Figure 1. TAK1 and MST3 directly phosphorylate Rab8A at Thr72.

(A) Summary analysis of kinase assay screen quantification to identify novel Rab8A kinases. One hundred and forty protein kinases from the MRC PPU Reagents and Services library were assessed for the phosphorylation of GDP-Rab8A. One hundred nanograms of protein kinase was incubated with 2 µg substrate and [γ-³²P] ATP for 30 min. Samples were subjected to SDS–PAGE and analysis with Cerenkov counting of Coomassie substrate bands, displayed graphically. Full kinase assay analysis by autoradiography and immunoblotting is described in Supplementary Figure S1. Proteins highlighted in red were identified to phosphorylate Rab8A at Thr72 by phospho-specific immunoblotting. *Thr72 is a minor MAP4K5 phosphorylation site and occurs in GDP-Rab8A but not GTP-Rab8A conformation. (B) Correlative analysis of kinase phosphorylation of GTP-Rab8A (y-axis) versus GDP-Rab8A (x-axis). The GTP-Rab8A kinase screen was performed in a similar manner to GDP-Rab8A kinase screen, with full analysis described in Supplementary Figure S2. Kinases that phosphorylate Thr72 as the major site of Rab8A are highlighted in red. (C) Mutation of Thr72Ala (T72A) reduces Rab8A phosphorylation by LRRK2, TAK1 and MST3 but not MAP4K5. The indicated kinases were incubated in the presence of WT or T72A GDP-Rab8A. Samples were subjected to SDS–PAGE with analysis by Coomassie staining and [γ-³²P] incorporation as visualized by autoradiography and immunoblotting analysis using the indicated antibodies.