Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues |
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Authors: | Melanie S Johnson James Simpson David J MacEwan Angela Ison Roger A Clegg Kevin Connor Rory Mitchell |
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Institution: | (1) MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, EH8 9JZ Edinburgh, UK;(2) Department of Biochemistry, University of Glasgow, G12 8QQ Glasgow, UK;(3) Hannah Research Institute, KA6 5HL Ayr, UK |
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Abstract: | 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 were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , 1, , , and / were all present in rat midbrain cytosolic extract, PKC , 1, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and 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 Ca2+-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 Ca2+-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca2+-dependent PKC isoform activity in a Ca2+ free environment. The greater potency of mezerein on PKC was confirmed using PKC and 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 Ca2+-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+. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, 3H]PDBu binding data using PKC purified from several sources gave very different IC50 values when DOG was used as a displacer, and in general these values correlated with the EC50 values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor. |
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Keywords: | protein kinase C activation phorbol ester diglyceride midbrain spleen COS 7 |
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