title: Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.
abstract: BACKGROUND
  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 PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ 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 (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. 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.
authors.affiliation: IHEM-CONICET, National Research Council, School of Medicine, University of Cuyo, Mendoza, Argentina.
authors.forename: Martín
authors.initials: M
authors.lastname: Pavarotti
authors.forename: Anahí
authors.initials: A
authors.lastname: Capmany
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vitale
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Colombo
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Damiani
mesh_terms: D019295:Computational Biology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D013058:Mass Spectrometry; D010766:Phosphorylation; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014168:Transferrin / Q000037:antagonists & inhibitors / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D007527:Isoenzymes; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D012694:Serine; D013755:Tetradecanoylphorbol Acetate; D014168:Transferrin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins