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

AbstractHere we describe the interaction of phosphorylated ∼40kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.Structured summaryMINT-7891324, MINT-7891304, MINT-7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006

Serine 62 is a phosphorylation site in folliculin, the Birt–Hogg–Dubé gene product

AbstractRecently, it was reported that the product of Birt–Hogg–Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5′-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN–AMPK complex. These results suggest that function(s) of FLCN–AMPK–FNIP complex is regulated by Ser62 phosphorylation.Structured summaryMINT-7298145, MINT-7298166: Flcn (uniprotkb:Q76JQ2) physically interacts (MI:0915) with AMPK alpha 1 (uniprotkb:P54645) by anti tag coimmunoprecipitation (MI:0007)MINT-7298267: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) tsc2 (uniprotkb:P49816) by protein kinase assay (MI:0424)MINT-7298182: FNIP1 (uniprotkb:Q8TF40) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)MINT-7298132: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) Flcn (uniprotkb:Q76JQ2) by protein kinase assay (MI:0424)MINT-7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007

〔報 文〕シイタケ子実体のスーパーオキシドジスムターゼの精製と諸性質

Crude extract of Lentinus edodes(L. edodes)contained more than three kinds of superoxide dismutases(SODs)which were distinguished electrophoretically. All of these were found to be insensitive to H2O2 and cyanide. A centrifugal study of the crude extract in 0.25M sucrose solution revealed that most of the SOD activities are located in the cytoplasmic fraction. We purified one of these superoxide dismutases from pilei of L. edodes to homogeneity by ammonium sulfate fractionation, DE-32 ion-exchange, Sephadex G-100 gel filtration and Butyl-toyopearl hydrophobic chromatographies. The purified enzyme showed that a single protein band coincided with the single SOD activity band in native polyacrylamide gel electrophoresis(PAGE). The purified SOD showed a single band in PAGE in the presence of sodium dodecyl sulfate(SDS-PAGE)and its subunit molecular weight was estimated to 23±0.5kDa. The subunit molecular weight of SOD was also estimated by LC-MS analysis to be 22,184Da. Using the sedimentation equilibrium centrifugation method the molecular mass of native SOD was estimated to be 84,240Da. These observations suggest that the enzyme is a tetramer composed of subunits of equal size. Metal analysis of the native enzymes revealed 0.64g-atoms Mn per mole subunit in the preparations whose specific activity were 3500U/mg. Direct analysis of N-terminal amino acid of this enzyme by Edman degradation using a protein sequencer cannot detect any amino acid residue, but amino acid residue of N-terminal appeared as serine residue after the treatment of the enzyme with tetrafluoroacetic acid in a vapor phase at 60℃ for 10min. Therefore, the N-terminal amino acid was modified with acetyl group. The modification of N-terminal amino acid is the first example of Mn-SOD. Some of other physiological and biochemical properties of the enzyme were also investigated

A novel and simple method for the generation of functional human dendritic cells from unfractionated peripheral blood mononuclear cells within 2 days: its application for induction of HIV-1-reactive CD4+ T cells in the hu-PBL SCID mice

Because dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. Generally, monocyte-derived DCs (MDDCs) were generated from purified monocytes by multiple steps of time-consuming physical manipulations for an extended period cultivation. In this study, we developed a novel, simple and rapid method for the generation of type-1 helper T cell (Th1)-stimulating human DCs directly from bulk peripheral blood mononuclear cells (PBMCs). PBMCs were cultivated in the presence of 20 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/ml of interleukin-4 (IL-4) and 1,000 U/ml of interferon-β (IFN-β) for 24 hours followed by 24 hour maturation with a cytokine cocktail containing 10 ng/ml of tumor necrosis factor-α (TNF-α), 10 ng/ml of IL-1β and 1 μg/ml of prostaglandin E2 (PGE2). The phenotype and biological activity of these new DCs for induction of allogeneic T cell proliferation and cytokine production were comparable to those of the MDDCs. Importantly, these new DCs pulsed with inactivated HIV-1 could generated HIV-1-reactive CD4+ T cell responses in humanized mice reconstituted with autologous PBMCs from HIV-1-negative donors. This simple and quick method for generation of functional DCs will be useful for future studies on DC-mediated immunotherapies