FIGURE 3.

PMA/PKD mediated phosphorylation of Syx at Ser⁹². A, Ser⁹² is a putative PKD phosphorylation site. The pMotif antibody recognizes a consensus motif that contains a leucine at the −5 position and an arginine at the −3 position. Ser⁹² and Ser⁸⁰⁶ are the only residues within Syx that conform to the consensus motif. B, Syx(1–630) is a PKD phosphorylation target. HeLa cells were co-transfected with the indicated constructs and subjected to DMSO (control) or PMA (100 nm, 10 min) treatment prior to immunoprecipitation. Protein samples were analyzed by Western blot. X-ray film was exposed to the same blot for different lengths of time to visualize the phosphorylation (by pMotif) of Syx(1–630) and Syx(791–1073). C, both control and PMA-induced 14-3-3 associations require Syx Ser⁹². As in B, transfected cells were subjected to immunoprecipitation after treatment with DMSO (control) or PMA (100 nm, 10 min). Protein samples were analyzed by Western blotting. Note that the mutation of Ser⁹² to alanine abrogated the PMA-induced increase in phosphorylation seen with pMotif in Syx(1–630). D, Ser⁹² is phosphorylated by PKD in addition to Ser⁸⁰⁶. A PMA-induced increase in pMotif phosphorylation and 14-3-3 binding was observed upon transfecting cells with full-length Syx mutant YFP-Syx S806A. As in B, transfected cells were subjected to immunoprecipitation, and protein samples were analyzed by Western blotting.