TLK1‐mediated MK5‐S354 phosphorylation drives prostate cancer cell motility and may signify distinct pathologies

Abstract

Metastases account for the majority of prostate cancer (PCa) deaths, and targeting them is a major goal of systemic therapy. We identified a novel interaction between two kinases: tousled-like kinase 1 (TLK1) and MAP kinase-activated protein kinase 5 (MK5) that promotes PCa spread. In PCa progression, TLK1-MK5 signalling appears to increase following antiandrogen treatment and in metastatic castration-resistant prostate cancer (mCRPC) patients. Determinations of motility rates (2D and 3D) of different TLK1-and MK5-perturbed cells, including knockout (KO) and knockdown (KD), as well as the use of specific inhibitors, showed the importance of these two proteins for in vitro dissemination. We established that TLK1 phosphorylates MK5 on three residues (S160, S354 and S386), resulting in MK5 activation, and additionally, mobility shifts of MK5 also supported its phosphorylation by TLK1 in transfected HEK 293 cells. Expression of MK5-S354A or kinase-dead MK5 in MK5-depleted mouse embryonic fibroblast (MEF) cells failed to restore their motility compared with that of wild-type (WT) MK5-rescued MK5 À/À MEF cells. A pMK5-S354 antiserum was used to establish this site as an authentic TLK1 target in androgen-sensitive human prostate adenocarcinoma (LNCaP) cells, and was used in immunohistochemistry (IHC) studies of age-related PCa sections from TRAMP (transgenic adenocarcinoma of the mouse prostate) mice and to probe a human tissue microarray (TMA), which revealed pMK5-S354 level is correlated with disease progression (Gleason score and nodal metastases). In addition, The Cancer Genome Atlas (TCGA) analyses of PCa expression and genome-wide association study (GWAS) relations identify TLK1 and MK5 as potential drivers of advanced PCa and as markers of mCRPC. Our work suggests that TLK1-MK5 signalling is functionally involved in driving PCa cell motility and clinical features of aggressiveness; hence, disruption of this axis may inhibit the metastatic spread of PCa.

The TLK1-MK5 axis regulates motility, invasion, and metastasis of prostate cancer cells

“…We previously reported that the TLK1>MK5 interaction is critical for the motility and invasive properties of all the PCa cell lines we tested, and this was most critically illustrated with the use of the specific MK5 inhibitor GLPG 0259 (in a dose-dependent manner) (28). However, as all instances of pharmacological inhibition, there was an issue of ascertaining specificityboth for MK5 and for its regulation/activation by TLK1.…”

“…IHC staining of mice lung tissue was conducted as previously described (7). Imaging was done using a 10X magnification.…”

“…Moreover, pMK5 S354 was elevated in the tissue sections of high-grade clinical samples and in a transgenic mice model of PCa (TRAMP), which also suggested that pMK5 S354 level maybe associated with increased aggressiveness of PCa. Finally, we observed that pharmacologic inhibition of MK5 leads to drastic reduction of motility in a panel of androgen dependent or independent PCa cell lines even at a low dose (1-5 μM concentration) (7).…”

“…We previously demonstrated that a serine/threonine (S/T) protein kinase, Tousled-like kinase 1 (TLK1) could promote PCa cell motility and clinical aggressiveness by phosphorylating another S/T kinase, MAPK-activated protein kinase 5 (MAPKAPK5/MK5) (7,8). TLK1 is known as a genome maintenance regulator, which functions in regulating replication, transcription, cell cycle checkpoint control, DNA damage response and repair through its direct and indirect substrates (reviewed in (9)(10)(11)(12)).…”

“…This is particularly important in PCa as androgen deprivation therapy (ADT) translationally upregulates TLK1B (a spliced variant of TLK1 with intact kinase domain as TLK1) through the activation of AKT>mTORC1>4EBP1>eIF4E pathway (reviewed in (9)). In our previous study, we demonstrated that TLK1/1B can directly phosphorylate MK5 in three novel residues (S160, S354, and S386) and increase MK5’s catalytic activity towards its downstream substrates (7). In fact, TLK1 alone cannot promote cellular motility in absence of MK5 as observed in a TLK1 overexpressing MK5 -/- MEF cell line model.…”