Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19.
The relationship between intracellular free calcium ([Ca2+]i) and the activation of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) was investigated in the NSC-19 motoneuron cell line. Increased extracellular calcium ([Ca2+]o) up to 10 mM resulted in sustained elevations of [Ca2+]i. Control cell cultures (1.3 mM [Ca2+]o, [Ca2+]i = 83 +/- 17 nM) contained Ca2+- and PS/DO lipid-dependent PKC activity predominantly in the cytosol. However, elevation of [Ca2+]o up to 5 mM ([Ca2+]i = 232 +/- 24 nM) resulted in almost complete loss of cytosolic PKC activity. Cells incubated in 10 mM [Ca2+]o ([Ca2+]i = 365 +/- 13 nM) showed increased levels of both cytosolic and membrane PKC activity compared to control. These alterations in PKC activity appeared to be translocation-independent, since PKC protein levels were unchanged as demonstrated by Western blotting analysis. When cells were exposed to 25 or 50 mM [Ca2+]o, [Ca2+]i rose transiently to over 600 and 900 nM, respectively, and then returned to near basal values. Under these conditions, total PKC activity decreased, and increased amounts of the catalytic fragment of PKC, protein kinase M, were generated. Extracts from cells exposed to [Ca2+]o between 1.3 and 25 mM did not differ significantly in the levels of measurable CaMKII activity 10 min following the change in [Ca2+]o.
Hasham
M I
MI
Department of Medicine, VHHSC, University of British Columbia, Vancouver, Canada.
Pelech
S L
SL
Koide
H B
HB
Krieger
C
C
D000818:Animals; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D051379:Mice; D009046:Motor Neurons / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*
D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium