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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−/− 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. Targeting metastasis in prostate cancer (PCa) remains a major challenge during systemic therapy. Here, we describe a novel interaction between the TLK1 and MK5 kinases, which resulted in activation and cytoplasmic relocalization of MK5 via MK5‐S354 phosphorylation. Knockout and pharmacological inhibition of MK5 impaired motility and invasion of PCa and MEF cells. Additionally, MK5‐S354 phosphorylation correlated with PCa progression in a TRAMP mouse model and correlated to disease progression in patients with PCa. Thus, disruption of the TLK1‐MK5 axis may inhibit the metastatic spread of PCa.

Multiple p38 MAP kinase inhibitors have been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, but their effectiveness has been limited due to toxicity and tachyphylaxis, leading to a lack of clinical benefit. Efforts have been made to circumvent this limitation by targeting individual substrates downstream of p38, including MK2 and MK5. This approach has failed to yield clinical benefit despite preclinical evidence of a therapeutic effect. We hypothesized that there is redundancy in the MAPK activating kinase family that would necessitate blocking multiple kinases to sufficiently impact inflammatory processes. We used heterobifunctional protein degraders that either specifically degraded MK2 selectively or degraded MK2/3/5 simultaneously to test the hypothesis, in addition to genetic approaches to enable knockdown. In human PBMCs, elimination of MK2/3/5 with heterobifunctional degraders resulted in full reduction of TLR4 or TLR7/8 induced TNFα, whereas MK2-specific degradation only attenuated TNFα biosynthesis. In contrast, both specific MK2 degradation and broad MK2/3/5 degradation inhibited TGF-β-induced collagen production in human fibroblasts. This observation was consistent with genetic deletions of MK2, MK3 and MK5 (singly and in combination) whereby single deletion of MK2, MK3 or MK5 attenuated lipopolysaccharide (LPS) induced TNFα production and had no effect on R848-induced TNFα production. Double deletion of MK2 and MK3 or MK2 and MK5 or MK2/3/5 triple deletion had a significantly greater effect on TNFα production regardless of stimulus. The combined data suggest cooperativity between MK2 and either MK3 or MK5 for efficient, cell context-dependent modulation of inflammatory responses.

Background Long noncoding RNAs (lncRNAs) are considered critical regulators in cancers; however, the clinical significance and mechanisms of MAPKAPK5-AS1 (hereinafter referred to as MK5-AS1) in colorectal cancer (CRC) remain mostly unknown. Methods In this study, quantitative real-time PCR (qPCR) and western blotting were utilized to detect the levels of MK5-AS1, let-7f-1-3p and MK5 (MAPK activated protein kinase 5) in CRC tissues and cell lines. The biological functions of MK5-AS1, let-7f-1-3p and MK5 in CRC cells were explored using Cell Counting Kit-8 (CCK8), colony formation and transwell assays. The potential mechanisms of MK5-AS1 were evaluated by RNA pull-down, RNA immunoprecipitation (RIP), dual luciferase reporter assay, chromatin immunoprecipitation (ChIP) and bioinformatics analysis. The effects of MK5-AS1 and MK5 on CRC were investigated by a xenotransplantation model. Results We confirmed that MK5-AS1 was significantly increased in CRC tissues. Knockdown of MK5-AS1 suppressed cell migration and invasion in vitro and inhibited lung metastasis in mice. Mechanistically, MK5-AS1 regulated SNAI1 expression by sponging let-7f-1-3p and cis -regulated the adjacent gene MK5. Moreover, MK5-AS1 recruited RBM4 and eIF4A1 to promote the translation of MK5. Our study verified that MK5 promoted the phosphorylation of c-Jun, which activated the transcription of SNAI1 by directly binding to its promoter. Conclusions MK5-AS1 cis -regulated the nearby gene MK5 and acted as a let-7f-1-3p sponge, playing a vital role in CRC tumorigenesis. This study could provide novel insights into molecular therapeutic targets of CRC.

Background: The spread of metastatic prostate cancer (PCa) is responsible for the majority of PCa-related deaths, yet the precise mechanisms driving this process remain unclear. We have identified a novel interaction between two distinct promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), which triggers a signaling cascade that promotes metastasis. In PCa, the TLK1-MK5 pathway may play a critical role, as androgen deprivation therapy (ADT) has been linked to increased expression of both TLK1 and MK5 in metastatic patients linked with poor survival. Objectives: In this study, we directly examined the effects of disrupting the TLK1>MK5 axis on the motility, invasiveness, and metastatic potential of PCa cells. Methods: To establish this, we used both pharmacologic and systemic approaches with genetically engineered mouse models and the use of IVIS. Results: The results of targeting the TLK1>MK5 axis support the notion that this axis is essential for the spread of metastatic cells and the development of age-related metastases.

Objective Microglial activation plays a crucial role in neuroinflammation following ischemic stroke. This study was conducted to investigate the role and potential mechanisms of MK5 within microglial cells in the inflammatory response following ischemic stroke in mice in vivo and in vitro. Methods Microglia‐specific conditional MK5 knockout (MK5 cKO) mice and their control mice (MK5f/f) were subjected to middle cerebral artery occlusion (MCAO). BV2 cells (a mouse microglial cell line) were transfected with small interfering RNA (siRNA) to knock down MK5 levels and subsequently exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to simulate ischemic conditions in vitro. Following MCAO, behavioral tests and infarct volume measurements were conducted. Levels of cytokines and microglial markers were evaluated using qPCR and Western blotting, while immunofluorescence was employed to observe microglial activation. Additionally, Western blotting was performed to assess the phosphorylation of HSP27 and NF‐κB. Results Compared to the control group, the knockout of the MK5 gene in microglia significantly exacerbated neurological deficits and increased infarct volume in MCAO mice. The loss of the MK5 promoted inflammation by upregulating pro‐inflammatory factors and downregulating anti‐inflammatory factors, while also enhancing microglial activation in both MCAO mice and BV2 microglial cells subjected to OGD/R. Furthermore, the knockout of the MK5 gene in microglia reduced the phosphorylation levels of HSP27 and increased the phosphorylation levels of NF‐κB in the aforementioned models. Conclusion Microglial MK5 plays a critical role in the ischemic neuroinflammatory response by regulating the phosphorylation of HSP27 and NF‐κB, positioning it as a potential target for stroke treatment. MK5 gene knockout promotes microglial activation by regulating the phosphorylation of HSP27 and NF‐κB, thereby exacerbating functional decline.

Background: Metastatic dissemination of prostate cancer (PCa) accounts for the majority of PCa-related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), that initiates a signaling cascade promoting metastasis. In PCa, TLK1–MK5 signaling might be crucial, as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients, but in this work, we directly investigated the motility, invasive, and metastatic capacity of PCa cells following impairment of the TLK1 > MK5 axis. Results: We conducted scratch wound repair and transwell invasion assays with LNCaP and PC3 cells to determine if TLK1 and MK5 can regulate motility and invasion. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration and invasion through a Matrigel plug. We further elucidated the potential mechanisms underlying these effects and found that this is likely due to the reorganization of the actin fibers at lamellipodia and the focal adhesions network, in conjunction with increased expression of some MMPs that can affect penetration through the ECM. PC3, a highly metastatic cell line when assayed in xenografts, was further tested in a tail-vein injection/lung metastasis model, and we showed that, following inoculation, treatment with GLPG0259 (MK5 specific inhibitor) or J54 (TLK1 inhibitor) resulted in the lung tumor nodules being greatly diminished in number, and for J54, also in size. Conclusion: Our data support that the TLK1–MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness; hence, disruption of this axis may inhibit the metastatic capacity of PCa.

Prostate cancer (PCa) is the second most diagnosed cancer in Western male population. In this study, we insert mK5 (the mutational kringle5 of human plasminogen) into a DD3-promoted (differential display code 3) oncolytic adenovirus to construct OncoAd.mK5.DD3. E1A.dE1B, briefly, O .DD3.mK5. DD3 is one of the most prostate cancer specific promoters which can transcriptionally control adenoviral replication. mK5 has been proved to be able to inhibit the tumor angiogenesis and inhibit cell proliferation. Our results suggested that targeting PCa with O .DD3.mK5 elicited strong antitumor effect.

Extracellular signal‐regulated kinase 3 (ERK3) is an atypical mitogen‐activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK‐activated protein kinase‐5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3‐dependent phosphorylation and activation of MK5 in vitro and in vivo . Endogenous MK5 activity is significantly reduced by siRNA‐mediated knockdown of ERK3 and also in fibroblasts derived from ERK3 −/− mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor‐induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC 50  = 37.5 μM; K i  = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.

The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD) simulations of: (1) MK5 alone; (2) MK5 in complex with an inhibitor; and (3) MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS) calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.