Interaction of JLP with Plk1 recruits FoxK1 to interact and form a ternary complex

JLP (JNK associated Leucine zipper protein) is a scaffolding protein, which has been shown to interact with and activate JNK/p38MAPK pathway. Its interaction with various signaling proteins is associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here we identified a mitotic Serine/Threonine kinase, Polo like kinase 1 (Plk1), as a novel interaction partner of JLP through a mass spectrometry based approach. We show 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 Thr 351 residue on its Nterminus, which is recognized by the PBD of Plk1 leading to phosphorylation of JLP at additional sites. Moreover, treatment of cells with the Plk1 inhibitor, BI2536 affects the interaction demonstrating the importance of Plk1 kinase activity in this process. Since JLP is a scaffolding protein that recruits proteins to mediate specific cell signaling events, we hypothesized that the interaction of JLP with Plk1 might result 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 transcription factor, FoxK1 as a Plk1-dependent JLP-interacting protein. Furthermore, we show that JLP, Plk1 and FoxK1 form a ternary complex, which occurs 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 dependent on Plk1. FoxK1 has been previously characterized as a transcriptional repressor of cyclin dependent kinase inhibitor, p21/WAF1. We observed that knockdown of JLP in U2OS cells results in increased protein levels of FoxK1 and a reduction of p21 expression. Moreover, immunofluorescence studies in asynchronous cells showed that FoxK1 is excluded from the nucleus during mitosis. Based on our observations, we propose that formation of the ternary complex between JLP, Plk1 and FoxK1 regulates the stability and/or localization of FoxK1.