Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells.
The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.
Goodall
A R
AR
Institute for Cardiovascular Research, University of Leeds, England.
Turner
N A
NA
Walker
J H
JH
Ball
S G
SG
Vaughan
P F
PF
D000409:Alanine / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009638:Norepinephrine / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010703:Phorbol Esters; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; D010770:Phosphotransferases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine; D009638:Norepinephrine