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  • The androgen receptor AR is a steroid


    The androgen receptor (AR) is a steroid hormone receptor that plays a crucial role in the normal development of male reproductive tissues, and its high expression and/or relaxation of its regulation are strongly implicated in prostate cancer (PCa) [11]. Androgen binding induces conformational changes of AR that influence its interactions with other proteins and DNA as well as its subcellular localization and transcriptional activity. AR is further regulated by numerous post-translational modifications (PTMs) that affect its physiological role, especially its transcriptional program. Most phosphorylation events are mediated by different kinases, including phosphorylation of serine, threonine or tyrosine residues, which are distributed along the whole AR sequence, unlike other types of PTMs [8], [10]. While the proximal kinases for some phosphosites have been identified, the function of several phospho-residues and the kinases responsible for their phosphorylation are still unknown. One of the most frequently studied phosphosites, serine 81, has been shown to be phosphorylated by the CDK1, CDK5 and CDK9 kinases [12], [13], [14], [15], [16]. Notably, overexpression of CDK1 and cyclin B have been suggested to contribute to increased AR activity in prostate cancer and its resistance to AR antagonists [16]. Several studies have suggested that CDK1 can also cooperate with CDK7 and CDK11 to phosphorylate the other two residues of the androgen receptor, serine 515 [17], [18], [19] and serine 308 [20], [21], respectively. Our study focuses on the phosphorylation of S81, the function of which was previously investigated using various approaches, including S81-phosphosite mutants, AR overexpression, inhibition, siRNA, chromatin-immunoprecipitation and cell reporters [12], [13], [14], [15], [16]. Previous studies have demonstrated that S81 phosphorylation is required for AR nuclear translocation and association with Lauric Acid and also regulates endogenous AR-regulated transcription in response to hormones, resulting in positive effects on cell growth. Previous reports were mostly supported by chemical inhibition of CDKs, but in our opinion, the observed effects are usually pharmacologically corroborated using non-selective CDK inhibitors or CDK inhibitors with unknown selectivity. The use of inhibitors with unclear selectivity might lead to misleading conclusions, especially when these inhibitors are used as a tool for evaluating certain biological processes that are linked with the function of a particular CDK. Roscovitine is an example, and it was initially believed to be a relatively specific inhibitor of CDK1, 2 and 5. However, subsequent studies have demonstrated that it also blocks transcription through inhibition of CDK7 and 9 [22], [23]. Importantly, at least two groups used roscovitine as a tool in studies related to the function of AR and showed its ability to block androgen-stimulated phosphorylation of S81 of AR in LNCaP cells [12], [15], but each group pointed to a different CDK as the responsible enzyme. Independently, CGP74514A was shown to explain the role of CDK1 in the phosphorylation of S81-AR [13]. Nevertheless, the available selectivity profile and our own data suggest that this compound is less selective and not suitable as a tool.
    Materials and methods
    Discussion CDK1 is responsible for the phosphorylation of several cellular substrates that are predominantly important in the progression of mitosis and cytokinesis. Recently, it was suggested that CDK1 may be able to provide a pool of activated AR during mitosis [13]. Protein expression of AR during the cell cycle remains unchanged, but transcription of most AR-responsive genes is suppressed in mitosis [20]. Surprisingly, one study indicated that an increased level of S81-phosphorylation can be observed in nocodazole-G2/M-arrested LNCaP cells independently on androgen addition [13]. Because S81-phosphorylation is connected with transactivation of AR, we investigated whether increased transcriptional activity of AR would be observed in synchronized G2/M cells after nocodazole treatment. In the experiments reported here, we observed neither S81-phosphorylation nor transcriptional activation of AR in synchronized cells, which contrasted with previously published data [13], [16]. Therefore, phosphorylation of S81-AR during mitosis requires further investigation. Possible differences of the abovementioned results may be explained by the different experimental conditions used during LNCaP cultivation, which can clearly affect AR signaling. For example, a recent study revealed that a high cell density, time of cultivation and presence of androgens during cultivation can deregulate expression of several cell cycle regulators and influence the proliferation status of PCa cells [32]. Other reports also showed that the same type of PCa cells can display different patterns of AR expression and that its target PSA is probably influenced by the concentration of androgens in the media or by the duration of cultivation in charcoal-stripped serum at the beginning of the experiment [24], [51].