PKC-ß modulates Ca2+ mobilization through Stim1 phosphorylation.

BACKGROUND: Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool. OBJECTIVES: Here, we investigated whether PKC-ß controls intracellular calcium dynamics through Stim1. METHODS: Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope. RESULTS: Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors. CONCLUSION: In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

Expression of TMBIM6 in Cancers: The Involvement of Sp1 and PKC.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) is upregulated in several cancer types and involved in the metastasis. Specific downregulation of TMBIM6 results in cancer cell death. However, the TMBIM6 gene transcriptional regulation in normal and cancer cells is least studied. Here, we identified the core promoter region (-133/+30 bp) sufficient for promoter activity of TMBIM6 gene. Reporter gene expression with mutations at transcription factor binding sites, EMSA, supershift, and ChIP assays demonstrated that Sp1 is an essential transcription factor for basal promoter activity of TMBIM6. The TMBIM6 mRNA expression was increased with Sp1 levels in a concentration dependent manner. Ablation of Sp1 through siRNA or inhibition with mithramycin-A reduced the TMBIM6 mRNA expression. We also found that the protein kinase-C activation stimulates promoter activity and endogenous TMBIM6 mRNA by 2- to 2.5-fold. Additionally, overexpression of active mutants of PKCι, PKCε, and PKCδ increased TMBIM6 expression by enhancing nuclear translocation of Sp1. Immunohistochemistry analyses confirmed that the expression levels of PKCι, Sp1, and TMBIM6 were correlated with one another in samples from human breast, prostate, and liver cancer patients. Altogether, this study suggests the involvement of Sp1 in basal transcription and PKC in the enhanced expression of TMBIM6 in cancer.

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells.

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser473 and Thr308, the insulin receptor at Tyr1158, IRS-1 at Tyr632, and GSK-3 at Ser9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser612, a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRAS(G12V) into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRAS(G12V) overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Bioinformatic identification of novel protein phosphatases in the dog genome.

Protein kinases and protein phosphatases constitute about 2-4% of the genes in a typical eukaryotic genome. Protein phosphatases are important players in many cellular processes such as proliferation, differentiation, cell adhesion, and motility. In this study, we identified, classified, and analyzed protein phosphatase complement of the dog genome. In this article, we report the identification of at least 178 putative protein phosphatases in dog which include 51 PSTPs, 112 PTPs, and 15 Asp-based protein phosphatases. Interestingly, we found at least five novel protein phosphatases in dog, namely DUSP5L, DUSP18L, MTMR9L, MTMR12L, and PPP6CL which are not present in human, mouse, rat, and cow. In addition, we found PTP4A1-rt, a retro-transposed copy of the PTP4A1 gene, in chromosome 27. Furthermore, we modeled three-dimensional structures of the catalytic domains of these putative protein phosphatases and aligned them to see the structural similarities between them. We docked PPP2CA with okadaic acid and calculated the value of affinity energy as -8.8 kcal/mol. Our nucleotide substitution rate study revealed that apparently none of the phosphatase family is under significantly higher evolutionary pressure.

FOXM1 expression mediates growth suppression during terminal differentiation of HO-1 human metastatic melanoma cells.

Induction of terminal differentiation represents a potentially less toxic cancer therapy. Treatment of HO-1 human metastatic melanoma cells with IFN-ß plus mezerein (MEZ) promotes terminal differentiation with an irreversible loss of growth potential. During this process, the transcription factor FOXM1 is down-regulated potentially inhibiting transactivation of target genes including those involved in G(2)/M progression and cell proliferation. We investigated the mechanism of FOXM1 down-regulation and its physiological role in terminal differentiation. Genetic and pharmacological studies revealed that FOXM1 down-regulation was primarily caused by MEZ activation of PKCα and co-treatment with IFN-ß plus MEZ augmented the effect of PKCα. Promoter analysis with a mutated E-box on the FOXM1 promoter, and in vitro and in vivo binding assays confirm a direct role of c-Myc on FOXM1 expression. Reduction of c-Myc and overexpression of Mad1 by IFN-ß plus MEZ treatment should cause potent and persistent reduction of FOXM1 expression during terminal differentiation. Overexpression of FOXM1 restored expression of cell cycle-associated genes and increased the proportion of cells in the S phase. Our experiments support a model for terminal differentiation in which FOXM1 down-regulation via activation of PKCα followed by suppression of c-Myc expression, are causal events in promoting growth inhibition during terminal differentiation.

The involvement of specific PKC isoenzymes in phorbol ester-mediated regulation of steroidogenic acute regulatory protein expression and steroid synthesis in mouse Leydig cells.

Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -µ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a -83/-67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells.

Cyclin-dependent kinase 5 phosphorylates endothelial nitric oxide synthase at serine 116.

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Visualization of the melanosome transfer-inhibition in a mouse epidermal cell co-culture model.

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

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

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

Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase Calpha and beta.

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway.

Induction of the nuclear proto-oncogene c-fos by the phorbol ester TPA and v-H-Ras.

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.

Cdk5-mediated phosphorylation of c-Myc on Ser-62 is essential in transcriptional activation of cyclin B1 by cyclin G1.

It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

Amino-acid sequence motifs for PKC-mediated membrane trafficking of the inhibitory killer Ig-like receptor.

Activation-induced upregulation of inhibitory killer Ig-like receptor (KIR) is regulated by protein kinase Cs (PKCs). Conventional PKCs increase KIR expression on the post-transcriptional level by increasing the recycling of surface molecules and endoplasmic reticulum (ER)-Golgi processing. PKCdelta plays a role in the secretion of cytoplasmic KIR through lytic granules. In this study, we identified amino acid sequence motifs associated with PKC-mediated KIR membrane trafficking by systematic mutagenesis. Mutations of Y(398) and HLWC(364) completely inhibited the PMA-induced increase of KIR molecules at surface as well as total protein levels, indicating that these are associated with ER-Golgi processing and sorting to plasma membrane through lytic granules. Mutations of Y-based motif, including Y(398), acidic region (PE(394)), dileucine motif-like region (IL(423)) and PKC-phosphorylatable S(415) caused a blockade of surface KIR endocytosis after PKC stimulation. Mutation of T(145) caused an accumulation of mutant proteins in late endosomes and lysosomes after PKC activation, suggesting that T(145) might be related to the recovery of endocytosed KIR to the surface membrane. We also demonstrated that PKCs could directly phosphorylate the KIR cytoplasmic tail by means of western blot and in vitro kinase assay, implying that phosphorylation status of KIR cytoplasmic tail can direct the fate of surface KIR molecules. Taken together, various sequence motifs are implicated in the PKC-mediated post-transcriptional upregulation of KIR, and each of these motifs work in different steps after PKC activation.

Celecoxib-induced growth inhibition in SW480 colon cancer cells is associated with activation of protein kinase G.

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs.

Bioinformatic prediction and analysis of eukaryotic protein kinases in the rat genome.

Eukaryotic protein kinases, containing a conserved catalytic domain, represent one of the largest superfamilies of the eukaryotic proteins and play distinct roles in cell signaling and diseases. Near completion of rat genome sequencing project enables the evaluation of a near complete set of rat protein kinases. Publicly accessible genetic sequence databases were searched for rat protein kinases, and 515 eukaryotic protein kinases, 40 atypical protein kinases and 45 kinase pseudogenes were identified. The rat has 509 putative protein kinases orthologous to human kinases. Unlike microtubule affinity-regulating kinases, the rat has a few more kinases, in addition to the orthologous pairs of mouse kinases. The comparison of 11 different eukaryotic species revealed the evolutionary conservation of this diverse family of proteins. The evolutionary rate studies of human disease and non-disease associated kinases suggested that relatively uniform selective pressures have been applied to these kinase classes. This bioinformatic study of the rat protein kinases provides a suitable framework for further characterization of the functional and structural properties of these protein kinases.

Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA.

Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specific PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKCalpha is localized mainly in the cytoplasm while PKCepsilon is localized mainly in the Golgi apparatus. We also observed that PKCalpha, beta1, beta2, gamma, delta, epsilon, and eta translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKCzeta and iota were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.

Inhibition of heat shock protein 27-mediated resistance to DNA damaging agents by a novel PKC delta-V5 heptapeptide.

Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase C delta (PKC delta)-V5 region with ensuing inhibition of PKC delta activity and PKC delta-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKC delta activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKC delta-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27.

Activation-induced upregulation of inhibitory killer Ig-like receptors is regulated by protein kinase C.

Inhibitory killer Ig-like receptor (KIR) expression was upregulated by protein kinase C (PKC) activation in stable Jurkat clones that express KIR or CD8KIR fusion proteins. PKC-induced KIR upregulation was mediated by the cytoplasmic tail of KIR and regulated at the post-transcriptional level. PKC inhibition, metabolic labeling and colocalization studies demonstrated that the activation of the conventional PKCs upregulated surface and cellular KIR levels by stimulating the maturation processes in endoplasmic reticulum-Golgi and by promoting the recycling of surface KIR through sorting endosomes. Similar studies also revealed that KIR was secreted to plasma membrane through lytic granules in a PKCdelta-dependent manner. Consequently, PKCdelta inhibition caused the formation of giant perinuclear granules, which trapped KIR and FasL as well as CPE and Lamp1.

Gonadotropin-releasing hormone couples to 3',5'-cyclic adenosine-5'-monophosphate pathway through novel protein kinase Cdelta and -epsilon in LbetaT2 gonadotrope cells.

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.