JBC Papers in Press. Published on October 14, 2015 as Manuscript M115.664649 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.664649 JLP forms a ternary complex with PLK1 and FOXK1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caltech Authors JNK associated leucine zipper protein functions as a docking platform for Polo like kinase 1 and regulation of the associating transcription factor Forkhead box protein K1 Poornima Ramkumar1*, Clement M. Lee1*, Annie Moradian3, Michael J. Sweredoski3, Sonja Hess3, Andrew D. Sharrocks4, Dale S. Haines2, E. Premkumar Reddy1§ 1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 2Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA, 3Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, 4Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom §To whom correspondence should be addressed: Department of Oncological Sciences, Icahn School of Downloaded from Medicine at Mount Sinai, 1425 Madison Avenue Rm 15-20C, New York, NY-10029. Tel: 212-659-5571; Fax: 212-849-2446; Email: [email protected] * Co-corresponding authors: [email protected]; [email protected] http://www.jbc.org/ Running Title: JLP forms a ternary complex with PLK1 and FOXK1 Keywords: JLP, Scaffold protein, Serine/Threonine protein kinase, PLK1, Mitosis, Phosphorylation, Transcription factor, FOXK1, Protein-protein interaction at CALIFORNIA INSTITUTE OF TECHNOLOGY on October 19, 2015 Background: The role of JLP in cell signaling is K1 (FOXK1) transcriptional repressor to JLP. JLP not well defined. was found to interact with PLK1 and FOXK1 Results: JLP interacts with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 during mitosis and ablation of JLP leads to affected the interaction between JLP and FOXK1. increased FOXK1 protein levels. FOXK1 is a known transcriptional repressor of the Conclusion: Phosphorylation of JLP by PLK1 CDK inhibitor p21/WAF1 and knockdown of JLP recruits FOXK1, whose expression and activity is resulted in increased FOXK1 protein levels and a regulated by JLP. reduction of p21 transcript levels. Our results Significance: Our results suggest a novel suggest a novel mechanism by which FOXK1 mechanism of regulation of FOXK1 by associating protein levels and activity are regulated by with JLP-PLK1 complex. associating with JLP and PLK1. ABSTRACT INTRODUCTION JLP (JNK associated Leucine zipper protein) is a Assembly of protein complexes that mediate scaffolding protein that interacts with various responses to extracellular stimuli is critical to signaling proteins associated with coordinated ensuring the execution of specific signaling regulation of cellular process such as endocytosis, pathways. Scaffolding proteins play key roles in motility, neurite outgrowth, cell proliferation and this process by recruiting and tethering the apoptosis. Here we identified Polo like kinase 1 proteins that convey these messages within the cell (PLK1) as a novel interaction partner of JLP (1). One such well-studied scaffolding protein through mass spectrometric approaches. Our family is the family of JNK interacting proteins results indicate that JLP is phospho-primed by (JIP)↵. The JIP family consists of four members, PLK1 on Thr 351, which is recognized by the JIP1, JIP2, JIP3 and JLP (2). Of all the members PBD of PLK1 leading to phosphorylation of JLP characterized in the JIP family, JLP is at additional sites. SILAC and quantitative LC- characterized by the presence of two leucine MS/MS analysis was performed to identify PLK1 zipper (LZ) domains and a JNK binding domain dependent JLP interacting proteins. Treatment of (JBD) in its N-terminus, and a conserved C- cells with the PLK1 kinase inhibitor BI2536 terminal domain all of which functions in binding suppressed binding of the Forkhead box protein to and forming specific cell signaling complexes 1 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. JLP forms a ternary complex with PLK1 and FOXK1 (3). Although JLP is ubiquitously expressed, transcription factor FOXK1 during mitosis and previous studies have shown that the protein plays this JLP regulated function is required for down- a role in regulating neurite outgrowth by regulation of FOXK1 protein expression and interacting with SCG10 (4); activating JNK/p38 activity. MAPK signaling (3); regulate cell migration by interacting with Gα12 and Gα13 (5,6), and EXPERIMENTAL PROCEDURES function in vesicle transport by binding to kinesin light chain (KLC1) and dynein microtubule motor Plasmids: The expression vectors encoding proteins (7). Recent studies have identified that murine S-tagged full length JLP, JLP domains and JLP and JIP3 can interact with ADP ribosylation