Potencies of protein kinase C inhibitors are dependent on the activators used to stimulate the enzyme.
The aim was to examine systematically the potencies of protein kinase C inhibitors as a function of the kinase activator. Protein kinase C is activated by at least four stimulators: calcium plus phosphatidylserine (Ca/PS), phorbol 12-myristate 13-acetate plus PS (PS/PMA), arachidonic acid plus calcium (Ca/AA) and the synthetic peptide activator PCK530-558. With histone or GS1-12 as substrates, protein kinase C was maximally activated by Ca/PS, or to maxima of 62%, 89% or 82% with PS/PMA, Ca/AA or PKC530-558, respectively. One group of inhibitors, including H-7 and staurosporine, were equipotent, regardless of the activator. All other inhibitors showed variable selectivity, dependent upon the activator. A second group of inhibitors, including sphingosine and lipophosphoglycan, were eight or 200 times more potent for inhibition of PS/PMA-stimulated activity (relative to Ca/PS) and a third group, including retinal and palmitoylcarnitine, were 14 or 262 times more potent towards Ca/PS-stimulated activity. A final group (rhodamine 6G) was nine times more potent when Ca/AA was the activator. Similar results were obtained using the endogenous substrates dephosphin or MARCKS in synaptosol. Phosphorylation of MARCKS was stimulated by PS/PMA or Ca/PS, while phosphorylation of dephosphin was stimulated only by Ca/PS. The phosphorylation of either by Ca/PS-activated kinase was nine times more potently inhibited by palmitoylcarnitine, while phosphorylation of MARCKS by PS/PMA-activated kinase was 10 times more potently inhibited by sphingosine. H-7 inhibited both at similar concentrations. A model encompasses these differences in potency if the inhibitors are divided into four groups (A-D) according to their competitive inhibition with the appropriate activator or at the active site. The non-selective inhibitors interact at the active sites of protein kinase C (group A). The compounds which preferentially inhibit PS/PMA-activated kinase (sphingosine and lipophosphoglycan) are competitive inhibitors of PMA and 1,2-diacylglycerol (group B), those selective for Ca/PS-activated kinase (palmitoylcarnitine and retinal) are competitive with PS (group C) and those selective for Ca-AA activation (rhodamine 6G) are likely to be competitive with fatty acid (group D). Therefore, the effectiveness of protein kinase C inhibitors is dependent upon the activator employed.
Robinson
P J
PJ
Endocrine Unit, John Hunter Hospital, New South Wales, Australia.
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D034281:Dynamins; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010718:Phosphatidylserines; D010750:Phosphoproteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D016718:Arachidonic Acid; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D034281:Dynamins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium