Reversible Acetylation Of SrGAP2F-BAR Domain And Its Functional Implications

The srGAP family (Slit-Robo GTPase-activating proteins) mainly consists offour family members: srGAP1, srGAP2, srGAP3and ARHGAP4. SrGAPs containN-terminal F-BAR domain mediating membrane deformation, the central RhoGAPdomain for specific hydrolysis of RhoGTPase, and C-terminal SH3domain regulatingprotein-protein interactions.The current research reveals that signaling adaptor protein srGAP2plays diversekey functions primarily through its F-BAR domain: srGAP1facilitates cell-celladhesion through its F-BAR domain during C. elegans morphogenesis; F-BARdomain of srGAP2induces neurite outgrowth and neuronal migration; srGAP3F-BAR domain facilitates the initiation of spine development. Further, this effect isproved to be closely associated with filopodia-inducing activity of F-BAR. It has to beconsidered that the mechanism underlying F-BAR is associated to numerouspositively-charged basic amino acids; And these lysines and argines could regulatemembrane deformation via electrostatic contacts with negatively-charged lipid on themembrane.This study focuses on key lysines reversible acetylation ofsrGAP2-F-BAR/IF-BAR domian and its effect on filopodia-inducing and endocytoticability.The main results are described as following:1. We transfected C-terminal GFP-tagged srGAP F-BAR/IF-BARs vectors intoCOS7L cells. Results show that GFP-tagged srGAP(1-3)-F-BARs mainlylocolized to the membrane and induce filopodia formation. Furthermore,srGAP(1-3)-F-BARs bind to several lipids, including PA, PtdIns(4)P,PtdIns(4,5)P2and PtdIns(3,4,5)P3by Membrane Lipid assays of puriedGST-tagged srGAP(1-3)-F-BARs.2. We performed multiple sequence alignment of different speices ofsrGAP2-F-BAR domain. Among all fifty lysines there are several highly-conserved key lysines during biological evolution. HEK293FT cellswere transfected with srGAP2-F-BAR vector and lyzed after24-48h. Afterdetection of lysates by using AceK antibody, acetylated lysines were foundin F-BAR domain and acetylation level raised after treatment of HDACinhibitor TSA/NAM. We select two key lysine sites to manufactureAc-K226and Ac-K234antibodies to further prove acetylation of K226andK234.3. We take advantage of Co-IP and Western Blot to screen histoneacetyltransferase and histone deacetylase which act on srGAP2and usesrGAP2as substrate. Data show that SIRT1and p300interact to srGAP2;co-transfection of SIRT1and srGAP2decreases srGAP2acetylation levelwhile co-transfection of p300and srGAP2increases.4. We acquire2KQ,5KQ acetylation-mimetic mutants and6KRdeacetylation-mimetic mutant through multi site-directed mutagenesis. Inthe functional studies, srGAP2-F-BAR-WT has filopodia-inducing and cellmigration-promoting ability; however, acetylation-mimetic5KQ abrogatesthe function of filopodia formation and cell migration but facilitatesendocytosis instead; deacetylation-mimetic6KR suppresses neuronaldifferentiation.To sum up, our study first confirms that reversible acetylation is observed insrGAP2-F-BAR domain; p300is its acetyltransferase and SIRT1is its deacetylase (Inother words, p300and SIRT1are involved in the regulation of srGAP2-F-BARacetylation). Further function-analysis proves that reversible acetylation ofsrGAP2-F-BAR might mediate functional transformation bewteen filopodia-inducingability and endocytosis. This study provides a new way to investigate diversefunctions and mechanisms underlying signal transducing adaptor protein srGAP2.