Screening of AAK1 interaction proteins and its role in regulating global translation level in cells

Abstract

Objective·To investigate noval interacting partners for adaptor-associated protein kinase 1 (AAK1) and AAK1-mediated biological functions besides clathrin-mediated endocytosis.Methods·The labeled AAK1 vector and the blank control vector were transfected in HEK-293T cells, and the potential AAK1 interacting proteins were obtained by co-immunoprecipitation with agar-specific gel and mass spectrometry. Further verifications were performed by CoIP and fluorescence-based imaging. Recombinant proteins were purified in vitro and the direct interaction between proteins were confirmed by glutathione-S-transferase pulldown (GST Pulldown) assay. The regulation of AAK1 in the global protein synthesis was explored by puromycin incorporation assay.Results·Mass spectrometry results showed that AAK1 was associated with a series of proteins, including fragile X mental retardation syndrome-related protein 1 (FXR1), FXR2 and fragile X mental retardation protein 1 (FMRP). Enriching with anti-FLAG agarose gels after exogenous transfecting of AAK1-3xFLAG and FMRP-MYC plasmids, the expression of FMRP-MYC was detected. The expression of FMRP could also be detected by CoIP with endogenous AAK1 antibodies. Fluorescence-based imaging showed that they were spatially colocalized in the cytoplasm. GST Pulldown assay showed that FMRP could pulldown recombinant HIS6-AAK1 protein. Puromycin incorporation assay showed that in the same amount of time, the number of newly synthesized peptides labeled with puromycin was positively correlated with AAK1 protein expression.Conclusion·AAK1 directly interacts with FMRP in cytoplasm and could up-regulate global protein synthesis level.

Keywords

• adaptor-associated protein kinase 1 (aak1)
• translation
• mass spectroscopy
• protein-protein interaction