Structural basis of RasGRP binding to high-affinity PKC ligands

The Ras guanyl releasing protein RasGRP belongs to the CDC25 class of guanyl nucleotide exchange factors that regulate Ras-related GTPases. These GTPases serve as switches for the propagation and divergence of signaling pathways. One interesting feature of RasGRP is the presence of a C-terminal C1 domain, which has high homology to the PKC C1 domain and binds to diacylglycerol (DAG) and phorbol esters. RasGRP thus represents a novel, non-kinase phorbol ester receptor. In this paper, we investigate the binding of indolactam(V) (ILV), 7-(n-octyl)-ILV, 8-(1-decynyl)benzolactam(V) (benzolactam), and 7-methoxy-8-(1-decynyl)benzolactam(V) (methoxylated benzolactam) to RasGRP through both experimental binding assays and molecular modeling studies. The binding affinities of these lactams to RasGRP are within the nanomolar range. Homology modeling was used to model the structure of the RasGRP C1 domain (C1-RasGRP), which was subsequently used to model the structures of C1-RasGRP in complex with these ligands and phorbol 13-acetate using a computational docking method. The structural model of C1-RasGRP exhibits a folding pattern that is nearly identical to that of C1b-PKCdelta and is comprised of three antiparallel-strand beta-sheets capped against a C-terminal alpha-helix. Two loops A and B comprising residues 8-12 and 21-27 form a binding pocket that has some positive charge character. The ligands phorbol 13-acetate, benzolactam, and ILV are recognized by C1-RasGRP through a number of hydrogen bonds with loops A and B. In the models of C1-RasGRP in complex with phorbol 13-acetate, benzolactam, and ILV, common hydrogen bonds are formed with two residues Thr12 and Leu21, whereas other hydrogen bond interactions are unique for each ligand. Furthermore, our modeling results suggest that the shallower insertion of ligands into the binding pocket of C1-RasGRP compared to C1b-PKCdelta may be due to the presence of Phe rather than Leu at position 20 in C1-RasGRP. Taken together, our experimental and modeling studies provide us with a better understanding of the structural basis of the binding of PKC ligands to the novel phorbol ester receptor RasGRP.     

Activation of human leukemia protein kinase C by tumor promoters and its inhibition by N-trifluoroacetyladriamycin-14-valerate (AD 32).

N-Trifluoroacetyladriamycin-14-valerate (AD 32), a lipophilic, DNA non-binding analog of Adriamycin (ADR), was found to be a potent inhibitor of the membrane-bound enzyme, protein kinase C (PKC). PKC was isolated and purified from human leukemia ML-1 cells, and the enzyme activity was shown to be activated by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). AD 32, nevertheless, inhibited the activation of PKC by TPA or PDBu. The IC50 values for AD 32 inhibition of PKC activation were 0.85 microM for TPA and 1.25 microM for PDBu. Under the same assay conditions, ADR demonstrated much higher IC50 values: 550 microM for TPA and greater than 350 microM for PDBu. The inhibition of PKC by AD 32 was further shown to be competitive in nature; AD 32 inhibited the binding of [3H]PDBu to PKC. Therefore, AD 32 competes with the tumor promoter for the PKC binding site and prevents the latter from both interacting with the phospholipid and binding to PKC. These effects of AD 32 were reproduced in situ; incubation of human leukemia ML-1 cells with TPA showed an increased phosphorylation of cellular proteins, and the TPA-induced protein phosphorylation was inhibited by the addition of AD 32 to the cultured cells.     

Probing the binding of indolactam-V to protein kinase C through site-directed mutagenesis and computational docking simulations.

Protein kinase C (PKC) comprises a family of ubiquitous enzymes transducing signals by the lipophilic second messenger sn-1, 2-diacylglycerol (DAG). Teleocidin and its structurally simpler congener indolactam-V (ILV) bind to PKC with high affinity. In this paper, we report our computational docking studies on ILV binding to PKC using an automatic docking computer program, MCDOCK. In addition, we used site-directed mutagenesis to assess the quantitative contribution of crucial residues around the binding site of PKC to the binding affinity of ILV to PKC. On the basis of the docking studies, ILV binds to PKC in its cis-twist conformation and forms a number of optimal hydrogen bond interactions. In addition, the hydrophobic groups in ILV form "specific" hydrophobic interactions with side chains of a number of conserved hydrophobic residues in PKC. The predicted binding mode for ILV is entirely consistent with known structure-activity relationships and with our mutational analysis. Our mutational analysis establishes the quantitative contributions of a number of conserved residues to the binding of PKC to ILV. Taken together, our computational docking simulations and analysis by site-directed mutagenesis provide a clear understanding of the interaction between ILV and PKC and the structural basis for design of novel, high-affinity, and isozyme-selective PKC ligands.     

Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C

The microbial alkaloid staurosporine is a member of a recently described family of protein kinase inhibitors. [N,N-dimethyl-3H]N-dimethylstaurosporine ([3H]DMS) was prepared from staurosporine by methylation with [3H]methyl iodide. Since staurosporine inhibits protein kinase C (PKC) most potently, the binding of [3H]DMS to this enzyme was examined. Unlike [20-3H(N)]phorbol-12,13-dibutyrate ([3H]PDBu) binding to PKC, [3H]DMS binding was not calcium or phosphatidylserine (PS) dependent. Binding was reversible, with a T1/2 of 69 min and a Koff of 0.01/min. Non-specific binding was defined by a 500-fold molar excess of staurosporine and was less than 10% of total [3H]DMS binding. Specific binding of [3H]DMS was consistent with a single class of binding sites with a Kd of 3.8 +/- 0.6 nM and a Bmax of 675 +/- 30 pmol/g tissue. In competition experiments, staurosporine inhibited [3H]DMS binding with a Ki of 4.7 +/- 0.6 nM, indicating that the two alkaloids had a similar potency for PKC. Also, unlabeled DMS and staurosporine inhibited [3H]DMS binding and PKC catalysis with equivalent potencies. Highly purified rat brain PKC bound equimolar amounts of [3H]PDBu and [3H]DMS. In contrast, crude rat brain PKC, which had been proteolysed to generate a PS and Ca2+ independent enzyme (PK-M) retained the ability to bind [3H]DMS, but not [3H]PDBu. In addition, the kinase inhibitors K-252a and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited [3H]DMS binding, whereas PDBu did not. These results indicate that [3H]DMS is a useful ligand to identify catalytic inhibitors of kinase activity and to explore their mechanisms of action.     

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

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

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.     

Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay

An alternative in vivo assay, based on growth inhibition of yeast expressing an individual mammalian protein kinase C (PKC) isoform (proportional to the degree of PKC activation), was used to characterize the activities of phorbol-12-myristate-13-acetate (PMA) and its analogues on classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC isoforms. Effects of PMA, 4alpha-PMA, phorbol-12-myristate-13-acetate-4-O-methyl-ether (MPMA), phorbol-12-monomyristate (PMM), phorbol-12,13-diacetate (PDA), phorbol-13-monoacetate (PA), phorbol-12,13-dibutyrate (PDB), phorbol-12,13-didecanoate (PDD) and 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA), on growth of yeast expressing individual PKC isoforms was determined. PMA-induced growth inhibition on all isoforms tested (except on PKC-zeta). PDD and PDB presented an efficacy similar to PMA; the other PMA-analogues presented lower efficacies. MPMA and 4alpha-PMA stimulated growth of yeast expressing classical PKCs and reduced the PMA-induced growth inhibition, effects similar to those exhibited by the PKC inhibitors chelerythrine and R-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide dihydrochloride (NPC 15437). This study reveals that phorbol esters differ on their potency to activate a given PKC isoform, and presents their isoform-selectivity. Furthermore, MPMA and 4alpha-PMA caused effects similar to those expected from PKC inhibition.     

Molecular models of N-benzyladriamycin-14-valerate (AD 198) in complex with the phorbol ester-binding C1b domain of protein kinase C-delta

N-Benzyladriamycin-14-valerate (AD 198) is a semisynthetic anthracycline with experimental antitumor activity superior to that of doxorubicin (DOX). AD 198, unlike DOX, only weakly binds DNA, is a poor inhibitor of topoisomerase II, and circumvents anthracycline-resistance mechanisms, suggesting a unique mechanism of action for this novel analogue. The phorbol ester receptors, protein kinase C (PKC) and beta2-chimaerin, were recently identified as selective targets for AD 198 in vitro. In vitro, AD 198 competes with [3H]PDBu for binding to a peptide containing the isolated C1b domain of PKC-delta (deltaC1b domain). In the present study molecular modeling is used to investigate the interaction of AD 198 with the deltaC1b domain. Three models are identified wherein AD 198 binds into the groove formed between amino acid residues 6-13 and 21-27 of the deltaC1b domain in a manner similar to that reported for phorbol-13-acetate and other ligands of the C1 domain. Two of the identified models are consistent with previous experimental data demonstrating the importance of the 14-valerate side chain of AD 198 in binding to the C1 domain as well as current data demonstrating that translocation of PKC-alpha to the membrane requires the 14-valerate substituent. In this regard, the carbonyl of the 14-valerate participates in hydrogen bonding to the deltaC1b while the acyl chain is positioned for stabilization of the membrane-bound protein-ligand complex in a manner analogous to the acyl chains of the phorbol esters. These studies provide a structural basis for the interaction of AD 198 with the deltaC1b domain and a starting point for the rational design of potential new drugs targeting PKC and other proteins with C1 domains.     

Role of protein kinase Calpha in signaling from the histamine H(1) receptor to the nucleus

Stimulation of histamine H(1) receptors produced a marked activation of inositol phospholipid hydrolysis, intracellular calcium mobilization, and stimulation of the c-fos promoter in CHO-H1 cells expressing the H(1) receptor at a level of 3 pmol/mg protein. The latter response was determined using a luciferase-based reporter gene (pGL3). This response to histamine was not sensitive to inhibition by pertussis toxin but could be completely attenuated by the protein kinase C (PKC) inhibitor Ro-31-8220, or by 24-h pretreatment with the phorbol esters phorbol 12,13-dibutyrate or phorbol-12-myristate-13-acetate. Several isoforms of PKC can be detected in CHO-H1 cells (alpha, delta, epsilon, mu, iota, zeta) but only PKCalpha and PKCdelta were down-regulated by prolonged treatment with phorbol esters. Of the two isoforms that were down-regulated, only protein kinase Calpha was translocated to CHO-H1 cell membranes after stimulation with either histamine or phorbol esters. The PKC inhibitor G? 6976, which inhibits PKCalpha but not PKCdelta, was also able to significantly attenuate the c-fos-luciferase response to histamine. The mitogen-activated protein kinase kinase inhibitor PD 98059 markedly inhibited the response to histamine, suggesting that the likely major target for PKCalpha was the mitogen-activated protein kinase pathway. These data suggest that the histamine H(1) receptor can signal to the nucleus via PKCalpha after activation of phospholipase Cbeta.     

Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells

Polychlorinated biphenyls (PCBs) induce apoptotic cell death of HL-60 cells. In the present study, we examined the possible involvement of protein kinase C (PKC) in PCB-induced apoptosis of HL-60 cells. Treatment of cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, suppressed DNA fragmentation induced by PCBs in HL-60 cells. Treatment with another active phorbol ester, phorbol-12,13-dibutyrate (PDBu), also suppressed PCB-induced DNA fragmentation, whereas 4alpha-phorbol-12,13-didecanoate (4alphaPDD), an inactive phorbol ester, did not affect PCB-induced apoptosis of HL-60 cell. Moreover, 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of PKC that is not a phorbol ester, also suppressed PCB-induced DNA fragmentation. However, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, increased DNA fragmentation induced by PCBs. These results demonstrate that the activation of PKC is responsible for the suppression of PCB-induced apoptosis of HL-60 cells. Furthermore, inhibition of PKC promotes DNA fragmentation of HL-60 cells treated with PCBs, thereby suggesting the involvement of PKC activity in PCB-induced apoptosis of HL-60 cells.     

In vitro model for intrinsic drug resistance: effects of protein kinase C activators on the chemosensitivity of cultured human colon cancer cells

We investigated the effects that phorbol ester and diacylglycerol protein kinase C (PKC) activators had on the chemosensitivity of the human colon cancer cell line KM12L4a to Adriamycin (ADR), vincristine (VCR), and vinblastine (VLB) and on the intracellular accumulation of those drugs. Exposure of the cells to the PKC activator phorbol-12,13-dibutyrate (PDBu) (15 nM) during a 96-hr in vitro chemosensitivity assay significantly reduced the sensitivity of KM12L4a cells to ADR, VCR, and VLB, but not to 5-fluorouracil. Because a 96-hr treatment with 15 nM PDBu did not down-regulate PKC activity in KM12L4a cells, activation of PKC appeared to be responsible for the observed protection conferred by PDBu. PDBu-induced alterations in drug accumulation may account for its protective effects against these cytotoxic drugs, because both PDBu and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate significantly reduced accumulation of [3H] VCR and [14C]ADR in the cultured human colon cancer cells. Unsaturated diacylglycerols are structural and functional analogues of phorbol ester PKC activators that are present in the lumen of the colon. We found that treatment of KM12L4a human colon cancer cells with the diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG) significantly reduced [14C]ADR and [3H]VCR accumulation in the cells. The effects of OAG were dose dependent at physiological diacylglycerol concentrations and were completely reversed by the protein kinase inhibitor H7. OAG, which is rapidly metabolized in cultured cells, did not protect KM12L4a cells against the cytotoxic drugs in our 96-hr in vitro chemosensitivity assay. However, rapid metabolism of diacylglycerols should not limit their capacity to activate PKC in the colonic epithelium in vivo, because that tissue is chronically exposed to replenished supplies of unsaturated diacylglycerols in the intestinal tract. Our results provide evidence that unsaturated diacylglycerols may be environmental factors that contribute to the intrinsic drug resistance of colon cancer in vivo by reducing drug accumulation in the cancer cells.     

Regional and temporal profiles of phorbol 12,13-dibutyrate binding after myocardial infarction in rats: effects of captopril treatment

Phosphoinositide turnover and protein kinase C (PKC) mediate the signaling of angiotensin II, which plays a pivotal role in ventricular remodeling after myocardial infarction (MI). To determine whether PKC is activated after MI, rat hearts after MI were subjected to in vitro quantitative autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu), which is highly selective for PKC. [3H]PDBu binding in the infarcted area increased significantly compared with the non-infarcted region 7 and 21 days after MI, but not 1 and 3 days and 10 months after MI. [3H]PDBu binding in the noninfarcted area was similar to that in the sham-operated rats. Immunohistochemical analysis revealed that abundant macrophages (7 days after MI), fibroblasts, and myofibroblasts (7 and 21 days after MI) occupied the infarcted region. To investigate whether myocardial [3H]PDBu binding is affected by captopril, hearts were subjected to in vitro autoradiography with [3H]PDBu after 1- or 3-week captopril treatment or no treatment. Captopril treatment significantly suppressed [3H]PDBu binding in the infarcted area 3 weeks after MI, but not 1 week after MI nor in the noninfarcted areas. These results suggest that PKC is upregulated during the healing and fibrogenic process after MI and that captopril treatment suppresses the upregulation in the infarcted area.     

Differential inhibition of epidermal growth factor binding by photoactivated psoralens and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in cells overexpressing protein kinase C.

A rat fibroblast cell line, R6PKC3, that stably overexpresses the beta-1 form of protein kinase C was used to analyze sensitivity to inhibitors of epidermal growth factor (EGF) binding. R6PKC3 cells overexpress protein kinase C activity 53-fold relative to non-overexpressing control R6C1 cells. Inhibition of EGF binding by the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) and photo-activated psoralens was compared in these cells. We found that 125I-EGF bound both of the cell lines and was rapidly internalized in a temperature-dependent process and metabolized. Binding of EGF to the R6 cells overexpressing protein kinase C was markedly less than binding to R6C1 control cells. In both of the cell lines, TPA and photoactivated psoralens inhibited 125I-EGF binding but the response of these cells to these inhibitors was distinct. R6PKC3 cells were markedly more sensitive to TPA and were resistant to recovery from TPA-induced inhibition of 125I-EGF binding when compared to control cells. These differences were not observed in other subclones of cells overexpressing protein kinase C, suggesting that they may be unique to R6PKC3 cells. In contrast, no major differences in sensitivity to photoactivated psoralens were observed in R6C1 and R6PKC3 cells. These data indicate that TPA and photoactivated psoralens inhibit 125I-EGF binding to these cell lines by distinct mechanisms.     

Mapping the binding of GluN2B-selective N-methyl-D-aspartate receptor negative allosteric modulators

We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists.     

