Interaction of pyrin with 14.3.3 in an isoform-specific and phosphorylation-dependent manner regulates its translocation to the nucleus

Objective. Pyrin, the familial Mediterranean fever gene product, exists in several isoforms of unknown functions. The recombinant full-length isoform (pyrin.fl) is cytoplasmic, whereas an alternatively spliced isoform lacking exon 2 (pyrin.Delta Ex2) concentrates in the nucleus. Native pyrin, mainly consisting of pyrin.fl, is also cytoplasmic in monocytes but is predominantly nuclear in other cell types. To understand pyrin-dependent biologic pathways and to decipher the mechanisms accounting for such different patterns of subcellular compartmentalization, binding partners and posttranslational modifications of pyrin were assessed. Methods. A yeast 2-hybrid screen was performed with pyrin.fl as the bait. The interaction identified between pyrin.fl and 14.3.3 proteins was confirmed by immunoprecipitation of S-35-radiolabeled protein complexes; similar experiments were performed with pyrin.Delta Ex2, pyrin.fl after alkaline phosphatase treatment, and pyrin.fl mutants in which several exon 2-encoded serine residues were replaced by nonphos-phorylatable alanines. The subeellular localization of the different wild-type and mutated pyrin proteins was assessed by immunofluorescence. Results. Two members of the 14.3.3 protein family were identified as pyrin partners. Whereas pyrin.fl interacted with 14.3.3 tau and 14.3.3 epsilon, these interactions did not occur with pyrin.Delta Ex2. Pyrin.fl was phosphorylated, and this modification mediated 14.3.3 binding. Serines 208, 209, and 242, within exon 2, acted as critical residues in the interaction between pyrin.fl and 14.3.3. When an S208-S209-S242A pyrin.fl triple mutant or wild-type pyrin.fl in the presence of an inhibitor of 14.3.3-ligand interactions was used, promotion of nuclear translocation of pyrin was observed. Conclusion. These results disclose the role played by 14.3.3 in the regulation of the subcellular compart-mentalization of pyrin in a phosphorylation- and isoform-dependent manner. They also reconcile the observations made in vitro with those made in vivo, while providing a direct link between 14.3.3-dependent pathways and pyrin.