TLR3- and TLR4-mediated DC activation lead to distinct function of mature DC, in which MAPKs pathway is involved. JNK-associated leucine zipper protein (JLP) associates with JNK/p38 phosphorylation, however the role of JLP in DC is not well understood. We previously found either LPS or poly(I:C) up-regulated JLP expression in BMDC. Therefore we hypothesized JLP plays a differential role in TLR3- and TLR4-mediated BMDC maturation and functions. We demonstrated JLP facilitated LPS- and poly(I:C)-induced CD86 and CD40 up-regulation, LPS-induced CD80 up-regulation and poly(I:C)-induced MHC-II up-regulation. We found JLP down-regulated IL-12 production in LPS-stimulated BMDC, and up-regulated IL-6 production in poly(I:C)-stimulated BMDC. Our data also showed JLP negatively regulated MHC-II antigen presentation in LPS- and poly(I:C)-stimulated BMDC and JLP was involved in promoting LPS-activated BMDC survival, but not in poly(I:C)-activated BMDCs. Therefore our current data suggested a multi-functional role of JLP in the regulation of TLR3- and TLR4-mediated DC maturation.

TLR3- and TLR4-mediated DC activation lead to distinct function of mature DC, in which MAPKs pathway is involved. JNK-associated leucine zipper protein (JLP) associates with JNK/p38 phosphorylation, however the role of JLP in DC is not well understood. We previously found either LPS or poly(I:C) up-regulated JLP expression in BMDC. Therefore we hypothesized JLP plays a differential role in TLR3- and TLR4-mediated BMDC maturation and functions. We demonstrated JLP facilitated LPS- and poly(I:C)-induced CD86 and CD40 up-regulation, LPS-induced CD80 up-regulation and poly(I:C)-induced MHC-II up-regulation. We found JLP down-regulated IL-12 production in LPS-stimulated BMDC, and up-regulated IL-6 production in poly(I:C)-stimulated BMDC. Our data also showed JLP negatively regulated MHC-II antigen presentation in LPS- and poly(I:C)-stimulated BMDC and JLP was involved in promoting LPS-activated BMDC survival, but not in poly(I:C)-activated BMDCs. Therefore our current data suggested a multi-functional role of JLP in the regulation of TLR3- and TLR4-mediated DC maturation.

JLP (JNK associated Leucine zipper protein) is a scaffolding protein that has been shown to interact with and activate the JNK/p38MAPK pathway. Its interaction with various signaling proteins is associated with coordinated regulation of cellular processes such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here, we undertook a mass spectrometric approach to identify novel interaction partners of JLP and identified the mitotic Ser/Thr kinase, Polo like Kinase 1 (PLK1) and the Fox transcription factor, Forkhead box protein K1 (FoxK1), as proteins that interact with and form a ternary complex with JLP during mitosis. Domain mapping studies showed that the N-terminal domain of JLP interacts with the polo-box domain (PBD) of PLK1 in a phosphorylation-dependent manner. Our results indicate that, JLP is phospho-primed on Thr351, which is recognized by the PBD of PLK1 and leads to phosphorylation of JLP at additional sites. Moreover, treatment of cells with the PLK1 inhibitor BI2536 affects this interaction, demonstrating the importance of PLK1 kinase activity in this process. Because JLP is a scaffolding protein that recruits proteins to mediate specific cell signaling events, the interaction of JLP with PLK1 likely results in the recruitment of other proteins to this complex. To test this hypothesis, we carried out SILAC labeling of proteins in mitotic cells in the presence or absence of BI2536. Through mass-spectrometry, we identified the FoxK1 transcription factor as a PLK1-dependent JLP-interacting protein. Furthermore, we show that JLP, PLK1 and FoxK1 form a ternary complex that is present only during mitosis. Knockdown of PLK1 and not JLP affected the interaction between JLP and FoxK1, indicating that the formation of the ternary complex is PLK1-dependent. FoxK1 is a known transcriptional repressor of the cyclin dependent kinase inhibitor, p21/WAF1. Knockdown of JLP in U2OS cells resulted in increased FoxK1 protein levels and a reduction of p21 expression. Moreover, immunofluorescence studies in asynchronous cells showed that FoxK1 is excluded from the nucleus during mitosis and that a fraction of FoxK1 localizes to the midbody region during cytokinesis. Analysis of FoxK1 protein in cells exiting S-phase suggests that FoxK1 is post-translationally modified during mitosis. In this study we characterized the ternary complex formed between JLP, PLK1 and FoxK1 during mitosis. Based on our observations, we propose that formation of the JLP/PLK1/FoxK1 ternary complex regulates the stability and/or transcriptional activity of FoxK1. / Molecular Biology and Genetics