Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.
Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.
Rey
O
O
Unit of Signal Transduction and Gastrointestinal Cancer, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90095, USA.
Young
S H
SH
Cantrell
D
D
Rozengurt
E
E
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D001839:Bombesin / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D018004:Receptors, Bombesin / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D000097573:Red Fluorescent Protein
D001839:Bombesin; D003545:Cysteine; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011493:Protein Kinase C; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C089212:protein kinase D