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Cell Biology and Toxicology

, Volume 24, Issue 1, pp 39–53 | Cite as

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

  • J. Vrba
  • Z. Dvořák
  • J. Ulrichová
  • M. Modrianský
Original Article

Abstract

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 IC50 values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. 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 ∼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.

Keywords

Chelerythrine HL-60 cells Oxidative burst Phosphoinositide-dependent protein kinase 1 Protein kinase C Sanguinarine 

Abbreviations

A23187

Ca2+ ionophore A23187

DAG

diacylglycerol

DMSO

dimethyl sulfoxide

fMLP

N-formyl-Met-Leu-Phe

LDH

lactate dehydrogenase

MCLA

2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride

MG132

Z-Leu-Leu-Leu-al

PDK1

phosphoinositide-dependent protein kinase 1

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

SDS

sodium dodecyl sulfate

SOD

superoxide dismutase

Notes

Acknowledgments

We thank Dr. Jaroslav Vičar for kindly providing the tested alkaloids. This work was supported by grant MSM 6198959216 from the Ministry of Education, Youth and Sports of the Czech Republic and by grant 303/06/P193 from the Grant Agency of the Czech Republic.

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • J. Vrba
    • 1
  • Z. Dvořák
    • 1
  • J. Ulrichová
    • 1
  • M. Modrianský
    • 1
  1. 1.Department of Medical Chemistry and Biochemistry, Faculty of MedicinePalacký UniversityOlomoucCzech Republic

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