Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H₂O₂, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase α chain is probably tyrosine-phosphorylated, but demonstrated that the β chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.Received 7 January 2005; received after revision 19 April 2005; accepted 22 April 2005     

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD⁺ and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD⁺ and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion. Received 27 July 2006; accepted 26 October 2006     

Ethanol inhibits insulin expression and actions in the developing brain

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.Received 30 December 2004; received after revision 1 March 2005; accepted 10 March 2005     

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, ³²P-poly(Glu-Tyr)_4:1 and ³²P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.Received 17 May 2004; received after revision 20 July 2004; accepted 26 July 2004     

The tyrosine phosphatase SHP-1 negatively regulates cytotrophoblast proliferation in first-trimester human placenta by modulating EGFR activation

Insulin-like growth factors (IGFs) influence placental cell (cytotrophoblast) kinetics. We recently reported that the protein tyrosine phosphatase (PTP) SHP-2 positively regulates IGF actions in the placenta. In other systems, the closely related PTP, SHP-1, functions as a negative regulator of signaling events but its role in the placenta is still unknown. We examined the hypothesis that SHP-1 negatively regulates IGF actions in the human placenta. Immunohistochemical (IHC) analysis demonstrated that SHP-1 is abundant in cytotrophoblast. SHP-1 expression was decreased in first-trimester placental explants using siRNA; knockdown did not alter IGF-induced proliferation but it significantly enhanced proliferation in serum-free conditions, revealing that placental growth is endogenously regulated. Candidate regulators were determined by using antibody arrays, Western blotting, and IHC to examine the activation status of multiple receptor tyrosine kinases (RTKs) in SHP-1-depleted explants; amongst the alterations observed was enhanced activation of EGFR, suggesting that SHP-1 may interact with EGFR to inhibit proliferation. The EGFR tyrosine kinase inhibitor PD153035 reversed the elevated proliferation seen in the absence of SHP-1. This study demonstrates a role for SHP-1 in human trophoblast turnover and establishes SHP-1 as a negative regulator of EGFR activation. Targeting placental SHP-1 expression may provide therapeutic benefits in common pregnancy conditions with abnormal trophoblast proliferation.     

Apolipoprotein CIII hyperactivates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca²⁺ (Ca_V) channels in the pancreatic β cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates β cell Ca_V channels. We now demonstrate that ApoCIII increased Ca_V1 channel open probability and density. ApoCIII enhanced whole-cell Ca²⁺ currents and the Ca_V1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of β1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating β cell Ca_V channels. These data reveal that ApoCIII hyperactivates β cell Ca_V1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src.     

Recruitment of Pyk2 to SHPS-1 signaling complex is required for IGF-I-dependent mitogenic signaling in vascular smooth muscle cells

In vascular smooth muscle cells, IGF-I stimulates SHPS-1/SHP2/Src complex formation which is required for IGF-I-stimulated cell proliferation. Using SHP2/Src silencing and a Pyk2/Y402F mutant, we showed that Pyk2 was also recruited to the SHPS-1 complex. Pyk2 recruitment to SHPS-1 is mediated via the interaction of Pyk2 Tyr402 and the Src in response to IGF-I. Following Src/Pyk2 association, Src phosphorylates Pyk2 on Tyr881 providing a binding site for Grb2. Cells expressing Pyk2/Y881F showed decreased Grb2 recruitment to SHPS-1 and impaired Shc/Grb2 association. This change led to reduced Erk1/2 (MAP kinase) activation and cell proliferation in response to IGF-I. Our results show that, following its recruitment to the SHPS-1 signaling complex, Pyk2 localizes Grb2 in close proximity to Shc thereby facilitating Shc/Grb2 association which leads to Erk1/2 activation in response to IGF-I. Thus, Pyk2 recruitment to SHPS-1 plays an important role in regulating the IGF-I-stimulated mitogenic response.     

Does an inhibitor of mitochondrial adenylate kinase also affect oxidative phosphorylation?

Adenylate kinase activity of intact mitochondria is strongly inhibited by Ap5A, i.e. p1,p5-Di (adenosine-5') pentaphosphate, whereas oxidative phosphorylation is not affected. Therefore, Ap5A is a useful tool to distinguish between oxidative and non oxidative ATP generating reactions.     

Protective effect of N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF9601N) on mitochondrial permeability transition

PF9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine, an monoamine oxidase (MAO) B inhibitor, has shown neuroprotective properties against dopaminergic toxins. To elucidate the mechanisms involved in this protection, the effect of PF9601N on mitochondria was assessed. PF9601N prevents mitochondrial swelling, drop in the electrical potential and oxidation of sulfhydryl groups, glutathione and pyridine nucleotides induced by Ca²⁺. These observations demonstrate the protective effect of PF9601N on the induction of mitochondrial permeability transition. This protection is due to the interaction of the secondary protonated amino group in the molecule with pore-forming structures and to its antioxidant property, rather than to inhibition of MAO B activity. PF9601N also prevents the release of cytochrome c from mitochondria, suggesting its potential inhibitory effect on mitochondria-mediated apoptosis. The low IC⁵⁰ value for this inhibition, in comparison with deprenyl, make it a more efficient compound than propargylamines and other amines in protecting the bioenergetic functions of mitochondria. Received 9 March 2006; received after revision 10 April 2006; accepted 21 April 2006     

Src activation and translocation from focal adhesions to membrane ruffles contribute to formation of new adhesion sites

Cell migration requires the coordinated turnover of focal adhesions, a process that involves FAK phosphorylation. Since Src is the major kinase implicated in FAK phosphorylation, we focus here on the role of Src activation on adhesion remodelling. In astrocytoma cells, constitutively activated Src induces both FAK phosphorylation and adhesion rearrangement. To evaluate how Src controls these processes, we used a recently described Src reporter to monitor the dynamics of Src phosphorylation. Upon Src activation, focal adhesions started to disassemble while Src appeared highly expressed at newly formed membrane ruffles. Kinetic analysis of time-lapse movies showed that loss of phospho-Src at focal adhesions was time-correlated with the appearance of membrane ruffles containing phospho-Src. Moreover, FLIP analysis revealed a dynamic equilibrium of Src between focal adhesions and membrane ruffles. We conclude that upon phosphorylation, Src is directly translocated from focal adhesions to membrane ruffles, thereby promoting formation of new adhesion complexes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 21 July 2008; received after revision 10 October 2008; accepted 03 November 2008     

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.     

Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

Mitochondrial permeability transition in cardiac ischemia–reperfusion: whether cyclophilin D is a viable target for cardioprotection?

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia–reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia–reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.     

Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.