Physiological and pathological role of serine 96 phosphorylation in the regulation of androgen receptor

Spinal and bulbar muscular atrophy (SBMA) is an X-linked neuromuscular disorder characterized by the progressive dysfunction and loss of lower motor neurons. SBMA is caused by the expansion of a CAG tandem repeat encoding a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which includes eight other neurological diseases caused by the same mutation in unrelated genes. PolyQ diseases share common features, such as that polyQ proteins are typically expressed throughout the body, yet they cause specific neuronal loss. It remains to be clarified why specific sub-populations of neurons degenerate in each polyQ disease. The well-known structure and function of AR make SBMA a good model to investigate polyQ disease pathogenesis. Androgen binding to AR results in its nuclear translocation and binding to androgen-responsive elements (AREs) to regulate gene expression. Moreover, AR is highly phosphorylated. Recently, we obtained evidence that phosphorylation of polyQ-AR by cyclin-dependent kinase 2 (CDK2) at serine 96 increases toxicity. This post-translational modification was enriched in neurons. Therefore, we hypothesized that phosphorylation of polyQ-AR at serine 96 modulates its function in response to activation of neuronal activity, a level of regulation altered in SBMA. We carried out a microarray analysis in resting and stimulated neurons in which AR was activated by androgens. Our preliminary results suggest that AR activation drives a differential gene expression program in stimulated neurons. In order to analyze the role of CDK2 and serine 96 phosphorylation in vivo, we deleted one or both CDK2 alleles in SBMA mice. Modulation of CDK2 expression reduced polyQ-AR phosphorylation at serine 96, decreased polyQ-AR accumulation in neurons, and attenuated disease manifestations in SBMA mice. Finally, we carried out an unbiased high-throughput screening of phosphatase and kinase inhibitors. As read-out, we analyzed polyQ-AR nuclear translocation induced by testosterone, in order to identify compounds to lower polyQ-AR toxicity. We isolated 6 phosphatase and 17 kinase inhibitors as modifiers of polyQ-AR nuclear shuttling. Among them, we found two compounds targeting Cdc25, a known activator of CDK2. Cdc25 modulation altered serine 96 phosphorylation, toxicity and transcriptional activity of polyQ-AR in cells. Our results support the idea that Cdc25 represents a potential candidate to develop new therapeutic strategies for SBMA. In summary, our findings show that serine 96 phosphorylation modifies AR physiological functions in neurons and polyQ-AR toxicity in SBMA.