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Activation of Protein Kinase C‐α and Translocation of the Myristoylated Alanine‐Rich C‐Kinase Substrate Correlate with Phorbol Ester‐Enhanced Noradrenaline Release from SH‐SY5Y Human Neuroblastoma Cells
Author(s) -
Goodall Anna R.,
Turner Neil A.,
Walker John H.,
Ball Stephen G.,
Vaughan Peter F. T.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68010392.x
Subject(s) - marcks , protein kinase c , cytosol , phorbol , myristoylation , chromosomal translocation , phosphorylation , biology , biochemistry , microbiology and biotechnology , chemistry , enzyme , gene
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 n M ) 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 α‐subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC‐ε from cytosolic and membrane‐associated to cytoskeleton‐ and membrane‐associated TPA had no effect on the cytosolic location of PKC‐ζ. 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 µ M ). Selective down‐regulation of PKC subtypes by prolonged exposure to phorbol 12,13‐dibutyrate (100 n M ) attenuated the TPA‐induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down‐regulation of PKC‐α (but not ‐ε or ‐ζ). 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‐α.