PAK1 phosphorylates RabGDIβ. (A) Sequence alignment of RabGDIα, RabGDIβ, and RhoGDI. PAK1 phosphorylation sites on RhoGDI are highlighted in green. Based on PAK1 phosphorylation motifs of known substrates (Fig. S3), putative phosphorylated site by PAK1 on RabGDIβ is highlighted (light blue). (B) In vitro kinase assay using purified proteins incubated with PAK1 kinase with and without radioactive ATP: RabGDIα, RabGDIβ, and MBP (positive control) or GST (negative control). PAK1 autophosphorylation is shown. (C) RabGDIβ WT or nonphosphorylatable mutant (S382A) were phosphorylated in vitro as described in B and separated using a Phostag gel to show mobility retardation of phosphorylated proteins. (D) Schematic diagram of RabGDIα/β domain structure showing shared conserved domains (sequence conserved region [SCR]; Schalk et al., 1996) and phosphorylated amino acids on RabGDIα or RabGDIβ known to modulate Rab binding. (E and F) Crystal structure of RabGDI-α (Protein Data Bank accession no. 1GND) mapping the different phosphorylation sites. Phosphorylated residue identified in RabGDIβ is shown in purple font (S382). (F) Zoom of the Rab binding platform on RabGDIα shows the proximity of residues identified in this work and in the literature.