NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

Abstract

Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.

Interaction of TLK1 and AKTIP as a Potential Regulator of AKT Activation in Castration-Resistant Prostate Cancer Progression

“…Unexpectedly, myristoylated AKT overexpressing LNCaP cells (LNCaP Myr-AKT), which are characterized by lipid raft-residing, constitutively active AKT, independent of PI3K pathway activation, can be equally inhibited in their proliferation rate upon TLK1 inhibition, by varying concentrations of J54 (Figure 4). This suggests that in addition to TLK1>AKTIP>AKT signaling, other pathways through TLK1 signaling, such as TLK1>NEK1>YAP, exist and regulate the survival and proliferation of PCa cells, as demonstrated by our previous work [12]. In support of this hypothesis, we monitored NEK1-T141 phosphorylation (the residue uniquely phosphorylated by TLK1) and could demonstrate its time-and dose-dependent inhibition by J54 in both the control and Myr-AKT expressing cells; it is clearly an independent substrate of TLK1 that does not involve active AKT (Figure 4b).…”

“…The knockdown of AKTIP was carried out using 10 nM of AKTIP specific siRNA (Thermo Scientific, Waltham, MA, USA, cat# 4392420) by a Lipofectamine 3000 (Thermo Scientific, Waltham, MA, USA, cat# L3000-015) transfection reagent, following manufacturer's protocol, for 48 h. After the transfection, cells were harvested for WB analysis. The shRNA-mediated knockdown of TLK1 was described by Khalil et al (2020) [12].…”

MINDY1 promotes bladder cancer progression by stabilizing YAP

“…No directly YAP/TAZ-targeting compounds have been identified so far [ 41 , 42 ]. In addition, using small molecule inhibitors against YAP cannot completely abolish YAP transcriptional activity and is not very effective in treating YAP-driven cancers [ 43 ]. Therefore, targeting MINDY1 may also provide a new means to inhibit tumor‐specific YAP activity.…”

Targeting the Hippo Pathway in Prostate Cancer: What’s New?

“…In support of MK5 s oncogenic role, enhanced MK5 levels correlate with YAP levels, resulting in a poor prognosis [75]. However, independent proteomic analysis of YAP1 in the presence of MK5 revealed no novel phosphorylation sites, suggesting MK5 may not directly phosphorylate YAP [76]. Interestingly, MK5 is up-regulated in cancer versus normal samples within the Grasso Prostate dataset [71], suggesting further investigation may be warranted in PC.…”

“…Interestingly, MK5 is up-regulated in cancer versus normal samples within the Grasso Prostate dataset [71], suggesting further investigation may be warranted in PC. In contrast, NEK1 was found to phosphorylate YAP at multiple sites to result in enhanced protein stability [76]. Similarly, PIN1, which is overexpressed in PC [77], has been shown to enhance the stability of YAP/TAZ in breast cancer to produce taxol resistance [78].…”