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  • Published results have revealed relationships between serine

    2019-07-31

    Published results have revealed relationships between serine 81 in AR and some CDKs, especially CDKs 1, 5 and 9 [12], [13], [14], [15], [16]. These results are often supported by pharmacological inhibition of a certain CDK. An effect of CDK1 inhibition on suppression of S81-phosphorylation was previously confirmed by many CDK inhibitors, including roscovitine and CGP74514A [12], [13], [16]. Nevertheless, the low selectivity of these inhibitors can influence the proper interpretation of the results. CGP74514A is a typical example of an incorrectly used CDK1 probe because it has other targets (Table 1) [36], [37]. RO3306 is widely considered to be a selective CDK1 inhibitor, but its kinase profile has not yet been published. We confirmed here for the first time that RO3306 selectively inhibits CDK1 over other CDKs and conclude that its effect on the phosphorylation of serine 81 is indeed caused by this kinase. Similarly, our experiments with two novel selective CDK9 inhibitors, LDC000067 and PHA767491, supported previous findings about the role of CDK9 in S81-phosphorylation of AR [13], [14], [16]. Refinement of the selectivity of CDK inhibitors led us to speculate on whether other interphase CDKs could be involved in this process. We focused on CDK2 and CDK4, which are known to interact with steroid receptors either directly or indirectly, but to the best of our knowledge, no previous reports have indicated a possible role of any G1-regulating CDK in S81-AR phosphorylation. The CDK2/A complex causes transcriptional activation of the estrogen receptor through its phosphorylation of serines 104 and 106 [52], [53]. Interaction of cyclin A with CDK2 also contributes to transactivation of the progesterone receptor via phosphorylation of multiple phosphosites [54], [55]. For the first time, we demonstrate a relationship between phosphorylation of S81-AR and CDK2 through siRNA-based silencing and pharmacological inhibition in PCa cells. The NS3694 NU6102 has been shown to have a high preference for CDK2 and inhibits it at the lowest concentrations (see Table 1 and references [41], [42], [43]), and therefore, it is a suitable tool for cellular studies. In C4-2 and LAPC-4 cells, NU6102 selectively inhibited CDK2 in subtoxic doses, diminished androgen-stimulated S81-phosphorylation and also supressed androgen-mediated transcription of AR-regulated genes in the reporter cell line 22Rv1-ARE14 in a concentration-dependent manner. Finally, similar results were observed with other CDK inhibitors with lower selectivity for CDK2 as well (Table 1, Fig. 7b). Taken together, these results from chemical and molecular inhibition indicated that CDK2 contributes to S81-phosphorylation and transcriptional activity of AR. Interestingly, cyclin D was reported to bind to AR and inhibit ligand-dependent AR activity [56], [57], [58]. Another study suggested that CDK6 can bind to AR in the presence of androgen and markedly enhance its transcriptional activity, but this process was documented as being independent of cyclin D1 and CDK6 kinase activity [59]. The stimulatory impact on AR may be linked to frequent overexpression of CDK6 in human PCa, while cyclin D amplification is observed only rarely [60], [61]. Our results provide no NS3694 evidence that CDK4 is involved in phosphorylation of AR at S81 and its transactivation. Silencing of CDK4 (as well as palbociclib and abemaciclib application) did not cause significant changes in the expression and phosphorylation of AR as well as of NKX3.1 and PSA expression. In addition, there was no effect on R1881-stimulated transcriptional activity in a reporter cell line, which also suggests that CDK4 has no effect on the transcriptional activity of AR in PCa cells. Based on published reports and our own findings, we propose to combine genetic inactivation with pharmacological inhibition in mechanistic studies because the selectivity of many commercially available inhibitors is often limited. Experiments with non-selective inhibitors easily lead to misleading conclusions, as discussed for SB203508 or dasatinib [62]. Fortunately, selectivity profiles of many CDK inhibitors are increasingly found in publications [36], [37], [48], [63], [64], [65], [66], which allow researchers to pick the most suitable chemical tools for their studies. We also suppose that the role of CDKs in the phosphorylation of AR cannot be integrated into a definite model yet mainly because several studies have shown that cyclins or CDKs can behave as coactivators of steroid receptors independently of their kinase activities. Furthermore, CDK/cyclin complexes have been shown to be able to substitute for each other in a compensatory mechanism in cells [67]. In this study, we confirmed the importance of CDK1 and CDK9 in AR activation and identified CDK2 as another kinase that phosphorylates S81 in AR.