Dynamic Positive Feedback Phosphorylation of Mixed Lineage Kinase 3 by JNK Reversibly Regulates Its Distribution to Triton-soluble Domains

Abstract

MLK3 (mixed lineage kinase 3) is a widely expressed, mammalian serine/threonine protein kinase that activates multiple MAPK pathways. Previously our laboratory used in vivo labeling/mass spectrometry to identify phosphorylation sites of activated MLK3. Seven of 11 identified sites correspond to the consensus motif for phosphorylation by proline-directed kinases. Based on these results, we hypothesized that JNK, or another proline-directed kinase, phosphorylates MLK3 as part of a feedback loop. Herein we provide evidence that MLK3 can be phosphorylated by JNK in vitro and in vivo. Blockade of JNK results in dephosphorylation of MLK3. The hypophosphorylated form of MLK3 is inactive and redistributes to a Triton-insoluble fraction. Recovery from JNK inhibition restores MLK3 solubility and activity, indicating that the redistribution process is reversible. This work describes a novel mode of regulation of MLK3, by which JNK-mediated feedback phosphorylation of MLK3 regulates its activation and deactivation states by cycling between Triton-soluble and Triton-insoluble forms.Protein kinases and their signaling cascades are dynamically regulated through protein-protein interactions, subcellular targeting, and phosphorylation. By differential integration of these regulatory mechanisms, a protein kinase and its signaling pathway can lead to distinct cellular responses. In addition, many protein kinases are subjected to feedback regulation, and biological signaling pathways are modulated by positive and negative regulatory feedback loops.MLK3 (mixed lineage kinase 3) is a widely expressed mammalian serine/threonine kinase that functions as a mitogenactivated protein kinase kinase kinase (MAPKKK) 2 and can activate multiple MAPK pathways (1). MLK3-induced JNK activation, through dual phosphorylation of MKK4 and/or JNKK2/MKK7 (2), is implicated in neuronal apoptosis in response to trophic factor withdrawal (3-6). Targeted gene disruption of mlk3 decreases tumor necrosis factor-␣-induced JNK activation (7), consistent with findings in human cell lines (8, 9). In addition, Slipper, the MLK3 homolog in Drosophila, is required for JNK-dependent dorsal closure in the fly embryo (10). MLK3 can also activate the p38 pathway (11), perhaps in a JNK-interacting protein-2-dependent fashion (12-14), and recently a scaffolding role for MLK3 in B-Raf-mediated ERK activation has emerged (15, 16). Our laboratory has focused on understanding the mechanisms that regulate MLK3 activity and signaling. In addition to its catalytic domain, MLK3 contains several other regions important for its regulation, including an N-terminal Src homology 3 domain and a centrally located zipper followed by a Cdc42/Rac-interactive binding motif. Notably, the COOH-terminal region of MLK3 is rich in Ser, Thr, and Pro residues, with the final 220 amino acids of MLK3 being composed of 12, 13, and 24%, respectively, of these amino acids. Activated forms of the small GTPases, Cdc42 and Rac, can bind to MLK3, cause retardation of MLK3 electrophoretic mobility, increas...

MLK3 is critical for breast cancer cell migration and promotes a malignant phenotype in mammary epithelial cells

“…The increased levels of active JNK and active ERK observed (Supplementary Figure 3), upon treatment with the p38 inhibitor, likely reflect the previously reported antagonistic crosstalk between the p38 pathway and other MAPKs (Hall and Davis, 2002;Schachter et al, 2006), as this phenomenon was also observed in MDA-MB-231 cells (Supplementary Figure 4). (-) or with 25 nM AP21967 and subjected to a transwell migration assay as described under Materials and methods.…”

“…In complementary experiments, we asked whether induced expression of active MLK3 could promote the migration of poorly invasive MCF-7 breast cancer cells (Zhang et al, 2004;Schachter et al, 2006) and MCF10A mammary epithelial cells. When a stable population of MCF-7-MLK3 cells was treated with the transcriptional inducer AP21967 and subjected to a transwell migration assay, a 2.5-fold increase in the migration of the MCF-7 cells ( Figure 4a) was observed.…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…However, transcriptional-level feedback is not the only option for MAPK pathways: the JNK pathway is also subject to feedback phosphorylation. It is known that MLK3 and DLK can be directly phosphorylated by activated JNK to exert positive feedback, although the exact sites and mechanisms differ greatly between these two proteins (44,96). Such positive feedback loops can result in bistable switches (97) that can ensure immediate and robust responses by rapid, local, and maximal kinase activation.…”

Critical Role for Mixed-Lineage Kinase 3 in Acetaminophen-Induced Hepatotoxicity

“…5A); however, the underlying mechanism is unclear. It was suggested that positive feedback phosphorylation of MLK3 by JNK may lead to sustained JNK activation (Schachter et al, 2006), which may result in amplification of oxidative stress. We observed decreased ROS production in APAPtreated MLK3-KO mice, compared with WT mice (Fig.…”