Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

Downregulation of Bid is associated with PKCepsilon-mediated TRAIL resistance

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use. We have previously shown that protein kinase C-epsilon (PKCepsilon) acts as an antiapoptotic protein in MCF-7 breast cancer cells. In the present study, we have investigated the mechanism(s) by which PKCepsilon contributes to TRAIL resistance. Overexpression of PKCepsilon inhibited caspase-8 and -9 activation, release of mitochondrial cytochrome c and cell death induced by TRAIL, but did not interfere with the recruitment of caspase-8 to the death-inducing signaling complex. Knockdown/inhibition of PKCepsilon resulted in enhanced sensitivity to TRAIL. The level of Bcl-2 was increased and Bid was decreased by PKCepsilon at both the protein and mRNA level but PKCepsilon had no effect on Bax. Knockdown of Bcl-2 by siRNA reversed TRAIL resistance in PKCepsilon-overexpressing cells, whereas depletion of Bid contributed to TRAIL resistance in MCF-7 cells. A decrease in Bid content was also associated with inhibition of TRAIL-induced caspase-8 activation. Furthermore, PKCepsilon depletion or overexpression of DN-PKCepsilon was associated with a decrease in Bcl-2 protein level. Thus, our results suggest that PKCepsilon acts upstream of mitochondria and mediates TRAIL resistance via both Bcl-2 and Bid in MCF-7 cells.     

IL-2 and related cytokines can promote T cell survival by activating AKT.

The regulated elimination of T cells serves to maintain normal immune function and prevents autoimmune responses. IL-2 family cytokines play an important role in controlling the survival of immature and mature T cells. These molecules activate the protein kinase, AKT/PKB. AKT has been shown to transduce an antiapoptotic signal in numerous cell types. In this study, we show that an active form of AKT can protect T cells from apoptosis following growth factor withdrawal and that IL-2 family cytokines can promote T cell survival by activating this kinase. We also provide evidence that AKT does not block death receptor-mediated killing of lymphocytes. These data suggest that AKT may serve as a common signaling element by which members of the IL-2 family of cytokines promote T cell survival.     

Identification of an antiapoptotic protein complex at death receptors

Stimulation of death receptors activates the extrinsic apoptotic signaling pathway that leads to cell death. Although many steps of this apoptotic signaling cascade are known, few mechanisms that counterbalance the death signal have been described. We identified an antiapoptotic protein complex associated with death receptors that contains glycogen synthase kinase-3 (GSK3), DDX3 and cellular inhibitor of apoptosis protein-1 (cIAP-1). GSK3, DDX3 and cIAP-1 are associated in cells with each other and with death receptors. Blocking the actions of GSK3 or DDX3 potentiated caspase-3 activation induced by stimulation of four different death receptors in several types of cells. GSK3 restrained apoptotic signaling by inhibiting formation of the death-inducing signaling complex and caspase-8 activation. Stimulated death receptors surmount the antiapoptotic complex by causing GSK3 inactivation and cleavage of DDX3 and cIAP-1 to enable progression of the apoptotic signaling cascade, but the antiapoptotic complex remains functional in cancer cells resistant to death receptor stimulation, a resistance that is overcome by GSK3 inhibitors. Thus, an antiapoptotic complex of GSK3, DDX3 and cIAP-1 caps death receptors, providing a checkpoint to counterbalance apoptotic signaling.     

The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis

The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.     

Functional proteomic analysis of a three-tier PKCepsilon-Akt-eNOS signaling module in cardiac protection

Cardiac protective signaling networks have been shown to involve PKCepsilon. However, the molecular mechanisms by which PKCepsilon interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKCepsilon-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKCepsilon, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKCepsilon transgenic mouse hearts, we demonstrate the following: 1) PKCepsilon, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKCepsilon or Akt enhances the formation of PKCepsilon-Akt-eNOS signaling modules. 2) PKCepsilon directly phosphorylates and enhances activation of Akt in vitro, and PKCepsilon activation increases phosphorylation and activation of Akt in PKCepsilon transgenic mouse hearts. 3) PKCepsilon directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKCepsilon in vivo increased phosphorylation of eNOS at Ser(1177), indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKCepsilon, Akt, and eNOS in the heart and implicates these PKCepsilon-Akt-eNOS signaling modules as critical signaling elements during PKCepsilon-induced cardiac protection.     

Treatment combinations targeting apoptosis to improve immunotherapy of melanoma

Immunotherapy based on T cell responses to the tumor is believed to involve killing of cancer cells by induction of apoptosis. The predominant mechanisms are death ligand-induced signaling mainly by TNF-related apoptosis-inducing ligand (TRAIL) mediated by CD4 T cells, monocytes and dendritic cells, and perforin/granzyme mediated apoptosis mediated by CD8 T cells and NK cells. Resistance against TRAIL involves loss of TRAIL death receptors and/or activation of the MEK and/or Akt signal pathways. Resistance to CD8 CTL responses also involves activation of the MEK and/or Akt pathways. Apoptosis induced by immune responses is regulated by the Bcl-2 family of proteins. Many reagents have been developed against the Bcl-2 antiapoptotic proteins and clinical trials combining them with immunotherapy are awaited. The second group of agents that regulate the Bcl-2 family of proteins are the signal pathway inhibitors. Clinical trials with inhibitors of RAS, RAF or MEK are in progress and would appear an exciting combination with immunotherapy. One of the main drivers of resistance to apoptosis are adaptive mechanisms that allow cancer cells to overcome endoplasmic reticulum (ER) stress. These adaptive mechanisms inhibit practically all known apoptotic pathways and create an acidic environment that may reduce infiltration of lymphocytes against the tumor. The signal pathway inhibitors may be effective against these adaptive processes but additional agents that target ER stress pathways are in development. In conclusion, combination of immunotherapy with agents that target antiapoptotic mechanisms in cancer cells offers a new approach that requires evaluation in clinical trials.     

Fas Ag-FasL coupling leads to ERK1/2-mediated proliferation of gastric mucosal cells

When cells within the gastric mucosa progress from metaplasia to dysplasia to cancer, they acquire a Fas Ag apoptosis-resistant phenotype. It is unusual to completely abolish the pathway, suggesting other forms of Fas Ag signaling may be important or even necessary for gastric cancer to progress. Little is known about alternate signaling of the Fas Ag pathway in gastric mucosal cells. Using a cell culture model of rat gastric mucosal cells, we show that gastric mucosal cells utilize a type II signaling pathway for apoptosis. Under conditions of low receptor stimulation or under conditions where apoptosis is blocked downstream of the death-inducing signal complex, Fas Ag signaling proceeds toward proliferative signaling. Under conditions favoring proliferative signaling, cFLIP is recruited to the Fas-associated death domain-like interleukin-1beta-converting enzyme at the death-inducing signal complex and activates ERK1/2. ERK1/2 in turn activates NF-kappaB. ERK1/2 stimulates proliferation, whereas NF-kappaB activation results in upregulation of the antiapoptotic protein survivin, further promoting proliferation over apoptosis. These results suggest that factors that inhibit apoptosis confer a growth advantage to the cells beyond the survival advantage of avoiding apoptosis and in effect convert the Fas Ag signaling pathway from a tumor suppressor to a tumor promoter.     

Apoptosis in the development of the immune system

Apoptosis is a conserved genetic program critical for the development and homeostasis of the immune system. During the early stages of lymphopoiesis, growth factor signaling is an essential regulator of homeostasis by regulating the survival of lymphocyte progenitors. During differentiation, apoptosis ensures that lymphocytes express functional antigen receptors and is essential for eliminating lymphocytes with dangerous self-reactive specificities. Many of these critical cell death checkpoints during immune development are regulated by the BCL-2 family of proteins, which is comprised of both pro- and antiapoptotic members, and members of the tumor necrosis factor death receptor family. Aberrations in the expression or function of these cell death modulators can result in pathological conditions including immune deficiency, autoimmunity, and cancer. This review will describe how apoptosis regulates these critical control points during immune development.     

Obestatin-mediated proliferation of human retinal pigment epithelial cells: regulatory mechanisms

In this work, we have evaluated the effect of the new discovered peptide obestatin on cell proliferation in primary cultures of human retinal epithelial cells (hRPE cells). The results showed that this peptide induced, in a dose-dependent manner, cell proliferation by MEK/ERK 1/2 phosphorylation. A sequential analysis of the obestatin transmembrane signaling pathway showed that the ERK 1/2 activity is partially blocked after preincubation of the cells with pertussis toxin (PTX), as well as by wortmannin (an inhibitor of PI3K), claphostin C (an inhibitor of PKC), and PP2 (which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon-, theta-, and micro-isoenzymes rise with different time courses, from which PKCepsilon might be responsible for ERK 1/2 response. Based on the experimental data, a signaling pathway involving the consecutive activation of Gi, PI3K, novel PKC (probably PKCepsilon), and Src for ERK 1/2 activation is proposed. These results incorporate a new mitogenic factor to the group of factors that regulate proliferation of hRPE cells.     

Radiation induces apoptosis primarily through the intrinsic pathway in mammalian cells

Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in “initiator” caspases, “effector” caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both “initiator” and “effector” caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.     

JunD mediates survival signaling by the JNK signal transduction pathway

The c-Jun NH(2)-terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. Here we demonstrate that JNK-stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF-kappaB to increase antiapoptotic gene expression. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Furthermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biological response to a specific challenge.     

The mechanism by which the mitochondrial ATP-sensitive K+ channel opening and H2O2 inhibit the mitochondrial permeability transition

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial K(ATP) channel (mitoK(ATP)). In this study, we explored the pathway that links mitoK(ATP) with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K(+) channel openers as well as the K(+) ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of K(ATP) channel openers, PKG, or valinomycin were mediated by a PKCepsilon. Activation of PKCepsilon by phorbol 12-myristate 13-acetate or H(2)O(2) resulted in mitoK(ATP)-independent inhibition of MPT opening, whereas activation of PKCepsilon by PKG or the specific PKCepsilon agonist psiepsilon receptor for activated C kinase caused mitoK(ATP)-dependent inhibition of MPT opening. Exogenous H(2)O(2) inhibited MPT, because of its activation of PKCepsilon, with an IC(50) of 0.4 (+/-0.1) microm. On the basis of these results, we propose that two different PKCepsilon pools regulate this signaling pathway, one in association with mitoK(ATP) and the other in association with MPT.     

Involvement of protein kinase Cepsilon (PKCepsilon) in thyroid cell death. A truncated chimeric PKCepsilon cloned from a thyroid cancer cell line protects thyroid cells from apoptosis.

The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCepsilon gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCepsilon (Tr-PKCepsilon) mRNA, coding for N-terminal amino acids 1-116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCepsilon protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCepsilon, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCepsilon and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCepsilon grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCepsilon were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCepsilon in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCepsilon mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Cell Fate Decisions Regulated by K63 Ubiquitination of Tumor Necrosis Factor Receptor 1

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding.