Protein kinase Cδ-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

Abstract

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKCdelta is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKCdelta by RNAi (sh-PKCdelta) or expression of the kinase-dead (Lys376Arg) mutant of PKCdelta (PKCdeltaKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (gamma-H2AX), a hallmark for DNA damage, was altered by sh-PKCdelta, and sh-PKCdelta completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKCdelta-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKCdelta and caspase-3, suggesting that PKCdeltaKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKCdelta-dependent and -independent manners. In summary, these findings suggest that the activation of a PKCdelta-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment.

Identification of an AAA ATPase VPS4B-Dependent Pathway That Modulates Epidermal Growth Factor Receptor Abundance and Signaling during Hypoxia

“…We therefore evaluated steady-state protein levels of these molecules under hypoxic stress. First, Pa-4 cells were treated with desferrioxamine (DFO), known to induce a hypoxic environment (9,11). Consistent with recent reports by us and others (10,41), sustained hypoxic stress decreased steady-state levels of BECLIN-1 and p62, while the UVRAG level increased during chronic hypoxic exposure (Fig.…”

“…For protein expression level and phosphorylation studies, whole-cell lysates were extracted with sodium dodecyl sulfate (SDS) lysis buffer as previously described (9,11), supplemented with both complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche). Equal amounts of whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently immunoblotted with antibodies recognizing VPS4B (Abnova), p62/ SQSTM1 (American Research Products, Inc.), BECLIN-1 and UVRAG (Abcam), hemagglutinin (HA) and Myc tags (Santa Cruz Biotechnologies), LC3 (MBL International Co.), pERK1/2, pJNK1/2, EGFR, pY1068-EGFR (recognizes human pY1092), pY1173-EGFR (recognizes human pY1197), and pY845-EGFR (recognizes human pY869) (all from Cell Signaling), and actin (Millipore).…”

Novel Roles for Protein Kinase C;-dependent Signaling Pathways in Acute Hypoxic Stress-induced Autophagy

“…PKC␦, PKC␦KD, is a dominant negative form of PKC␦ (17). In contrast, EBSS rendered a comparable LC3 processing in both Pa-4 and Pa-4/PKC␦KD cells ( Fig.…”

“…Cell Lines and Chemicals-Rat parotid epithelial cell lines, Pa-4 and Pa-4/PKC␦KD, were generated and cultured as previously described (17). MEF/WT, MEF/JNK1 Ϫ/Ϫ , MEF/ JNK2 Ϫ/Ϫ , MEF/Atg5…”

“…Hypoxic Stress-We previously reported that treatment of cells with DFO, a hypoxia-mimetic agent, confers a cross-talk between the activation of PKC␦ and apoptotic caspase-3 pathways (17). Since signaling pathways that regulate cell proliferation and apoptosis are frequently associated with the regulation of autophagy, we hypothesized that hypoxia or DFO treatment induces autophagy.…”