Fulzele, A. | bioRxiv Science

bioRxiv2018

Deacetylation of catalytic lysine in CDK1 is essential for Cyclin-B binding and cell cycle

Cyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.

cell biology↗

bioRxiv2018

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

Many protein homeostasis stressors induce the formation of membraneless cytoplasmic stress granules (SGs) that contain large assemblies of repressed mRNAs and associated RNA binding proteins. Similar stressors have been shown to globally alter the function of the ubiquitin proteasome system (UPS) resulting in the accumulation of ubiquitylated proteins. Previous studies have demonstrated that ubiquitin and specific UPS components co-localize with SGs and that reducing the abundance or activity of ubiquitin pathway proteins can inhibit SG formation. These studies suggest that SG dynamics and composition may be regulated by ubiquitylation of SG resident proteins. Using ubiquitin-specific proteomic approaches, we demonstrate that many proteins, including some SG proteins are dynamically ubiquitylated upon SG-inducing sodium arsenite treatment. We utilized potent and selective inhibitors of the ubiquitin activating enzyme (UAE) or the NEDD8 activating enzyme (NAE) to directly test if active protein ubiquitylation or neddylation was required for SG dynamics. Using ubiquitin-site specific proteomics, we establish that UAE inhibition results in the rapid loss of nearly all protein ubiquitylation regardless of ubiquitin chain type. Addition of UAE or NAE inhibitors to cells did not alter arsenite-induced SG formation or dissolution. While we confirmed that ubiquitin co-localizes with both sodium arsenite and thapsigargin-induced SGs, antibodies that recognize all forms of ubiquitin more strongly co-localize with SGs compared to antibodies that preferentially recognize polyubiquitin or specific polyubiquitin-linkages. Interestingly, ubiquitin itself co-localizes with SGs in a UAE independent manner suggesting that the ubiquitin present within SGs is likely unconjugated ubiquitin. Our findings clearly demonstrate that active protein ubiquitylation or neddylation is not required for SG dynamics. These results suggest that ubiquitin-binding SG proteins may recruit free ubiquitin into SGs to modulate SG protein interactions.

cell biology↗