title: [14-3-3/HIP-55 complex increases the stability of HIP-55].
abstract: OBJECTIVE
  To further demonstrate the interaction of a new 14-3-3 interaction protein hematopoietic progenitor kinase 1[HPK1]-interacting protein (HIP-55) and 14-3-3 proteins and its potential biological function in HEK293 cells.
  
  
  METHODS
  PDEST-N-Venus-HIP-55WT (wild type),PDEST-N-Venus-HIP-55AA (mutants, S269A/T291A, abolishing the binding of HIP-55 to 14-3-3),PDEST-GST-HIP-55WT and PDEST-C-Venus-14-3-3τ plasmids were constructed by gateway system. Their expressions were demonstrated by Western blotting method. Then we used Bimolecular Fluorescence Complementation (BiFC) and co-immunoprecipitation (co-IP) methods to demonstrate the interaction of HIP-55 and 14-3-3 in HEK293 cells. Moreover, the 14-3-3 antagonist peptide, R18 and HIP-55 protein mutant plasmid HIP-55AA were used to detect the protein synthesis of HIP-55 at different time points induced by puromycin, an inhibitor of protein production.
  
  
  RESULTS
  The HEK293 cells expressed HIP-55 protein respectively, after being transected with PDEST-N-Venus-HIP-55WT,PDEST-N-Venus-HIP-55AA,PDEST-GST-HIP-55WT plasmids and expressed 14-3-3 protein after being transected with PDEST-C-Venus-14-3-3τ plasmids. We could detect venus fluorescence of venus-HIP-55 protein via confocal microscopy in HEK 293 cells transfected with N-Venus-HIP-55 and C-14-3-3τ plasmids by BiFC, but not in HEK 293 cells transfected with N-Venus-HIP-55 AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results of BiFC suggested that 14-3-3 interacted with HIP-55 through HIP-55 S269/T291 sites. At the same time, the data of co-IP showed that there were endogenous interactions between 14-3-3 and HIP-55. Furthermore, puromycin had no influence in HIP-55 protein synthesis at hours 0, 4, or 8 in HEK 293 cells expressing GST-HIP-55WT and 14-3-3 plasmids, while puromycin blocked HIP-55 protein synthesis in HEK 293 cells transfected with N-Venus-HIP-55AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results indicated that the 14-3-3/HIP-55 complex could contributed to the stability of HIP-55.
  
  
  CONCLUSIONS
  HIP-55 forms a complex with 14-3-3 and 14-3-3/HIP-55 interaction increases the stability of HIP-55.
authors.affiliation: Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Research; Beijing 100191,China.
authors.forename: Ai-ju
authors.initials: AJ
authors.lastname: Tian
authors.affiliation: Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Research; Beijing 100191,China.
authors.forename: Zi-jian
authors.initials: ZJ
authors.lastname: Li
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry*; D015153:Blotting, Western; D057809:HEK293 Cells; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids; D014176:Protein Biosynthesis; D055550:Protein Stability; D011691:Puromycin / Q000737:chemistry; D014162:Transfection; D018909:src Homology Domains
chemical_list: D048948:14-3-3 Proteins; C117063:DBNL protein, human; D008840:Microfilament Proteins; D011691:Puromycin