Figure 3.
ARF1 specifically interacts with the second cysteine-rich zinc finger domain (C1b) of PKD2 and significance of C1b for Golgi targeting of PKD2. (A) Schematic representation of the PKD2 mutants used in this study. WT, wild-type kinase; D695A, kinase dead; S706/710E, constitutively active; Δ1-137, deletion of the first 138 aa; Δ CRD, deletion of the entire cysteine-rich zinc finger domain; ΔC1a, deletion of the first cysteine-rich zinc finger domain; ΔC1b, deletion of the second cysteine-rich zinc finger domain; Δ323-368, deletion of aa 323-368, which includes the acidic domain (AC); ΔPH, deletion of the pleckstrin homology domain; Δ KD, deletion of the kinase domain. (B) HEK293-T-cells expressing EGFP-PKD2 wild type or various mutants as indicated were lysed, and the lysates were incubated with anti-GFP antibody to determine the expression level of the mutants. (C) HEK293-T-cells expressing EGFP-PKD2 WT or various mutants were lysed, and the lysates were incubated with GST-ARF1 immobilized on glutathione-Sepharose beads and retained PKD2-WT and mutants were assessed by Western blotting with GFP antibody, respectively (D) The purified His-tagged C1b domain of PKD2 was incubated with purified GST-ARF1, GST-ARF6, or inactive and active ARF1 and ARF6 mutants immobilized on glutathione-Sepharose beads in an in vitro binding assay. Bound proteins were resolved by Western blotting with anti-His and anti-GST antibodies, respectively. Quantification of the band intensities of His-C1b bound to GST-ARF1 and GST-ARF6 wild type or mutants is represented in the bottom panel. Data are means ± SEM of two independent experiments. HeLa cells coexpressing (E) EGFP-PKD2-WT (top), or EGFP-PKD2-ΔC1b (bottom) and ARF1-mRFP. (F) HeLa cells expressing EGFP-PKD2-WT (top), EGFP-PKD2-ΔC1a (middle), or EGFP-PKD2-ΔC1b (bottom) were fixed followed by anti-TGN46/Alexa-594 immunostaining. Bars, 20 μm.