Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation

Background information. Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Results. Sustained phorbol ester stimulation induces the translocation of the classical PKC alpha and PKC beta II isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKC epsilon and atypical PKC zeta isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKC alpha and PKC beta II but not PKC beta I) directly phosphorylate Rab11 in vitro. In addition, novel PKC epsilon and PKC eta but not PKC delta isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKC beta II or PKC epsilon. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. Conclusions. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking.