Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells.
Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.
Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA; Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Electronic address: jin.ouchi@jefferson.edu.
Jin
J
O-Uchi
Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Jaime
J
Sorenson
Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Bong Sook
BS
Jhun
Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Jyotsna
J
Mishra
Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Stephen
S
Hurst
Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Kaleef
K
Williams
Center for Translational Medicine, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Shey-Shing
SS
Sheu
Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Electronic address: coeli_lopes@urmc.rochester.edu.
Coeli M B
CM
Lopes
D002462:Cell Membrane / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D020033:Protein Isoforms; D018340:Receptors, Adrenergic, alpha-1; D011493:Protein Kinase C
Adrenoceptor; Confocal microscopy; G-protein coupled receptor; Protein kinase C; Signal transduction