Palmitoylcarnitine modulates interaction protein kinase C delta-GAP-43

Palmitoylcarnitine, reported previously to promote neuronal differentiation, was observed to affect distribution of protein kinase C (PKC) isoforms in neuroblastoma NB-2a cells, leading to retardation in cytoplasm of high molecular weight species of PKCbeta and delta. Growth cone protein-GAP-43, a PKC substrate, was co-immunoprecipitated with all the conventional and novel PKCs: palmitoylcarnitine, however, decreased its amount exclusively in the complex with PKCdelta. Administration of palmitoylcarnitine, although did not change the subcellular distribution of GAP-43, decreased its phosphorylation, which could regulate other signal transduction pathways (calmodulin and G(0)-dependent).     

A novel 2,4-diaminopyrimidine derivative as selective inhibitor of protein kinase C theta prevents allograft rejection in a rat heart transplant model

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.     

Design and synthesis of tamoxifen derivatives as a selective estrogen receptor down-regulator

We designed and synthesized an estrogen receptor (ER) down-regulator (5), which is a derivative of tamoxifen with a long alkyl side chain. Compound 5 effectively reduced ER protein levels in MCF-7 cells and had an antagonistic effect.     

蛋白激酶C与吗啡耐受

蛋白激酶C(protein kinase C,PKC)属于AGC蛋白激酶家族(即PKA/PKG/PKC激酶家族),在吗啡介导的μ-阿片受体脱敏及吗啡耐受中具有重要作用,因此研究PKC的细胞信号传导机制对吗啡耐受的治疗具有重要的临床意义。本文综述了PKC在吗啡耐受中的作用。     

Design,synthesis and biological evaluation of novel triaryl (Z)-olefins as tamoxifen analogues

Tamoxifen (TAM) is used for the treatment and prevention of estrogen receptor positive breast cancer. However, the limited activity, toxicity and the development of resistance raised the current need for new potent nontoxic antiestrogen. Six novel TAM analogues 5a–f were synthesized using McMurry olefination reaction. Replacement of the dimethylamino group in TAM by piperidino, piperazino or N-methyl piperazino, substituting the phenyl ring with florine atom at p-position and changing the ethyl group by methyl, afforded compounds showing comparable activity to TAM (1). Compounds 5c and 5e showed significant increase in antiproliferative activity in two breast cancer cell lines (MCF-7 and MDA-MB-231) compared to tamoxifen, while other compounds showed similar activity. The increased anticancer activity of compounds 5c and 5e was attributed to their ability to induce ER-independent cell death.     

Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model.     

Regulation of protein kinase C inactivation by Fas-associated protein with death domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.     

Synthesis and biological evaluation of novel tamoxifen analogues

A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction was used as the key synthetic step in the preparation of these analogues and the structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel compounds were compared with those of the untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay and compared to 17β-estradiol (E2). Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerases I and II was assayed.     

三苯氧胺和雌二醇对大鼠子宫雌激素受体的竞争性结合作用

利用改进的葡聚糖活性炭饱和分析法（ＤＣＣ法）,对卵巢切除大鼠注射雌二醇（Ｅ组）或同时注射雌二醇和三苯氧胺（Ｅ＋Ｔ组）后６ｈ至６４ｄ,进行子宫ＥＲ含量测定,发现,Ｅ＋Ｔ组比Ｅ组的ＥＲ值小并且增加缓慢;Ｅ组的ＥＲ值在注射后３０ｄ达到最大值而且数值超过对照组（Ｃ组）。计算机曲线拟合Ｅ组或Ｅ＋Ｔ组与Ｃ组的ＥＲ比值求得：Ｅ组开始注射雌二醇时ＥＲ水平为Ｃ组的２．０３３倍,即＝２．０３３,恢复过程的时间常数τ＇为５１．５５ｄ,３￣Ｈ－雌二醇与ＥＲ结合过程的时间常数τ为１３．０ｄ;Ｅ＋Ｔ组的＝１．３１５,τ＇＝３８．７６ｄ,τ＝２１．５５ｄ,说明同时给予三苯氧胺和雌二醇情况下,三苯氧胺有抑制雌二醇诱导ＥＲ水平升高的作用,并减缓３￣Ｈ－雌二醇与ＥＲ的结合过程。另外,还求得Ｅ＋Ｔ组中三苯氧胺和ＥＲ和结合率约占４３．７％,表明三苯氧胺与雌二醇对ＥＲ有竞争性结合作用。     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

蛋白激酶C抑制剂Staurosporine对HeLa细胞周期的影响

用蛋白激酶C的抑制剂Staurosporine(10nmol/L)处理HeLa细胞,明显抑制HeLa细胞的增殖。这种抑制作用不是由于引起细胞死亡,而是因为细胞被阻断在G2期。这种阻断作用伴随着HeLa细胞多倍体的形成,提示Staurosporine抑制了HeLa细胞蛋白激酶C活性后引起的细胞阻滞,对细胞核的周期运转没有影响。进一步的探讨发现这种抑制作用可能是通过干扰细胞骨架的正确分布形成的,表明蛋白激酶C对于HeLa细胞由G2到M期正确过渡起重要作用。     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

