Fig. 4: SIRT2 removes ARF6 K3 myristoylation.

a Alk12 labeling results on overexpressed ARF6 WT or K3R mutant (KR) showing that SIRT2 demyristoylates ARF6 in HEK293T cells as indicated by the disappearance of the dimyristoylated species with SIRT2 OE. b SIRT2 demyristoylates ARF6 and ARF6 G2A in vitro. ARF6 WT and G2A were isolated from Alk12 treated cells with NMT2 overexpression and were treated with recombinant SIRT2 followed by TAMRA azide conjugation and in-gel fluorescence. c ³²P-NAD⁺ assay scheme. d SIRT2 demyristoylates ARF6 K3 purified from cells with endogenous NMT in vitro (detected using the ³²P-NAD⁺ assay). e SIRT2 inhibition with TM in cells increases lysine fatty acylation of ARF6 (detected using the ³²P-NAD⁺ assay on ARF6 isolated from HEK293T cells treated with TM). f Alk12 labeling in HEK293T cells transiently overexpressing ARF6 G2A showing that SIRT2 KD or inhibition increase but SIRT2 OE removes the labeling. g SIRT2 removes double and single lysine myristoylation produced by in vitro acylation with NMT1/2. The indicated ARF6 proteins were isolated from cells with NMT inhibition and were first modified by NMT with Alk12-CoA in vitro and then reacted with SIRT2 in vitro. h Endogenous ARF6 has lysine myristoylation in HEK293T cells. Fifteen-hour treatment with 10 mM nicotinamide or 2 μM DDD85646 was used. Endogenous myristoylated proteins were labeled with Alk12 followed by conjugation to biotin azide via click chemistry and streptavidin pull down. The pull down products were analyzed by western blot for ARF6. Data points represent biological replicates analyzed by unpaired two-tailed t test. Error bars represent SEM. i Model showing that SIRT2 can remove ARF6 K3 myristoylation.