Involvement of protein kinase C-epsilon in activity-dependent potentiation of large dense-core vesicle exocytosis in chromaffin cells.
Neurotransmitter release is modulated in an activity-dependent manner. We showed previously that repetitive stimulation of nicotinic acetylcholine receptor (nAChR) induced activity-dependent potentiation (ADP) of large dense-core vesicle (LDCV) exocytosis in chromaffin cells. Here we report that protein kinase C (PKC)-epsilon is critically involved in ADP. Stimulation of nAChR induced activation of PKC-epsilon, and inhibition of PKC-epsilon by expression of the dominant-negative mutant of PKC-epsilon (DN-PKC-epsilon) or short interfering (siRNA) against PKC-epsilon abolished ADP via decreasing the frequency and quantal size of fused vesicles without affecting basal exocytosis, suggesting that PKC-epsilon is specifically involved in ADP. Electron microscopy revealed that inhibition of PKC-epsilon disrupts activity-induced vesicle translocation required for ADP. We also suggest the involvement of myristoylated alanine-rich C kinase substrate (MARCKS), which is known as a downstream target of PKC-epsilon, in ADP of LDCV exocytosis. The level of phospho-MARCKS correlated with the time course of ADP and was reduced by transfection with DN-PKC-epsilon. Actin filament disassembly induced by MARCKS phosphorylation was also significantly blocked by transfection of DN-PKC-epsilon. Furthermore, knockdown of MARCKS by siRNA resulted in inhibition of ADP and reduction of the number of fused vesicles. Together, we provide evidence that ADP of LDCV exocytosis is regulated by PKC-epsilon and its downstream target MARCKS via modulating vesicle translocation.
Park
Yong-Soo
YS
Department of Life Science, Division of Molecular and Life Sciences, Systems Biodynamics National Core Research Center, Pohang University of Science and Technology, Pohang, 790-784, South Korea.
Hur
Eun-Mi
EM
Choi
Bo-Hwa
BH
Kwak
Eunyee
E
Jun
Dong-Jae
DJ
Park
Su-Jin
SJ
Kim
Kyong-Tai
KT
D000199:Actins / Q000737:chemistry / Q000502:physiology; D000200:Action Potentials; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000378:metabolism
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051744:Protein Kinase C-epsilon