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, butnot  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.Conclusion: HIP-55 forms a complex with 14-3-3 and 14-3-3/HIP-55 interaction increases the stability of HIP-55.

Key words: HIP-55 protein, human, 14-3-3 proteins, Protein interaction