Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 5. Dok-6 binds to Ret via the phosphorylated Ret Tyr1062 residue. A, Dok-6 associates with ligand-activated Ret. Neuro2A cells stably transfected with an expression vector for GFR1 (N2A-1) were deprived of serum for 2 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Cleared cellular extracts (1 mg of protein/assay) were subjected to pull-down assays using immobilized GST fusion proteins containing the PTB domains of Dok-5 (amino acid residues 110–240), Dok-6 (amino acid residues 110–240), or GST as a control. Cellular protein complexes bound to the fusion proteins were eluted and subjected to SDS-PAGE followed by immunoblotting with Ret and phosphotyrosine (P-Tyr) antibodies. B, Dok-6 PTB domain binding to Ret requires the Ret Tyr1062 residue. CHP126 cells stably expressing wild type Ret9 or Ret51 isoforms (Ret9 and Ret51, respectively), isoforms containing tyrosine 1062 to phenylalanine (Y1062F) point mutations, or a kinase inactive Ret51 (Ret51 K758M) were treated and used in pull-down assays as described in A, above. Wild-type CHP126 cells (CHP) do not express endogenous Ret. The experiments in A and B were performed three times each with similar results. P-Ret, phospho-Tyr905 Ret; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Residue, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, SDS Page, Western Blot, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 6. Ret phosphorylates the C terminus of Dok-6 via Src. A, Dok-6 is a substrate of the Ret signaling cascade. N2A-1 cells were transiently transfected with expression vectors encoding HA-tagged versions of Dok-5 or Dok-6 or with an empty expression vector (Control). Two days after transfection, cells were deprived of serum for 1 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Cellular extracts were immunoprecipitated using anti-HA antibodies. The eluted immunocomplexes were then subjected to SDS-PAGE followed by HA and phosphotyrosine (P-Tyr) immunoblotting. B, the Dok-6 C terminus is tyrosine-phosphorylated in response to activation of the Ret signaling cascade. N2A-1 cells were transiently transfected with expression vectors encoding HA-tagged versions of Dok-6 or C-terminally truncated Dok-6 (amino acids 1–260, Dok-6C) or with an empty expression vector (Control). Two days after transfection, cells were treated as described above and anti-HA immunoprecipitates were subjected to SDS-PAGE followed by HA and P-Tyr immunoblotting. C, Ret phosphorylates Dok-6 through Src. N2A-1 cells were transiently transfected with an expression vector encoding HA-tagged Dok-6. Two days after transfection, cells were deprived of serum for 1 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Where indicated, cells were preincubated with either PP2 (1 M), PP3 (1 M), or SU6656 (SU, 2 M) for 30 min prior to GDNF stimulation. Anti-HA immunoprecipitates prepared from cellular extracts were subjected to SDS-PAGE followed by HA and P-Tyr immunoblotting. P-Ret, phospho-Tyr905 Ret; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, SDS Page, Western Blot, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 7. Dok-6 promotes Ret-mediated neurite outgrowth. N2A-1 cells were infected with lentiviruses to permit the stable expression of HA-tagged Dok-5, Dok-6, C-terminally truncated Dok-6 (Dok-6C), or an empty expression cassette (Control). Virus infected cells selected by FACS against the Venus reporter gene were used in neurite outgrowth assays. A, expression of Dok proteins in lentivirus-infected N2A-1 cells. Cell extracts were immunoprecipitated using anti-HA antibodies. Immunoprecipitates were subjected to SDS-PAGE followed by HA immunoblotting (bottom panel). Ret activation is not affected by the expression of the Dok proteins used in this study (top panel). B, N2A-1 cells expressing the indicated Dok gene construct or empty cassette were placed in low serum medium (0.5% FBS), treated with () or without () GDNF (30 ng/ml) for 24 h, and photographed. C and D, quantification of neurite outgrowth. In C, cells were placed in low serum medium (0.5% FBS) and treated with the indicated concentration of GDNF for 24 h. Neurite outgrowth was assessed by evaluating cells in randomly chosen fields and calculating the percentage of neurite-bearing cells. Expression of Dok-6 significantly enhanced neurite outgrowth compared with N2A-1 cells expressing Dok-6C or an empty expression cassette (p 0.0001). There was no significant difference in neurite outgrowth induced by 5 ng/ml versus 30 ng/ml GDNF treatments within any given cell line, between Dok-6- and Dok-5-expressing N2A-1 cells, or between empty expression cassette and Dok-6C-expressing N2A-1 cells. In D, neurite lengths were measured in each cell line treated with 30 ng/ml GDNF for 24 h. Average neurite length in Dok-6-expressing N2A-1 cells was significantly greater than that of N2A-1 cells expressing Dok-6C or an empty expression cassette (p 0.002). Results in C and D were obtained from 3–5 independent experiments each. P-Ret, phospho-Tyr905 Ret; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Infection, Expressing, Control, Virus, Immunoprecipitation, SDS Page, Western Blot, Activation Assay, Construct, Concentration Assay