Irritant contact dermatitis in humans from phorbol and related esters

Nine compounds, based on four biogenetically-related polycyclic diterpene skeletons, were subjected to open and closed patch testing on human volunteer subjects. The tigliane esters phorbol-12, 13, 20-triacetate, 12-O-2Z-4E-octadienoyl-4-deoxyphorbol-13-acetate and 12-O-tigloyl-4-deoxyphorbol-13-isobutyrate, in increasing order of potency, produced symptoms of toxicity in closed patch tests, with the dose of the most potent compound in this series being 0.5 μg in 5 μl acetone. Phorbol, a tigliane alcohol, was inactive in closed tests at a dose level of 50 μg/5 μl. The daphnane derivative, daphnetoxin, produced bullae and vesiculation in closed patch tests, but daphnetoxin-5,20-diacetate was devoid of these effects when applied at 10 times the dose of daphnetoxin. The ingenane compounds, ingenol-3,5,20-triacetate and 20-deoxy-16-hydroxyingenol-3,5,16-triacetate, and the lathyrane compound, ingol-3,7,8,12-tetraacetate, were obtained from the hydrolyzed, acetylated irritant latex of Euphorbia hermentiana. At the doses tested, ingenol-3,5,20-triacetate was the only compound derived from this plant to exhibit irritant activity in closed patch tests. In contrast, this compound is inactive as an irritant to the mouse ear at doses up to 250 μg/ear. Only three compounds, 12-O-2Z-4E-octadienoyl-4-deoxyphorbol-13-acetate, 12-O-tigloyl-4-deoxyphorbol-13-isobutyrate and daphnetoxin, produced dermatological toxicity in open patch tests at the doses used. Inflammatory signs and symptoms for several of the compounds under test persisted for over four days in open patch tests and for a week or more after application in closed patch testing.     

Protein kinase C delta is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24h of treatment, and pre-treatment with (1 microM) of either a general PKC inhibitor (BisI) or PKCdelta-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCalpha, beta, epsilon, tau, lambda and RACK1. PKCgamma, eta, theta and DGKtheta were not detected. TCDD (1 nM) increased PKCdelta protein approximately eight-fold after 24h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCdelta, from the cytoplasm and the nucleus to the perinuclear area after 2h of treatment, however, after 24h of treatment PKCdelta was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCdelta-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCdelta-specific activities increased after 1-2h of treatment. Then TCDD increased the total PKC activity again after 12h of treatment, whereas, PKCdelta-specific activity resurged at 24h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCdelta protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCdelta as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.     

Glycine hinges with opposing actions at the acetylcholine receptor-channel transmitter binding site

The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (α subunit loop B) with regard to both of these parameters. αGly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. αGly153 is a "deactivation" hinge that maintains low values for these parameters. αTrp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions αTrp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse.     

Branched diacylglycerol-lactones as potent protein kinase C ligands and alpha-secretase activators

Using as our lead structure a potent PKC ligand (1) that we had previously described, we investigated a series of branched DAG-lactones to optimize the scaffold for PKC binding affinity and reduced lipophilicity, and we examined the potential utility of select compounds as alpha-secretase activators. Activation of alpha-secretase upon PKC stimulation by ligands causes increased degradation of the amyloid precursor protein (APP), resulting in enhanced secretion of sAPPalpha and reduced deposition of beta-amyloid peptide (Abeta), which is implicated in the pathogenesis of Alzheimer's disease. We modified in a systematic manner the C5-acyl group, the 3-alkylidene, and the lactone ring in 1 and established structure-activity relationships for this series of potent PKC ligands. Select DAG-lactones with high binding affinities for PKC were evaluated for their abilities to lead to increased sAPPalpha secretion as a result of alpha-secretase activation. The DAG-lactones potently induced alpha-secretase activation, and their potencies correlated with the corresponding PKC binding affinities and lipophilicities. Further investigation indicated that 2 exhibited a modestly higher level of sAPPalpha secretion than did phorbol 12,13-dibutyrate (PDBu).     

MD studies on neuraminidase for probing binding pose of its inhibitors

Influenzal neuraminidase (NA) has proven to be potential drug target in designing anti-influenza drugs. The active site residues of group-1 and group-2 NA exhibit similarities, such that as structure-based drug design on group-2 NA has given potential anti-influenza drugs showing activity against group-1 as well as group-2 NA. The 150 (Gly147 to Asp151)-loop of NA has been reported to be the cause for the structural differences in the group-1 and group-2 NA enzymes. A cavity arises from an open conformation of the 150 loop which closes upon ligand binding has been reported for group-1 NA. Also recent studies have illustrated that mutation not only causes variation in the active site geometry but also changes the binding orientation of the NA inhibitors. To understand and examine the dynamic nature of ligand binding to NA, we have performed molecular dynamics simulations on NA. The docking study for the reported influenza inhibitors has been performed on averaged structure obtained from three minimum energy conformations of molecular dynamics (MD) trajectory. These results were compared with docking studies done on reported crystal structure of NA (PDB ID 3B7E). The information obtained from these studies can be used for the rational design of novel NA inhibitors.     

PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters

RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG