Takeshi Baba

Actin Tyrosine Dephosphorylation by the Src Homology 1-Containing Protein Tyrosine Phosphatase Is Essential for Actin Depolymerization After Membrane IgM Cross-Linking

Src homology protein 1 (SHP-1) plays an important role in B cell Ag receptor (BCR) differentiation, proliferation, survival, and apoptosis. After BCR stimulation in apoptotic cells, SHP-1 has been shown to be recruited to phosphorylated immunoreceptor tyrosine-based inhibitory motifs present in receptors such as CD22 and CD72. However, the substrates of SHP-1 in the chicken B cell line, DT40, have remained undefined. To identify SHP-1 substrates in DT40, we used a trapping mutant, SHP-1 C/S (a catalytically inactive form). Cross-linking of BCR induced hyperphosphorylation of ∼44-kDa protein in C/S transfectants. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed that this was actin (cytoplasmic type 5) carrying three immunoreceptor tyrosine-based inhibitory motif-like sequences. SHP-1 was shown to bind to one of these sequences in synthetic peptide binding experiment. Thus, actin is a direct SHP-1 substrate. Furthermore, more SHP-1 molecules translocate into lipid rafts, and their association with actin was increased after BCR stimulation. In C/S transfectants, actin polymerization induced by membrane IgM ligation was sustained to a greater extent for a longer time compared with wild-type transfectants. Therefore, actin dephosphorylation by SHP-1 is essential for actin depolymerization after BCR stimulation. Our data suggest that SHP-1 plays a pivotal role in reorganization of cytoskeletal architecture inducing actin dephosphorylation. These results clearly demonstrate the direct interaction of SHP-1 with actin.

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

MINT‐7891324, MINT‐7891304, MINT‐7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)

Mass spectrometric identification of tryptophan nitration sites on proteins in peroxynitrite-treated lysates from PC12 cells.

One of the important sites of peroxynitrite action that affects cellular function is known to be nitration of tyrosine residues. However, tryptophan residues could be another target of peroxynitrite-dependent modification of protein function, as we have shown previously using a model protein (F. Yamakura et al., J. Biochem. 138:57-69; 2005). Here, we report the identification of several proteins that allowed us to determine the position of nitrotryptophan in their amino acid sequences in a more complex system. We modified lysates from PC12 cells with and without nerve growth factor (NGF) by treatment with peroxynitrite (0.98 or 4.9 mM). Western blot analyses using anti-6-nitrotryptophan antibody showed several immunoreactive bands and spots, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. We identified several tryptic peptides including nitrotryptophan residues, which were derived from L-lactate dehydrogenase A, malate dehydrogenase 1, M2 pyruvate kinase, and heat-shock protein 90 α, in peroxynitrite-treated lysates from PC12 cells, and l-lactate dehydrogenase A, malate dehydrogenase 1, transaldorase, and lactoylglutathione lyase, in peroxynitrite-treated lysates from NGF/PC12 cells. The molar ratio of 3-nitrotyrosine to 6-nitrotryptophan in protease-digested PC12 cell lysates treated with peroxynitrite was determined to be 5.8 to 1 by using an HPLC-CoulArray system. This is the first report to identify several specific sites of nitrated tryptophan on proteins in a complex system treated with peroxynitrite and to compare the susceptibility of nitration between tryptophan and tyrosine residues of the proteins.

Dual regulation of BCR‐mediated growth inhibition signaling by CD72

CD72 has been reported to regulate BCR‐mediated signals both positively and negatively. SHP‐1 and Grb2 bind, respectively, to ITIM1 and ITIM2 of CD72. We generated transformed B cell lines with an immature phenotype following J2 virus infection of splenocytes from CD72–/– and wild‐type (Wt) mice. The transformed lines were infected with retroviral vectors carrying Tyr (Y) to Phe (F) substitutions in the ITIM sequences (ITIM1 mutated: Y7/F; ITIM2 mutated: Y39/F; and both ITIM mutated: Y7,39/F). Cross‐linking of the BCR induced growth inhibition in transfectants expressing Wt CD72, but this response was less sensitive in transfectants with Y7,39/F. The Y7/F transfectants demonstrated the least sensitive response. We were not able to obtain transfectants with Y39/F, suggesting that CD72 associated with SHP‐1, but not with Grb2, delivers a strong negative signal. Pre‐ligation of CD72, which induces dephosphorylation of the molecule, partially rescued the Wt transfectants from growth inhibition, leading to a growth response profile similar to that of Y7,39/F transfectants. These results suggest that ITIM1/SHP‐1 delivers a very strong negative signal that is down‐modulated by signals through ITIM2/Grb2, leading to delivery of an attenuated negative signal. Thus, pre‐ligation of CD72 results in the manifestation of an ostensible positive signal.

Nitration of tryptophan in ribosomal proteins is a novel post-translational modification of differentiated and naïve PC12 cells.

Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels (p<0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC-ESI-MS-MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.

Myosin is an in vivo substrate of the protein tyrosine phosphatase (SHP-1) after mIgM cross-linking

SHP-1 plays an important role in negative signaling in many cell types. For example, after BCR stimulation in apoptotic B cells, SHP-1 has been shown to be recruited to phosphorylated ITIMs present in receptors such as CD72. However, the SHP-1 substrates in the chicken B cell line, DT40, have been poorly undefined. To identify SHP-1 substrates in DT40, we used a trapping mutant SHP-1 C/S (a catalytically inactive form). BCR stimulation induced hyper-phosphorylation of 230 kDa protein in C/S transfectants. MALDI-TOF/MS analysis revealed that this was myosin carrying ITIM. SHP-1 was shown to bind to this ITIM in synthetic peptide binding experiment. Thus, myosin is a direct SHP-1 substrate in B cells. The results suggest that SHP-1 plays a critical role in the reorganization of cytoskeletal architecture mediated via BCR stimulation.

Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor‐A (VEGF) biologics, termed VEGF “Sticky‐traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky‐traps could locally inhibit angiogenesis to at least the same extent as the original VEGF‐trap that also gains whole‐body access. Sticky‐traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky‐trap remained localized to various regions of the eye, such as the inner‐limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky‐trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre‐maturity.

Highly efficient site-specific transgenesis in cancer cell lines

BackgroundTransgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells.MethodsAccording to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31.ResultsUsing the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format.ConclusionsThe novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes.

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Accumulation of immunoglobulin G against Dermatophagoides farinae tropomyosin in dorsal root ganglia of NC/Nga mice with atopic dermatitis-like symptoms

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.