JLP forms a ternary complex with PLK1 and FOXK1

1

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 Medicine at Mount Sinai, 1425 Madison Avenue Rm 15-20C, New York, NY-10029. Tel: 212-659-5571; Fax: 212-849-2446; Email: ep.reddy@mssm.edu * Co-corresponding authors: poornima.ramkumar@ucsf.edu; clement.lee@mssm.edu 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 Background: The role of JLP in cell signaling is not well defined. Results: JLP interacts with PLK1 and FOXK1 during mitosis and ablation of JLP leads to increased FOXK1 protein levels. Conclusion: Phosphorylation of JLP by PLK1 recruits FOXK1, whose expression and activity is regulated by JLP. Significance: Our results suggest a novel mechanism of regulation of FOXK1 by associating with JLP-PLK1 complex. ABSTRACT JLP (JNK associated Leucine zipper protein) is a scaffolding protein that interacts with various signaling proteins associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here we identified Polo like kinase 1 (PLK1) as a novel interaction partner of JLP through mass spectrometric approaches. Our results indicate that JLP is phospho-primed by PLK1 on Thr 351, which is recognized by the PBD of PLK1 leading to phosphorylation of JLP at additional sites. SILAC and quantitative LC-MS/MS analysis was performed to identify PLK1 dependent JLP interacting proteins. Treatment of cells with the PLK1 kinase inhibitor BI2536 suppressed binding of the Forkhead box protein

K1 (FOXK1) transcriptional repressor to JLP. JLP was found to interact with PLK1 and FOXK1 during mitosis. Moreover, knockdown of PLK1 affected the interaction between JLP and FOXK1. FOXK1 is a known transcriptional repressor of the CDK inhibitor p21/WAF1 and knockdown of JLP resulted in increased FOXK1 protein levels and a reduction of p21 transcript levels. Our results suggest a novel mechanism by which FOXK1 protein levels and activity are regulated by associating with JLP and PLK1. INTRODUCTION Assembly of protein complexes that mediate responses to extracellular stimuli is critical to ensuring the execution of specific signaling pathways. Scaffolding proteins play key roles in this process by recruiting and tethering the proteins that convey these messages within the cell (1). One such well-studied scaffolding protein family is the family of JNK interacting proteins (JIP)↵. The JIP family consists of four members, JIP1, JIP2, JIP3 and JLP (2). Of all the members characterized in the JIP family, JLP is characterized by the presence of two leucine zipper (LZ) domains and a JNK binding domain (JBD) in its N-terminus, and a conserved C-terminal domain all of which functions in binding to and forming specific cell signaling complexes

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.664649The latest version is at JBC Papers in Press. Published on October 14, 2015 as Manuscript M115.664649

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

2

(3). Although JLP is ubiquitously expressed, previous studies have shown that the protein plays a role in regulating neurite outgrowth by interacting with SCG10 (4); activating JNK/p38 MAPK signaling (3); regulate cell migration by interacting with Gα12 and Gα13 (5,6), and function in vesicle transport by binding to kinesin light chain (KLC1) and dynein microtubule motor proteins (7). Recent studies have identified that JLP and JIP3 can interact with ADP ribosylation factor 6 (ARF6), a small G protein that function in the regulating cytokinesis by transporting recycling endosomes to the cleavage furrow (8). This study showed that binding of ARF6 to JLP or JIP3 causes a switch in the binding to dynein as opposed to kinesin and that this pathway functions in the transport of dynein-cargo proteins out of the midbody during cytokinesis (8). Studies following this using JIP3:JLP double knockout mouse embryonic fibroblasts (MEF) have shown that these cells, when cultured in vitro, have an increased frequency of binucleate cells (9). In addition this study demonstrated the importance of JLP in ARF6 localization to the midbody and efficient cytokinesis (9). These studies uncovered a role for JLP in the cell cycle, which was not previously reported for other JIP proteins. In this study, we used a mass-spectrometry based approach to identify JLP-associated proteins and identified the mitotic kinase, Polo like kinase 1 (PLK1), as a novel interaction partner of JLP. PLK1 belongs to the Polo like kinase family of Ser/Thr kinases and functions as a proto-oncogene, with increased expression in various cancer types (10,11). The protein has an N-terminal conserved kinase domain which functions in phosphorylating substrates by promoting a transient interaction with the protein (12,13). In addition, the C-terminal polo box domain (PBD), which recognizes and binds phospho-primed substrates, regulates substrate specificity and localization of the protein during mitosis (14-18). PLK1 has been characterized to phosphorylate and regulate the function of many proteins during G2/M phase ensuring smooth progression of mitosis (19). Here we show that JLP interacts with the polo box domain of PLK1 in a phosphorylation-dependent manner. In addition, we provide evidence that the interaction of JLP with PLK1 results in the recruitment of the

transcription factor FOXK1 during mitosis and this JLP regulated function is required for down-regulation of FOXK1 protein expression and activity. EXPERIMENTAL PROCEDURES Plasmids: The expression vectors encoding murine S-tagged full length JLP, JLP domains and shJLP have been described previously (3,7). Expression vectors for FLAG-hJLP were constructed by sub-cloning PCR fragments into the EcoRI/XhoI sites of the pCMV-Tag2B (Agilent technologies) vector. PLK1 expression vectors were constructed by sub-cloning PCR fragments of the PLK1 cDNA IMAGE clone into the pHM6-HA vector. Site directed mutagenesis of JLP (T351A, T334A, 2TA) and PLK1 (H538A/K540M) was performed using Quickchange II XL kit according to manufacturer’s instructions (Agilent Technologies). Wild-type FLAG-FOXK1, R127A, H355A and the wild-type FLAG-FOXK2 plasmids have been described previously (20,21). The FOXK1 domain mutants (I, II, III) were constructed by sub-cloning PCR amplified fragments of the FLAG-FOXK1 cDNA into the parental vector. Antibodies and Reagents: The JLP-specific antibody has been described previously (3). Antibodies against PLK1, GAPDH, HA-tag, FLAG-tag, Cyclin E, S-tag, Myc-tag and p21 were obtained from Santa Cruz Biotechnology. Antibodies against FOXK1, FOXK2, BubR1 and T7-tag were obtained from Cell signaling technology and Abcam. On-Target plus Smart pool siPlk1, siFOXK1, siFOXK2 and siScramble were purchased from GE Dharmacon. Stock solutions of BI2536 (Selleckchem), RO3306 (Tocris) and nocodazole (Noc) (Sigma) were prepared in tissue culture grade DMSO. Cell lines and Transfection: HEK293T, HeLa and U2OS cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Sigma) and Penicillin/Streptomycin (Invitrogen). Transient transfections of exponentially growing cells were performed using Lipofectamine 2000 (Invitrogen). Cells were harvested 36-48 hrs post-transfection. For siRNA experiments, cells were

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

3

transfected with 30nmol of siRNA using Lipofectamine RNAimax (Invitrogen) and the cells were harvested 24-36 hrs post-transfection. Mass Spectrometry, Immunoprecipitation and Western blotting: To identify novel interaction partners through mass spectrometry, exponentially growing HEK293T cells were transfected with S-tagged JLP or vector control plasmid and harvested 36 hrs post-transfection. The cells were lysed in lysis buffer (50mM HEPES, pH 7.5; 70mM potassium acetate; 5mM magnesium acetate, 35mM sodium chloride, 0.2% β-D maltoside, supplemented with EDTA-free protease inhibitor tablet (Roche), 0.2mM sodium orthovanadate) and 5mg of protein lysate was incubated with S-protein agarose (69704, Millipore) for 2 hrs at 4 ºC. Protein complexes were resolved by SDS-PAGE and bands that were detected specifically in JLP-S sample were excised and subjected to microcapillary Liquid Chromatography-Mass spectrometry (LC-MS/MS) at the Taplin Mass Spectrometry Facility at Harvard Medical School. For interaction studies, cells were lysed in IP buffer (50mM HEPES, pH 7.5; 70mM potassium acetate; 5mM magnesium acetate, 35mM NaCl, 0.2% TritonX-100) for 30 mins. FLAG- and HA-tagged proteins were precipitated from 1mg protein lysate using FLAG-M2 beads (Sigma) or HA-beads (Piercenet) for 2 hrs at 4 ºC and washed well with the IP buffer. For immunoprecipitation using antibodies, 1mg of pre-cleared lysate was incubated with 2 μg of antibody for 2 hrs at 4 ºC and the protein complexes were precipitated using protein-G or protein-A sepharose (GE healthcare) for 2 hrs. The eluted proteins were resolved by SDS-PAGE gel and subjected to Western blot analysis. Proteins were analyzed using the Li-Cor Odyssey imaging system. Proximity Ligation Assay: HeLa cells were grown on an 8-well chamber slide and fixed using 4% paraformaldehyde. The cells were washed with PBS containing glycine and permeabilized using 0.1% Triton X 100 in PBS for 20 minutes. The cells were then blocked in blocking solution (PBS/10% FBS,/0.1% Triton X-100 and incubated with anti-JLP and anti-PLK1 antibodies diluted in blocking solution. The subsequent steps of the assay were performed according to the

