Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2

Programmed cell death 6 (PDCD6) was originally found as a pro-apoptotic protein, but its molecular mechanism is not well understood. In this study, we have attempted to investigate the effects of PDCD6 on the inhibition of angiogenesis-mediated cell growth as a novel anti-angiogenic protein. Purified recombinant human PDCD6 inhibited cell migration in a concentration-time-dependent manner. We also found that overexpressed PDCD6 suppressed vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro. PDCD6 suppressed phosphorylation of signaling regulators downstream from PI3K, including Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β(GSK-3β), ribosomal protein S6 kinase (p70S6K), and also decreased cyclin D1 expression. We found binding PDCD6 to VEGFR-2, a key player in the PI3K/mTOR/P70S6K signaling pathway. Taken together, these data suggest that PDCD6 plays a significant role in modulating cellular angiogenesis.     

果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .     

Protein Kinase D1 (PKD1) Phosphorylation Promotes Dopaminergic Neuronal Survival during 6-OHDA-Induced Oxidative Stress

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1^WT) or constitutively active PKD1 (PKD1^S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.     

程序性细胞死亡因子4与单纯疱疹病毒US3蛋白相互作用及参与细胞凋亡

程序性细胞死亡因子-4（programmed celld eath-4,PDCD4）通过阻断相关基因的转录与翻译从而抑制肿瘤发生,单纯疱疹病毒-1（herpes simplex virus-1,HSV-1）US3蛋白激酶可有效调控病毒基因产物或外源因素引致的细胞凋亡。近期研究证明PDCD4在病毒感染细胞中以US3依赖及非依赖两种模式被磷酸化修饰,其中受US3修饰的PDCD4仍定位细胞核并随之被降解,这可能是细胞凋亡被抑制的主要原因之一,此外,PDCD4沉默可阻断复制不完全病毒引致的细胞凋亡,表明PDCD4与HSV-1 US3阻断细胞凋亡途径直接相关。本文综述了这两种蛋白及其作用关系的研究进展,为解析病毒与细胞相互作用机理提供新方向。     

F-Box Only Protein 31 (FBXO31) Negatively Regulates p38 Mitogen-activated Protein Kinase (MAPK) Signaling by Mediating Lysine 48-linked Ubiquitination and Degradation of Mitogen-activated Protein Kinase Kinase 6 (MKK6)

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1·Cul1·F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.     

Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.     

细胞程序性死亡因子家族分子在细胞凋亡中的调控作用

细胞程序性死亡因子(programmed cell death,PDCD)是一类与肿瘤发展相关并在进化上高度保守的蛋白质。PDCD家族由多个成员构成。其中,研究较为深入的包括PDCD1、PDCD2、PDCD4、PDCD5、PDCD6、PDCD7、PDCD8及PDCD10。PDCD在人类的各组织及细胞中广泛分布,其主要功能是对细胞凋亡的调控。目前研究发现,PDCD家族成员可通过不同信号通路实现对肿瘤细胞活力的调控,且某些家族成员的缺失或过表达都会引起机体发生病变,证明其在多种疾病当中具有重要作用。本文汇总了PDCD1、PDCD2、PDCD4、PDCD5、PDCD6、PDCD7、PDCD8、PDCD9、PDCD10、PDCD11、PDCD12的基因结构与蛋白质结构,介绍了各家族成员在细胞程序性死亡过程中的关系,并总结目前所报道的PDCD家族成员在肿瘤,以及多种疾病中所发挥的调控作用,以期帮助科研工作者了解其在细胞凋亡中的作用,以及为肿瘤和相关疾病发生发展的分子机制提供参考。     

Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5–STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31–PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.     

Suppression of programmed cell death 4 (PDCD4) protein expression by BCR-ABL-regulated engagement of the mTOR/p70 S6 kinase pathway

There is accumulating evidence that mammalian target of rapamycin (mTOR)-activated pathways play important roles in cell growth and survival of BCR-ABL-transformed cells. We have previously shown that the mTOR/p70 S6 kinase (p70 S6K) pathway is constitutively activated in BCR-ABL transformed cells and that inhibition of BCR-ABL kinase activity by imatinib mesylate abrogates such activation. We now provide evidence for the existence of a novel regulatory mechanism by which BCR-ABL promotes cell proliferation, involving p70 S6K-mediated suppression of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein that acts as an inhibitor of cap-dependent translation by blocking the translation initiation factor eIF4A. Our data also establish that second generation BCR-ABL kinase inhibitors block activation of p70 S6K and downstream engagement of the S6 ribosomal protein in BCR-ABL transformed cells. Moreover, PDCD4 protein expression is up-regulated by inhibition of the BCR-ABL kinase in K562 cells and BaF3/BCR-ABL transfectants, suggesting a mechanism for the generation of the proapoptotic effects of such inhibitors. Knockdown of PDCD4 expression results in reversal of the suppressive effects of nilotinib and imatinib mesylate on leukemic progenitor colony formation, suggesting an important role for this protein in the generation of antileukemic responses. Altogether, our studies identify a novel mechanism by which BCR-ABL may promote leukemic cell growth, involving sequential engagement of the mTOR/p70 S6K pathway and downstream suppression of PDCD4 expression.     

Expression and function of PDCD1 at the human maternal-fetal interface

The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3+ cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8 bright, CD4+, and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P 相似文献   

有丝分裂诱导基因6(MIG-6)可诱导人成纤维细胞发生提前衰老

年老细胞许多基因表达发生变化,其中有些基因的表达可以诱导成纤维细胞早衰.癌基因诱导的衰老(oncogene-induced senescence,OIS)是由一些连续癌症基因的信号,以阻止细胞增殖造成的反应.有丝分裂诱导基因6(mitogen-inducible gene-6,MIG-6)是一个抑癌基因,是ErbB RTK通路的负调控因子,抑制肿瘤细胞增殖.本文以人胚肺二倍体成纤维细胞为对象,研究MIG-6蛋白在细胞衰老中的作用.通过Western印迹发现,在老年细胞中MIG-6表达升高.利用逆转录病毒载体将MIG-6基因转入年轻的人胚肺二倍体成纤维细胞中,通过Western印迹方法检测是否过表达MIG-6,然后通过SA-β-gal染色检测其阳性率,发现转染MIG-6基因后的成纤维细胞SA-β-gal染色率明显高于对照组,生长缓慢.实验结果证实,MIG-6蛋白可以诱导人二倍体成纤维细胞提前衰老.     

PDCD5-regulated cell fate decision after ultraviolet-irradiation-induced DNA damage

Programmed cell death 5 (PDCD5) is a human apoptosis-related molecule that is involved in both the cytoplasmic caspase-3 activity pathway (by regulating Bax translocation from cytoplasm to mitochondria) and the nuclear pathway (by interacting with Tip60). In this study, we developed a mathematical model of the PDCD5-regulated switching of the cell response from DNA repair to apoptosis after ultraviolet irradiation-induced DNA damage. We established the model by combining several hypotheses with experimental observations. Our simulations indicate that the ultimate cell response to DNA damage is dependent on a signal threshold mechanism, and the PDCD5 promotion of Bax translocation plays an essential role in PDCD5-regulated cell apoptosis. Furthermore, the model simulations revealed that PDCD5 nuclear translocation can attenuate cell apoptosis, and PDCD5 interactions with Tip60 can accelerate DNA damage-induced apoptosis, but the final cell fate decision is insensitive to the PDCD5-Tip60 interaction. These results are consistent with experimental observations. The effect of recombinant human PDCD5 was also investigated and shown to sensitize cells to DNA damage by promoting caspase-3 activity.     

PDCD6 additively cooperates with anti-cancer drugs through activation of NF-κB pathways

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53^−/− and p21^−/−. At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.     

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.     

