pkd3  (Carna Inc)

Journal: Nature communications

Article Title: Protein kinase D regulates positive selection of CD4 + thymocytes through phosphorylation of SHP-1.

doi: 10.1038/ncomms12756

Figure Lengend Snippet: Figure 2 | Generation of PKD-deficient mice. (a,b) Genomic structures and targeting constructs of PKD2 (a) and PKD3 (b). Exons encoding the DxxxN motif and the phosphorylation sites required for kinase activity are flanked by loxP sites (indicated by open triangles). (c) Total thymocytes from WT, PKD2fl/fl Lck-Cre Tg (PKD2DT), PKD3fl/fl Lck-Cre Tg (PKD3DT) and PKD2fl/fl PKD3fl/fl Lck-Cre Tg (PKD2/3DT) mice were analysed for PKD expression by western blotting using anti-PKDs antibody. b-actin was used as a loading control. (d) PKD phosphorylation in thymocytes on TCR stimulation by CD3 cross-linking for 2 min was detected using anti-pPKDs. Data are representative of two independent experiments (c,d).

Article Snippet: For in vitro kinase assay using [g-32P]ATP, 2 mg GST-SHP-1, GST-Gads and GST-NCK1 were incubated with 50 ng active PKD2 or PKD3 (Carna Bioscience) and 100 mM [g-32P]ATP (5 mCi) in 20 ml kinase buffer (5mM MgCl2 and 20mM Tris–HCl, pH7.5) for 30min at 30 C. The reaction was stopped by addition of sample buffer followed by heating at 95 C for 5min.

Techniques: Construct, Phospho-proteomics, Activity Assay, Expressing, Western Blot, Control

Journal: Nature communications

Article Title: Protein kinase D regulates positive selection of CD4 + thymocytes through phosphorylation of SHP-1.

doi: 10.1038/ncomms12756

Figure Lengend Snippet: Figure 8 | Identification of PKD2 and PKD3 substrates. (a) A gel image of 2D-DIGE of phosphoproteins from unstimulated WT thymocytes (Cy2-labelled, blue), WT thymocytes stimulated by CD3 cross-linking (Cy3-labelled, red) and stimulated PKD2/3DT thymocytes (Cy5-labelled, green). Red spots are indicated by arrowheads as spot #1 to #6. (b) The summary of the protein identity, accession number, calculated molecular weight, calculated pI, Mascot score, sequence coverage and query match for each of six spots in a. Spots #1, #2 and #3, which have the same molecular weight with different pI’s were identified as pro-IL-16. (c) Phos-tag immunoblot analysis of cell lysates from unstimulated- and TCR-stimulated-WT thymocytes for 2 min and stimulated PKD2/3DT thymocytes using anti-SHP-1, anti-NCK1 and anti-Gads. (d) In vitro kinase assays were performed in the presence of [g-32P]ATP with active, recombinant PKD2 or PKD3 and GST-fused substrates as indicated (upper panel). CBB staining shows that equivalent amounts of proteins were used for the assays (lower panel). Red arrowheads indicate GST-fused substrates. (e) Identification of phosphorylation sites. In vitro kinase assays were performed with PKD2 or PKD3 and, as substrates, WTor mutant SHP-1, Gads and NCK1. Mutant substrates carry serine to alanine substitution at the indicated amino acid positions. CBB staining is shown as a loading control. (f) Comparison of the PKD consensus phosphorylation motif and phosphorylation sites identified in e. Data are representative of two independent experiments (a–e).

Article Snippet: For in vitro kinase assay using [g-32P]ATP, 2 mg GST-SHP-1, GST-Gads and GST-NCK1 were incubated with 50 ng active PKD2 or PKD3 (Carna Bioscience) and 100 mM [g-32P]ATP (5 mCi) in 20 ml kinase buffer (5mM MgCl2 and 20mM Tris–HCl, pH7.5) for 30min at 30 C. The reaction was stopped by addition of sample buffer followed by heating at 95 C for 5min.

Techniques: Molecular Weight, Sequencing, Western Blot, In Vitro, Recombinant, Staining, Phospho-proteomics, Mutagenesis, Control, Comparison

Journal: Nature communications

Article Title: Protein kinase D regulates positive selection of CD4 + thymocytes through phosphorylation of SHP-1.

doi: 10.1038/ncomms12756

Figure Lengend Snippet: Figure 9 | SHP-1 is phosphorylated by PKD2 and PKD3 at Ser557 in thymocytes. (a) Phosphorylation of SHP-1 on peptide stimulation. OT-I DP thymocytes were stimulated with the designated OVA peptides for the indicated times. Cell lysates were subjected to Phos-tag immunoblot analysis using anti-SHP-1. Arrowheads indicate phosphorylated SHP-1 that showed stimulation-dependent mobility shift. (b) The extracted ion chromatogram of m/z 496.23096 corresponds to the quadruply charged SHP-1 phosphopeptide (TSpS557KHKEEVYENVHSK), which was increased by TCR stimulation in tryptic phosphopeptides from WT thymocytes and was almost undetectable in those from stimulated PKD2/3DT thymocytes. The relative values of the peak area are indicated. Note that the hextuply charged ion peak at a retention time of 21.70 min was unrelated to this SHP-1 phosphopeptide. (c) Phosphorylation of Ser557 was demonstrated by the MS/MS spectrum of the m/z 496.23 ion at a retention time of 7.52 min in tryptic phosphopeptides from stimulated-WT thymocytes. (d) Phosphorylation of Ser557 of SHP-1 was analysed by immunoblotting. Total thymocytes were stimulated by CD3 cross-linking for 2 min and analysed by anti-phospho-SHP-1 (Ser557) Abs. Total SHP-1 was blotted as a control. (e) Schematic structure of SHP-1 and sequence alignment of the C-terminal region of SHP-1 that contains Ser residue corresponding to Ser557 of murine SHP-1 (shown in red). Data are representative of two independent experiments (a–d).

Article Snippet: For in vitro kinase assay using [g-32P]ATP, 2 mg GST-SHP-1, GST-Gads and GST-NCK1 were incubated with 50 ng active PKD2 or PKD3 (Carna Bioscience) and 100 mM [g-32P]ATP (5 mCi) in 20 ml kinase buffer (5mM MgCl2 and 20mM Tris–HCl, pH7.5) for 30min at 30 C. The reaction was stopped by addition of sample buffer followed by heating at 95 C for 5min.

Techniques: Phospho-proteomics, Western Blot, Mobility Shift, Tandem Mass Spectroscopy, Control, Sequencing, Residue