Abstract 2294: NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output

Abstract

Abstract Background: Despite recent development in prostate cancer (PCa) therapeutics, castration-resistant prostate cancer still accounts for high morbidity and mortality in patients, partly because of incomplete knowledge of the major players that contribute to the androgen independent growth of prostate tumor. NIMA related kinase 1 (NEK1), a dual specificity kinase, mainly functions in maintaining genomic stability by regulating DNA damage response (DDR) and G2/M transition of the cells. We previously reported that tousled-like kinase 1 (TLK1) mediated phosphorylation of NEK1 on T141 can lead to rapid androgen independent progression of PCa cells upon anti-androgen treatment by activating DDR. However, activating DDR alone may not be sufficient to trigger androgen insensitive growth of PCa cells. We hypothesize that TLK1>NEK1 axis may also regulate hippo pathway oncoprotein YAP as a previous study reported that NEK1 can phosphorylate YAP homolog TAZ and upregulate YAP/TAZ transcriptional targets. Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of hippo kinases that binds to transcription factor TEAD and promotes many oncogenic activities in the cells. Methods and Results: To determine if NEK1 can regulate Hippo pathway, our western blotting (WB) analysis revealed higher cleaved YAP (cl-YAP) and lower total YAP level in the NEK1-T141A hypoactive variant overexpressing LNCaP than wt-NEK1 overexpressing cells treated with bicalutamide (anti-androgen) and/or THD (a specific inhibitor of TLK1) which suggest that NEK1 may play a role in stabilizing YAP. TLK1 inhibition also resulted in the reduced expression of some YAP target genes such as CTGF, CDH2, Twist1, and P53AIP1. Co-IP assay revealed that NEK1 can interact with YAP regardless of its kinase activity. We generated a CRISPR-Cas9 mediated NEK1 knockout NT1 (a mouse PCa cell line) cells which showed reduced level of YAP in NEK1 KO clones as well as downregulation of YAP target genes such as CTGF, Zeb1, Twist1, and Cyr61. TLK1 knockdown and inhibition also resulted in reduced pNEK1 (T141) and total YAP level which suggested TLK1 mediated NEK1 activation is necessary for YAP stabilization. An IVK assay using [γ-32P] ATP demonstrated that catalytically active purified NEK1 can phosphorylate purified YAP1. MS analysis determined eight NEK1 mediated phospho-acceptor sites of YAP1: T83, T361, S366, S388, Y407, and T493. Y407 and T493 are the first ever reported phosphorylation sites on YAP1 especially by NEK1 which lies in the transactivation and PDZ binding domain. Our bioinformatics analysis revealed mRNA downregulation of YAP1, while upregulation of YAP protein level was observed in high-grade PCa, which is consistent with our model of post-transcriptional protein stabilization. Conclusion: Our data support that TLK1>NEK1 signaling promotes YAP stabilization and may contribute to castration resistant growth of PCa during AR blockade. Citation Format: Md Imtiaz Khalil, Ishita Ghosh, Vibha Singh, Jing Chen, Haining Zhu, Arrigo De Benedetti. NEK1 phosphorylation of YAP promotes its stabilization and transcriptional output [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2294.