Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.