Figure 6: ARHGAP36 induces PKAC ubiquitylation and engagement with the ESCRT pathway.

(a) HEK293T cells were transfected with PKAC-YFP or YFP-Cherry control, Flag-ARHGAP36 and His-Ubiquitin as indicated. Lysates were subjected to GFP IP. (b) SILAC-labelled HEK293T cells were transfected with PKAC-Flag in the presence or absence of YFP-ARHGAP36. Lysates were subjected to Flag IP. Normalized heavy/light ratio plot of the peptide evidences matching PRKAC. Only one peptide, highlighted in red, was upregulated in the presence of ARHGAP36 and was the only ubiquitylated PRKAC peptide identified. (c) The same as in a except where indicated PKAC-K285R-YFP was transfected and a Flag-Cherry control for Flag-ARHGAP36 was used. (d) The same as in c except lysates were subjected to His IP and immunoblotted with anti-GFP. (e) HEK293T cells were transfected with PKAC-Flag and CFP-ARHGAP36, the RRV mutant or CFP-Cherry control. Lysates were immunoblotted with the indicated antibodies. (f) Confocal micrographs of MDCK cells transfected with PKAC-YFP or PKAC-K285R-YFP and Flag-ARHGAP36. Twelve hours after transfection, cells were pretreated for 30 min with bafilomycin (100 nM), before cycloheximide (CHX; 1 μg ml⁻¹) addition for a further 5 h. Cells were fixed and subjected to immunofluorescence using anti-GFP and anti-Flag. Scale bars, 10 μm. (g) HEK293T cells were transfected with PKAC-YFP in the presence or absence of Flag-ARHGAP36 and subjected to GFP IP followed by SRM-MS-based relative quantification of polyubiquitin linkage-type peptides. Data were normalized using the signal intensity of PKAC derived from a shotgun MS analysis of the same samples. Samples were measured in duplicate and the top two transitions used for quantification. The error bars represent the s.d. of the four calculated area ratios. (h) HEK293T cells were transfected with GFP-Vps4-WT or GFP-Vps4-EQ, PKAC-Flag and Cherry-ARHGAP36 or Cherry control as indicated. Cells were treated with CHX (50 μg ml⁻¹) and collected at the indicated time points. Flag-PKAC levels were densitometrically evaluated and normalized to the first lane shown.