title: Interacting domains of P14-3-3 and actin involved in protein-protein interactions of living cells.
abstract: 14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76-108 amino acids and the C-terminal 207-216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1-54 amino acids and the C-terminal 326-376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 → Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76-108 and C-terminal 207-216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1-54 or C-terminal 326-376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.
authors.affiliation: College of Life Sciences, Shenzhen University, China.
authors.forename: Daliang
authors.initials: D
authors.lastname: Luo
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Zhongzhen
authors.initials: Z
authors.lastname: Guo
authors.forename: Shide
authors.initials: S
authors.lastname: Liu
authors.forename: Shengli
authors.initials: S
authors.lastname: Tian
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D020798:Two-Hybrid System Techniques
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; D010455:Peptides