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Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser͞Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G 1 and G2͞M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5͞eIF2͞eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation. C asein kinase 2 (CK2) (1-4) is composed of two subunits, ␣ or ␣Ј and ␤, which combine to form a native ␣ 2 ␤ 2 tetramer. Disruption of the catalytic subunits (␣ and ␣Ј) is lethal in Saccharomyces cerevisiae (5) and disruption of the regulatory ␤ subunit in mice leads to early embryonic lethality (6). CK2 phosphorylates a range of cellular targets in a variety of subcellular sites and appears to be highly pleiotropic; it is involved in many key biological functions, including growth and cell cycle control (7), signal transduction (3), circadian rhythms (8, 9), and gene expression (10, 11). CK2 is also a stress-activated kinase and might participate in the transduction of survival signals to avoid damage by mutagenic UV radiation (12, 13). An important role for CK2 in promoting cell proliferation and transformation has been indicated by several studies. In mammalian systems, its targeted overexpression in mice results in the development of T cell lymphoma and mammary tumorigenesis (5). Despite these findings, there is still much uncertainty regarding the activation of CK2 in response to stimuli (14). The mechanism by which it is regulated and its precise function in cell cycle progression and proliferation is still poorly understood.CK2 activity and stability are believed to be regulated in part by holoenzyme formation via a self-assembly mechanism and by phosphorylation. Phosphorylation by p34 cdc2 of the catalytic ␣ subunit at the C-terminal domain occurs in ...

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Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown etiology. We previously revealed increased oxidative stress and high expression of antioxidant proteins in culture cell lines established from lesional lung tissues with IPF (Kabuyama Y, Oshima K, Kitamura T, Homma M, Yamaki J, Munakata M, Homma Y. Genes Cells 12: 1235-1244, 2007). In this study, we show that IPF cells contain high levels of free cholesterol and its peroxidized form as compared with normal TIG7 lung fibroblasts, suggesting that radical oxygen species (ROS) are generated within specific organelles. To understand the molecular basis underlying the generation of ROS in IPF cells, we performed proteomic analysis of mitochondrial proteins from TIG and IPF cells. This analysis shows that the phosphorylation of Ser586 of very long chain acyl-CoA dehydrogenase (VLCAD) is significantly reduced in IPF cells. Similar results are obtained from immunoblotting with anti-pS586 antibody. Kinase activity toward a peptide containing Ser586 from IPF cells is significantly lower than that from TIG cells. Furthermore, a phosphorylation-negative mutant (S586A) VLCAD shows reduced electron transfer activity and a strong dominant-negative effect on fatty acid beta-oxidation. The ectopic expression of the S586A mutant induced human embryonic kidney (HEK) 293 cells to produce significantly high amounts of oxidized lipids and hydrogen peroxide. HEK293 cells expressing the S586A mutant exhibit a reduction in cell growth and an enhancement in apoptosis. These results suggest a novel regulatory mechanism for homeostatic VLCAD activity, whose dysregulation might be involved in the production of oxidative stress and in the pathogenesis of IPF.

Development of new immunosuppressing agents is necessary in organ transplantation or immune diseases. Because Ppc-1 exhibits a suppressing effect on interleukin-2 (IL2) production in Jurkat cells, we synthesized and screened Ppc-1 derivatives that preserve prenylated quinolinecarboxylic acid (PQA) structure, and identified compound 18 (PQA-18) as a novel molecule with immunosuppressing effect. PQA-18 suppressed not only IL2 but also IL4, IL6, and tumor necrosis factor-α production in human peripheral lymphocytes without affecting cell viability. Two-dimensional gel electrophoresis analysis and in vitro kinase assay revealed that PQA-18 inhibits kinase activity of p21-activated kinase 2 (PAK2). Administration of PQA-18 by intraperitoneal injection suppressed the population of a subset of regulatory T cells and the immunoglobulin (Ig) production against T cell-dependent antigens in mice. Treatment with the PQA-18 ointment on Nc/Nga mice, a model of human atopic dermatitis, improved skin lesions and serum IgE levels. These results suggest that PQA-18 is a unique PAK2 inhibitor with potent immunosuppressing effects in vitro and in vivo. PQA-18 may be a valuable lead for the development of novel immunosuppressants.

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Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B-cell function and development, we created transgenic (Tg) mice expressing a phosphorylation-defective mutant of succinate dehydrogenase A in B cells (bSDHA ). Splenic B cells in male, but not female, bSDHA mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG , and IgG , and affinity maturation of IgG against T-cell-dependent antigens. Following immunization, the male bSDHA mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca mobilization. Notably, the expression levels of B-cell co-receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHA mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B-cell immunity may be more labile to oxidative stress in male mice than in female mice.