manufacturer’s instructions (DUO92101, Sigma). Coverslips were mounted on the slide using DUOLINK in situ mounting medium with DAPI. Images were obtained using a Carl Zeiss AxioImager Z1 and processed with Zen pro 2012 imaging software (Zeiss). Kinase assays: Kinase assays were performed as described previously (22). Briefly, 10ng of His-PLK1 (PV3501, Invitrogen) or GST-PLK2 (PV4204, Invitrogen) was incubated with 2 μg of purified substrate, and ATP mix (10 μM cold ATP, 30 μCi P32γATP) for 30 mins at 30 ºC. Where required, kinase was pre-incubated with indicated concentration of inhibitor for 30 mins at room temperature before addition of substrate and ATP mix. Reactions were terminated by the addition of sample buffer and resolved by SDS-PAGE. The gels were coomassie stained, dried and subjected to autoradiography. Cell synchronization and flow cytometry: To synchronize cells in the G1 phase, cells grown to 60% confluence were washed with PBS and released in media containing 0.1% serum for 24 hrs. Where indicated, the cells were treated with 2μg/ml aphidicolin (A0781, Sigma) or 1 μM nocodazole overnight to synchronize cells in the S and G2/M phases, respectively. For double thymidine block, HeLa cells were treated with 2mM thymidine for 18 hrs and released into complete media for 9hrs followed by a second round of treatment with 2mM thymidine for 18hrs. Cells were released into complete media and harvested at the indicated time points. For flow cytometric analysis, cells were harvested by trypsinization and fixed for 15 mins using 0.5% paraformaldehyde at room temperature. Fixed cells were resuspended in ice-cold 90% methanol added dropwise. Cells were then blocked in 2% BSA in PBS and stained with anti-pHis H3 antibody (Millipore) overnight at room temperature. The cells were then stained for 2 hours using an Alexa Fluor 488-conjugated secondary antibody prior to staining with propidium iodide to quantitate DNA. DNA content was measured using a BD-FACS Calibur (BD Biosciences). Cell cycle distribution was analyzed using Flowjo (Treestar).

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

4

SILAC labeling: HEK293T cells were grown for 6-generations in Heavy and Light amino acid medium prior to transfection with FLAG-JLP plasmid. Cells were subjected to treatment as indicated and lysed in a Maltoside-based Lysis Buffer. 2 mg of cell lysate was incubated with FLAG-conjugated beads for 1 hour at 4 °C and bound proteins were eluted through treatment with 10M Urea. The eluted protein was digested for 4 hrs with 0.2 μg Lys-C (Wako) and then for 14hrs with 0.2 μg Trypsin (Promega). The samples were desalted using Vivapure C18 microspin columns and lyophilized. Mass spectrometric analysis of the lyophilized peptides was performed by the Proteome Exploration Laboratory, California Institute of Technology as described previously (23). Raw files were analyzed by MaxQuant (v. 1.4.1.2) (24,25) in a manner similar to that previously described (26). Protein ratios were normalized to equalize bait (JLP) levels. Quantitative PCR: Total RNA was isolated from cell lines using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. cDNA was generated from 0.5 µg of total RNA using Superscript III Reverse transcriptase (Invitrogen) according to manufacturers instructions. For quantitative PCR (qPCR), cDNA was mixed with 1X Power SYBR Green PCR master mix (4367659, Applied Biosystems) and gene-specific primers. Amplification was performed using the ABI Step One Plus system. qPCR values were analyzed using the comparative Ct method to obtain relative gene expression levels. Normalization was performed using ß-actin. Sequences of primer pairs used for qPCR analysis are as follows: ACTB: 5’-ACCTTCTACAATGAGCTGCG-3’ and 5’-CCTGGATAGCAACGTACATGG-3’; JLP: 5’-TCACGAGAAAATCCAGCCATG-3’ and 5’-GAGTAACATGAGACGTGGGTG-3’; CDKN1A: 5’-TGTCACTGTCTTGTACCCTTG-3’ and 5’-GGCGTTTGGAGTGGTAGAA-3’; FOXK1: 5’-ACATCACCAAGCATTACCCC-3’ and 5’-TCTATTCGCCAAAAGGACCC-3’; FOXK2: 5’-ACCATCAACATTCCAGACACC-3’ and 5’-CCACCTTGTACCCTGAAGAC-3’

RESULTS Polo-like kinase 1 (PLK1) is a novel interaction partner of JLP We undertook a mass spectrometry-based approach to identify novel JLP interaction partners. To that end, pull-down assays were performed from cells transfected with an expression vector encoding S-tagged JLP or a parental control plasmid. The protein complexes were resolved by SDS-PAGE, stained with coomassie blue. As shown in Fig 1A, there were a number of bands specifically detected in the JLP S-tagged precipitates. These bands were excised and protein constituents determined by liquid chromatography-mass spectrometry (LC-MS/MS) (Fig 1A). The predominant peptides identified in bands labeled 1, 2 and 4 were JLP itself, likely due to proteolytic cleavage of the full length protein. However, band 3 was made up of peptides derived from the mitotic Serine/Threonine kinase, Polo-like kinase 1 (PLK1). To validate the novel JLP-PLK1 interaction, we subjected the S-tag pull-down precipitates to immunoblotting using a PLK1-specific antibody. As shown in Fig 1B, PLK1 was specifically precipitated by JLP but not in the vector control sample. To determine whether endogenous JLP associates with PLK1, HeLa cell extracts were incubated with either JLP or PLK1-specific antisera and the resulting immunoprecipitates subjected to Western blot analysis. The results of this study (Fig 1C) show that both proteins associate in reciprocal co-immunoprecipitation assays, thereby confirming that endogenous JLP and PLK1 associate in cells. The JIP family of scaffolding proteins contains four members, JIP1, JIP2, JIP3 and JLP. Of these, JIP3 is highly homologous to JLP in its sequence and domain structure (3), which suggests that it might interact with PLK1. We therefore performed FLAG pull-down assays from cells over-expressing either FLAG-tagged JIP3 or JLP and subjected the precipitates to Western blot analysis using PLK1-specific antiserum. While JLP readily precipitated PLK1, we failed to detect any associated PLK1 protein in the JIP3 sample (Fig 1D). To determine whether JLP interacts with PLK1 during mitosis, HeLa cells were synchronized in

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

5

S-phase using a double thymidine block and released into the cell cycle in the presence of DMSO or nocodazole. Cells were then harvested at the indicated time points to determine the level of JLP-PLK1 complexes as a function of cell cycle progression. The results of this study showed that interaction of JLP with PLK1 increases significantly as cells enter the mitotic phase (Fig 2A-B). To further demonstrate that JLP and PLK1 associate during mitosis we also performed an in situ proximity ligation assay (PLA), which detects protein-protein interactions with high degrees of specificity and sensitivity. HeLa cells incubated with JLP and PLK1 antisera that recognize the endogenous proteins emitted a strong PLA signal only in mitotic cells (Fig 2C). Background signals observed in interphase cells were comparable to the negative control (Fig 2C), suggesting that JLP and PLK1 proteins interact predominantly during mitosis. The JLP protein has several functional domains, including two leucine zipper domains, a JNK binding domain and a carboxy-terminal domain, which mediate protein-protein interactions (3,27). To map the region of JLP that interacts with PLK1, HEK293T cells were transfected with S-tagged wild-type (WT) and domain mutants of JLP (Fig 1E). Pull down experiments showed that in addition to the full-length (WT) protein, PLK1 interacts with the N-terminal domain (NTD) of JLP, which spans two leucine zipper domains and the JNK binding domain (amino acids 1-463), but not with any of the individual domains within the N-terminal region of JLP or with the C-terminus of the protein (domain III and CTD) (Fig 1E-F). In order to map the region of PLK1 that interacts with JLP, cells expressing HA-tagged wild-type and domain mutants of PLK1 (Fig 1G) were subjected to pull-down assays using HA-agarose and subsequent immunoblotting with JLP-specific antisera. Our results show that JLP interacts with the full length (WT) PLK1 protein and the domain mutant expressing the PLK1 polo-box domain (PBD aa 306-603) (Fig 1G-H), but not with the kinase domain (PKD aa 1-316). To confirm that the JLP N-terminal domain and the PLK1 Polo box domain interact, we performed additional immunoprecipitation assays using extracts derived from cells over-expressing a S-tagged version of the JLP N-terminal domain (aa 1-463) and a HA-

