Differential modulation of mitochondrial OXPHOS system during HIV-1 induced T-cell apoptosis: up regulation of Complex-IV subunit COX-II and its possible implications

Human Immunodeficiency Virus-1 (HIV-1) infection leads to CD4+ T cell depletion primarily by apoptosis employing both intrinsic and extrinsic pathways. Although extensive literature exists about the role of mitochondrial proteins in HIV induced T cell apoptosis, there is little understanding about the role of different components of mitochondrial oxidative phosphorylation (OXPHOS) system in apoptosis. The OXPHOS system comprises of five enzyme complexes (Complex I, II, III, IV, V), subunits of which have been implicated in various functions in addition to their primary role in energy generating process. Here using differential gene expression analysis, we report that Cytochrome Oxidase-II (COX-II), a subunit of Complex-IV is induced in HIV infected apoptotic T-cells. We also observe a temporal up regulation of this subunit across different T-cell lines and in human PBMCs. Further analysis indicates increase in expression of majority of Complex-IV subunits with concomitant increase in Complex-IV activity in HIV infected T cells. Silencing of COX-II expression leads to reduced apoptosis in infected T-cells, indicating its importance in apoptosis. Furthermore, our results also show that the activities of enzyme complexes I, II and III are decreased while those of Complex IV and V are increased at the time of acute infection and apoptosis. This differential regulation in activities of OXPHOS system complexes indicate a complex modulation of host cell energy generating system during HIV infection that ultimately leads to T cell apoptosis.     

HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.     

Fyn kinase regulates translation in mammalian mitochondria

BackgroundMitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known.MethodsMitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches.ResultsHere, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes.Conclusions and general significanceOur findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.     

In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis

Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress, OXPHOS     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

The cell death regulator GRIM-19 is involved in HIV-1 induced T-cell apoptosis

One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-β and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.     

Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3-L1 cells.

Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.     

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 ρ(0) cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ(0) cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.     

Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes

Src-family kinases (SFKs) are co-expressed with multiple combinations of each member in a single cell and involved in various signalings. Recently, we showed by sucrose-density gradient fractionation that the subcellular distribution of c-Src is distinct from that of Lyn. However, little is known about the trafficking of c-Src in living cells. Here, we show by time-lapse monitoring combined with photobleaching techniques that c-Src, a non-palmitoylated SFK, is rapidly exchanged between the plasma membrane and intracellular organelles representing late endosomes/lysosomes possibly through its cytosolic release. Although Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi apparatus along the secretory pathway, lack of palmitoylation directs Lyn away from the exocytotic transport to the c-Src-type trafficking between the plasma membrane and late endosomes/lysosomes. Intriguingly, c-Src and a non-palmitoylated Lyn mutant are efficiently delivered and immobilized to focal adhesions when their SH2 domains are able to mediate protein-protein interactions in place of intramolecular bindings. However, palmitoylation of Lyn inhibits its recruitment to focal adhesions. These results suggest that palmitoylation of SFKs is critical for SFK localization and trafficking and implicate that two distinct trafficking pathways for SFKs may be involved in SFKs' specific functions.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

Metabolic rewiring in cancer cells overexpressing the glucocorticoid-induced leucine zipper protein (GILZ): Activation of mitochondrial oxidative phosphorylation and sensitization to oxidative cell death induced by mitochondrial targeted drugs

Cancer cell metabolism is largely controlled by oncogenic signals and nutrient availability. Here, we highlighted that the glucocorticoid-induced leucine zipper (GILZ), an intracellular protein influencing many signaling pathways, reprograms cancer cell metabolism to promote proliferation. We provided evidence that GILZ overexpression induced a significant increase of mitochondrial oxidative phosphorylation as evidenced by the augmentation in basal respiration, ATP-linked respiration as well as respiratory capacity. Pharmacological inhibition of glucose, glutamine and fatty acid oxidation reduced the activation of GILZ-induced mitochondrial oxidative phosphorylation. At glycolysis level, GILZ-overexpressing cells enhanced the expression of glucose transporters in their plasmatic membrane and showed higher glycolytic reserve. ¹H NMR metabolites quantification showed an up-regulation of amino acid biosynthesis. The GILZ-induced metabolic reprograming is present in various cancer cell lines regardless of their driver mutations status and is associated with higher proliferation rates persisting under metabolic stress conditions. Interestingly, high levels of OXPHOS made GILZ-overexpressing cells vulnerable to cell death induced by mitochondrial pro-oxidants. Altogether, these data indicate that GILZ reprograms cancer metabolism towards mitochondrial OXPHOS and sensitizes cancer cells to mitochondria-targeted drugs with pro-oxidant activities.     

The promoting role of lysosome-localized c-Src in autophagosome-lysosome fusion

Src-family kinases (SFKs), such as c-Src, Lyn and Fyn, belong to non-receptor-type tyrosine kinases and play key roles in cell proliferation, adhesion, and migration. SFKs are anchored to the plasma membrane, Golgi membranes and lysosomal membranes through lipid modifications. Although the functions of SFKs being localized to the plasma membrane are intensively studied, those of SFKs being localized to organelle membranes are poorly understood. Here, we show that, among SFKs, c-Src in particular is involved in a decrease in the amount of LC3-II. c-Src and non-palmitoylated Lyn [Lyn(C3S) (cysteine-3 → serine-3)], which are localized onto lysosomes, decrease the amount of LC3-II and treatment with SFK inhibitors increases the amount of LC3-II, suggesting the importance of SFKs' lysosomal localization for a change of autophagic flux in a kinase activity-dependent manner. Colocalization of LC3-II with the lysosome-associated membrane protein LAMP1 shows that lysosome-localized SFKs promote the fusion of autophagosomes with lysosomes. Lysosome-localized SFKs play a positive role in the maintenance of cell viability under starvation conditions, which is further supported by knockdown of c-Src. Therefore, our results suggest that autophagosome-lysosome fusion is promoted by lysosome-localized c-Src, leading to cell survival under starvation conditions.     

Essential roles of mitochondrial and heme function in lung cancer bioenergetics and tumorigenesis

Contrary to Warburg’s hypothesis, mitochondrial oxidative phosphorylation (OXPHOS) contributes significantly to fueling cancer cells. Several recent studies have demonstrated that radiotherapy-resistant and chemotherapy-resistant cancer cells depend on OXPHOS for survival and progression. Several cancers exhibit an increased risk in association with heme intake. Mitochondria are widely known to carry out oxidative phosphorylation. In addition, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in OXPHOS and oxygen consumption. Further, lung cancer cells exhibit heme accumulation and require heme for proliferation and invasion in vitro. Abnormalities in mitochondrial biogenesis and mutations are implicated in cancer. This review delves into mitochondrial OXPHOS and lesser explored area of heme metabolism in lung cancer.     

Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.     

Interaction between the SH3 domain of Src family kinases and the proline-rich motif of HTLV-1 p13: a novel mechanism underlying delivery of Src family kinases to mitochondria

The association of the SH3 (Src homology 3) domain of SFKs (Src family kinases) with protein partners bearing proline-rich motifs has been implicated in the regulation of SFK activity, and has been described as a possible mechanism of relocalization of SFKs to subcellular compartments. We demonstrate in the present study for the first time that p13, an accessory protein encoded by the HTLV-1 (human T-cell leukaemia virus type?1), binds the SH3 domain of SFKs via its C-terminal proline-rich motif, forming a stable heterodimer that translocates to mitochondria by virtue of its N-terminal mitochondrial localization signal. As a result, the activity of SFKs is dramatically enhanced, with a subsequent increase in mitochondrial tyrosine phosphorylation, and the recognized ability of p13 to insert itself into the inner mitochondrial membrane and to perturb the mitochondrial membrane potential is abolished. Overall, the present study, in addition to confirming that the catalytic activity of SFKs is modulated by interactors of their SH3 domain, leads us to hypothesize a general mechanism by which proteins bearing a proline-rich motif and a mitochondrial localization signal at the same time may act as carriers of SFKs into mitochondria, thus contributing to the regulation of mitochondrial functions under various pathophysiological conditions.     

