FIGURE 2.

Ser⁹² and Ser⁹³⁸ mediate 14-3-3 binding to Syx. A, Ser⁹² is a putative 14-3-3-binding residue. HeLa cells were co-transfected with plasmids encoding HA-14-3-3β and YFP, YFP-Syx, YFP-Syx(1–630), or one of three YFP-Syx(1–630) mutants in which a single serine was mutated to alanine. Cells were subjected to immunoprecipitation, and protein samples were analyzed by Western blotting. Only the replacement of Ser⁹² with alanine abolished the interaction between Syx(1–630) and 14-3-3β. B, Ser⁹³⁸ is a putative 14-3-3-binding residue. As in A, HeLa cells were co-transfected with plasmids encoding HA-14-3-3β and YFP, YFP-Syx, YFP-Syx(791–1073), or one of four YFP-Syx(791–1073) in which a single serine was mutated to alanine. Only the replacement of Ser⁹³⁸ with alanine abolished the interaction between Syx(791–1073) and 14-3-3β. Note that Ser⁸⁰⁶ was previously shown as a PKD-mediated phosphorylation target; mutating Ser⁸⁰⁶ to alanine, however, has no effect on the binding of 14-3-3β. C and D, Ser⁹² and Ser⁹³⁸ are essential residues for 14-3-3 interaction. HeLa cells were co-transfected with the indicated constructs and subjected to immunoprecipitation. Protein samples were analyzed by Western blot analysis. Mutating Ser⁹² and Ser⁹³⁸ on Syx either abolished or diminished its interaction with 14-3-3 isoforms. Note that mutation of Ser⁹³⁸ alone in C has no clear effect on 14-3-3 binding, but resulted in a decrease or absence of 14-3-3 co-immunoprecipitates when combined with S92A mutation. E, endogenous 14-3-3 proteins fail to co-immunoprecipitate with Syx S92A/S938A. HeLa cells were transfected with the indicated constructs and subjected to immunoprecipitation. Protein samples were analyzed by Western blotting. Mutating Ser⁹² and Ser⁹³⁸ on Syx significantly diminished its interaction with endogenous 14-3-3 proteins.