Screening And Identification Of DHHC5 Substrates By BioID

Protein S-palmitoylation is the covalently attachment of a 16-carbon palmitate to a thiol group of cysteine,and it is widely involved in many physiological processes,such as Ras signaling pathway,proteins trafficking,ion channels activity,cytoskeleton dynamics,pathogens infection,and so on.Palmitoylation is catalyzed by DHHC family proteins,which are originally discovered in yeast with a characteristic DHHC(Asp-His-His-Cys)motif.The specificity of the interaction between the DHHC and the substrates is very complex and poorly understood.Due to the lack of the specific recognition signal on the substrates,it is difficult to study the substrates profile of a DHHC.DHHC5 is highly expressed in various tissues,such as brain and heart,and DHHC5 knockout mice is embryonic lethal,indicating that the physiological function DHHC5 is very important.In this study,we used BioID technique combining with biochemical and cellular biological methods to screen and identify DHHC5 substrates.We found that the palmitoylation of Plakophilin-2(PKP2),Fibroblast growth factor receptor substrate 2(FRS2),Desmoglein-2(DSG2),Ephrin type-A receptor 2(EPHA2)and AP2-associated protein kinase 1(AAK1)is dependent on DHHC5.PKP2 plays an important role in Arrhythmogenic right ventricular cardiomyopathy.PKP2 is a component of desmosomes on cell membrane,but it also has an important function in nucleus.We found that palmitoylation of PKP2 mediated by DHHC5 is very important for the localization of PKP2 on cell membrane.In DHHC5 knockout cells,PKP2 is located in the nucleus;however,in WT cells,PKP2 is located on or in the vicinity of the plasma membrane,suggesting that palmitoylation and depalmitoylation might regulate the transport of PKP2 between the cytoplasm and the nucleus.Palmitoylation also regulates the location of AAK1.In WT 293T cells,AAK1 is located on the nuclear membrane,but in DHHC5 knockout 293T cells AAK1 aggregates as a speck in cytoplasm or near plasma membrane.Our application of the BioID technique in searching for DHHC substrates provides a way to identify substrates of other DHHC family palmitoyltransferases.In addition,the discovery of a series of DHHC5 substrates provides new important information for future researches in DHHC5 function.