Journal:

Article Title: Caveolin-1 Maintains Activated Akt in Prostate Cancer Cells through Scaffolding Domain Binding Site Interactions with and Inhibition of Serine/Threonine Protein Phosphatases PP1 and PP2A

doi: 10.1128/MCB.23.24.9389-9404.2003

Figure Lengend Snippet: The expression of cav-1 leads to increased phosphorylation of multiple Akt substrates. The phosphorylation state of Akt substrates was analyzed using Tg-treated cav-1-expressing and control cells. Data used were from two independent viral infection experiments (indicated as #1 and #2). The numbers under each gel band are the ratios of phosphorylated protein (indicated by P- prefix before the protein name) to β-actin in the same sample. The protein expression of corresponding molecules in the same cell lysates are also shown. Note significantly increased phosphorylation levels of GSK3α/β, FKHR, and MDM2 in cav-1-expressing cells. Marginally increased phosphorylation of BAD and IKKα/β were also observed.

Article Snippet: Rabbit polyclonal antibodies against IKKα, IKKβ, phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-IKKα (Ser180)/IKKβ (Ser181), phospho-MDM2 (Ser166), phospho-FKHR (Thr24)/FKHRL1 (Thr32), phospho-tyrosine (P-Tyr-100), monoclonal antibody against phospho-CREB (Ser133), and Akt kinase assay kit were from Cell Signaling.

Techniques: Expressing, Infection

Journal: Nature Communications

Article Title: The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization

doi: 10.1038/s41467-018-03193-0

Figure Lengend Snippet: Sequence motifs and the structure of iPLA 2 β. a Domain composition of iPLA 2 β. ARs are shown in orange with the novel AR 1 in dark orange, catalytic residues are in magenta, poly-Gly region is in green, and putative CaM-binding motifs in blue. Black lines underneath mark INAD and PD mutations. b Cartoon representation of the iPLA 2 β monomer color-coded in a rainbow scheme with the N terminus in blue and the C terminus in red. The catalytic dyad is shown by magenta spheres. The location of the unstructured loop between ANK and CAT domains is indicated by the dashed gray line and of the disordered membrane-interacting loop by the black dotted line. The position of the proline-rich insert in the long variant is shown by the grey arrow in this panel and by the red triangle in panel a

Article Snippet: Se-Met-labeled iPLA 2 β was produced in Sf9 cells with the same procedure as native protein, except for using methionine-deficient medium (Expression Systems, Davis, CA, USA) and supplementing with 100 mg/L l -selenomethionine 16 h after infection.

Techniques: Sequencing, Binding Assay, Variant Assay

Journal: Nature Communications

Article Title: The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization

doi: 10.1038/s41467-018-03193-0

Figure Lengend Snippet: Configuration of the iPLA 2 β dimer in the crystal structure. a The CAT and ANK domains of a dimer are shown in cyan and light navy, respectively, in monomer A and in yellow and orange in monomer B. Putative CaM-binding 1-9-14 motifs in both monomers are shown in dark blue. Catalytic dyads are shown by magenta spheres. b Same dimer rotated by 90° around horizontal axis. The schematic drawing of a membrane illustrates the orientation of the membrane-binding surface of iPLA 2 β

Article Snippet: Se-Met-labeled iPLA 2 β was produced in Sf9 cells with the same procedure as native protein, except for using methionine-deficient medium (Expression Systems, Davis, CA, USA) and supplementing with 100 mg/L l -selenomethionine 16 h after infection.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization

doi: 10.1038/s41467-018-03193-0

Figure Lengend Snippet: iPLA 2 β dimerization and mechanism of CaM inhibition. a Sedimentation velocity s(w) distributions of wild-type iPLA 2 β shown in blue (peak MW: 152 kDa; theoretical MW: 170 kDa), of the W695E mutant in orange (83 kDa), and of W695E in the presence of CaM/Ca 2+ in red (138 kDa). Lower MW can be due to unresolved contribution of minor fraction of monomeric species in the latter case. b Inhibition of iPLA 2 β enzymatic activity by CaM in the presence (black) and absence (blue) of Ca 2+ . c Interaction of FAM-CaM/Ca 2+ with iPLA 2 β as measured by fluorescence anisotropy upon titration by wild-type iPLA 2 β (black) and the W695E mutant (red). Error bars represent average ± s.e.m of triplicate experiments, which were performed at least twice independently. d , e Two orthogonal views of the iPLA 2 β dimer with monomers color coded in cyan and yellow. CaM in the conformation as reported in the 3SJQ PDB structure is placed next to 1-9-14 motifs (highlighted in blue) to illustrate the possibility of CaM interaction with two 1-9-14 motifs of a dimer

Article Snippet: Se-Met-labeled iPLA 2 β was produced in Sf9 cells with the same procedure as native protein, except for using methionine-deficient medium (Expression Systems, Davis, CA, USA) and supplementing with 100 mg/L l -selenomethionine 16 h after infection.

Techniques: Inhibition, Sedimentation, Mutagenesis, Activity Assay, Fluorescence, Titration

Journal: Nature Communications

Article Title: The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization

doi: 10.1038/s41467-018-03193-0

Figure Lengend Snippet: The proposed mechanism of iPLA 2 β regulation and macromolecular interactions. a Schematic representation of the iPLA 2 β dimer in a hypothetical inhibited state bound to CaM. CAT domains are shown in blue and yellow, ANK domains in navy and orange, and a single CaM molecule is represented by two connected circles in pink. Active site cavities are represented by narrow channels (gray lines) leading from the solvent-exposed surface to the Ser/Asp catalytic dyad depicted by magenta. b An active conformation of the dimer. CaM dissociation leads to the opening of the active sites. ANK domains are available for interactions with protein partners as illustrated for CAMKIIβ (light cyan transparent sphere), known to interact with ANK domain, and with transmembrane Cnx (shown as transmembrane helix with the C-terminal cytosolic peptide in pale yellow), which could recruit iPLA 2 β to the membrane. The Cnx-binding site of iPLA 2 β is not known and the hypothetical interaction with ANK domain is based on similar interaction of AnkB and sodium channel peptide. ATP binding (red) in the middle of the ANK domain could trigger additional conformational changes of the AR. Acylation of C651 by oleoyl-coA (green) can facilitate interaction with the membrane and/or opening of active site channels. Other conformational states are feasible as well, such as CaM-bound inhibited protein at the membrane or an open conformation of active sites in CaM-free form in cytosol, corresponding to the crystallized form

Article Snippet: Se-Met-labeled iPLA 2 β was produced in Sf9 cells with the same procedure as native protein, except for using methionine-deficient medium (Expression Systems, Davis, CA, USA) and supplementing with 100 mg/L l -selenomethionine 16 h after infection.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The structure of iPLA 2 β reveals dimeric active sites and suggests mechanisms of regulation and localization

doi: 10.1038/s41467-018-03193-0

Figure Lengend Snippet: Positions of selected INAD and PD mutations. Residues mutated in INAD and PD patients are shown as green and red sticks, respectively, on the cartoon of the iPLA 2 β monomer. Position of the second monomer is shown as a pale transparent surface. Active site Ser/Asp residues are shown by magenta spheres. Mutations previously tested for enzymatic activity are labeled. Numbers correspond to the human long variant (L-iPLA 2 β) sequence with corresponding numbers of the short variant structure in parentheses

Article Snippet: Se-Met-labeled iPLA 2 β was produced in Sf9 cells with the same procedure as native protein, except for using methionine-deficient medium (Expression Systems, Davis, CA, USA) and supplementing with 100 mg/L l -selenomethionine 16 h after infection.

Techniques: Activity Assay, Labeling, Variant Assay, Sequencing