Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues.
The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca2+ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC alpha were included for comparison. The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C alpha, beta 1, delta, epsilon, and zeta/iota were all present in rat midbrain cytosolic extract, PKC alpha, beta 1, delta, and zeta/iota were present in spleen cytosol, and PKC alpha and zeta/iota were present in COS 7 cell cytosol. The predominance of alpha and beta activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay. The PKC activity assay was configured such that the Ca2+ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca(2+)-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca2+, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca2+ whereas in the presence of Ca2+, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca2+. Maximum values for Ca(2+)-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca(2+)-dependent PKC isoform activity in a Ca2+ free environment. The greater potency of mezerein on PKC alpha was confirmed using PKC alpha and beta further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca2+ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca(2+)-dependent isoforms in Ca2+ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Johnson
M S
MS
MRC Brain Metabolism Unit, University Department of Pharmacology, Edinburgh, UK.
Simpson
J
J
MacEwan
D J
DJ
Ison
A
A
Clegg
R A
RA
Connor
K
K
Mitchell
R
R
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008636:Mesencephalon / Q000201:enzymology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000201:enzymology; D014018:Tissue Distribution
D004075:Diglycerides; D007527:Isoenzymes; D011994:Recombinant Proteins; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C