Journal: Frontiers in Neuroscience

Article Title: Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy

doi: 10.3389/fnins.2018.00044

Figure Lengend Snippet: Phosphorylation sites in tau molecule. (A) The longest human tau isoform is composed of 441 amino acids with four microtubule-binding (MTB) repeats in the C-terminal half. Phosphorylation sites are indicated by black arrowheads. AT8 (Ser202 and Thr205), AT180 (Thr231 and Ser235) and PHF1 (Ser396 and Ser404) are phosphospecific antibodies frequently used for the postmortem diagnosis of tauopathy and their epitopes are indicated. The number of phosphorylation combination, if all sites are phosphorylated independently, is indicated below. (B) Amino acid sequences conforming to the GSK3β consensus sequences, (S/T)xx(x)p(S/T), in tau. There are 25 such sequences and 12 sites are reported to be phosphorylated (orange). The site in the C-terminal sides known to be phosphorylated are indicated by green. (C) Ser/Thr-Pro {(S/T)P} sequences in tau targeted by proline-directed protein kinases (PDPK). Arrow indicates Ser or Thr in (S/T)P sequences. Orange is the reported phosphorylation sites, blue is proline (P) conforming to the consensus sequence {Px(S/T)P or P(S/T)P} for MAPK, and magenta is basic amino acids at the C-terminal site which makes Ser or Thr phosphorylation sites favorable for Cdk5.

Article Snippet: We have newly identified the phosphorylation sites of several proteins, ataxin 2, drebrin and GRAB, by applying this method (Asada et al., ; Tanabe et al., ; Furusawa et al., ). (3) Once you have determined the phosphorylation states of each band, you may determine the combination of phosphorylated sites even in vivo by comparing with the standard profile of phosphoisotypes. (4) The relative ratio of each phosphoisotype can be measured as a percent ratio of the total by densitometric scanning of the blots.

Techniques: Phospho-proteomics, Binding Assay, Biomarker Discovery, Sequencing

Journal: Frontiers in Neuroscience

Article Title: Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy

doi: 10.3389/fnins.2018.00044

Figure Lengend Snippet: Separation of phosphoisotypes of a protein on Phos-tag SDS-PAGE. If there are two phosphorylation sites, A and B, in a protein, there would be 4 phosphoisotypes, doubly phosphorylated (blue), two single site-phosphorylated (green and red) and nonphosphorylated (left). These four phosphoisotypes can be separated into four bands on Phos-tag SDS-PAGE by shifting upward differently depending on the number and site of phosphorylation (Phos-tag). Phospho-specific antibodies, αpA and αpB, are required to detect the respective phosphorylation in Laemmli's SDS-PAGE, in contrast, the separation in Phos-tag indicates not only phosphorylation but also the degree of phosphorylation detected with a phosphorylation-independent antibody alone (Pind).

Article Snippet: We have newly identified the phosphorylation sites of several proteins, ataxin 2, drebrin and GRAB, by applying this method (Asada et al., ; Tanabe et al., ; Furusawa et al., ). (3) Once you have determined the phosphorylation states of each band, you may determine the combination of phosphorylated sites even in vivo by comparing with the standard profile of phosphoisotypes. (4) The relative ratio of each phosphoisotype can be measured as a percent ratio of the total by densitometric scanning of the blots.

Techniques: SDS Page, Phospho-proteomics

Journal: Frontiers in Neuroscience

Article Title: Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy

doi: 10.3389/fnins.2018.00044

Figure Lengend Snippet: Phosphoisotypes of tau on Phos-tag SDS-PAGE. (A) Immunoblotting of tau expressed in COS-7 cells with tau5 in the presence (+) or absence (−) of Cdk5-p35 after Phos-tag-SDS-PAGE, and the phosphorylation sites in each band are indicated by the amino acid number according to the human longest isoform of tau. Y is an unknown phosphorylation site. (B) Immunoblotting of tau in human brains after Phos-tag SDS-PAGE. NC, normal control; AD V and VI, Braak stage V and VI of Alzheimer's disease (AD); CBD Tl and Pg, temporal lobe (Tl) and prefrontal gyrus (Pg) of corticobasal degeneration (CBD). We propose that the banding pattern created using Phos-tag SDS-PAGE could be read by a bar code reader and result in a simple method for diagnosing tauopathy. (A,B) are reproduced and modified from figures reported previously by Kimura et al. ( , ), respectively.

Article Snippet: We have newly identified the phosphorylation sites of several proteins, ataxin 2, drebrin and GRAB, by applying this method (Asada et al., ; Tanabe et al., ; Furusawa et al., ). (3) Once you have determined the phosphorylation states of each band, you may determine the combination of phosphorylated sites even in vivo by comparing with the standard profile of phosphoisotypes. (4) The relative ratio of each phosphoisotype can be measured as a percent ratio of the total by densitometric scanning of the blots.

Techniques: SDS Page, Western Blot, Phospho-proteomics, Control, Modification

Journal: Frontiers in Neuroscience

Article Title: Phospho-Tau Bar Code: Analysis of Phosphoisotypes of Tau and Its Application to Tauopathy

doi: 10.3389/fnins.2018.00044

Figure Lengend Snippet: Cartoons representing the images of immunoblotting with many phospho-specific antibodies (A) and a phosphorylation-independent single antibody after Phos-tag SDS-PAGE (B) . Phosphospecific antibodies detect only part of phosphorylation at each site and some sites can be masked by another phosphorylated site nearby (here the site for αpTau6 is masked by a phosphorylated site which reacts with αpTau4). Phos-tag shows the whole phosphorylation profile at once. An illustration of an elephant was downloaded from the free illustration site at: http://illpop.com/png_animalhtm/elephant_a04.htm .

Article Snippet: We have newly identified the phosphorylation sites of several proteins, ataxin 2, drebrin and GRAB, by applying this method (Asada et al., ; Tanabe et al., ; Furusawa et al., ). (3) Once you have determined the phosphorylation states of each band, you may determine the combination of phosphorylated sites even in vivo by comparing with the standard profile of phosphoisotypes. (4) The relative ratio of each phosphoisotype can be measured as a percent ratio of the total by densitometric scanning of the blots.

Techniques: Western Blot, Phospho-proteomics, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *

doi: 10.1074/jbc.M116.760330

Figure Lengend Snippet: In vitro screening of kinases that phosphorylate a PRMT5 C-terminal peptide. A, the activity of 295 protein kinases was tested against a PRMT5 C-terminal peptide. These assays were based on the direct quantification of radiolabeled phosphate from ATP (γ-33P) on to the peptide substrate. These assays were performed by Kinexus. In vitro kinase activity was ranked as excellent (>5.5 pmol/min), good (4.6–5.4 pmol/min), low (1.8–4.5 pmol/min), and non-phosphorylated (<1.7 pmol/min). B, the 18 best kinases (excellent and good ranking) were used to validate the initial in vitro phosphorylation screen using PRMT5 WT and T634A C-terminal peptides as substrates.

Article Snippet: In vitro phosphorylation assay were performed on the PRMT5 WT peptide by Kinexus, using a radiometric assay [ γ - 33 P]ATP and 295 different recombinant kinases.

Techniques: In Vitro, Activity Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *

doi: 10.1074/jbc.M116.760330

Figure Lengend Snippet: PDZ domain and 14-3-3 share a common binding motif. A, comparison of 14-3-3 versus PDZ domain binding motifs. The consensus binding sequences of the PDZ-binding motif I and 14-3-3-binding motif III are very similar, differing only in the phosphorylation-dependent characteristic of 14-3-3 interactions. Single-letter amino acid codes are used; X, any residue; φ, a hydrophobic residue; P, phosphorylation. B, schematic diagram of proteins harboring overlapping PDZ and 14-3-3 binding motifs, displaying predicted (*) phosphorylation sites (ERBB4 and PGHS2) and reported phosphorylation sites (PRMT5, IRK1, and E6). Predictions were done using iGPS version 1.0 software.

Article Snippet: In vitro phosphorylation assay were performed on the PRMT5 WT peptide by Kinexus, using a radiometric assay [ γ - 33 P]ATP and 295 different recombinant kinases.

Techniques: Binding Assay, Comparison, Phospho-proteomics, Residue, Software

Journal: The Journal of Biological Chemistry

Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *

doi: 10.1074/jbc.M116.760330

Figure Lengend Snippet: Phosphorylation triggers switching between 14-3-3 and PDZ interactions. A, PRMT5, ERBB4, E6 (HPV16), PGHS2, and IRK1 unphosphorylated and phosphorylated peptides were labeled with Cy5 (red) and Cy3 (green), respectively, and used to probe a protein microarray containing PDZ domains and 14-3-3 GST fusion proteins. The bottom right panel shows the array probed with α-GST for the loading control. The PDZ domains (red) and 14-3-3 proteins (green) are blocked. B, graphical depiction of the interactions observed in A. Red and green squares, interactions with unphosphorylated and phosphorylated peptides, respectively.

Article Snippet: In vitro phosphorylation assay were performed on the PRMT5 WT peptide by Kinexus, using a radiometric assay [ γ - 33 P]ATP and 295 different recombinant kinases.

Techniques: Phospho-proteomics, Labeling, Microarray, Control