shJLP have been described previously (3,7). factor 6 (ARF6), a small G protein that function in Expression vectors for FLAG-hJLP were the regulating cytokinesis by transporting constructed by sub-cloning PCR fragments into Downloaded from recycling endosomes to the cleavage furrow (8). the EcoRI/XhoI sites of the pCMV-Tag2B This study showed that binding of ARF6 to JLP or (Agilent technologies) vector. PLK1 expression JIP3 causes a switch in the binding to dynein as vectors were constructed by sub-cloning PCR opposed to kinesin and that this pathway functions fragments of the PLK1 cDNA IMAGE clone into http://www.jbc.org/ in the transport of dynein-cargo proteins out of the the pHM6-HA vector. Site directed mutagenesis of midbody during cytokinesis (8). Studies following JLP (T351A, T334A, 2TA) and PLK1 this using JIP3:JLP double knockout mouse (H538A/K540M) was performed using embryonic fibroblasts (MEF) have shown that Quickchange II XL kit according to these cells, when cultured in vitro, have an manufacturer’s instructions (Agilent at CALIFORNIA INSTITUTE OF TECHNOLOGY on October 19, 2015 increased frequency of binucleate cells (9). In Technologies). Wild-type FLAG-FOXK1, R127A, addition this study demonstrated the importance of H355A and the wild-type FLAG-FOXK2 plasmids JLP in ARF6 localization to the midbody and have been described previously (20,21). The efficient cytokinesis (9). These studies uncovered FOXK1 domain mutants (I, II, III) were a role for JLP in the cell cycle, which was not constructed by sub-cloning PCR amplified previously reported for other JIP proteins. fragments of the FLAG-FOXK1 cDNA into the parental vector. In this study, we used a mass-spectrometry based approach to identify JLP-associated proteins and Antibodies and Reagents: The JLP-specific identified the mitotic kinase, Polo like kinase 1 antibody has been described previously (3). (PLK1), as a novel interaction partner of JLP. Antibodies against PLK1, GAPDH, HA-tag, PLK1 belongs to the Polo like kinase family of FLAG-tag, Cyclin E, S-tag, Myc-tag and p21 were Ser/Thr kinases and functions as a proto- obtained from Santa Cruz Biotechnology. oncogene, with increased expression in various Antibodies against FOXK1, FOXK2, BubR1 and cancer types (10,11). The protein has an N- T7-tag were obtained from Cell signaling terminal conserved kinase domain which functions technology and Abcam. On-Target plus Smart in phosphorylating substrates by promoting a pool siPlk1, siFOXK1, siFOXK2 and siScramble transient interaction with the protein (12,13). In were purchased from GE Dharmacon. Stock addition, the C-terminal polo box domain (PBD), solutions of BI2536 (Selleckchem), RO3306 which recognizes and binds phospho-primed (Tocris) and nocodazole (Noc) (Sigma) were substrates, regulates substrate specificity and prepared in tissue culture grade DMSO. localization of the protein during mitosis (14-18). PLK1 has been characterized to phosphorylate and Cell lines and Transfection: HEK293T, HeLa regulate the function of many proteins during and U2OS cells were maintained in DMEM G2/M phase ensuring smooth progression of (Invitrogen) supplemented with 10% FBS (Sigma) mitosis (19). Here we show that JLP interacts with and Penicillin/Streptomycin (Invitrogen). the polo box domain of PLK1 in a Transient transfections of exponentially growing phosphorylation-dependent manner. In addition, cells were performed using Lipofectamine 2000 we provide evidence that the interaction of JLP (Invitrogen). Cells were harvested 36-48 hrs post- with PLK1 results in the recruitment of the transfection. For siRNA experiments, cells were 2 JLP forms a ternary complex with PLK1 and FOXK1 transfected with 30nmol of siRNA using manufacturer’s instructions (DUO92101, Sigma). Lipofectamine RNAimax (Invitrogen) and the Coverslips were mounted on the slide using cells were harvested 24-36 hrs post-transfection. DUOLINK in situ mounting medium with DAPI. Images were obtained using a Carl Zeiss Mass Spectrometry, Immunoprecipitation and AxioImager Z1 and processed with Zen pro 2012 Western blotting: To identify novel interaction imaging software (Zeiss). partners through mass spectrometry, exponentially growing HEK293T cells were transfected with S- Kinase assays: Kinase assays were performed as tagged JLP or vector control plasmid and described previously (22). Briefly, 10ng of His- harvested 36 hrs post-transfection. The cells were PLK1 (PV3501, Invitrogen) or GST-PLK2 lysed in lysis buffer (50mM HEPES, pH 7.5; (PV4204, Invitrogen) was incubated with 2 μg of 70mM potassium acetate; 5mM magnesium purified substrate, and ATP mix (10 μM cold Downloaded