Miwako K. Homma

Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein

Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2α- and β-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G2/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.

Involvement of EphB1 receptor/EphrinB2 ligand in neuropathic pain.

Study Design. We investigated involvement of EphB/ephrinB system in neuropathic pain. Objective. Using immunoblotting, immunohistochemistry, and RNA interference techniques, we examined the expression levels of EphB receptors and ephrinB ligands in neuropathic pain. We also explored the effect of ephrinB siRNA for neuropathic pain. Summary of Background Data. It has been reported that EphB2 regulates the development of synaptic plasticity in the hippocampus by interacting with N-methyl-D-aspartate (NMDA) receptors. In acute pain models, it has been clear that EphB1/ephrinB2 interactions via the NMDA receptor modulates synaptic efficacy in spinal cord. Methods. Adult female Sprague-Dawley rats were used in this study. A crush injury model was prepared by crushing the left L5 spinal nerve distal to dorsal root ganglions (DRG) under deep anesthesia. The sham operation was subjected as control. Expression of ephrinB2 and EphB1 were examined by immunoblotting and immunohistochemical analyses with anti-EphB and anti-ephrinB antibodies. To assess involvement of ephrinB in neuropathic pain, we examined the effect of small interference RNA (siRNA) on mechanical allodynia. Results. Among EphB and ephrinB isoforms tested, ephrinB2 and EphB1 were predominant in DRG and spinal cord. Results showed that the expression of ephrinB2 was enhanced in neurons in DRG and spinal cord by the injury in a time-dependent manner. EphB1 was expressed in neurons of spinal cord. Administration of ephrinB2 siRNA reduced the expression of ephrinB2 and mechanical allodynia. Conclusion. Expression of ephrinB2 is enhanced by nerve injury in neurons in DRG and spinal cord, while its receptor EphB1 is expressed in spinal cord. These results suggest that induction of ephrinB2 might activate EphB1/ephrinB2 signaling pathway to regulate synaptic plasticity and reorganization, and that ephrinB2 siRNA could be a potential therapeutic agent for neuropathic pain.

Involvement of ErbB-2 in rheumatoid synovial cell growth

OBJECTIVE The synovial tissue affected by rheumatoid arthritis (RA) is characterized by hyperproliferation of synovial cells. High amounts of epidermal growth factor (EGF) in the synovial fluid of RA patients contribute to the growth of rheumatoid synovial cells. To characterize the receptor for EGF in rheumatoid synovial cells, the expression and function of ErbB family members were examined. METHODS Synovial tissues were obtained from surgical excisions. The expression of ErbB products was examined by immunohistochemistry and immunoblotting by using specific antibodies. Primary cultures were established from the surgical materials. Cell growth was measured using MTT. The levels and phosphorylation state of the ErbB-2 protein were analyzed by immunoprecipitation and immunoblotting. RESULTS The expression of ErbB-2, but not other ErbB-related products, was detected in synovium with RA as compared with that with osteoarthritis (OA) and ligament injury. Growth of primary synovial cells with RA was inhibited by genistein, a tyrosine kinase inhibitor, and herceptin, a specific monoclonal antibody against ErbB-2. Herceptin showed a small effect on growth of primary synovial cells with OA. EGF stimulated the phosphorylation of ErbB-2 in primary synovial cells with RA. This EGF-stimulated phosphorylation was completely abrogated by genistein and herceptin. CONCLUSION ErbB-2 is expressed in rheumatoid synovial cells and may function as the receptor for EGF. Our data suggest that mitotic signals from EGF family members are transduced by ErbB-2 in these cells. Inhibition of ErbB-2 may provide a new approach to the effective treatment for RA.

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression.

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr 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 G1 and G2/M 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 eIF5/eIF2/eIF3 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.

Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components.

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.

Cell cycle and activation of CK2

Casein kinase 2 (CK2) is a highly conserved and ubiquitous eukaryotic Ser/Thr protein kinase. Genetic, biochemical, and cell biological studies have indicated the involvement of this enzyme in the control of cell proliferation and in signal transduction. The regulation of CK2 is not well defined, and it has been considered a constitutively non-regulated protein kinase. However, we show that CK2 activation occurred during the progression of cell cycle in response to FBS stimuli of G0 arrested cells. Importantly, we show that as the downstream target for CK2, the phosphorylation of eukaryotic translation-initiation factor eIF5 by CK2 may play a critical role in cell cycle progression. We find that eIF5 is associated with CK2 when the kinase activity is at the highest level in vivo, and is phosphorylated at Ser389 and Ser390 by CK2. Expression of eIF5 mutants that lack those phosphorylation sites reveals that these mutants have a dominant-negative effect on phosphorylation of endogenous eIF5, as well as a significant reduction in the formation of the mature complex, in the growth rate, and the expression of cell cycle-regulated proteins. Also, a pool of CK2 translocates into the nuclear fraction following its activation during the progression of the cell cycle. Consistent with these findings, we report that CK2 may be involved in the regulation of cell cycle progression through the phosphorylation of a key molecule for translation initiation and of nuclear substrates upon activation of CK2 by itself.

Involvement of thioredoxin reductase 1 in the regulation of redox balance and viability of rheumatoid synovial cells.

Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patients with RA by oligonucleotide microarrays. Gene expression profiles clearly suggested that oxidative stress is enhanced in RA synovial cells, which was confirmed by measuring cellular levels of reactive oxygen species. One of the highly up-regulated proteins in RA synovial cells was thioredoxin reductase 1 (TRXR1), a protein that plays an important role in antioxidant defense system. Subsequent analysis demonstrated that TRXR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Thus, our results reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.

Involvement of selenoprotein P in the regulation of redox balance and myofibroblast viability in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease of unknown etiology, is characterized by the expansion of myofibroblasts and abnormal deposition of extracellular matrix in the lung parenchyma. To elucidate the molecular mechanisms that lead to IPF, we analyzed myofibroblasts established from patients with IPF by oligonucleotide microarrays. Gene expression profiles clearly suggested that lipid peroxidation is enhanced in myofibroblasts, which was confirmed by measuring cellular lipid hydroperoxides. One of the most highly up‐regulated proteins in myofibroblasts was selenoprotein P, an antioxidant protein not previously associated with IPF. Subsequent analysis demonstrated that selenoprotein P reduces lipid hydroperoxides and maintains the viability of myofibroblasts. Thus, our results reveal a novel pathophysiologic function of myofibroblasts as a generator of lipid hydroperoxides, and a self‐defense mechanism against self‐generated oxidative stress.

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation.

Abstract The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal–regulated kinase was specifically activated within 1 min after UV irradiation in the range 320–360 nm. In contrast, P38 kinase was activated by 270–280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.

Dysregulation of very long chain acyl-CoA dehydrogenase coupled with lipid peroxidation.

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.