MK5 promotes cell motility in both non-malignant and neoplastic cell lines
We have utilized a novel proteomic screening assay from Life Technologies to identify the interactome of TLK1B [45]. TLK1B is a spliced variant of TLK1 that lacks the first 238 amino acids in the N-terminal domain of TLK1. TLK1B is translationally regulated, shares the identical C-terminal kinase domain and is believed to have similar substrate specificity with TLK1 [8, 41]. Briefly, biotinylated TLK1B was used to hybridize 9000 human full-length proteins spotted in duplicates on glass slides, after which, fluorophore conjugated streptavidin was used to generate the signals from the interactions between TLK1 and arrayed proteins. This assay identified 165 human proteins that interact with TLK1 with high confidence that are involved in various cellular functions such as cell cycle regulation, DNA damage repair, DNA replication, and cell motility. MK5 was identified as one of the top interactor proteins as shown in Fig. 1A where the top 20 TLK1 interactors were plotted according to their affinity p-values. Additional information of these interacting proteins including signal intensity, negative control signal (streptavidin only), Z-score, signal variance between duplicated spots, and CI -value (Chebyshev’s Inequality р- value) can be found in the supplementary section of Singh et al. (2017) [45]. Since MK5 promotes cellular motility in both non-malignant (e.g., PC12 [17], HUVEC [16]) and neoplastic cells (e.g., HeLa [20]), we tested whether MK5 overexpression or depletion alters the motility rate of mouse embryonic fibroblast (MEF). MK5 was overexpressed in wild type (WT) MEF cells using eGFP-MK5 mammalian construct and exogenous MK5 level was determined by western blotting (Fig. S1A). We compared the scratch healing rate among MK5−/− MEF [43], WT MEF, and MK5 overexpressing MEF cells by scratch repair assay. Scratch repair appeared to be the fastest in the MK5 overexpressing MEF and slowest in the MK5−/− MEF cells (Fig. 1B and S1B).
It is noteworthy that MK5−/− MEF cells was generated in a mixed genetic background (129/Ola X C57BL/6), whereas wild type MEF cells were isolated from C57BL/6 mice [43]. To rule out the possibility that the discrepancy in the genetic background of MK5−/− MEF and WT MEF cells can affect their migration rate in scratch wound repair assay, we reconstituted MK5 in the MK5−/− MEF cells (Fig. S2A). In addition, we tested if TLK1 alone in absence of MK5 can enhance their migration rate, by stably overexpressing TLK1 in the MK5−/− MEF Cells (Fig. S2B). When we compared the wound healing rates among these three cell lines, namely, MK5 rescued MEF, MK5−/− MEF, and TLK1 overexpressing MK5−/− MEF cells in a scratch repair assay, we observed that while MK5 rescued cells migrated significantly faster, there was no significant difference in the migration rate between TLK1 overexpressing MK5−/− MEF and MK5−/− MEF cells (Fig. 1C and S2C). The latter results indicate that without MK5, TLK1 cannot exert its function in motility promotion. To confirm our observations from the scratch assay, we utilized the 3D chemotactic trans-well migration assay in the Incucyte machine using three different extra cellular matrices (ECMs). We observed that in fibronectin, MK5 rescued cells migrated significantly faster and no difference in the migration rate of TLK1 overexpressing MK5−/− vs. MK5−/− MEF cells (Fig. 1D and S3). Similar trends in migration were also observed using Matrigel and collagen I (Fig. S4 and S5). To examine whether reconstitution of MK5 increases the proliferation rate of the cells which might contribute to enhanced migration rate that we observed in the scratch and chemotactic migration assays, we conducted a proliferation assay between MEF MK5−/− and MEF MK5−/− GFP-MK5 cells. MK5 reconstitution did not significantly increase the proliferation rate compared to MK5−/− MEF cells (Fig. 1E and S6). We conducted another scratch experiment with LNCaP cells, an androgen dependent PCa cell line. LNCaP cells were transfected with either eGFP-MK5 or TLK1 kinase dead (KD) dominant negative mammalian construct (Fig. S7A) [41]. We observed significantly faster wound healing in the MK5 overexpressing LNCaP cells compared to the TLK1 KD and WT LNCaP cells (Fig. 1F and S7B). Overall, these findings suggest MK5 as a promotility factor, in addition, TLK1 alone in absence of MK5 cannot increase the migration rate of the cells.
