Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine.
The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC(50) values not exceeding 4.6 micromol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 micromol/L, sanguinarine and chelerythrine prevented phosphorylation of approximately 80 kDa protein and sequestered approximately 60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCalpha/betaII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCalpha/betaII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of approximately 70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.
Vrba
J
J
Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacký University, Olomouc, Czech Republic. vrbambv@seznam.cz
Dvorák
Z
Z
Ulrichová
J
J
Modrianský
M
M
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000470:Alkaloids / Q000737:chemistry / Q000494:pharmacology; D053119:Benzophenanthridines / Q000737:chemistry / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D016897:Respiratory Burst / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
D000470:Alkaloids; D053119:Benzophenanthridines; D007527:Isoenzymes; D007546:Isoquinolines; C005705:sanguinarine; C016299:chelerythrine; D019255:NADPH Oxidases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C