Regulation of p38, PKC/Foxo3a/p73 Signaling Network by GTP During Erythroid Differentiation in Chronic Myelogenous Leukemia

It is well established that Foxo3a is a fundamental module of signal transduction pathways regulating erythropoiesis; however, precise mechanism which regulates its physiological function still remains unclear. Here, our results revealed that the nuclear localization and stability of Foxo3a were modulated by the physical interaction of PKC and p38 signaling elements and that direct interactions led to phosphorylation of threonine residue(s) in Foxo3a. In addition, our findings revealed that the sequential activity of Foxo3a by guanosine 5′-triphosphate can impede cellular proliferation and suppress p73 expression as oncoprotein in K562 cells; thus identifying Foxo3a as a tumor suppressor in these p53 null cells. However, down-regulation of Foxo3a-dependent p73 expression causes cell differentiation along the erythroid lineage. Collectively, our findings suggest that restoration of Foxo3a function by pharmacological agents under the influence of specific activated protein kinases might constitute a potential therapeutic strategy for combating the CML disease.     

Death-associated protein 3 localizes to the mitochondria and is involved in the process of mitochondrial fragmentation during cell death

Death-associated protein 3 (DAP3) was previously isolated in our laboratory as a positive mediator of cell death. It is a 46-kDa protein containing a GTP binding domain that was shown to be essential for the induction of cell death. DAP3 functions downstream of the receptor signaling complex, and its death-promoting effects depend on caspase activity. Recent reports have suggested that DAP3 is localized to the mitochondria, but no functional significance of this localization has been reported so far. Here, we study the sub-cellular localization and cellular function of human DAP3 (hDAP3). We found that hDAP3 is localized to the mitochondria and, in contrast to cytochrome c, is not released to the cytoplasm following several cell death signals. Overexpression of hDAP3 induced dramatic changes in the mitochondrial structure involving increased fragmentation of the mitochondria. Both the mitochondrial localization of hDAP3 and its GTP-binding activity were essential for the fragmentation. The punctiform mitochondrial morphology was similar to that observed upon treatment of HeLa cells with staurosporine. In fact, reduction of endogenous hDAP3 protein by RNA interference partially attenuated staurosporine-induced mitochondrial fission. Thus, hDAP3 is a necessary component in the molecular pathway that culminates in fragmented mitochondria, probably reflecting its involvement in the fission process. These results, for the first time, provide a specific functional role for hDAP3 in mitochondrial maintenance.     

Bnip3-mediated defects in oxidative phosphorylation promote mitophagy

The Bcl-2 proteins are best known as regulators of the intrinsic mitochondrial pathway of apoptosis. However, recent studies have demonstrated that they can also regulate autophagy. For many years, autophagy was considered to be a nonselective process where the autophagosomes randomly sequestered contents in the cytosol to supply the cells with amino acids and fatty acids during nutrient deprivation. However, it is now clear that autophagy is important for cellular homeostasis under normal conditions, and that it can be a selective process where specific protein aggregates or organelles, such as mitochondria, are targeted for removal by the autophagosomes. Removal of damaged mitochondria is essential for cellular survival, and defects in this process lead to accumulation of dysfunctional mitochondria and cell death. However, the molecular mechanism underlying the selective removal of mitochondria in cells is still poorly understood. A recent study from our laboratory demonstrates that the BH3-only protein Bnip3 is a specific activator of mitochondrial autophagy (mitophagy) and that this process is independent of its role in apoptotic signaling. Here, we discuss how Bnip3-mediated impairment of mitochondrial oxidative phosphorylation facilitates mitochondrial turnover via autophagy in the absence of permeabilization of the mitochondrial membrane and apoptosis.     

Bnip3-mediated defects in oxidative phosphorylation promote mitophagy

The Bcl-2 proteins are best known as regulators of the intrinsic mitochondrial pathway of apoptosis. However, recent studies have demonstrated that they can also regulate autophagy. For many years, autophagy was considered to be a nonselective process where the autophagosomes randomly sequestered contents in the cytosol to supply the cells with amino acids and fatty acids during nutrient deprivation. However, it is now clear that autophagy is important for cellular homeostasis under normal conditions, and that it can be a selective process where specific protein aggregates or organelles, such as mitochondria, are targeted for removal by the autophagosomes. Removal of damaged mitochondria is essential for cellular survival, and defects in this process lead to accumulation of dysfunctional mitochondria and cell death. However, the molecular mechanism underlying the selective removal of mitochondria in cells is still poorly understood. A recent study from our laboratory demonstrates that the BH3-only protein Bnip3 is a specific activator of mitochondrial autophagy (mitophagy) and that this process is independent of its role in apoptotic signaling. Here, we discuss how Bnip3-mediated impairment of mitochondrial oxidative phosphorylation facilitates mitochondrial turnover via autophagy in the absence of permeabilization of the mitochondrial membrane and apoptosis.     

Dual Function of Mitochondrial Nm23-H4 Protein in Phosphotransfer and Intermembrane Lipid Transfer: A CARDIOLIPIN-DEPENDENT SWITCH*?

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes. Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur. A protein of almost identical size was also found to be labelled in mitochondria from yeast.     

Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.     

Nicotinamide enhances mitochondria quality through autophagy activation in human cells

Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (ΔΨ m ) by accelerating autophagic degradation of mitochondria. In the NAM-treated cells, the level of LC3-II as well as the number of LC3 puncta and lysosomes co-localizing with mitochondria substantially increased. Furthermore, in the NAM-treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ΔΨ m . All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD⁺. Taken together with our previous findings, these results suggest that up-regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy-mediated mitochondria quality maintenance.     

Phosphorylation of rat liver mitochondrial glycerol-3-phosphate acyltransferase by casein kinase 2

We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed.     

Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin

Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.