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.
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This work was supported primarily by DoD-PCRP grant W81XWH-17-1-0417 to A.D.B. LC–MS/MS equipment was acquired using a National Center for Research Resources High-End Instrumentation grant (1S10RR029127 to H.Z.).
The following are available online at http://www.mdpi.com/2072-6694/12/12/3666/s1: Figure S1: MS/MS spectrum of the phosphor-peptide N70AVMNPKTANVPQTVPMRL88 to determine pT83, Figure S2. MS/MS spectrum of the phosphor-peptide R161QSSFEIPDDVPLPAGW177 to determine pS163/pS164, Figure S3. MS/MS spectrum of the phosphor-peptide A347LRSQLPTLEQDGGTQNPVSSPGmSQEL374 to determine pT361/pS366, Figure S4. MS/MS spectrum of the phosphor-peptide R375TMTTNSSDPFLNSGTY391 to determine pS388, Figure S5. Densitometry of the original uncropped blots and gel images with their respective numbers from the main figures, Table S1. Assigned phosphorylated sites.
The above supplementary materials are also available for download as the additional file listed at the end of this record.
Khalil, Md Imtiaz; Ghosh, Ishita; Singh, Vibha; Chen, Jing; Zhu, Haining; and De Benedetti, Arrigo, "NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output" (2020). Molecular and Cellular Biochemistry Faculty Publications. 181.