Complete Inhibition of Anisomycin and UV Radiation but Not Cytokine Induced JNK and p38 Activation by an Aryl-substituted Dihydropyrrolopyrazole Quinoline and Mixed Lineage Kinase 7 Small Interfering RNA

Abstract

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC 50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1␤ or tumor necrosis factor-␣-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stressactivated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.The mitogen-activated protein kinases (MAPK) 1 are a highly conserved family of signal transduction molecules that transmits extracellular signals from the membrane to the nucleus. There are three major branches of MAPK signaling that include ERK, c-Jun N-terminal kinase (JNK) and p38. The JNK and p38 branches of the MAPK family are activated by stress stimuli including cytokines, osmotic stress, mitogens, UV irradiation, chemotherapeutic agents, and anisomycin (1, 2). There are three kinases that form a MAPK signaling module where a MAPK is activated by a MAPK kinase (MAPKK), which in turn is regulated by a MAPKK kinase (MAPKKK) (Fig. 1). The upstream activation of JNK and p38 is complex, allowing for activation of this pathway in multiple cells and by multiple stimuli. Cellular and receptor specificity of the pathway is conferred by protein-protein interactions where the MAPK and a MAPKK assemble with a specific MAPKKK on scaffold proteins such as JNK-interacting protein (3, 4) or ␤-arrestin (5). The resulting signaling module acts as a bridge joining the appropriate receptor to the downstream effectors, enabling activation of the stress-activated MAPK.The most distal point at which signal and cell specificity for JNK and p38 activation is conferred is at the MAPKKK level. Mixed lineage kinases (MLKs) are a family of MAPKKKs activating JNK and p38. There are currently seven mammalian kinases belonging to the MLK family that have recently been reviewed (6, 7). These kinases can be divided into three subclasses based on sequence similarity and domain structure, and they include MLK1-4, dual leucine zipper kinases, and the zipper sterile ␣-motif kinases (ZAKs). Although much is known about the mechanisms re...

MAP3Ks as central regulators of cell fate during development

“…This strongly suggests a role for MLK7 in regulating cardiac function and in stress responses (Christe et al, 2004). In vitro, MLK7 is activated in response to stressors, such as anisomycin and ultraviolet irradiation, and leads to pro-apoptotic pathways through p38 and JNK (Wang et al, 2005a). Saturated free fatty acid (FFA) also causes metabolic stress and activates JNK through MLK3, MLK4, and MLK7, while JNK activity in knockout mouse models of each of these MLKs is decreased in response to FFA.…”

ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

“…17,18 SiRNA-mediated knockdown of ZAK or administration of a small molecule inhibitor (DHP-2) of ZAK, suppress anisomycin-and UV-induced apoptosis and the phosphorylation of p38 MAPK and JNK. 17 Inhibition of ZAK also suppresses Shiga toxin-and ricin-induced SAPK activation and improves cell viability.…”

“…17,18 SiRNA-mediated knockdown of ZAK or administration of a small molecule inhibitor (DHP-2) of ZAK, suppress anisomycin-and UV-induced apoptosis and the phosphorylation of p38 MAPK and JNK. 17 Inhibition of ZAK also suppresses Shiga toxin-and ricin-induced SAPK activation and improves cell viability. 18 By employing siRNA mediated knockdown of ZAK or administration of sorafenib and nilotinib, kinase inhibitors that have a high affinity for ZAK, we provide evidence that ZAK is required for doxorubicin-induced proinflammatory and apoptotic responses in HaCaT cells, a pseudo-normal human keratinocyte cell line, but not in HeLa cells.…”

“…15 In many cell types, ZAK, a MAP3K, is required for ribotoxic stressors to be capable of activating the MAPKs, through which these kinases modulate the transcription of downstream genes. 17,18 In order to determine whether doxorubicin activates JNK and p38 MAPK, we exposed HaCaT cells to varying concentrations of doxorubicin and detected the increased phosphorylation of JNK and p38 MAPK by immunoblotting against an antibody specific for the phosphorylated form of JNK and p38 MAPK. Phosphorylation of these MAPKs was detected 24 h after addition of doxorubicin (5 to 50 µM; Fig.…”

Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma

“…Recently, however, the MRKa mRNA has been shown to be abnormally spliced in gastric tumors as well as in colorectal, bladder, and breast cancers with consequent overexpression of the respective protein (13). MRKb is activated by stress (9,(14)(15)(16), including ionizing radiation (IR; refs. 7,12,17) upon which it contributes to activation of Chk2 and p38 proteins, which leads to IR-induced cell-cycle arrest.…”