tagged PLK1 polo box domain mutant (aa 306-603). As shown in Fig 1I-J, reciprocal pull-down experiments followed by Western blot analyses confirmed the association between the two domains. The interaction between JLP and PLK1 is phosphorylation dependent It has previously been established that the PBD of PLK1 is involved in recognizing and interacting with phospho-primed substrates (15). Because we determined that JLP binds to the PLK1 PBD (Fig 1H), we performed experiments to determine if the interaction is phosphorylation dependent. JLP was immunoprecipitated from asynchronous cell lysates and the immunocomplex was treated with λ-phosphatase to dephosphorylate the proteins. Following treatment with phosphatase, the precipitate was washed rigorously to remove any unbound proteins and the level of associated PLK1 was determined by Western blot analysis. As can be seen in Fig 3A, treatment with λ-phosphatase greatly reduced the interaction between JLP and PLK1, suggesting that the interaction is dependent on phosphorylation. We next performed additional interaction studies using HA-tagged wild type (WT) PLK1 and a PBD mutant in which two residues that are critical for recognizing phospho-primed substrates, His538 and Lys540, were mutated to Ala and Met, respectively. Figure 3B shows that while WT PLK1 readily associated with JLP, the H538A/K540M PBD mutant (H/K) failed to do so. These experiments provide additional evidence that the JLP-PLK1 interaction is phosphorylation dependent. The polo box domain of PLK1 is known to initiate interactions by recognizing phospho-primed sites on its substrates with a consensus binding motif of Ser-[pSer/pThr]-Pro (15,16). The protein sequence of JLP was analyzed for the presence of this consensus site and a corresponding motif was identified at amino acid residues Thr334 and Thr351 within the JLP N-terminal region. To test whether this motif functions as a PLK1 docking site, these two residues were mutated to Ala either individually (T334A and T351A) or in combination (2TA) in the JLP-NTD plasmid and tested for their ability to bind to PLK1 in pull-down assays. The results of this study showed that

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

6

while mutation of JLP at residue T334 did not affect binding to PLK1, mutation at Thr351 (T351A), either alone or in the 2TA mutant, abolished this association (Fig 3C). Similar results were obtained using cells expressing FLAG-tagged, full-length versions of the wild type or T351A JLP proteins (Fig 3D). In addition, the reciprocal experiment, whereby endogenous PLK1 was immunoprecipitated from cells expressing FLAG-tagged WT and JLP-T351A mutant plasmids, also confirmed that wild-type JLP, but not the T351A mutant, associated with PLK1 (Fig 3E). Taken together, these studies suggest that phospho-priming of JLP at Thr351 is required for its interaction with PLK1. Previous studies have shown that CDK1 serves as a priming kinase for PLK1 (15). There have also been several reports suggesting that PLK1 can also serve as a priming kinase which stabilizes its interaction with other proteins (28-30). To determine if these kinases play a role in priming JLP for PLK1 binding, HeLa cells synchronized in mitosis were treated with BI2536 (PLK inhibitor), RO3306 (CDK1 inhibitor), CX4945 (CK2 inhibitor) or DMSO for 2 hrs. Nocodazole was used to synchronize cells in mitotic phase of the cell cycle, where PLK1 and CDK1 are expressed in their active state (31-33). Whole cell extracts were prepared and subjected to immunoprecipitation using a PLK1 antibody. Treatment with BI2536, but not the other kinase inhibitors, inhibited the association of JLP with PLK1 (Fig 3F), suggesting that PLK1 creates its own docking site on JLP to initiate the interaction. PLK1, when bound to substrates through its PBD, catalyzes their phosphorylation on the D/E-X-S/T-Φ-X-D/E (X, any amino acid; Φ, a hydrophobic amino acid) consensus sequence (13). We performed in vitro kinase assays using recombinant His-PLK1 and GST-PLK2 to determine whether JLP is a substrate of one or both kinases. Kinase assays showed that PLK1, but not PLK2, can specifically phosphorylate JLP (Fig 3G). Furthermore, this phosphorylation is inhibited by BI2536 (Fig 3G). Because these studies indicated that JLP is a substrate of PLK1, we mapped the phosphorylation sites on JLP using a series of GST-tagged JLP deletion mutants as substrates in in vitro kinase assays. As shown in Fig 3H, JLP fragments - aa 355-559 and aa 763-

966 were phosphorylated by PLK1, suggesting that JLP contains multiple PLK1 phosphorylation sites. Interaction of JLP with PLK1 recruits FOXK1 to the complex Having determined that JLP interacts with and undergoes phosphorylation by PLK1, we next sought to determine if PLK1 promoted phosphorylation of JLP regulates the association of this scaffolding protein with other binding factors. To that end, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and a quantitative solution based LC-MS/MS interaction proteomics analysis (34,35) was performed in cells grown in media containing heavy or light isotope amino acids (Fig 4A). HEK293T cells over-expressing FLAG-JLP were synchronized in the G2/M phase with nocodazole prior to treatment with 0.1 µM BI2536 or DMSO as a control. FLAG-tagged JLP was precipitated from the cell lysates and the protein complexes subjected to digestion using Lys-C and trypsin and processed for mass spectrometric analysis as described previously (36). To identify non-specific proteins, light cells expressing the parental plasmid and heavy cells expressing FLAG-tagged JLP plasmid were subjected to FLAG-pull down and further processed for mass spectrometric analysis. Mass spectral counts derived from cells expressing the parental plasmid were filtered out as non-specific interactions (data not shown). Further, a ratio-based filter was used to identify proteins that were differentially pulled down in the presence or absence of BI2536. Using this technique we were able to reconfirm that PLK1 co-precipitated with JLP in mock-treated cells but not in BI2536 treated sample, verifying the interaction by another unbiased quantitative proteomics approach (Table1). When we further examined the data, we identified two transcription factors, FOXK1 and FOXK2, that specifically co-precipitated with JLP in mock treated cells (Table 1; 22 and 5 spectra, respectively) but not in cells that were treated with PLK inhibitor, BI2536 (0 spectra for both FOXK1 and FOXK2). To validate association of JLP with FOXK1 and FOXK2, we performed pull-down assays from cell lysates derived from asynchronous and G2/M-arrested HEK293T cells expressing FLAG-tagged FOXK1 and FOXK2

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

7

proteins. Immunoblotting showed that FLAG-FOXK1 and FOXK2 interact with both JLP and PLK1, predominantly in cells arrested in the G2/M phase (Fig 4B) which express highest levels of PLK1. Similar results were obtained using nocodazole-treated cells that over-expressed Myc-tagged JLP and FLAG-tagged FOXK1 or FOXK2 (Fig 4C-D), which confirmed that JLP and PLK1 interact with FOXK1 or FOXK2 in G2/M-phase cells. To determine if the interaction between JLP and FOXK1 is a cell-cycle phase specific event, HeLa cells over-expressing FLAG-tagged FOXK1 were synchronized in the G1, S or G2/M phases and subjected to pull-down assay using FLAG beads (Fig 5A). Immunoblotting showed a significant increase in the formation of the PLK1-JLP-FOXK1 complex in cells synchronized in mitotic phase as compared to those in the G1 or S-phases. In addition, we performed FLAG pull down assays from HeLa cells that were released into cell cycle following a double thymidine S-phase block. Our results show formation of the ternary complex between JLP, PLK1 and FOXK1 as the cells progress into mitotic phase (TT8; Fig 5B). FOXK1 has a conserved DNA-binding domain (DBD 305-400 aa) and an N-terminal Forkhead association domain (FHA 123-175 aa), the latter of which functions in recognizing and binding phospho-Thr residues (37,38). In order to map the region(s) with which JLP and PLK1 interact, we over-expressed wild-type (WT) and domain mutants of FOXK1 (I, II, III) and subjected the cell lysates to pull down assays using FLAG-beads. Our results show that while JLP strongly interacts with the domain mutant I containing the FHA domain and to a lesser extent with the DBD containing domain mutant II (Fig 5C-D), PLK1 associates with the FOXK1 domain II which contains the DBD (Fig 5C-D). PLK1 has been shown to interact with and phosphorylate several FOX transcription factors, including FOXO1, FOXO3 and FOXM1 (39-41). To determine whether FOXM1 can also form a ternary complex with JLP and PLK1, we performed pull down assays using cell extracts derived from cells expressing FOXM1, PLK1 and JLP. The results of this study showed that PLK1, but not JLP, could interact with FOXM1 (Fig 5E), suggesting that the

interaction of FOXK1 with the JLP-PLK1 complex is highly specific. Interaction of JLP with FOXK1 is PLK1-dependent Mass spectrometric data from the FLAG-JLP pull-down assay showed a loss of FOXK1 in the BI2536-treated sample (Table 1). To further investigate this observation, mitotic cells over-expressing FLAG-tagged FOXK1 were either mock-treated or treated with BI2536 for 2 hrs prior to being subjected to pull-down assays using FLAG beads. Western blotting of the resulting protein complexes showed that interaction of FOXK1 with both JLP and PLK1 is affected in the presence of the PLK1 inhibitor, BI2536 (Fig 6A) suggesting that BI2536 inhibits PLK1-mediated phosphorylation of JLP, which in turn, affects its association with FOXK1. We have previously shown that phosphorylation of JLP at Thr351 is required for its interaction with PLK1 (Fig 3C-E). To determine whether Thr351 phosphorylation affects its recruitment to the FOXK1-PLK1 complex, we performed a pull-down experiment using cells that over-express Myc-tagged wild-type and the JLP T351A mutant. As shown in Fig 6B, FOXK1 associated with wild-type but not with the T351A mutant JLP protein. Because treatment with the PLK1 inhibitor BI2536 and mutation of JLP at Thr351 adversely affect binding of JLP to PLK1 and the FOXK1 transcription factor, it is highly likely that the formation of this ternary complex is phosphorylation dependent. Within the FOX family of transcription factors, FOXK1 and FOXK2 are the only two proteins that have an amino-terminal Forkhead association domain (FHA) in addition to the highly conserved winged helix DNA binding domain (WHD). Because the FHA domain functions in recognizing and binding phospho-Thr residues (42) and because our results show that the FHA domains interact with JLP (Fig 5C-D), we sought to determine if disrupting function of the FHA domain would result in changes in its ability to form the ternary complex. To that end, Arg127 in the FOXK1 FHA domain, which mediates the recognition and association with phospho-Thr residues, was mutated to Ala (R127A). As a control, His355 in the winged helix domain of

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

8

FOXK1, which is important for the protein’s DNA-binding function, was mutated to Ala (H355A) (Fig 6C). Pull-down assays from cells over-expressing wild type and mutant FLAG-FOXK1 proteins showed that while the WT and H355A-FOXK1 proteins retained the ability to form a complex with PLK1 and JLP, the R127A mutant failed to do so (Fig 6C). This suggests that the FOXK1 FHA domain recognizes a phosphorylation site(s) on JLP when interacting with these proteins. Taken together, our results suggest that interaction of JLP with FOXK1 is dependent on phosphorylation of JLP by PLK1. To test the hypothesis that the formation of the ternary complex is PLK1 dependent, we performed interaction studies in cells where the expression of JLP or PLK1 was knocked down using shRNA or siRNA technology. HeLa cells expressing shJLP or scramble control (shS) were transfected with FLAG-tagged FOXK1, synchronized in mitosis using nocodazole and subjected to pull-down assay using FLAG beads. Knockdown of JLP did not affect the ability of PLK1 to interact with FOXK1 (Fig 6D). However, a parallel experiment whereby HeLa cells that over-express FLAG-tagged FOXK1 were transfected with a PLK1 siRNA or control siRNA for 24 hrs showed that the level of associated JLP with FLAG-FOXK1 was significantly reduced in the siPlk1 cells (Fig 6E), confirming that PLK1 is required for the formation of a JLP-PLK1-FOXK1 ternary complex. Knockdown of JLP leads to an increase in FOXK1 protein levels FOXK1 has been shown to promote myocyte proliferation by recruiting transcriptional co-repressor complexes to regulate gene transcription (43) as well as by binding to and inhibiting transcription factors involved in differentiation (44). To determine whether JLP affects FOXK1 activity, we knocked-down JLP expression in U2OS osteosarcoma cells using shRNA and examined the effects on FOXK1 activity. Loss of JLP expression (shJLP) resulted in an increase in and a reduction in FOXK1 and p21 protein levels, respectively (Fig 7A). Quantitative-PCR (qPCR) analysis showed that there is a significant reduction in p21 mRNA levels (P≤0.02) in shJLP cells compared to the control cells (Fig 7B).

Furthermore, no significant differences in FOXK1 mRNA levels were observed (Fig 7B), suggesting that knockdown of JLP affects FOXK1 protein levels. We also performed qPCR analysis in siControl and siFOXK1 cells, which showed a significant increase in p21 relative expression levels (P≤0.0005) upon knockdown of FOXK1 (Fig 7C). These results are consistent with previous reports that demonstrated FOXK1-mediated repression of p21 gene expression and increased proliferation in the C2C12 myoblast cell line (45). Furthermore, we performed immunoblotting of cell lysates derived from asynchronous and mitotic cells expressing control (shS) and shJLP. As shown in Fig 7D, knockdown of JLP resulted in increased levels of FOXK1 and a reduction in p21 levels in both cell types. Because our results show an increase in FOXK1 protein levels in shJLP cells (Fig 7A,D), they suggest that JLP can potentially regulate the stability and/or degradation of FOXK1 protein in turn regulating the levels of its transcriptional targets. DISCUSSION JLP is a JNK scaffolding protein that binds to signaling proteins and regulates various developmental process such as neurite outgrowth, myogenesis, TGN transport and cytokinesis (2). In this study, using a mass spectrometric approach, we have identified the mitotic Ser/Thr kinase, PLK1 as a JLP-associated protein. Our results presented here show that the interaction of JLP with PLK1 is phosphorylation dependent (Fig 3A-B, 7E) and that phosphorylation of JLP Thr351 is required for this association (Fig 3C-E, 7E). This interaction of PLK1 with JLP appears to be unique amongst the JIP family of scaffolding proteins, as JIP3, a member of this family which is highly homologous to JLP (3) fails to interact with PLK1 (Fig 1D). Furthermore our results suggest that PLK1 serves as the priming kinase that phosphorylates JLP (Fig 3F) and uses this phosphorylated site for binding and phosphorylating additional sites in JLP (Fig 3G-H, 7E). Since JLP knockdown by shRNA did not result in an interruption of cell cycle progression (data not shown), we reasoned that the interaction between

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

9

JLP and PLK1 might result in the recruitment of additional protein partners to the JLP/PLK1 complex. SILAC and quantitative LC-MS/MS analysis was performed to identify PLK1 dependent JLP interacting proteins (Table 1) and we identified that the FOXK1 transcription factor interacts with the JLP/PLK1 complex in a phosphorylation dependent manner during mitosis. Our pull down assays show that PLK1 interacts with the FOXK1 DNA binding domain whereas JLP interacts with the FHA domain (Fig 5D) suggesting that PLK1 phosphorylates JLP and this phosphorylation site is recognized by FOXK1 FHA domain. Our observation that inhibition of PLK1 kinase activity by BI2536 (Fig 6A) or knock-down by siRNA (Fig 6E), affects the interaction between JLP and FOXK1 highlights the importance of PLK1 kinase activity in the formation of the JLP, PLK1 and FOXK1 ternary complex (Fig 7E). Recent studies have shown that the FOX transcription factors can interact with different protein complexes when they are “on” or “off” the chromatin (46,47). This study suggests that the recruitment of co-regulatory proteins to the transcription factors determine their transcriptional-dependent and independent functions. Furthermore, FOXK1 has been shown to interact with and function in the nuclear translocation of the WNT scaffolding protein, Dishevelled (DVL), resulting in the activation of WNT/β-catenin pathway (48). Our results show that JLP interacts with FOXK1 predominantly during mitosis and knockdown of JLP results in an increase in FOXK1 protein levels and a reduction in p21 mRNA and protein levels (Fig 7A-B). This suggests that the increase in the FOXK1 protein

levels could be the result of changes in the stability or degradation of the protein, which is mediated by JLP, but the detailed mechanism remain to be elucidated. PLK1 has been shown to phosphorylate and regulate the localization and transcriptional function of various FOX transcription factors, FOXM1(39), FOXO1(40), FOXO3 (41). Although FOXK1 is implicated as a regulator of myogenic proliferation (21,43-45), little is known about its function during mitosis. Previous studies have shown that CYCLIN-CDK complexes phosphorylate FOXK2, a protein that is highly homologous to FOXK1, during mitosis and that such phosphorylation regulates its stability and transcriptional function (20). Because our studies show the mitotic kinase, PLK1 to interact with FOXK1 and FOXK2, and we observed potential phosphorylation of FOXK1 during mitosis (data not shown), a similar mechanism of regulation might exist. In summary, we report for the first time that JLP can interact with a mitotic kinase, PLK1, and that this interaction results in recruitment of a transcription factor, FOXK1, to form a ternary complex during mitosis (Fig 7E). Although there are many reports suggesting a role for FOXK1 in proliferation and maintenance of myogenic progenitor cells (21,43,44), the mechanism by which FOXK1 functions during this process is not well defined. Our study describes a potentially novel mechanism by which FOXK1 regulates cell cycle dependent gene expression via association with a scaffolding protein, JLP.

CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article. AUTHOR CONTRIBUTIONS PR designed the study, conducted most of the experiments and wrote the paper. EPR designed the study, helped in the interpretation of results and wrote the paper. CML constructed the JLP-S plasmids and contributed to Figures 3, 5, 7. AM, MJS, SH and DSH performed SILAC labeling, mass spectrometry, data analysis and writing of the paper. ADS provided the FOXK vectors and helped in the interpretation of FOXK1 results, drafting and critical review of the manuscript. All authors reviewed the results and approved final version of the manuscript.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

10

REFERENCES 1. Buday, L., and Tompa, P. (2010) Functional classification of scaffold proteins and related

molecules. Febs j 277, 4348-4355 2. Dhanasekaran, D. N., Kashef, K., Lee, C. M., Xu, H., and Reddy, E. P. (2007) Scaffold proteins

of MAP-kinase modules. Oncogene 26, 3185-3202 3. Lee, C. M., Onesime, D., Reddy, C. D., Dhanasekaran, N., and Reddy, E. P. (2002) JLP: A

scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors. Proc Natl Acad Sci U S A 99, 14189-14194

4. Xu, H., Dhanasekaran, D. N., Lee, C. M., and Reddy, E. P. (2010) Regulation of neurite outgrowth by interactions between the scaffolding protein, JNK-associated leucine zipper protein, and neuronal growth-associated protein superior cervical ganglia clone 10. J Biol Chem 285, 3548-3553

5. Gantulga, D., Tuvshintugs, B., Endo, Y., Takino, T., Sato, H., Murakami, S., and Yoshioka, K. (2008) The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13). J Biochem 144, 693-700

6. Takino, T., Nakada, M., Miyamori, H., Watanabe, Y., Sato, T., Gantulga, D., Yoshioka, K., Yamada, K. M., and Sato, H. (2005) JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration. J Biol Chem 280, 37772-37781

7. Nguyen, Q., Lee, C. M., Le, A., and Reddy, E. P. (2005) JLP associates with kinesin light chain 1 through a novel leucine zipper-like domain. J Biol Chem 280, 30185-30191

8. Montagnac, G., Sibarita, J. B., Loubery, S., Daviet, L., Romao, M., Raposo, G., and Chavrier, P. (2009) ARF6 Interacts with JIP4 to control a motor switch mechanism regulating endosome traffic in cytokinesis. Curr Biol 19, 184-195

9. Tuvshintugs, B., Sato, T., Enkhtuya, R., Yamashita, K., and Yoshioka, K. (2014) JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis. Genes Cells 19, 692-703

10. Takahashi, T., Sano, B., Nagata, T., Kato, H., Sugiyama, Y., Kunieda, K., Kimura, M., Okano, Y., and Saji, S. (2003) Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancers. Cancer Sci 94, 148-152

11. Weichert, W., Kristiansen, G., Schmidt, M., Gekeler, V., Noske, A., Niesporek, S., Dietel, M., and Denkert, C. (2005) Polo-like kinase 1 expression is a prognostic factor in human colon cancer. World J Gastroenterol 11, 5644-5650

12. Kothe, M., Kohls, D., Low, S., Coli, R., Cheng, A. C., Jacques, S. L., Johnson, T. L., Lewis, C., Loh, C., Nonomiya, J., Sheils, A. L., Verdries, K. A., Wynn, T. A., Kuhn, C., and Ding, Y. H. (2007) Structure of the catalytic domain of human polo-like kinase 1. Biochemistry 46, 5960-5971

13. Nakajima, H., Toyoshima-Morimoto, F., Taniguchi, E., and Nishida, E. (2003) Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate. J Biol Chem 278, 25277-25280

14. Cheng, K. Y., Lowe, E. D., Sinclair, J., Nigg, E. A., and Johnson, L. N. (2003) The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex. Embo j 22, 5757-5768

15. Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299, 1228-1231

16. Elia, A. E., Rellos, P., Haire, L. F., Chao, J. W., Ivins, F. J., Hoepker, K., Mohammad, D., Cantley, L. C., Smerdon, S. J., and Yaffe, M. B. (2003) The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115, 83-95

17. Hanisch, A., Wehner, A., Nigg, E. A., and Sillje, H. H. (2006) Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting. Mol Biol Cell 17, 448-459

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

11

18. Lee, K. S., Park, J. E., Kang, Y. H., Zimmerman, W., Soung, N. K., Seong, Y. S., Kwak, S. J., and Erikson, R. L. (2008) Mechanisms of mammalian polo-like kinase 1 (Plk1) localization: self- versus non-self-priming. Cell Cycle 7, 141-145

19. de Carcer, G., Manning, G., and Malumbres, M. (2011) From Plk1 to Plk5: functional evolution of polo-like kinases. Cell Cycle 10, 2255-2262

20. Marais, A., Ji, Z., Child, E. S., Krause, E., Mann, D. J., and Sharrocks, A. D. (2010) Cell cycle-dependent regulation of the forkhead transcription factor FOXK2 by CDK.cyclin complexes. J Biol Chem 285, 35728-35739

21. Freddie, C. T., Ji, Z., Marais, A., and Sharrocks, A. D. (2007) Functional interactions between the Forkhead transcription factor FOXK1 and the MADS-box protein SRF. Nucleic Acids Res 35, 5203-5212

22. Jatiani, S. S., Cosenza, S. C., Reddy, M. V., Ha, J. H., Baker, S. J., Samanta, A. K., Olnes, M. J., Pfannes, L., Sloand, E. M., Arlinghaus, R. B., and Reddy, E. P. (2010) A Non-ATP-Competitive Dual Inhibitor of JAK2 and BCR-ABL Kinases: Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition. Genes Cancer 1, 331-345

23. Kalli, A., Smith, G. T., Sweredoski, M. J., and Hess, S. (2013) Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers. J Proteome Res 12, 3071-3086

24. Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V., and Mann, M. (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10, 1794-1805

25. Cox, J., and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367-1372

26. Pierce, N. W., Lee, J. E., Liu, X., Sweredoski, M. J., Graham, R. L., Larimore, E. A., Rome, M., Zheng, N., Clurman, B. E., Hess, S., Shan, S. O., and Deshaies, R. J. (2013) Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153, 206-215

27. Kelkar, N., Standen, C. L., and Davis, R. J. (2005) Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways. Mol Cell Biol 25, 2733-2743

28. Kang, Y. H., Park, C. H., Kim, T. S., Soung, N. K., Bang, J. K., Kim, B. Y., Park, J. E., and Lee, K. S. (2011) Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores. J Biol Chem 286, 19744-19757