Transforming Potential of Src Family Kinases Is Limited by the Cholesterol-Enriched Membrane Microdomain

The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.Src family kinases (SFKs) are membrane-associated, non-receptor protein tyrosine kinases involved in a variety of intracellular signaling pathways (5). SFKs are comprised of eight members in mammals: c-Src, Fyn, c-Yes, Lyn, Lck, Hck, c-Fgr and Blk. Among these, c-Src, Fyn, and c-Yes are ubiquitously expressed, whereas the others are relatively concentrated in hematopoietic cell lineages. The intracellular distribution of each SFK also varies depending on their unique N-terminal sequences and acyl modifications (5, 27). These distinctive features of SFKs suggest that each SFK member plays a unique role in particular tissues or cells. In contrast, it is also known that SFKs have redundant and pleiotropic functions in regulating critical cellular events, such as cell division, motility, adhesion, angiogenesis, and survival (26). In a variety of human cancers, protein levels and/or specific activities of c-Src and c-Yes are frequently upregulated (13, 35). Upregulation of Lyn, Lck, Hck, c-Fgr, or Blk is also observed in some leukemias and lymphomas (10, 16, 26). These observations imply a role for SFKs in cell transformation, tumorigenesis, and metastasis (31). However, because SFK genes are rarely mutated in human cancers (31), the mechanisms underlying their upregulation in these cancers remain unclear. Furthermore, the distinctive expression patterns and functional redundancy among SFK members have hampered concurrent analyses of their intrinsic transforming abilities and contribution to cancer progression.In normal cells, the kinase activity of SFKs is negatively regulated by the phosphorylation of its C-terminal regulatory Tyr residue by C-terminal Src kinase (Csk) (21, 22). The cytoplasmic Csk requires Csk-binding scaffold proteins to gain efficient access to membrane-bound SFKs. Previously, we identified a transmembrane adaptor protein, Cbp (also known as PAG1), as a specific Csk-binding protein. Cbp/PAG1 is exclusively localized to a membrane microdomain enriched by cholesterol and sphingolipids and plays a scaffolding role for Cbp/PAG1 in Csk-mediated negative regulation of SFKs (3, 15). We also reported that expression of Cbp/PAG1 is noticeably downregulated by c-Src transformation and in some human cancer cells and that reexpression of Cbp/PAG1 can suppress c-Src-induced transformation and tumorigenesis (23). In addition, we showed that Cbp/PAG1 suppressed c-Src function independently of Csk by directly sequestering activated c-Src in the membrane microdomain. These findings suggest a potential role for Cbp/PAG1 as a suppressor for c-Src-mediated cancer progression. However, whether Cbp/PAG1 would serve as a suppressor for other SFK members and whether other microdomain adaptors, such as LIME (4, 11), would also contribute to the suppression of SFK-mediated transformation have yet to be examined.The membrane microdomain has been regarded as a signaling platform that harbors various signaling molecules and positively transduces cell signaling evoked by activated receptors (29). This model has been best exemplified in immunoreceptor-mediated signaling (8). Moreover, it was reported that SFKs could function positively when bound to Cbp/PAG1 in the microdomain (30, 32). Such positive roles of the microdomain in cell signaling are apparently inconsistent with its suppressive role in Src-mediated transformation. However, this discrepancy rather raises the possibility that the membrane microdomain would function to segregate or protect the normal signaling pathway from the transforming pathways. To prove this hypothesis, more extensive analysis of the role of the membrane microdomain in controlling cell transformation remains to be performed (28).To elucidate the role of the membrane microdomain in regulating the functions of SFKs, we first compared the transforming abilities of all SFK members using Csk-deficient cells, a reconstitution system in which wild-type SFKs can induce cell transformation (24), and we evaluated the relevance of the membrane distribution of SFKs to their transforming activities. We then investigated the role of the microdomain by disrupting or enhancing its function using methyl-β-cyclodextrin (MβCD) and a microdomain-specific adaptor, Cbp/PAG1, respectively. Our results show that the membrane microdomain and Cbp/PAG1 spatially limit the oncogenic potential of SFKs by sequestering them away from the transforming pathways.     

A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential

Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions.