Báo cáo khoa học: Protein kinase Ce: the mitochondria-mediated signaling pathway pot

MINIREVIEW Protein kinase Ce: the mitochondria-mediated signaling pathway Hiromichi Yonekawa and Yoshiko Akita Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, Japan PKCe and apoptosis Two isoforms of a novel type of protein kinase C (PKC), PKC d and PKCe, show opposing effects on apoptosis. For example, activation of PKCd induces and ⁄ or enhances the apoptotic events that occur dur- ing ischemic reperfusion and malignant progression of cancer cells, whereas activation of protein kinase Ce (PKCe) inhibits and ⁄ or reduces these events [1]. Later in this review, we will focus much more on the effects of PKCe in apoptosis. PKCe acts as an oncogene with anti-apoptotic effects when PKCe is overexpressed in cancer cells. Overexpression of PKCe in rat fibroblasts has indi- cated that those clones expressing very high activity display a number of transformed cell-specific pheno- mena, including growth in soft agar and tumor form- ation in nude mice, providing direct evidence that only PKCe in this enzyme family can exert full oncogenic effects on malignant transformation in the same cell type [2]. PKCe is also oncogenic in colon epithelial cells via its interaction with ras signal transduction, where ras acts upstream of PKCe [3]. Involvement of phosphatidylinositol 3-kinase (PI3K) in PKCe-medi- ated oncogenic signaling has been demonstrated in the same cells [4]. Well-known characteristics identified in the oncogene are gene amplification and gene rear- rangement, as seen in the oncogene myc, src, etc. [5]. Keywords apoptosis; Bcl-2 family proteins; cardiolipin; ischemia; mitochondria; PKCe; PMPT; phospholipid scramblase 3; ROS; TRAIL Correspondence H. Yonekawa, Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan Fax: +81 3 3824 7445 Tel: +81 3 3823 2105 E-mail: yonekawa-hr@igakuken.or.jp (Received 12 February 2008, revised 23 May 2008, accepted 30 May 2008) doi:10.1111/j.1742-4658.2008.06558.x Mitochondria, which are the cellular energy plants, also act as the integra- tion center of cellular signaling pathways. Apoptosis is a well-known pathway in which mitochondria are involved. Protein kinase Ce has been classified as a novel type of protein kinase C and is involved in many cellular events regulating mitochondrial function. Much evidence has accumulated regarding the relationships between mitochondria-mediated apoptosis and protein kinase Ce. Therefore, by focusing on these relation- ships, in particular the anti-apoptotic effects of protein kinase Ce on mitochondrial function, we highlight the importance and significance of protein kinase Ce in cell survival and death. Abbreviations Apaf-1, apoptosis protease activating factor-1; CAD, caspase-activated DNase; CL, cardiolipin; COIV, cytochrome c oxidase subunits IV; DNA-PK, DNA-dependent protein kinase; IAP, inhibitor of apoptosis; IPC, ischemic preconditioning; IR, ischemia ⁄ perfusion; MIM, mitochondrial inner membrane; mitoK ATP, mitochondrial ATP-sensitive K channels; MOM, mitochondrial outer membrane; MPT, mitochondrial permeability transition; MPTP, pore of MPT; PDK, phospholipid-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLS3, phospholipid scramblase 3; PPC, pharmacologic preconditioning; ROS, reactive oxygen species; SCLC, small cell lung cancer cells; tBid, the truncated C-terminal of the Bid; TRAIL, tumor necrosis factor-related apoptosis inducing legend; Tr-PKCe, chimeric ⁄ truncated PKCe. FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS 4005 Similar to other oncogenes, the amplification of PKCe has been reported in thyroid neoplasm. This genetic alteration is also associated with a rearrangement of the PKC e gene. This results in the overexpression of a chimeric ⁄ truncated PKCe (Tr-PKCe) mRNA, coding for N-terminal amino acids 1–116 of an isozyme fused to an unrelated sequence [6]. Expression of Tr-PKCe acts as a dominant negative effect of the mutant protein on activation-induced translocation of wild- type PKCe. Cells expressing Tr-PKCe are resistant to apoptosis, and therefore the cells become malignant. This anti-apoptotic effect is also associated with higher Bcl-2 levels, a marked impairment in p53 stabilization [6] and dampened expression of Bax [6–13]. These find- ings show that a dominant negative PKCe mutant can block cell death triggered by a variety of stimuli, including cancer therapeutic agents. Taken together, the evidence described above reveals that PKCe is intimately involved in anti-apoptotic effects. Structure and function of mitochondria with special reference to apoptosis The discovery of the involvement of mitochondria in apoptosis makes this organelle the most important key player supporting apoptotic signaling. Apoptosis is the phenomenon also known as fated cell death. The regu- lation of apoptosis plays a crucial role in development and neoplastic transformation. Two major pathways are involved in apoptotic signaling: intrinsic and extrinsic [14]. Briefly, the intrinsic pathway is activated by DNA damage, overproduction of reactive oxygen species (ROS) and ⁄ or of reactive nitrogen species, etc., which cause instability of mitochondrial outer membranes. As a result, such phenomena trigger mitochondrial cytochrome c release and activation of apoptosis protease activating factor-1 (Apaf-1) and, subsequently, caspase-9 [15]. On the other hand, the extrinsic pathway is activated by tumor necrosis factor-related apoptosis inducing legend (TRAIL), which activates caspase-8 and its downstream caspases. TRAIL-mediated apoptosis also induces mitochondria- mediated apoptosis to enhance the apoptotic pathway. Namely, Bid, a BH3-only Bcl-2 family member, is cleaved by caspase-8, and the truncated C-terminal of the Bid (tBid) translocates to mitochondria to promote apoptosis [16] (Fig. 1). Before we describe how mitochondria contribute to apoptosis, we will outline the structures and functions of mitochondria. Electron microscopy studies have led to the well- known and typical view of mitochondria as comprising bean-shaped organelles, although the recent develop- ment of bioimaging has drastically changed the view that mitochondria move in a dynamic manner, chang- ing their shapes with frequent fusions and fissions depending on external and internal stimuli to the cell [17]. Mitochondria consist of four subcompartments: two membranes, the mitochondrial outer membrane (MOM) and the inner membrane (MIM), and two spaces, the intermembrane space and the matrix. MIM also possesses unique structures called cristae, which are invaginations of MIM exserted into the matrix. These subcompartments divide their own functions, harboring the factors necessary for the functions within them (Table 1). As will be described below, MOM and MIM are the most important compart- ments participating in apoptotic events. Mitochondria consist of two protein groups with dif- ferent genetic origins. Approximately 1500 proteins are encoded on the nuclear genome, whereas only 13 proteins are encoded on the mitochondrial genome (mtDNA). These mtDNA-encoded proteins are exclu- sively localized on MIM and function as components of oxidative phosphorylation to produce ATP. On the other hand, the nuclear DNA-encoded proteins are synthesized in the cytosol and are initially recognized by receptors on MOM to translocate into the mito- chondrial compartments noted above. These mitochon- drial membrane systems are tightly guarded as ‘double ramparts’ from free mixing of any lipid-insoluble macromolecules such as proteins to generate an electric membrane potential (Dwm). Therefore, any protein translocated in mitochondria should possess mitochon- dria-translocation signal sequence(s) on the N-terminal of its amino acid sequences. Translocases in MOM and MIM recognize the signal sequence and then mediate the import and intramitochondrial sorting of these proteins; ATP and the membrane potential are used as energy sources. Chaperones and auxiliary factors assist in the folding and assembly of mitochondrial proteins into their native, 3D structures [18]. The existence of PKCe in mitochondria is puzzling, in particular on the MIM, a topic that will be discussed below. Role of a mitochondria-specific lipid cardiolipin on mitochondria-mediated signaling pathway Mitochondria also possess a special major lipid called cardiolipin (CL) [19]. The well-known function of mitochondria is energy (ATP) production via oxidative phosphorylation as ‘cellular energy plants’. Oxidative phosphorylation also generates ROS, which have strong reactivity to oxidize macromolecules, such as proteins, lipids and nucleic acids. The partial degra- dation of CL caused by ROS, which is generated by PKCe and mitochondria H. Yonekawa and Y. Akita 4006 FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS dysfunction of oxidative phosphorylation, is now a recognized trigger of mitochondria-mediated apoptosis [20]. PKCe is the only PKC isoform that is activated by CL [21]. Recently, Bax ⁄ Bak activation and cardio- lipin peroxidation were reported to be essential for cytochrome c release during apoptosis. Specifically, the sequential events for apoptosis to occur in mouse embryonic fibroblast cells follow the order: Bax trans- location fi superoxide production fi CL peroxida- tion fi cytochrome c release fi apoptosis [22]. Involvement of PKC e in the translocation of Bax has also been reported [8,23,24] (Fig. 1). Major involvement of PKCe in extrinsic apoptotic pathway As noted above, the extrinsic pathway in apoptosis is triggered by the interaction between ligands belonging to the tumor necrosis factor family and its cell-surface receptors. The trigger signal is transmitted to caspase-8, and then, downstream of the signaling pathways, branches into two pathways: the mitochondria-inde- pendent path way and the mitochondria-mediated pathway. The former pathway involves the proteolytic activation of caspase-3 by caspase-8 fi release of caspase-activated DNase (CAD) by inactivation of inhibitor of CAD fi translocation of CAD into the nucleus fi chromatin condensation and DNA frag- mentation fi apoptosis. On the other hand, the latter pathway is started by proteolytic activation of Bid by caspase-8 (formation of tBid) fi binding of tBid to MOM fi activation of BAX and BAK to translocate from cytoplasm to MOM fi destabilizing of MOM fi release of cytochrome c from mitochondria to the cytoplasm fi Apaf-1 fi proteolytic activation of caspase-9 by Apaf-1 fi apoptosis. Fig. 1. Mitochondria-mediated apoptosis pathway and involvement of PKCe. The outline of two main apoptosis pathways (TRAIL-mediated and mitochondria-mediated) is shown. The TRAIL-mediated apoptosis pathway is triggered by activation of TRAIL receptors on the cell mem- brane, followed by activation of caspase-8 fi caspase-3 fi apoptosis. Mitochondria-mediated apoptosis is triggered by intrinsic stimuli such as hypoxia during ischemia, etc., which activate 7 transmembrane receptors (¢TMR), or so called G-protein-coupled receptors (GPCRs). The signals follow the order: PI3K fi Akt fi ERK fi GC (guanylyl cyclase) fi PGK fi inhibition of MTPT fi release of cytochrome c fi caspase-9 fi caspase-3 fi apoptosis. PKCe is shown in a red circle. The reaction regulated by PKCe is shown by a red line. H. Yonekawa and Y. Akita PKCe and mitochondria FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS 4007 We will discuss the mitochondria-mediated pathway in intrinsic and extrinsic apoptosis in more detail. As noted above, mitochondria are deeply involved in the apoptotic signaling pathways via the release of mito- chondrial proteins into the cytoplasm. In particular, once cytochrome c, a key component of electron trans- port in mitochondria, is released from mitochondria, its function is drastically changed into an apoptotic signal, leading to activation of Apaf-1, which is a protease also released from mitochondria to form the cytochrome c ⁄ Apaf-1. Activated Apaf-1 activates caspase-9, a trig- ger to activate the caspase cascade, and finally causes apoptosis. On the other hand, Smac ⁄ Diablo is also released from mitochondria when MOM is desensi- tized, and Smac ⁄ Diablo inhibits inhibitor of apoptosis (IAP) proteins that normally interact with caspase-9 to inhibit apoptosis. Bcl-2 family proteins (Bcl-2, Bid, Bax, Bak, Bcl-x1, etc.) are cytoplasmic proteins that act as regulators of apoptosis to bind mitochondria. The Bcl-2 family proteins are divided into two groups: the first comprises anti-apoptotic proteins, including Bcl-2 and Bcl-x1, whereas the second comprises pro-apopto- tic proteins. Such Bcl-2 family proteins interact with each other to form complexes that stimulate apoptosis to enter MOM to regulate the release of cytochrome c, Smac ⁄ Diablo and other proteins from mitochondria, or inhibit apoptosis to inactivate the apoptotic functions of the pro-apoptotic proteins to form a protein com- plex. Recent studies show that pro-apoptotic factor Bax is inactivated by Ku70 to form ‘Baxosome’ [25–29] and that Bax is regulated by Ku70-dependent deubiqui- tylation [27]. Similar to p53 [30], Ku70 is a molecule that functions in DNA damage (Fig. 1). In the cyto- plasm of a normal healthy cell, Bax forms a complex with Ku70 to inactivate Bax function. The release of Ku70 from the complex induces a conformational change of Bax, and the change allows tBid to bind to Bax [25]. The activated Bax then inserts into MOM to destabilize it [26]. By these processes, cells are progress- ing towards apoptosis. Involvement of PKCe in the apoptotic pathways has been disclosed both in cancer research and in cardiol- ogy [8,9,24,31,32]. TRAIL is a promising anticancer agent because it selectively kills tumor cells but spares normal cells. On the other hand, tumor cells are prone to be resistant to anti-cancer agents because resistance to TRAIL by tumor cells limits its therapeutic use. Using breast cancer MCF-7 cells, PKCe has been shown to be a major causative agent for resistance. Namely, overexpression of wild-type PKCe in MCF-7 Table 1. Structures and functions in mitochondira. mtDNA, mitochondrial DNA, pol c, DNA polymerase c. Mitochondrial subcompartments Compartment-specific processes Compartment-specific substances a MOM 1) Protein import 2) Metabolite influx ⁄ efflux 3) Fission, fusin and distribution 4) Apoptotic factors 5) Signaling molecules Bcl-2, Bcl-xL, Bax, tBid, p53(?) b TOM (translocase of MOM) and SAM (sorting and assembly machinery) c PLS 3 CL Intermembrane space 1) Electron transfer 2) Cristae remodeling 3) Redox enzymes 4) Protein inport 5) Apoptosis factors Cytochrome c, Smac ⁄ Diablo b sTIMs (small translocase of MIM) c MIM 1) Oxidative phosphorylation 2) Metabolite transport 3) Protein inport 4) Protein asembly 5) Protein degradation TIM (translocase of MIM) b,c COIV b ROS b Matrix 1) Tricarboxylic acid cycle 2) Fatty acid oxidation 3) mtDNA replication 4) mtDNA transcription ⁄ translation 5) SFe-S biogenesis 6) Protein holding and degradation 7) Urea cycle (liver and small intestine) 8) Gluconeogenesis (liver and kidney) mtDNA b ROS b p53 b pol c b a Only substances related to apoptosis and translocation are shown. b Proteins involved in mitochondria-mediated apoptosis. c Proteins involved in mitochondrial import. PKCe and mitochondria H. Yonekawa and Y. Akita 4008 FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS cells inhibits activation of caspases-8 and -9 and decreases tumor necrosis factor-induced mitochondrial depolarization, which leads to the release of mitochon- drial cytochrome c and cell death induced by TRAIL [32]. The level of an anti-apoptotic protein Bcl-2 increased, whereas that of an apoptotic protein Bid decreased by PKCe at both the protein and mRNA levels. The reverse is also true. Knockdown using small intefering RNA and inhibition by inhibitors specific to PKCe results in enhanced sensitivity to TRAIL. PKCe was also coimmunoprecipitated with Bax in MCF-7 cells, but had no effect on it. Namely, knockdown of Bcl-2 by small intefering RNA reverses TRAIL resis- tance in PKCe-overexpressing cells, whereas depletion of Bid contributes to TRAIL resistance in MCF-7 cells [8,9]. A decrease in Bid content is also associated with inhibition of TRAIL-induced caspase-8 activation. Furthermore, PKCe depletion or overexpression of a dominant negative PKCe is associated with a decrease in Bcl-2 protein levels. These findings offer clear evi- dence that PKC e mediates its anti-apoptotic effect via the mitochondria by regulating the activities of pro- apoptotic and anti-apoptotic proteins and transloca- tion of these proteins to the mitochondria [8,9,24,32]. Involvement of PKCe in intrinsic apoptotic pathway The intrinsic pathway depends on damage to mitochondria and on the release of apoptosis-inducing proteins (e.g. mitochondria-mediated apoptosis). Mitochondria-targeted intrinsic stimuli include hypoxia, and overproduction of ROS and reactive nitrogen species, etc. [20] (Fig. 1). Using MCF-7 cells, another important involvement of PKCe in the apoptosis pathway comprises the intrinsic pathway, which is triggered by DNA damage. DNA-dependent protein kinase (DNA-PK), which is fundamentally involved in this pathway, is a nuclear serine ⁄ threonine protein kinase, and is a member of the PI3K-related kinase subfamily of protein kinases. DNA-PK is activated upon DNA damage, and plays an important role in DNA repair and protects cells from apoptosis induced by DNA damaging agents, such as ionizing [30] or UV radiation [24]. On the other hand, a catalytic subunit of DNA-PK (DNA-PKcs) can colocal- ize with Akt on the cell membrane and phosphorylate Akt at Ser473 in a PI3K-dependent manner. Although Akt plays a critical role in cell survival, the involvement of DNA-PK in the anti-apoptotic function of Akt has not been investigated. In this pathway, PKCe activates Akt by enhancing the interaction between DNA-PK and Akt, resulting in phosphorylation of Akt at Ser473. Thus, PKCe acts upstream of Akt to regulate anti-apop- totic signaling in breast cancer cells [24]. In small cell lung cancer cells (SCLC), PKCe also shows an anti-apoptotic effect [31]. A high percentage of patients with SCLC die because of resistance to anti-cancer chemotherapeutic agents (chemoresistance). This may be due to the increased expression of anti- apoptotic proteins, X chromosome-linked IAP and Bcl-x1, triggering chemoresistance in SCLC cells [33]. These effects are mediated through the formation of a specific multiprotein complex comprising PKCe, B-Raf and S6K2. S6K1, Raf-1 and other PKC isoforms do not form similar complexes [31]. With regard to the correlation between PKCe activa- tion and prevention of apoptotic cell death, many other lines of evidence have accumulated. For exam- ple, in Jarkat cells (T lymphocytes) [34] and cardio- myocytes [35], PKCe inhibits apoptosis through phosphorylation and inactivation of Bad, whereas, in prostate cancer cells, PCKe activity blocks Bax activa- tion and mitochondrial integration [13]. By contrast, in glioma cells, PKCe regulates Akt expression and is essential for their survival, suggesting that the cleavage of PKCe and its down-regulation play important roles in the apoptotic effect of TRAIL [12]. In human vascular endothelial cells, PKCe is involved in the vascular endothelial growth factor-activated signaling pathway through a physical interaction between PKCe and Akt [36]. The interaction results in cooperative induction of Bcl-2 and enhanced protection against apoptotic cell death via inhibition of caspase-3 clea- vage [7,10,11,37,38]. Apoptosis triggered by DNA damage starts by sequential activation as follows: induction of ataxia- telangiectasia mutated (ATM) fi induction of Chk2 fi stabilization of p53[30] fi up-regulation of NOXA, p53 upregulated modulator of apoptosis (PUMA) and other apoptotic- (BAX, BAK, BID, etc.) and anti- apoptotic related proteins (Bcl2, Bcl-xL, etc.) fi translocation of NOXA, PUMA fi inhibition of anti-apoptotic protein Bcl2 fi destabilization of mitochondrial membrane potential fi release of cyto- chrome c fi activation of Apaf-1, and then caspase- 9 fi induction of apoptosis. In this pathway from ATM to PUMA and NOXA, only two molecules, p53 and MDM2, are affected by PKCe [6,39,40]. Signaling pathway towards mitochondria during apoptosis Because Bad, Bax, Akt and Bcl-2 are signaling elements for mitochondria-mediated apoptosis, it is evident that PKCe is intimately involved in the H. Yonekawa and Y. Akita PKCe and mitochondria FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS 4009 signaling pathway of apoptosis. If so, how is PKCe involved in mitochondria-mediated apoptosis? Ischemia ⁄ perfusion (IR) and preconditioning studies have disclosed the mechanism. IR causes severe cellu- lar injury in the heart, brain and other organs. How- ever, Murry et al. [41] demonstrated that ischemic preconditioning (IPC) prevents severe injury (cell death) in the heart (cardiomyocytes). IPC can occur by brief ischemic episodes before subjecting the heart, brain and other organs to prolonged ischemia in exper- imental animals. The discovery by Murry et al. [41] provided a breakthrough for disclosing the molecular mechanisms during IR. It has subsequently been confirmed that IR and IPC are controlled by various signaling pathways, and that IPC can also be mimicked by pharmacologic preconditioning (PPC) by using inhibitors, agonists and antagonists specific to the pathways; with regard to PPC, see Pacher and Hasko [42]. Some of these substances have been devel- oped as pharmaceutical drugs and are applied to save the lives of patients with cardiac ischemia. The devel- opment of PPC enables us to pinpoint the signaling pathways. The steps in the pathway have mainly been disclosed in trials using experimental animals. In IPC, key signaling elements are mitochondrial ATP-sensitive K channels (mitoK ATP ), G-protein cou- pled receptors and PKCe (Fig. 1). Therefore, consider- able attention has been paid to mitochondria as the target of IPC. During IPC, the heart releases brady- kinin and an endogenous opioid, and produces adeno- sine as the metabolic breakdown product of ATP. The binding of these three ligands to their respective G-protein coupled receptors triggers their downstream signaling pathways [43]. Both bradykinin and the opioid activate PI3K. The product of PI3K activates PDK which phosphorylates Akt to activate it. Akt phosphorylates endothelial NO synthetase, causing it to generate NO. NO activates soluble guanylyl cyclase to generate cGMP. cGMP then activates protein kinase G, which acts on mitochondria to open mitoK ATP on MIM [44]. When mitoK ATP open, two known actions occur: swelling of the matrix and generation of ROS [45]. Intramitochondrial signaling pathway As noted above, several triggers have been proposed for IPC leading to the activation of several intracellular pathways that ultimately prevent cardiomyocytes from cell death (cardioprotection) [43–45]. Although the details of these pathways are not totally understood, it is evident that mitochondria are key mediators of IPC. This is because pharmacological studies have impli- cated a role of mitoK ATP channel opening in cardio- protection and IPC has been shown to preserve mitochondrial function and to reduce mitochondrial cytochrome c release during IR and thus prevent apop- tosis of cardiomuscular cells during IR. With these phe- nomena, the activation of PKCe was first demonstrated to be critical for the protective phenotype by Jaburek et al. [46]. Furthermore, experiments using activators (weRACK) and inhibitors (eV 1–2 ) specific to PKCe, reconstitution procedures for mitoK ATP , and other functional analyses, indicated that PKCe is actually bound to MIM and associated with mitoK ATP and that the association is fully functional. For example, PKCe enhances mitoK ATP opening by phosphorylation [46,47]. The opening of mitoK ATP causes an increase in ROS production. Based on several lines of evidence, Costa et al. [45] suggested that two PKCe pools exist in mitochondria: one that acts to inhibit mitochondrial permeability transition (MPT) downstream of ROS generation and the other that acts to open mitoK ATP , which is upstream of ROS production. Under physio- logical conditions, each PKCe is activated indepen- dently and phosphorylates its unique substrate and therefore both are distinguishable. However, it will be difficult to confirm this phenomenon biochemically because both PKCe are biochemically identical. To date, due to the many studies performed on IR and IPC, the accumulating evidence demonstrates that PKCe interacts with mitochondrial components. Colo- calization and coimmunoprecipitation of PKCe and mitoK ATP protein have demonstrated the direct inter- action of both proteins, suggesting that the activation of PKCe intrinsically involves the opening of mitoK ATP . Additional evidence demonstrating the direct interactions between PKCe and mitochondrial components consists of coimmunoprecipitation to PKCe (COIV) [48] and components of the pore of MPT (MPTP) such as voltage dependent anion-selec- tive channel (VDAC), adenine nucleotide translocase (ANT) and hexokinase II [49]. Phosphorylation by PKCe enhances the activity of COIV, whereas the physical interaction of PKCe with components of the cardiac mitochondrial pore inhibits MPTP. Again, these lines of evidence provide clear molecular confir- mation of the anti-apoptotic effect of PKCe. Because mitoK ATP and COIV are localized in MIM, colocaliza- tion and coimmunoprecipitation of PKCe with such proteins evidently demonstrates that PKCe does exist on MIM. However, no mitochondrially-importing signaling peptides have yet been discovered in PKCe protein molecules or in the PKCe gene. Therefore, the question of how PKCe can exist on MIM remains unsolved. PKCe and mitochondria H. Yonekawa and Y. Akita 4010 FEBS Journal 275 (2008) 4005–4013 ª 2008 The Authors Journal compilation ª 2008 FEBS Further perspectives As noted above, IR and IPC prevent cardiac myoblasts from apoptotic cell death [43–46]. PKCe plays important roles in this process via activation of MPTP components and mitoK ATP to inhibit the open- ing of MPTP [45–47]. During ischemia, cells are exposed by severe hypoxia and acidosis, leading to apoptotic cell death. Hypoxia mediates the accumula- tion of hypoxia-inducible factor-1a and the activated form induces transcription of BNIP3, a pro-apoptotic Bcl-2 family member protein. BNIP3 is loosely bound to cellular membranes, including mitochondria at physiological pH, but translocates into the membrane when the pH level is decreased. The translocation stim- ulates opening of MPTP, releasing pro-apoptotic factors, cytochrome c and calcium, and, as a result, the myocyte dies. This cell death demonstrates DNA fragmentation and nuclear condensation as in usual apoptosis. However, this death is not associated with the activation of caspases, and cell death is thus unu- sual [50]. Although there is no experimental evidence supporting the involvement of PKCe in this signaling pathway, it will be interesting to examine the BNIP3- stimulated opening of MPTP and activation of PKCe. Reperfusion of ischemic cardiac tissue is associated with increased apoptosis, resulting in diminished heart function [43–46]. As noted above, IPC protects the heart from injury mediated by reperfusion. Two calcium-insensitive isoforms of the novel PKC subfam- ily, PKCd and PKCe, play opposing roles in IR injury [1]. Activation of PKCd during reperfusion induces cell death via the regulation of mitochondrial function and induction of apoptosis. A recent discovery on the effect of novel PKC isoforms on apoptosis is that phospholipid scramblase 3 (PLS3) is phosphorylated by PKCd at Thr21 and is the mitochondrial target of PKCd-induced apoptosis. PLS3 is a member of the phospholipid scramblase family present in mitochon- dria. PLS3 plays an important role in the regulation of mitochondrial morphology, respiratory function and apoptotic responses. Detailed analyses of this issue indicate that phosphorylation of PLS3 by PKCd induces PLS3 activation to facilitate mitochondrial targeting of tBid [23,51]. By contrast, activation of PKCe before ischemia protects mitochondrial function and diminishes apoptosis, as previously noted in detail [45–47]. Thus, the issue of how two highly homologous PKC isoforms can play such opposing roles through the regulation of mitochondrial function remains to be solved. Finally, it should be noted that mitochondrial dysfunction and PKCe have been implicated in the pathogenesis of insulin resistance and type 2 diabetes. There is much experimental evidence suggesting that mitochondrial dysfunction is involved in diabetes [52]. Linked to this topic, Zhang et al. 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MINIREVIEW Protein kinase Ce: the mitochondria-mediated signaling pathway Hiromichi Yonekawa and Yoshiko Akita Department of Laboratory Animal Science, The Tokyo