Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function

Abstract

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle–regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

Rational optimization of a transcription factor activation domain inhibitor

“…1a,d and 7a–e ) was verified using high-resolution microscopy and quantitative reverse transcription PCR (qRT–PCR). HEK293T cells (ATCC; CRL-3216) and AR-eGFP Hela stable cells 69 (a gift from the M. Pennuto lab) were maintained in DMEM containing 4.5 g L –1 glucose supplemented with 10% (vol/vol) charcoal-stripped FBS and antibiotics. LNCaP95 cells were obtained from S. R. Plymate (University of Washington) and cultured in phenol-red-free RPMI supplemented with 10% (vol/vol) charcoal-stripped FBS (Gibco) and antibiotics.…”

C9orf72poly(PR) mediated neurodegeneration is associated with nucleolar stress

“…One day old adults were collected, anesthetized and external images of the eye were taken using LEICA M205C microscope equipped with a LEICA DFC450 camera. The eye phenotype of > 20 animals per genotype was scored in an unblinded manner (Casci et al ., 2019; Fortuna et al , 2021; Piol et al ., 2023; Ramesh et al ., 2020a; Ramesh et al ., 2020b) and the differences between the genotypes were assessed using One Way ANOVA followed by Tukey’s multiple comparison test.…”

“…The copyright holder for this preprint this version posted February 16, 2023. ; https://doi.org/10.1101/2023.02.16.528809 doi: bioRxiv preprint GMR GAL4 driver line was obtained from Bloomington Drosophila Stock Center. Transgenes and markers were inserted at defined locations in the genome using phiC31 mediated transgenesis by the company Bestgene Inc as previously reported (Casci et al, 2019;Kour et al, 2021;Piol et al, 2023;Ramesh et al, 2020a;Ramesh et al, 2020b).…”

“…Plasmids: To make the NES and NLS plasmids, cassettes were PCR amplified from the pUAST-Flag-DPR50-eGFP plasmids (Wen et al, 2014) markers were inserted at defined locations in the genome using phiC31 mediated transgenesis by the company Bestgene Inc as previously reported (Casci et al, 2019;Kour et al, 2021;Piol et al, 2023;Ramesh et al, 2020a;Ramesh et al, 2020b).…”

“…The eye phenotype of > 20 animals per genotype was scored in an unblinded manner (Casci et al, 2019;Fortuna et al, 2021;Piol et al, 2023;Ramesh et al, 2020a;Ramesh et al, 2020b) and the differences between the genotypes were assessed using One Way ANOVA followed by Tukey's multiple comparison test .…”