Stress Activated Protein Kinase Pathway Modulates Homologous Recombination in Fission Yeast

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.     

BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.     

Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines

In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoidsall trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells.atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARα, RARβ, and RARγ were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARα does not mediate the growth stimulatory effects of retinoids, for a selective RARα antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RARα, RARβ, and RARγ mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.     

Contribution of Protein Kinase C to p53-dependent WAF1 Induction Pathway after Heat Treatment in Human Glioblastoma Cell Lines

To examine whether protein kinase C (PKC) contributes to p53-dependent WAF1 induction after heat treatment, the effects of calphostin C (CAL), a specific inhibitor of PKC, on WAF1 induction were analyzed by PKC activity and gel mobility-shift assays and Western blot analysis in human glioblastoma cell lines. Heat-induced accumulation of WAF1 in A-172 cells carrying wild-typep53(wtp53) was suppressed by CAL in a dose-dependent manner. In T98G cells carrying mutantp53(mp53), no significant accumulation of WAF1 was observed after heat treatment and CAL exerted no significant effects on this response of T98G cells. In accordance with the accumulation of WAF1, heat-induced activation of the binding ability of p53 to p53 consensus sequence (p53 CON) was suppressed by CAL in A-172 cells but no DNA-binding activity was observed in the mp53 in T98G cells. PKC in A-172 cells was activated rapidly (within 5 min) after heat treatment in the membrane fraction but not in the cytosolic fraction. When the cell lines were treated with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), WAF1 was accumulated in A-172 cells in a dose-dependent manner but not in T98G cells. In addition, the cellular contents of WAF1 after heating did not increase in A-172 cells transformed with mp53.These results suggest that PKC contributes to heat-induced signal transduction leading to p53-dependent WAF1 induction in a way that PKC is involved in the specific DNA-binding activation of p53.     

The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein

Dishevelled (Dvl) is a key component in the canonical Wnt signaling pathway and becomes hyperphosphorylated upon Wnt stimulation. Dvl is required for LRP6 phosphorylation, which is essential for subsequent steps of signal transduction, such as Axin recruitment and cytosolic β-catenin stabilization. Here, we identify the HECT-containing Nedd4-like ubiquitin E3 ligase ITCH as a new Dvl-binding protein. ITCH ubiquitinates the phosphorylated form of Dvl and promotes its degradation via the proteasome pathway, thereby inhibiting canonical Wnt signaling. Knockdown of ITCH by RNA interference increased the stability of phosphorylated Dvl and upregulated Wnt reporter gene activity as well as endogenous Wnt target gene expression induced by Wnt stimulation. In addition, we found that both the PPXY motif and the DEP domain of Dvl are critical for its interaction with ITCH, as mutation in the PPXY motif (Dvl2-Y568F) or deletion of the DEP domain led to reduced affinity for ITCH. Consistently, overexpression of ITCH inhibited wild-type Dvl2-induced, but not Dvl2-Y568F mutant-induced, Wnt reporter activity. Moreover, the Y568F mutant, but not wild-type Dvl2, can reverse the ITCH-mediated inhibition of Wnt-induced reporter activity. Collectively, these results indicate that ITCH plays a negative regulatory role in modulating canonical Wnt signaling by targeting the phosphorylated form of Dvl.     

经典Wnt信号通路与人类肿瘤

近年来,随着对肿瘤的深入研究,Wnt信号的研究也受到了高度的关注．Wnt信号通路是一条在进化上保守的信号途径,在控制胚胎发育,调节细胞生长、迁移、分化,调控正常组织重建等生命活动中发挥重要的作用,其异常活化与众多人类肿瘤的发生、发展密切相关．Wnt信号途径异常的核心是β-catenin在细胞内累积,并通过其下游途径引起特异靶基因的转录．本文着重介绍Wnt/β-catenin信号转导通路的研究进展及其与肿瘤的关系,了解该通路在肿瘤发生过程中的具体分子机制有助于为临床诊断提供依据,为早期干预治疗提供方法．     

经典Wnt信号途径与结直肠肿瘤发生

癌发生的主要原因是控制细胞生长和存活的信号转导途径被异常激活,而这些信号途径在正常胚胎发育中起重要作用.经典的Wnt信号转导途径是其中最重要的通路之一,目前认为其成分主要包括：细胞外因子（Wnt）、跨膜受体（卷曲蛋白Frizzled,Fz）、辅助受体（低密度脂蛋白受体相关蛋白-5和-6,LRP5/6）、β-链蛋白（β-catenin,β-cat）、结肠腺瘤样息肉病（adenomatous polyposis coli,APC）蛋白、T细胞因子（TCF）等一系列蛋白质.多数结直肠癌患者的转化细胞存在APC基因缺失、突变或失活,导致β-cat在核内的累积,从而影响相关基因转录,这是结直肠癌发生的关键因素之一.对Wnt途径的进一步研究将为探索结直肠癌的发病机制以及寻找有效的诊治手段提供新视觉.     

Protein Kinase R Modulates c-Fos and c-Jun Signaling to Promote Proliferation of Hepatocellular Carcinoma with Hepatitis C Virus Infection

Double-stranded RNA-activated protein kinase R (PKR) is known to be upregulated by hepatitis C virus (HCV) and overexpressed in hepatocellular carcinoma (HCC). However, the precise roles of PKR in HCC with HCV infection remain unclear. Two HCV replicating cell lines (JFH-1 and H77s), generated by transfection of Huh7.5.1 cells, were used for experiments reported here. PKR expression was modulated with siRNA and a PKR expression plasmid, and cancer-related genes were assessed by real-time PCR and Western blotting; cell lines were further analyzed using a proliferation assay. Modulation of genes by PKR was also assessed in 34 human HCC specimens. Parallel changes in c-Fos and c-Jun gene expression with PKR were observed. Levels of phosphorylated c-Fos and c-Jun were upregulated by an increase of PKR, and were related to levels of phosphorylated JNK1 and Erk1/2. DNA binding activities of c-Fos and c-Jun also correlated with PKR expression, and cell proliferation was dependent on PKR-modulated c-Fos and c-Jun expression. Coordinate expression of c-Jun and PKR was confirmed in human HCC specimens with HCV infection. PKR upregulated c-Fos and c-Jun activities through activation of Erk1/2 and JNK1, respectively. These modulations resulted in HCC cell proliferation with HCV infection. These findings suggest that PKR-related proliferation pathways could be an attractive therapeutic target.     

经典Wnt信号通路在牙齿发育中的研究新进展

经典Wnt信号通路是参与胚胎及器官发育的四大信号传导途径之一,在牙齿发育中扮演了重要的角色。本文对其中的β-catenin,Left,Ape,Axin2这4个关键因子在牙齿发育中研究的新进展做了简要的概述：β-catenin在间充质中会调控多个信号,影响牙上皮和间充质相互作用;Left会和Tcf家族一道调控上皮细胞命运;Ape能抑制多余牙齿的形成;Axin2在牙晚期发育中影响牙本质的形成。通过这些因子的研究,希望人们能在牙齿再生等生物医学工程上有新的突破。     

Effects of Interferon and PKC Modulators on Human Glioma Protein Kinase C,Cell Proliferation,and Cell Cycle

The in-vitro effects of human interferon -2b (HuIFN -2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN -2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC₅₀ 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN -2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN -2b is similar to TPA (potent PKC activation and S phase arrest).     

Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor

Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn²⁺ enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn²⁺ increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with ⁶⁵Zn²⁺ revealed that Zn²⁺ bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn²⁺ binding induces conformational changes in protein C/APC. Zn²⁺ binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca²⁺. Zn²⁺ increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca²⁺ but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg²⁺ with Ca²⁺. Zn²⁺ had no effect on the anticoagulant activity of APC. Zn²⁺ enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn²⁺ binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR.     

经典Wnt 信号通路在牙齿发育中的研究新进展

经典Wnt信号通路是参与胚胎及器官发育的四大信号传导途径之一,在牙齿发育中扮演了重要的角色。本文对其中的β-catenin,Lef1,Apc,Axin2这4个关键因子在牙齿发育中研究的新进展做了简要的概述:β-catenin在间充质中会调控多个信号,影响牙上皮和间充质相互作用;Lef1会和Tcf家族一道调控上皮细胞命运;Apc能抑制多余牙齿的形成;Axin2在牙晚期发育中影响牙本质的形成。通过这些因子的研究,希望人们能在牙齿再生等生物医学工程上有新的突破。     

The Roles of Protein Kinase C βI and βII in Vascular Smooth Muscle Cell Proliferation

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-βI and -βII isozymes. PKC-βI and -βII are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-βI and PKC-βII (βI-A10 and βII-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in βI-A10 cells decreased by 12 h, and, in contrast, the doubling time of βII-A10 cells increased by 12 h compared to A10 cells. The increase of [³H]thymidine (TdR) incorporation was accelerated and increased in βI-A10 cells, but slowed and diminished in βII-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of βI-A10 cells showed an acceleration of S phase entry while βII-A10 cells slowed S phase entry. These results suggest that PKC-βI and PKC-βII regulate cell proliferation bidirectionally and that PKC-βI and PKC-βII may have distinct and opposing functions as cell cycle check point mediators during late G₁phase and may regulate S phase entry in A10 VSMC.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

Translocation of Protein Kinase C in Anterior Pituitary Tumor Cells

Previous studies have shown that phorbol esters and lithium each stimulate the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary tumor cell line AtT20/D16-16. Pretreatment with either lithium or phorbol ester desensitizes the cells to subsequent stimulation by phorbol ester. An early consequence of phorbol ester action in other systems is the translocation of protein kinase C from cytosol to membranes. We have assayed protein kinase C activity in cytosol and membranes of AtT20 cells after treatment with phorbol dibutyrate, lithium, or other agents that stimulate secretion of ACTH in these cells. Phorbol dibutyrate clearly induced translocation of protein kinase C, but lithium treatment did not cause translocation itself, nor did pretreatment with lithium affect the translocation induced by phorbol dibutyrate. These results are consistent with a role for translocation of protein kinase C in the stimulatory and desensitizing effects of phorbol esters but fail to implicate translocation in the actions of lithium on AtT20 cells.     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

Background

Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.

Methods

The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.

Results

Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.

Conclusion

NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.