A protein kinase inhibitor, staurosporine, enhances the expression of phorbol dibutyrate binding sites in human polymorphonuclear leucocytes

Abstract

Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.

NADPH Oxidase-Dependent Reactive Oxygen Species Mediate Amplified TLR4 Signaling and Sepsis-Induced Mortality in Nrf2-Deficient Mice

“…We did not observe any significant changes in PKC activity in LPS-treated Nrf2 −/− macrophages with or without BSO pretreatment. Finally, to determine whether PKC activity was responsible for higher ROS generation in Nrf2 −/− macrophages, we measured ROS levels after LPS stimulation in the presence of the PKC inhibitor, staurosporine (34). Staurosporine significantly attenuated LPS-induced ROS generation in Nrf2 −/− and Nrf2 +/+ macrophages (Figure 1F).…”

μ-Calpain Activation in β-Lapachone-Mediated Apoptosis

“…5B and Fig. 5D); STS is a protein kinase inhibitor 53 that initiates apoptosis via a caspase-mediated pathway. 54 Collectively, these data implicate µ-calpain activation in β-lap-mediated apoptosis and µ-calpain translocation to the nucleus upon its activation.…”

Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells

“…The InsP 3 receptor is fully activated in the presence of 10 Ϫ8 to 10 Ϫ7 M InsP 3 (Mak et al, 1998), which corresponds to a number of 12,000-120,000 molecules in a given cell of 2 picoliter volume. In various cell types, phorbol ester binding experiments have revealed a number of 40,000-800,000 binding sites per cell (Trilivas and Brown, 1989;Obeid et al, 1990;Combadière et al, 1993). Although these binding sites presumably include other DAG receptors, such as PKD, chimaerins, Munc13-1, or Ras-GRP, the total number of DAG acceptors, including classical and novel PKCs, appears not to be in vast excess over the number of DAGs formed during receptor stimulation.…”