EGF rapidly translocates tight junction proteins from the cytoplasm to the cell-cell contact via protein kinase C activation in TMK-1 gastric cancer cells

Tight junctions are commonly disrupted in cancer cells, including gastric cancer. Various growth factors have been reported to affect the localization of tight junction-associated proteins such as ZO-1 and occludin. We investigated the effect of epidermal growth factor (EGF), a growth factor that is often overexpressed in gastric cancer, and fetal bovine serum (FBS) on the localization of ZO-1 and occludin in a gastric cancer cell line. In the poorly differentiated gastric cancer cell line TMK-1, immunohistochemistry demonstrated that ZO-1 and occludin were predominantly localized to the cytoplasm, although there was some weak expression at the cell-cell contact. When the medium was replaced with fresh medium containing 10% FBS, ZO-1 and occludin were rapidly translocated from the cytosol to the cell-cell contact. A similar effect was seen in EGF exposure. These effects induced by FBS or EGF were attenuated in the presence of protein kinase C (PKC) inhibitors calphostin C and bisindolylmaleimide I, but not another PKC inhibitor G?6976, PD98059 (MAPK inhibitor), LY294002 (PI3 kinase inhibitor) or KT5720 (protein kinase A inhibitor). These results suggest that serum-derived factors, including EGF, can rapidly alter the localization of ZO-1 and occludin via a protein kinase C signaling pathway in TMK-1 gastric cancer cells.     

Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.     

Prolonged Alzheimer-like tau hyperphosphorylation induced by simultaneous inhibition of phosphoinositol-3 kinase and protein kinase C in N2a cells

Co-injection of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K) and GF109203X(inhibitor of protein kinase C, PKC) into the rat brain was found to induce spatial memory deficiency and enhance tau hyperphosphorylation in the hippocampus of rat brain. To establish a cell model with durative Alzheimer-like tau hyperphosphorylation in this study, we treated N2a neuroblastoma cells with wortmannin and GF109203X separately and simultaneously, and measured the glycogen synthase kinase 3 (GSK-3)activity by y-32p-labeling and the level of tau phosphorylation by Western blotting. It was found that the application of wortmannin alone only transitorily increased the activity of GSK-3 (about 1 h) and the level of tau hyperphosphorylation at Ser^396/Ser^404 and Ser^199/Ser^202 sites (no longer than 3 h); however, a prolonged and intense activation of GSK-3 (over 12 h) and enhanced tau hyperphosphorylation (about 24 h) were observed when these two selective kinase inhibitors were applied together. We conclude that the simultaneous inhibition of PI3K and PKC can induce GSK-3 overactivation, and further strengthen and prolong the Alzheimerlike tau hyperphosphorylation in N2a cells, suggesting the establishment of a cell model with early pathological events of Alzheimer‘s disease.     

Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent regulation

Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.     

Molecular docking of 1H-pyrazole derivatives to receptor tyrosine kinase and protein kinase for screening potential inhibitors

Tyrosine kinase receptor and protein kinases drawn much attention for the scientific fraternity in drug discovery due to its important role in different cancer, cardiovascular diseases and other hyper-proliferative disorders. Docking studies of pyrazole derivatives with tyrosine kinase and different serine/threonine protein kinases were employed by using flexible ligand docking approach of AutoDock 4.2. Among the molecules tested for docking study, 2-(4-chlorophenyl)-5-(3-(4-chlorophenyl)-5-methyl-1- phenyl-1H-pyrazol-4-yl)-1,3,4-thiadiazole (1b), 2-(4-methoxyphenyl)-5-(3-(4-methoxyphenyl)-5-methyl-1-phenyl-1H-pyrazol-4-yl)- 1,3,4-thiadiazole (1d) and 2-(4-chlorophenyl)-5-(3-(4-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-thiadiazole (2b) revealed minimum binding energy of -10.09, -8.57 and -10.35 kJ/mol with VEGFR-2 (2QU5), Aurora A (2W1G) and CDK2 (2VTO) protein targets, respectively. These proteins are representatives of plausible models of interactions with different anticancer agents. All the ligands were docked deeply within the binding pocket region of all the three proteins, showing reasonable hydrogen bonds. The docking study results showed that these pyrazole derivatives are potential inhibitor of all the three protein targets; and also all these docked compounds have good inhibition constant, vdW + Hbond + desolv energy with best RMSD value.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Structure of human protein kinase C eta (PKCeta) C2 domain and identification of phosphorylation sites

Protein kinase C eta (PKCeta) is one of several PKC isoforms found in humans. It is a novel PKC isoform in that it is activated by diacylglycerol and anionic phospholipids but not calcium. The crystal structure of the PKCeta-C2 domain, which is thought to mediate anionic phospholipid sensing in the protein, was determined at 1.75 A resolution. The structure is similar to that of the PKC epsilon C2 domain but with significant variations at the putative lipid-binding site. Two serine residues within PKC eta were identified in vitro as potential autophosphorylation sites. In the unphosphorylated structure both serines line the putative lipid-binding site and may therefore play a role in the lipid-regulation of the kinase.     

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.