The evolution and function of two phospho-sites in the androgen receptor

The androgen receptor (AR) is an essential gene, member of the steroid hormone receptor family (SHRs)1. AR mediates androgen signaling activating transcription of target genes crucial for development, sexual differentiation and homeostasis; however, this signaling becomes dysregulated in disease contexts, such as sex-biased cancer. It is well established taht androgen binding promotes AR nuclear translocation and induces ligand-dependent N-terminal to C-terminal (N/C) interactions that enhance DNA binding, cofactor recruitment, and transcriptional output. However, how these spatial and conformational transitions are regulated before ligand binding, how they are integrated with post-translational modifications, in particular phosphorylation, and how such mechanisms could affect AR function is still to be clarified. This thesis investigates the evolutionary origin, molecular function, and pathological relevance of two AR phosphorylation sites, S792 and S215, which represent distinct regulatory sites embedded within different structural regions of the receptor. Evolutionary analyses reveal that S792 resides within a highly conserved eukaryotic linear motif (RxRxxSxxF) that has been maintained across vertebrate evolution, from fish to mammals, indicating strong selective pressure. In contrast, the phospho-motif surrounding S215 is a recent evolutionary acquisition: while the motif emerges in early mammals, the phosphorylatable serine residue is largely restricted to primates. This divergence reflects their localization in AR structure, with S792 embedded in the ligand-binding domain (LBD) and S215 located within the intrinsically disordered N-terminal region (NTR), highlighting how structural constraints and intrinsic disorder shape regulatory evolution. Molecular dynamics simulations demonstrate that S792 is a cryptic phosphosite that becomes transiently exposed during early folding intermediates of the AR LBD but is buried in the ligand-bound conformation. We show that S792 phosphorylation controls AR subcellular localization by regulating a nuclear export signal embedded in the LBD, providing a mechanistic basis for its suppressive role. Conversely, phosphorylation at S215 does not act as an on/off switch but modulates AR dynamics in a ligand-dependent manner. Importantly, the functional role of S215 phosphorylation is dependent on the phosphorylation competence of S792, revealing a hierarchical phospho-control system: when S792 remains accessible to this modification, S215 phosphorylation attenuates AR activity; once ligand binding renders S792 inaccessible, S215 phosphorylation promotes physiological AR functions. This coordinated crosstalk is mechanistically mediated, at least partially, by 14-3-3 proteins. Indeed, we identified a phosphorylation-dependent mode of AR N-terminal/C-terminal interaction mediated by 14-3-3 proteins. Unlike the canonical ligand-induced N/C interactions, this 14-3-3-mediated intramolecular interaction occurs in the unliganded state and is driven primarily by S792 phosphorylation, with further stabilization when both phospho-sites are modified. This mechanism adds a previously unrecognized regulatory layer linking phosphorylation to AR conformation, localization, and signaling integration. In addition, we investigate whether this phospho-regulatory type of control has a key role not only at the physiological level, but also in pathologic condition such as in colorectal (CRC) and renal cell (RCC) cancer, two types of sex-biased cancer that affect more men the women. In vivo xenograft models demonstrated that loss of coordinated phosphorylation at S215 and S792 promotes tumor growth. Consistently, analysis of large CRC and RCC patient cohorts revealed that high AR phosphorylation correlates with improved overall survival. In CRC, S215 phosphorylation associates with favorable prognosis, immune activation, and reduced epithelial–mesenchymal transition, while S792 phosphorylation correlates