DAPk1 inhibits NF-κB activation through TNF-α and INF-γ-induced apoptosis

Recent studies have shown DAPk as a molecular modulator induced by the second messenger, responsible for controlling cell destiny decisions, but the detailed mechanism mediating the role of DAPk1 during cell death is still not fully understood. In this present report, we attempted to characterize the effects of TNF-α and INF-γ on DAPk1 in human ovarian carcinoma cell lines, OVCAR-3. Both TNF-α and INF-γ significantly induce DAPk1 levels in a time-dependent manner. At the same time, they both arrested cell cycle progression in the G(0)-G(1) and G2/M phase, down-regulated cyclin D1, CDK4 and NF-κB expression, while also up-regulating p27 and p16 expression. Subsequently, the efficacy of the combined treatment with DAPk1 was investigated. In the presence of DAPk1, TNF-α or INF-γ-induced apoptosis was additively increased, while TNF-α or INF-γ-induced NF-κB activity was inhibited. Conversely, TNF-α or INF-γ-dependent NF-κB activity was further enhanced by the inhibition of DAPk1 with its specific siRNA. The activity of NF-κB was dependent on the level of DAPk1, indicating the requirement of DAPk1 for the activation of NF-κB. Low levels of DAPk1 expression were frequently observed in different human patient's tissue and cancer cell lines compared to normal samples. In addition, over-expression of DAPk1 from either TNF-α or INF-γ-treatment cells suppressed the anti-apoptosis protein XIAP as well as COX-2 and ICAM-1, more than control. Taken together, our data findings suggest that DAPk1 can mediate the pro-apoptotic activity of TNF-α and INF-γ via the NF-κB signaling pathways.     

Programmed cell death protein 5 (PDCD5) is phosphorylated by CK2 in vitro and in 293T cells

CK2 is a multifunctional kinase, involved in cell growth, apoptosis, DNA integrity preservation, viral infection, and many other biological processes. Based on an analysis of phosphopeptides database derived from phosphoproteomic studies we previously identified a list of potential new CK2 substrates, including, among others, Programmed Cell Death 5 (PDCD5), a protein involved in cell death and down-regulated in different forms of human tumors. Here we provide experimental evidence that PDCD5 is indeed a bona fide substrate of CK2. PDCD5 is phosphorylated in vitro by both CK2α subunit and by the CK2 holoenzyme at a residue, S118, which is found phosphorylated in vivo. We also show that PDCD5 is phosphorylated by CK2 in 293T cells. Transfection of the non-phosphorylatable mutant (S118A) impairs the PDCD5 acceleration of either doxorubimicin- or UV-induced apoptosis in U2OS cells. Our results suggest a functional link between the CK2 phosphorylation and the apoptotic potential of PDCD5.     

Regulatory effects of programmed cell death 4 (PDCD4) protein in interferon (IFN)-stimulated gene expression and generation of type I IFN responses

The precise mechanisms by which the activation of interferon (IFN) receptors (IFNRs) ultimately controls mRNA translation of specific target genes to induce IFN-dependent biological responses remain ill defined. We provide evidence that IFN-α induces phosphorylation of programmed cell death 4 (PDCD4) protein on Ser67. This IFN-α-dependent phosphorylation is mediated by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) in a cell-type-specific manner. IFN-dependent phosphorylation of PDCD4 results in downregulation of PDCD4 protein levels as the phosphorylated form of PDCD4 interacts with the ubiquitin ligase β-TRCP (β-transducin repeat-containing protein) and undergoes degradation. This process facilitates IFN-induced eukaryotic translation initiation factor 4A (eIF4A) activity and binding to translation initiation factor eIF4G to promote mRNA translation. Our data establish that PDCD4 degradation ultimately facilitates expression of several ISG protein products that play important roles in the generation of IFN responses, including IFN-stimulated gene 15 (ISG15), p21(WAF1/CIP1), and Schlafen 5 (SLFN5). Moreover, engagement of the RSK/PDCD4 pathway by the type I IFNR is required for the suppressive effects of IFN-α on normal CD34(+) hematopoietic precursors and for antileukemic effects in vitro. Altogether, these findings provide evidence for a unique function of PDCD4 in the type I IFN system and indicate a key regulatory role for this protein in mRNA translation of ISGs and control of IFN responses.