29. Kang, Y. H., Park, J. E., Yu, L. R., Soung, N. K., Yun, S. M., Bang, J. K., Seong, Y. S., Yu, H., Garfield, S., Veenstra, T. D., and Lee, K. S. (2006) Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. Mol Cell 24, 409-422

30. Lee, K. S., Oh, D. Y., Kang, Y. H., and Park, J. E. (2008) Self-regulated mechanism of Plk1 localization to kinetochores: lessons from the Plk1-PBIP1 interaction. Cell Div 3, 4

31. Seki, A., Coppinger, J. A., Jang, C.-Y., Yates, J. R., and Fang, G. (2008) Bora and the Kinase Aurora A Cooperatively Activate the Kinase Plk1 and Control Mitotic Entry. Science 320, 1655-1658

32. Nurse, P. (1990) Universal control mechanism regulating onset of M-phase. Nature 344, 503-508 33. O'Farrell, P. H. (2001) Triggering the all-or-nothing switch into mitosis. Trends Cell Biol 11,

512-519 34. Haines, D. S., Lee, J. E., Beauparlant, S. L., Kyle, D. B., den Besten, W., Sweredoski, M. J.,

Graham, R. L., Hess, S., and Deshaies, R. J. (2012) Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking. Mol Cell Proteomics 11, M111.016444

35. Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol Cell Proteomics 1, 376-386

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

12

36. Lee, J. E., Sweredoski, M. J., Graham, R. L., Kolawa, N. J., Smith, G. T., Hess, S., and Deshaies, R. J. (2011) The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation. Mol Cell Proteomics 10, M110.006460

37. Katoh, M., and Katoh, M. (2004) Human FOX gene family (Review). Int J Oncol 25, 1495-1500 38. Katoh, M., and Katoh, M. (2004) Identification and characterization of human FOXK1 gene in

silico. Int J Mol Med 14, 127-132 39. Fu, Z., Malureanu, L., Huang, J., Wang, W., Li, H., van Deursen, J. M., Tindall, D. J., and Chen,

J. (2008) Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression. Nat Cell Biol 10, 1076-1082

40. Yuan, C., Wang, L., Zhou, L., and Fu, Z. (2014) The function of FOXO1 in the late phases of the cell cycle is suppressed by PLK1-mediated phosphorylation. Cell Cycle 13, 807-819

41. Bucur, O., Stancu, A. L., Muraru, M. S., Melet, A., Petrescu, S. M., and Khosravi-Far, R. (2014) PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor. Discoveries 2, e16

42. Durocher, D., Henckel, J., Fersht, A. R., and Jackson, S. P. (1999) The FHA domain is a modular phosphopeptide recognition motif. Mol Cell 4, 387-394

43. Shi, X., and Garry, D. J. (2012) Sin3 interacts with Foxk1 and regulates myogenic progenitors. Mol Cell Biochem 366, 251-258

44. Shi, X., Wallis, A. M., Gerard, R. D., Voelker, K. A., Grange, R. W., DePinho, R. A., Garry, M. G., and Garry, D. J. (2012) Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2. J Cell Sci 125, 5329-5337

45. Hawke, T. J., Jiang, N., and Garry, D. J. (2003) Absence of p21CIP rescues myogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice. J Biol Chem 278, 4015-4020

46. Li, X., Wang, W., Wang, J., Malovannaya, A., Xi, Y., Li, W., Guerra, R., Hawke, D. H., Qin, J., and Chen, J. (2015) Proteomic analyses reveal distinct chromatin-associated and soluble transcription factor complexes. Mol Syst Biol 11, 775

47. Ji, Z., and Sharrocks, A. D. (2015) Changing partners: transcription factors form different complexes on and off chromatin. Mol Syst Biol 11, 782

48. Wang, W., Li, X., Lee, M., Jun, S., Aziz, Kathryn E., Feng, L., Tran, My K., Li, N., McCrea, Pierre D., Park, J.-I., and Chen, J. (2015) FOXKs Promote Wnt/β-Catenin Signaling by Translocating DVL into the Nucleus. Dev Cell 32, 707-718

FOOTNOTES ↵The abbreviations used are: JIP, JNK interacting protein; JLP, JNK associated leucine zipper protein; PLK1, Polo like kinase 1; FOXK1, Forkhead box protein K1; aa, amino acid; PD, pull down; PBD, Polo box domain; FHA, Forkhead associated; qPCR, quantitative PCR; NTD, N-terminal domain; Noc, Nocodazole. EPR is supported by a grant from NIH RO1CA158209 AS is supported by a grant from the Wellcome Trust. The AM, MJS, SH is supported by the Gordon and Betty Moore Foundation through grant GBMF775 and the Beckman Institute. Acknowledgement: We thank Stacey J. Baker for critical reading of the manuscript.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

13

FIGURE LEGENDS: Figure 1: Polo like kinase 1 (PLK1) is a novel interaction partner of JLP A) HEK293T cells transfected with the parental vector control (V) or a plasmid encoding S-tagged JLP (JLP-S) were subjected to pull-down (PD) with S-beads. Proteins were resolved by SDS-PAGE and the indicated bands were excised and subjected to mass spectrometric analysis. B) Immunoblot analysis of the pull-down samples from (A), which shows binding of endogenous PLK1 to JLP-S. C) HeLa cell lysates were subjected to immunoprecipitation (IP) using JLP and PLK1 specific antibodies or their corresponding control IgGs and subjected to Western blot analysis using the indicated antibodies. D) HEK293T cells over-expressing FLAG-tagged JIP3 and JLP were subjected to pull-down assays using FLAG beads and subjected to immunoblotting using a PLK1-specific antibody. E) Schematic representation of the JLP protein domains used in the pull-down assays. Amino acid numbers are indicated. F) Whole cell extracts isolated from HEK293T cells transfected with S-tagged wild-type JLP and various domain mutants were subjected to pull-down assays using S-beads. The level of associated PLK1 was determined using immunoblotting. G) Schematic representation of the PLK1 protein domain mutants used in the pull-down assays. H) Immunoblot analysis of pull-down samples from cells over-expressing HA-PLK1 wild-type and domain mutants. *- Bands corresponding to HA-tagged PLK1 are indicated. I-J) HEK293T cells were transfected with expression vectors encoding HA-tagged PLK1-PBD and FLAG-tagged JLP-NTD and whole cell extracts were subjected to pull-down and immunoblotting assays. Abbreviations: V-Vector, WT-Wild type, CTD-C-terminal domain, LZ-Leucine zipper, NTD-N-terminal domain, PKD- Protein Kinase domain, PB – Polo box, PBD – Polo-box domain. Figure 2: PLK1 interacts with JLP during mitosis A&B) HeLa cells were subjected to double thymidine S-phase block and released into cell cycle in the presence of 1μM nocodazole or DMSO as a control. Cells were harvested at the indicated time points for immunoprecipitation and cell cycle analysis. (A) Cell lysates were subjected to immunoprecipitation using JLP antibody. The immune complexes and cell lysates were analyzed by western blotting. (B) Cells were double stained for pHistone H3 Ser10 and propidium iodide and subjected to flow cytometry to determine cell cycle progression. C) PLA analysis of endogenous JLP and PLK1 in HeLa cells. Cells representing mitotic and interphase stages of the cell cycle are shown. Combination of antibodies used is indicated for each panel, IgG represents a non-specific antibody. Figure 3: Interaction of JLP with PLK1 is phosphorylation dependent A) JLP was immunoprecipitated from HeLa cell lysates and the resulting immunocomplexes were treated with 400U of λ-phosphatase (where indicated), washed to remove any unbound protein and the level of associated PLK1 determined by Western blot analysis. B) HEK293T cells were transfected with wild-type (WT) HA-PLK1 or the PLK1 H538A/K540M (H/K) mutant. HA-PLK1 was precipitated using HA-beads and immunoblotted with JLP-specific antiserum. C) HEK293T cells were co-transfected with plasmids encoding HA-tagged PLK1 Polo Box Domain (PBD) and the indicated S-tagged JLP N-terminal domain plasmids. Lysates were subjected to pull-down assay using HA-beads and the level of associated S-tagged JLP N-terminal domain was determined by immunoblotting. D-E) Lysates from HEK293T cells transfected with FLAG-tagged wild-type JLP and the T351A mutant were subjected to pull-down assays using FLAG-beads (D) or immunoprecipitation using a PLK1 antibody (E) or a control IgG antibody. The immunocomplexes were analyzed by western blotting using the indicated antisera. F) HeLa cells were were treated overnight with 0.5μM nocodazole followed by a 2hr treatment with 2 μM BI2536, 9 μM RO3306, 2 μM CX4945 or DMSO prior to harvesting. PLK1 was immunoprecipitated from the cell lysates and the level of associated JLP analyzed by western blotting. G) Purified His-tagged JLP protein was subjected to in vitro kinase assay using recombinant PLK1 or PLK2 in the presence of 0.1 μM BI2536 or DMSO. The reactions were resolved by SDS-PAGE, fixed in coomassie stain and subjected to autoradiography. (H) Individual regions of JLP protein (200 amino acids in length) were expressed as bacterial GST-fusion proteins. The purified GST-tagged JLP protein domains were subjected to in vitro

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

14

kinase assays using recombinant PLK1. The reactions were resolved on a SDS-PAGE and subjected to autoradiography. Figure 4: FOXK1 and FOXK2 interact with JLP A) HEK293T cells were grown in normal (light) DMEM or in media containing heavy amino acids, Arg6 (U-13C6) and Lys8 (U-13C6, U-15N2). Cells were transfected with plasmid encoding FLAG-tagged JLP and treated with 1 μM nocodazole overnight and 0.1 μM BI2536 or DMSO as indicated for 1 hr prior to harvesting. Cell lysates were subjected to pull-down assays using FLAG-agarose. The protein complexes were eluted and subjected to digestion using Lys-C and trypsin. The digested peptides from corresponding light and heavy samples were mixed, desalted and lyophilized using a SpeedVac. The samples were then analyzed using an Orbitrap mass spectrometer followed by MaxQuant analysis. Peptides from heavy samples can be differentiated from the light samples by the change in their mass to charge (m/z) ratio. Potential hits were validated by immunoblotting. B) HEK293T cells were transfected with FLAG vector control and plasmids encoding FLAG-tagged FOXK1 or FOXK2 and treated with 1 μM nocodazole or DMSO overnight. Cell lysates were subjected to pull-down assays using FLAG beads and the level of associated JLP and PLK1 analyzed by immunoblotting. C-D) HEK293T cells were transfected with Myc-tagged JLP and FLAG-tagged FOXK1 (C) or FLAG-FOXK2 (D). 24 hrs post-transfection, cells were treated with nocodazole or DMSO overnight prior to harvesting. FLAG-tagged proteins were precipitated using FLAG-agarose and protein complexes were analyzed by western blotting. Figure 5: FOXK1 forms a ternary complex with JLP and PLK1 during mitosis A) HeLa cells transfected with FLAG-tagged FOXK1 were synchronized in the G1, S or G2/M phases as described in the Experimental procedures section. Cell lysates were subjected to pull-down assays using FLAG-agarose and the precipitates analyzed by immunoblotting. B) HeLa cells over-expressing FLAG-FOXK1 were synchronized in S-phase by double thymidine block and released into cell cycle for the indicated time points. Cell lysates were subjected to FLAG pull down assay and the levels of associated JLP and PLK1 were determined by immunoblotting. C) Schematic representation of the FOXK1 protein domains used in the pull-down assays. Amino acid numbers are indicated. D) Whole cell extracts isolated from HEK293T cells transfected with Myc-tagged JLP, wild-type FLAG-tagged FOXK1 and the indicated domain mutants were subjected to pull-down assays using FLAG-beads. The levels of associated Myc-JLP and PLK1 were determined using immunoblotting. E) HEK293T cells over-expressing FLAG-tagged JLP, HA-tagged PLK1 and T7-tagged FOXM1 were lysed and subjected to pull-down assay using HA- and FLAG-beads. Protein complexes were resolved by SDS-PAGE and subjected to immunoblotting using the indicated antisera. V-Vector, WT-wild-type, FHA-Forkhead association domain, DBD-DNA binding domain. Figure 6: FOXK1 interacts with JLP in a PLK1-dependent manner A) HeLa cells over-expressing FLAG-tagged FOXK1 were synchronized in mitosis and treated with 0.1μM BI2536 or DMSO for 2 hrs. Cell lysates were subjected to pull down assay using FLAG beads and the levels of associated JLP and PLK1 were determined by Western blot analysis. B) Interaction of FLAG-FOXK1 with MYC tagged wild-type (WT) and JLP T351A in mitotic cells were analyzed by pull-down assays using FLAG-beads and followed by immunoblotting with the indicated antibodies. C) Schematic representation of the FOXK1 protein. The FHA and DBD domains are indicated. HEK293T cells over-expressing FLAG-tagged WT, R127A and H355A FOXK1 proteins were treated with nocodazole overnight and subjected to pull-down assay using FLAG-beads. The resulting precipitates were subjected to immunoblotting using the indicated antisera. D) HeLa cells expressing shScramble (shS) and shJLP were transfected with a FLAG-FOXK1 expression vector, arrested in mitosis and subjected to pull-down assay using FLAG-beads. The levels of associated proteins were determined by Western blot analysis using the indicated antisera. E) siControl (siCnt) and siPlk1 cells were transfected with a FLAG-FOXK1 expression plasmid, treated with nocodazole for 16hrs prior to harvesting and

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

15

subjected to pull-down assay using FLAG beads. Associated proteins were examined by immunoblot analysis. Figure 7: Knockdown of JLP results in an increase in FOXK1 protein levels A) U2OS cells were transduced with shScramble (shS) or shJLP lentiviruses. Protein lysates from these cells were immunoblotted using the indicated antisera. B&C) Quantitative mRNA expression (qPCR) of differentially expressed genes in (B) control (shS) and shJLP cells and in (C) siControl and siFOXK1 cells. All results were normalized to β-actin expression and graphed as average fold change (±SEM) for two independent experiments performed in triplicate. *P≤0.02 **P≤0.0005 D) shS and shJLP U2OS cells were treated overnight with 1μM nocodazole or DMSO as a control. Nocodazole treated cells were subjected to mitotic shake-off and cell lysates from the mitotic cells and DMSO-treated cells were analyzed by immunoblotting using the indicated antisera. E) Model depicting the interaction of JLP with PLK1 and FOXK1. PLK1 serves as the priming kinase of JLP and interacts with the N-terminal domain of JLP resulting in phosphorylation of JLP at additional sites. FOXK1 is recruited to bind JLP in a PLK1-dependent manner which regulates FOXK1 protein levels and the transcript levels of its downstream targets.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

16

TABLES: Table 1:

Protein names Gene names

Normalized Log2FC

Std Error p-value DMSO

#MS2 BI2536 #MS2

Serine/threonine-protein kinase Polo like Kinase 1 PLK1 -2.02 0.347 5.48E-09 45 7

Protein arginine N-methyltransferase 5 PRMT5 0.41 0.176 1.85E-02 42 17

Heat shock cognate 71 kDa protein HSPA8 0.24 0.115 3.98E-02 65 39

RCC1 domain-containing protein 1 RCCD1 -0.29 0.217 1.77E-01 21 4

C-Jun-amino-terminal kinase-interacting protein 4 JLP 0.00 0.043 1.00E+00 1022 531

Forkhead box protein K1 FOXK1 - - - 22 0

Forkhead box protein K2 FOXK2 - - - 5 0

Table 1: Effects of PLK1 inhibition on JLP interacting proteins in G2/M arrested cells HEK293T cells expressing FLAG-JLP were synchronized in G2/M phase using nocodazole and treated with DMSO or BI2536 for 1 hour prior to harvesting. Cell lysates were subjected to pull down assay using FLAG-beads and the eluted proteins were subjected to proteomic analysis as described in materials and methods section. FOXK1 and FOXK2 did not have any quantifiable SILAC ratios and were only detected with spectra in the DMSO treated sample.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

17

FIGURE 1

JLP

V JLP-S

PD: S-tag

3-PLK1 Coomassie

V JLP-S PD: S-tag

IB: JLP-S

IB: PLK1

IgG JLP

IB: JLP

IB: PLK1

IP Ab: IgG PLK1

IB: PLK1 PD: FLAG

IB: PLK1

IB: FLAG

IB: FLAG

IB: GAPDH

Lysate

FLAG: V JLP JIP3

PD: HA IB: FLAG-JLP

IB: FLAG-JLP (Lysate)

HA-PBD: - + FLAG-JLP-NTD: + +

IB: HA-PBD PD: FLAG

IB: HA-PBD

IB: HA-PBD (Lysate)

IB: FLAG

HA-PBD: + + FLAG-JLP-NTD: - +

1 1321

463 110

164 160 398 160 463

479 1018 793 1321

WT NTD

I I-LZI

II II-LZII

III CTD

V WT I I-L

ZI II II-

LZII

NTD III

CTD

IB:PLK1

IB:PLK1 (Lysate)

JLP-S domains

IB: S-tag

PD: S

-tag

IB: JLP

IB: HA tag

* *

*

IB: JLP (Lysate)

WT PKD

PBD

HA-V

HA-PLK1

PD: H

A

A.

B.

C.

D.

E.

F.

G.

H.

I. J.

1 2

4

WT

PBD PKD

1 603

603 316

306

PKD PB1 PB2

1

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

18

FIGURE 2

IB:JLP

IB:PLK1

IgG As 0 3 6 8 10 12 24 3 6 8 9 10 11 12hrs

Double thymidine - Release

+ 1µM Nocodazole DMSO

IB:PLK1

IB: BUBR1

IB: CYCLIN E

IB:JLP

LYSATE

IP: JLP

As 0 3 6 8 10 12 24 3 6 8 9 10 11 12hrs

Double thymidine - Release

+ 1µM Nocodazole DMSO

A.

B.

ASY

N

TT0

TT3

TT6

TT8

TT10

TT

12

TT24

TT

3+N

OC

TT

6+N

OC

TT

8+N

OC

TT

9+N

OC

TT

10+N

OC

TT

11+N

OC

TT

12+N

OC

M

G2

G1/S

C.

Cel

ls in

mito

sis

PLA DAPI MERGE

Inte

rpha

se

JLP

+IgG

PL

K1+

IgG

JLP+

PLK

1 A

b

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

19

FIGURE 3

IP Ab IgG JLP JLP λPpase - - +

IB: JLP

IB: PLK1

(Lysate)

WT

T334A

T35

1A

2TA

IB: HA-PLK1 PBD

IB: JLP-S NTD

IB: JLP-S NTD (Lysate)

PD: H

A

JLP-S-NTD:

PD: F

LA

G

IB: FLAG-JLP

IB: PLK1

FLAG: V WT T351A

Lysa

te

IB: PLK1

IB: FLAG-JLP

IB: GAPDH

PLK1

IB: PLK1

IB: FLAG-JLP

FLAG: WT V WT T351A

IP Ab: IgG

IB: HA-PLK1

IB: JLP

HA: V WT H/K

PLK1

IB: JLP (Lysate)

PD: HA

PLK1: - + + - PLK2: - - - + BI2536: - - + -

p-JLP (Autoradiogram) His-JLP (Coomassie)

A.

D.

F.

B. C.

E.

G.

pGST-JLP (Autoradiogram)

1-188

18

8-355

35

5-559

55

9-763

76

3-966

96

6-115

2 11

52-13

11

PLK1: - + - + - + - + - + - + - +

JLP (aa):

H.

DMSOBI2536

RO3306

CX4945

IB: JLP

IB: JLP (Lysate)

IB: PLK1

+ Nocodazole

IP: P

LK

1

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

20

FIGURE 4

SILAC labeling schematic

FLAG-JLP +Noc + BI2536

FLAG-JLP + Noc

PD using FLAG-M2 beads

Mix elute 1:1

Tryptic peptides were generated, purified and lyophilized for mass-spectrometric

analysis Inte

nsity

m/z

Light Heavy

Validation

A. B.

FLAG-FOXK2: - + + Noc: - - +

MYC-JLP

IB: MYC-JLP

IB: PLK1

IB: FLAG-FOXK2

IB: MYC-JLP

IB: PLK1

IB: FLAG-FOXK2

IB: GAPDH

PD: FLAG

Lysate

PD: FLAG

IB: MYC-JLP

IB: PLK1

IB: FLAG-FOXK1

FLAG-FOXK1: - + + Noc: - - +

MYC-JLP

IB: MYC-JLP

IB: PLK1

IB: GAPDH

Lysate IB: FLAG-FOXK1

C. D.

V FOXK1

IB: JLP

IB: PLK1PD: FLAG

Lysate

FLAG-FOXK1

IB: JLP

IB: PLK1

IB: FLAG

FOXK2V FOXK1

FOXK2

FLAG:

FLAG-FOXK2

Noc: - - - + + +

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

21

FIGURE 5

IB: HA-PLK1

IB: T7-FOXM1

IB: T7-FOXM1

PD: H

A

IB: FLAG-JLP

IB: GAPDH

IB: HA-PLK1

IB: FLAG-JLP

FLAG-JLP : + - + HA-PLK1 : - + +

+ T7-FOXM1

PD:F

LA

G

IB: T7-FOXM1

IB: FLAG-JLP

IB: HA-PLK1

Lysa

te

C.

IB: MYC-JLP

IB: PLK1

IB: GAPDH

IB: MYC-JLP

IB: PLK1

IB: FLAG PD: F

LA

G

Lysa

te

FLAG-FoxK1: V WT I II III

MYC-JLP

IB: JLP

IB: PLK1

IB: FLAG-FOXK1

IB: JLP

IB: PLK1

IB: FLAG-FOXK1

IB: GAPDH

PD: F

LA

G

Lysa

te

G1

IB: CYCLIN E

G2/M

S

A. B.

1 733 195

400 733

190 400

WT I

II III

DBD FHA

1

IB: FLAG-FOXK1

IB: FLAG-FOXK1

IB: JLP

IB: PLK1

PD: F

LA

GIB: JLP

IB: PLK1

Lysa

te

IB: CYCLIN E *

TT0 TT3 TT8hrs

Double thymidine - release

IB: α-TUBULIN

D.

E.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

22

FIGURE 6

FLAG:

shS shJLP

IB: JLP

IB: JLP

IB: PLK1

IB: PLK1

PD: FLAG

Lysate

IB: FLAG-FOXK1

V FOXK1 V FOXK1

siCnt siPlk1 siCnt siPlk1

Lysate PD: FLAG

IB: JLP

IB: PLK1

IB: FLAG-FOXK1

FHA DBD

R127A H355A

IB: JLP

IB: JLP

IB: PLK1

IB: PLK1

R127A

H355A

WT

V FLAG-FOXK1:

PD: FLAG

Lysate

IB: FLAG-FOXK1

IB: FLAG-FOXK1

A.

B.

C. D.

E.

IB: JLP

IB: PLK1

BI2536: - + - + Lysate PD: FLAG

IB: FLAG-FOXK1

IB: GAPDH Lysate

IB: MYC-JLP

IB: FLAG-FOXK1

IB: MYC-JLP

PD: FLAG

MYC-JLP : WT WT T351A FLAG-FOXK1: - + +

IB: PLK1

IB: PLK1

IB: GAPDH

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from

JLP forms a ternary complex with PLK1 and FOXK1

23

FIGURE 7

IB: PLK1

IB: JLP

IB: p21

IB: Actin

IB: FOXK1

shS shJLP

U2OS A.

**

** C.

JLP FoxK1 FoxK2 p21

Rel

ativ

e E

xpre

ssio

n (m

RN

A)

siControl

siFoxK1

B.

*

*

JLP FoxK1 FoxK2 p21

Rel

ativ

e E

xpre

ssio

n (m

RN

A)

shS

shJLP

PLK1 phosphorylates JLP at Thr 351 and interacts with the N-terminus of

JLP through its Polo-box-domain

BI2536

FOXK1 interacts with JLP/PLK1 forming a ternary complex

T351A-JLP BI2536 siPlk1

R127A-FOXK1

PLK1

JLP

PLK1

JLP

FOXK1

E. JLP PLK1

IB: FOXK1

IB: JLP

IB: p21

IB: PLK1

IB: β-ACTIN

shS shJLP shS shJLP

DMSO + Noc

Band intensity(%): 100 262 100 175

Band intensity(%): 100 70 100 54

D.

at CA

LIFO

RN

IA IN

STIT

UT

E O

F TE

CH

NO

LO

GY

on October 19, 2015

http://ww

w.jbc.org/

Dow

nloaded from