TLK1 knockdown/inhibition results in reduced cell migration rate
Emerging evidence suggest that TLK2 can promote cancer invasiveness by enhancing the migration and invasion of breast cancer and glioblastoma cell. Since TLK1 and TLK2 share high homology in the amino acid sequence and are believed to have partly redundant functions, we hypothesize that TLK1 can also regulate cellular motility. To examine whether TLK1 acts a potential factor to regulate cell motility, we took both genetic and pharmacologic approach. Both siRNA mediated knockdown and pharmacologic inhibition of TLK1 using small molecule inhibitor (J54) resulted in the reduced scratch healing rate in WT MEF cells compared to the control cells (Fig. 2A, 2B, and 2C). J54 is a potent second generation phenothiazine derivative specific for TLK1 [44]. Scratch healing rate of TLK1 depleted and TLK1 inhibited WT MEF cells was similar and there is no statistical difference between these two experimental groups. Therefore, we opted to use J54 for our next experiments. J54 treatment also significantly reduces the wound healing rate of MK5 rescued MK5−/− MEF cells compared to the vehicle (DMSO) treated MK5 rescued cells (Fig. 2D and 2E). These findings establish both TLK1 and MK5 as promotility factors and we hypothesize that MK5 may function as a downstream effector of TLK1 to promote cellular motility. Unexpectedly, TLK1 inhibition by J54 also resulted in reduced wound closure in MK5−/− MEF cells which suggest that TLK1 may promote cellular migration through additional downstream effectors even in the absence of MK5 (Fig. S8A and S8B).
TLK1 interacts with MK5 both in vitro and in cultured cells
Results obtained from our protoarray assay demonstrated that TLK1B interacts with MK5 in vitro (Fig. 1A). To test whether TLK1 interacts with MK5 in cultured cells, we overexpressed MK5 in HEK 293 cells using eGFP-MK5 mammalian construct and conducted a co-immunoprecipitation (co-IP) experiment. Immunoprecipitations of TLK1 using TLK1 specific antibodies brought down both exogenous and endogenous MK5, which was confirmed by western blotting (Fig. 3A). Reciprocal immunoprecipitation of MK5 using GFP specific antibody also revealed the presence of TLK1 in the co-IP (Fig. 3B). These data suggest that TLK1 and MK5 form a complex in cells. We confirmed this observation performing His- and GST- pull down experiments independently. Incubation of purified recombinant his- tagged TLK1B with MK5 overexpressing HEK cell lysates precipitated both endogenous MK5 and exogenously expressed GFP-MK5, as determined by WB (Fig. 3C). Similarly, incubation of purified recombinant GST-tagged MK5 with TLK1 & MK5 co-expressing cell lysate precipitated both TLK1 (Fig. 3D, left panel) and GFP-MK5 (Fig. 3D, right panel). Together, these results indicate that TLK1 interacts directly with MK5. The observation that endogenous MK5 was also pulled down in both co-IP (Fig. 3B) and GST- pull down assay (Fig. 3D, right panel) suggest that endogenous MK5 may dimerize with exogenously expressed GFP-MK5 and/or GST-MK5.
TLK1 Phosphorylates MK5 both in vitro and in cultured cells and increases its catalytic activity
We examined whether TLK1 possesses the ability to phosphorylate MK5 in vitro and/or in cultured cells. We first purified recombinant his- tagged TLK1B and recombinant his-tagged MK5 following previously published protocol (Fig. 4A and 4B) [12, 40]. We confirmed the catalytic activity of MK5 using MK5 specific protein and peptide substrate, HSP27 (Fig. 4C) and PRAKtide (Fig. S9), respectively by ADP hunter assay. Incubation of purified recombinant his-tagged TLK1B with both purified recombinant his-tagged MK5 and HSP27 in an IVK assay synergistically increases the phosphorylation level of MK5 and intrinsic catalytic activity of MK5 towards its known substrate, HSP27 (Fig. 4D and 4E). To determine whether TLK1 can phosphorylate MK5 in cultured cells, we transfected HEK 293 cells either with GFP-MK5 alone or co-transfected the cells either with GFP-MK5 +TLK1 or GFP-MK5+ TLK1-kinase dead (KD) expression construct. WB analysis revealed a hyperphosphorylated form of GFP-MK5 only in MK5+ TLK1 co-expressed cell lysate that migrated slower than the main form of GFP-MK5 (Fig. 4F). Lambda protein phosphatase (LPP) treatment reduces this slower migrating band which suggest that TLK1 can phosphorylate MK5 in cultured cells. We also conducted another IVK assay by incubating both recombinant His-TLK1B and GST-MK5 in the presence of non-radioactive cold ATP. The reactions were run in an SDS-PAGE gel and the corresponding MK5 bands were excised for mass spectrometric analysis (Fig. 4G).
TLK1 Phosphorylates MK5 in vitro on three unique residues which are mapped to the functional domains of MK5
To determine the phosphopeptides of MK5 by TLK1, MK5 bands were digested by trypsin and subjected to shot-gun proteomic analysis using an LTQ-Orbitrap mass spectrometer. MS data sets are searched with MASCOT against a custom database containing human MAPKAPK5. The summary of the analysis as are follows: 1) MAP kinase-activated protein kinase 5 (MAPKAPK5 or MK5) was detected with high protein score (4686- 8482) and good peptides coverage (70.40-80.55%) in both samples. 2) Phosphorylated sites were detected in both samples. After comparing MK5 mock and MK5+TLK1B samples, a few unique phosphorylation sites were detected in MK5+TLK1B sample: S160, S348, S354, and S386 (Fig. 5 and Table S1). The constitutive phosphorylation sites present in both MK5 mock and MK5+TLK1B samples can be explained as the autophosphorylation sites of MK5 (Fig. S10). 3) S348 and S354 are within the same peptide with single phosphorylation event. After comparing the spectra, S354 was determined to be the more likely phosphorylation site. 4) Thus, we conclude that there are three unique phosphorylation sites in MK5+TLK1B sample: S160, S354, and S386. We mapped these serine residues to the domains of MK5 and identified that they are located in the kinase domain (in the activation loop near the residue Thr182, which needs to be phosphorylated for the catalytic activation of MK5 [46, 47]), in the nuclear localization signal (NLS) domain, and in the ERK3 binding domain (rev. in [37]) (Fig. S11).
Anti-androgen treatment increases pMK5 Ser354 level and can be inhibited by small molecule inhibitor specific to TLK1
Anti-serum raised against one of the phosphoresidue of MK5 (Ser354), one of the sites that we identified as being target of TLK1, has recently become commercially available and we tested it to determine if this specific MK5 phosphoprotein is present in several androgen dependent and independent PCa cell lines (Fig. 6A and 6D) [48, 49]. MK5−/− MEF cells were used as a negative control. We also detected TLK1 in those PCa cell lines (Fig. 6A). Western blotting analysis revealed the presence of pMK5 S354 level relative to the total MK5 in all PCa cell lines tested, while no pMK5 S354 was detected in MK5−/− MEF cells. We also previously reported that treatment of LNCaP and other androgen sensitive PCa cells with Bicalutamide results in increased expression of TLK1B[9, 10]. We now show that anti-androgen treatment also results in a corresponding dose-dependent increase in pMK5(S354) (Fig. 6B and 6E), suggesting that TLK1/1B may be the kinase responsible for this phosphorylation. Moreover, we treated LNCaP cells with two specific small molecule inhibitors of TLK1 (THD or J54), which resulted in the reduction of pMK5 S354 level compared to the DMSO treated control LNCaP cells, which further supports TLK1 role in MK5 Ser354 phosphorylation (Fig. 6C and 6F).
Expression of pMK5-S354 in PCa progression of TRAMP mice
We wanted to study if the presence of pMK5 presented with a pattern of progressive intensity during progression to invasive PCa in the TRAMP model, given that we have already reported the expression of TLK1 increases greatly from 12 to 30 weeks of age, particularly after castration [10] . At 12 weeks, TRAMP mice present feature of early carcinogenic progression of the prostate, with a mix of hyperproliferative lesions such as, prostatic intraepithelial neoplasia (PIN), to well differentiated adenocarcinomas, and in some case some areas of invasiveness with breakdown of the basement membrane. These features are remarkably well represented in Fig. 7A (top panels), which clearly shows that the areas of PIN, confined Ca, and locally invasive Ca are stained progressively darker upon pMK5-IHC, compared to normal acini (Fig. 7A and 7B). At 26 weeks the PCa has progressed to fully invasive, and the cancer cells are spread thought the glands (Fig. 7A, bottom panels).
pMK5 Ser354 is elevated in patients with high grade metastatic prostate cancer
We interrogated our institutional PCa TMA (with African American prevalence) to establish possible clinicopathologic correlations based on the Gleason score/s (GS), regional lymph node metastasis, and the levels of pMK5 Ser354. pMK5 Ser354 staining intensity was progressively higher in tumor samples with increasing Gleason scores and lymph nodes metastases. For instances, our representative IHC images revealed higher positive staining of pMK5 ser354 of several GS 8-9 tumors compared to GS 6 (Fig. 8). Similar increase was also observed in the tumors with 1-3 lymph nodes metastasis (N1). A very notable feature was that, regardless of GS (the key token of pathology determination to date) the nuclear to cytoplasm ratio of pMK5 was decreased in several samples, including some N1 of not the highest GS grade (Table S2). MK5 is generally localized to the nuclei, but it shuttles to the cytoplasm upon MAPK activation [42, 50], where it is believed to regulate actin cytoskeletal reorganization. While our TMA does not report on patients’ outcome, it would be intriguing to study this in the future to establish if the pMK5 nuclear/cytoplasm ratio can be used as a prognostic indicator. Generally, tumors with GS≤ 6 are considered as low risk tumors with well-differentiated PCa cells, GS=7 is considered as moderate risk, and GS≥ 8 is considered as high risk tumors with poorly differentiated PCa cells that have higher migratory and invasive potentials. Higher phosphorylation of MK5 may suggest TLK1-MK5 signaling as a key driver of metastasis in PCa subjects.
Pharmacologic inhibition of MK5 strongly reduces wound healing of both androgen dependent and
independent PCa cells
To establish that MK5 is indeed important for cell motility, we tested a panel of PCa cell lines, including LNCaP, C4-2B, PC3, DU145, and 22RV1 with the specific MK5 inhibitor GLPG-0259 (Fig. 9, S12, and S13) using the scratch repair assay via the Incucyte. In all cell lines tested, GLPG resulted in a strong immobilization effect with no apparent toxicity in terms of loss of viable cells. This effect was noticeable within few hours after addition of the drug, with already maximal effect at 10 μM, which is close to the established IC50 for GLPG0259 [51, 52]. These findings further confirm the promotility role of MK5 in PCa cells, where TLK1 interaction and phosphorylation of MK5 promotes motility.
Bioinformatic analysis revealed genomic amplification and upregulation of both TLK1 and MK5 in metastatic tumors
Previous studies reported TLK1 as one of the major drivers of CRPC progression after ADT[9, 10, 12]. Androgen ablation also results in the activation of some MAPK pathways which will further increase tumor aggressiveness. We interrogated publicly available databases to determine TLK1 and MK5 status in actual PCa patients with advanced tumors. Analysis of TCGA, SU2C, Broad/Cornell, and other PCa datasets revealed genomic copy number increase of MK5 in CRPC patients compared to localized tumors (Fig. 10A). We analyzed the mRNA expression of both TLK1 and MK5 of PCa tumors with different nodal metastatic status using the UALCAN online bioinformatic tool. Tumors with 1-3 regional nodal metastatic lesions (N1) showed slightly higher expression of TLK1, but significant upregulation of MK5, compared to tumors with no nodal metastatic lesion (N0) (Fig. 10B and 10C) was observed. Similarly, consistent upregulation of both TLK1 and MK5 was observed in tumors with increasing Gleason scores (Fig. 10D and 10E). These may suggest that genomic amplification or higher mRNA expression of TLK1 and MK5 may have association in increasing tumor aggressiveness and metastatic potential.