed to interact with p23 triggered a strong apoptotic response when expressed in LNCaP cells. In summary, our data compellingly support the concept that C1 domains have dual roles both in lipid and protein associations and provide strong evidence that p23 acts as an anchoring protein that retains PKCδ at the perinuclear region, thus limiting the availability of this kinase for activation in response to stimuli.     

Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C

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 anti-cancer 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 in a manner 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.     

Binding mode prediction of aplysiatoxin,a potent agonist of protein kinase C,through molecular simulation and structure–activity study on simplified analogs of the receptor-recognition domain

Aplysiatoxin (ATX) is a naturally occurring tumor promoter isolated from a sea hare and cyanobacteria. ATX binds to, and activates, protein kinase C (PKC) isozymes and shows anti-proliferative activity against human cancer cell lines. Recently, ATX has attracted attention as a lead compound for the development of novel anticancer drugs. In order to predict the binding mode between ATX and protein kinase Cδ (PKCδ) C1B domain, we carried out molecular docking simulation, atomistic molecular dynamics simulation in phospholipid membrane environment, and structure–activity study on a simple acyclic analog of ATX. These studies provided the binding model where the carbonyl group at position 27, the hydroxyl group at position 30, and the phenolic hydroxyl group at position 20 of ATX were involved in intermolecular hydrogen bonding with the PKCδ C1B domain, which would be useful for the rational design of ATX derivatives as anticancer lead compounds.     

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.     

Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Mutation of the hydrophobic motif in a phosphorylation-deficient mutant renders protein kinase C delta more apoptotically active

Protein kinase C delta (PKCδ) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCδ is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCδ. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCδ activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCδ (PKCδ-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCδ apoptotically more active. Furthermore, we found that the mutant PKCδ-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of protein kinase Cδ in induction of apoptosis by cationic liposomes in macrophage-like RAW264.7 cells

We have recently demonstrated that reactive oxygen species (ROS) play an important role in RAW264.7 cell apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). In this study, we investigated whether protein kinase Cδ PKCδ) is involved in apoptosis induced by cationic liposomes. Tyrosine phosphorylation, nuclear localization, and cleavage of PKCδ were observed following the treatment of cells with SA-liposomes, suggesting that SA-liposomes activate PKCδ. Rottlerin, a specific inhibitor of PKCδ, inhibited ROS generation and also suppressed apoptosis. Cell surface proteoglycans may contribute to PKCδ activation by SA-liposomes. These findings suggest that PKCδ is strongly associated with apoptosis induced by SA-liposomes.     

Fluorescence-quenching screening of protein kinase C ligands with an environmentally sensitive fluorophore

A novel fluorescence-quenching screening method for protein kinase C (PKC) ligands was developed utilizing solvatochromic fluorophores. Solvatochromic dyes, highly sensitive to the presence or the absence of competitive ligands in their binding to the C1b domain of PKCδ (δC1b), were combined with a known pharmacophoric moiety of 1,2-diacylglycerol (DAG) lactones, PKC ligands. Addition of δC1b to the fluorescent compounds caused a gradual increase in the fluorescent intensity in proportion to the increase of δC1b. As a competitive ligand was added to the complex of δC1b domain and fluorescent compounds, a gradual decrease in the fluorescent intensity was observed. The relative binding affinities of known ligands were successfully determined by this fluorescent method and corresponded well to the K(i) values measured by a radioisotope method. These results indicate that washing, which is a laborious step in binding evaluations, is not required for this environmentally sensitive fluorophore based system. Screening with the system was performed for 2560 preselected library compounds with possible pharmacophores, and some lead compounds were found. This fluorescence-based method could be applied widely to known ligand-receptor combinations.     

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.     

High glucose induces apoptosis in embryonic neural progenitor cells by a pathway involving protein PKCδ

Diabetic-induced neural tube defects in embryos are caused by apoptosis of neural progenitor cells (NPCs); however, the underlying mechanisms are poorly understood. The present study is aimed to investigate the specific cellular proteins that may be involved in apoptosis of NPCs. We show here that hyperglycemia-induced apoptosis of NPCs was through a PKCδ-dependent mechanism. Tyrosine phosphorylation of PKCδ was required for PKCδ binding to c-Abl in the cytoplasm, and inhibition of c-Abl by STI571 or knock-down of c-Abl by RNAi decreased the phosphorylation of PKCδ. Moreover, translocation of PKCδ and c-Abl complex from the cytoplasm to the nucleus, was blocked by down-regulation of PKCδ or c-Abl. Furthermore, we found that interaction of PKCδ and c-Abl played a crucial role in p53 accumulation in the nucleus, which was linked to the apoptosis of NPCs in response to high glucose.