Figure 6.

Binding of 14-3-3θ to TRPM7 requires autophosphorylation of the channel at Ser-1403. A, three highly conserved potential 14-3-3 binding motifs (underlining) were identified on TRPM7's C terminus. Two of the motifs contain TRPM7 autophosphorylation sites (red). B, GFP–TRPM7–Cterm–WT, S1403A, S1567A, and S1588A were expressed in HEK-293T cells and subjected to a GST-pulldown assay using GSH agarose bound with 20 μg of GST–14-3-3θ or GST proteins. GFP–TRPM7–Cterm was examined by Western blotting using an anti-GFP antibody. GST proteins were visualized by SDS–PAGE and Coomassie staining. Ser-1403 was identified as the major 14-3-3θ binding site on GFP–TRPM7–Cterm. C, GFP–TRPM7–Cterm–WT and Ser-1403 mutants (S1403A, S1403D, and S1403E) expressed in HEK-293T cells were subjected to the same GST-pulldown assay. GST–14-3-3θ failed to pulldown GFP–TRPM7–Cterm carrying mutations at Ser-1403, demonstrating a requirement of Ser-1403 phosphorylation for TRPM7 and 14-3-3θ interaction.