Mitogen-activated protein (MAP) kinases are involved in fundamental biological processes, such as cell proliferation, differentiation, migration, and death, as well as in various aspects of embryonic development, morphogenesis, inflammation, wound healing, and cellular responses to stress [1–11]. The mammalian MAPK superfamily encompasses the extracellular signal-regulated kinases (ERK1 and ERK2), the p38 MAP kinases (p38α, β, γ, and δ), the big MAP kinase (BMK1/ERK5), and the c-Jun NH2-terminal kinases (JNK1, JNK2, and JNK3) [1–7]. The JNK family of kinases is of particular importance for cellular responses to stress [8–11]. Most somatic cells express JNK1 and JNK2, the expression of JNK3 being restricted predominantly to the brain [12, 13]. While the single homozygous deletions of either jnk1 or jnk2 in the mouse revealed a significant functional redundancy between JNK1 and JNK2, the compound jnk1-/-/jnk2-/- mutant mice die before birth, thus revealing the non-redundant role of the JNK family as a whole in development . In addition to development, JNKs play important roles in mediating cellular responses to oxidative, genotoxic, ribotoxic, and hyperosmotic stresses [3, 15, 16]. JNKs are activated via phosphorylation by two upstream JNK kinases (JNKK), MKK4 and MKK7 [8, 17]. In turn, JNKK are activated via phosphorylation by JNK kinase kinases (JNKKK). To date, at least 13 mammalian JNKKK have been identified, namely: (i) the MEK kinase (MEKK) family, containing 4 members, MEKK1, 2, 3, and 4 , (ii) the apoptosis signal-regulating kinase 1 (ASK1) , (iii) the transforming growth factor β-activated kinase 1 (TAK1) , and (iv) the family of mixed lineage kinases (MLK), containing three subfamilies, namely the MLK subfamily (MLK 1, 2, 3, and 4), the dual-leucine-zipper-bearing kinase subfamily (DLK and LZK), and the zipper sterile-α-motif kinase (ZAK) . Clearly, the complexity and specificity of JNK activation is executed mainly at the level of JNKKK, as the number of different JNKKK exceeds by far the number of JNKK. Upstream of JNKKK, the regulation of the JNK pathways is thought to be dependent on the activity of various small GTP-binding proteins (such as Rho, Rac, cdc42, Ras, and Ral) [22–25]. Thus, elucidation of the mechanisms of activation of JNK by a stressor of interest necessarily involves the identification of GTPase(s) and JNKKK relaying a stress-generated signal to JNKK and JNK.
Toxic metals and their compounds (e.g. cadmium and arsenite) are among the most potent activators of JNK [26–28]. Antioxidants prevent or reduce the activation of JNK by both cadmium and arsenite, suggesting that toxicant-induced oxidative stress is operative in the activation of JNK by these agents . However, the identification of specific mammalian JNKKK-JNKK modules involved in the activation of JNK by cadmium and arsenite has been difficult due to the complexity and potential redundancy of mammalian JNKKK.
The genome of the fruit fly Drosophila melanogaster possesses all JNKKK, JNKK, and JNK families present in the mammalian genome, but represented, typically, by fewer genes. For instance, the mammalian MEKK family is represented in Drosophila by a single member, mekk1/D-MEKK1 . The mammalian MLK family is represented in Drosophila by 2 members, slpr/D-MLK and CG8789/D-DLK . The fruit fly orthologues of mammalian ASK1 and TAK1 are pk92B/D-ASK and tak1/D-TAK1, respectively . The fruit fly orthologues of MKK4 and MKK7 are mkk4/D-MKK4 and hep/D-MKK7, respectively . Finally, Drosophila contains only one JNK gene, bsk/D-JNK . This evolutionary conservation of JNK signal transduction pathways among metazoans underscores the fundamental importance of JNK in mediating inducible stress responses.
With the above rationale in mind, we employed Drosophila S2 cells and the RNA interference (RNAi) technique (also known as "knock down" technique) for gene silencing [30, 31] to investigate the signal transduction pathways mediating the activation of D-JNK by cadmium and arsenite. We knocked down 13 upstream regulators of D-JNK, either singly or in combinations of up to 7 at a time. As a result of this approach, we demonstrate the involvement of D-MEKK1 and D-MKK7 in the activation of D-JNK by cadmium and arsenite.