Conserved cysteine-switches for redox sensing operate in the cyclin-dependent kinase inhibitor p21(CIP/KIP) protein family

Abstract

The cell cycle is a tightly regulated, dynamic process controlled by multiple checkpoints. When the prevention of cell cycle progression is needed, key effectors suchas members of the p21 (CIP/KIP) inhibit cyclin-dependent kinases (CDKs). It is accepted that p21 does not sense DNA damage and that stress signals affect p21indirectly. A plethora of DNA damaging events activate the tumor suppressor p53, which in turn transcriptionally activates p21, steeply changing its levels to reachCDK inhibition. The levels of p21 are also controlled by phosphorylation and ubiquitination events, which are relevant as they modulate p21 activity, localization,and stability. Intriguingly, here we report the first evidence of the direct control of p21 cell proliferation inhibition by DNA damaging signals. Specifically, we haveidentified a redox regulating mechanism that controls p21 capacity to reduce cell proliferation. Using the human p21 protein, we identified two cysteine-switchesthat independently regulate its cyclin-binding and linker (LH) modules respectively. Additionally, we provide a mechanistic explanation of how reactive cysteinesembedded in unstructured regions of intrinsically disordered proteins respond to ROS without the guidance of protein structure, contributing to a vastly unexploredarea of research. Cellular experiments utilizing p21KID mutants that disrupt disulfide-based switches demonstrate their impact on the capacity of p21 to inhibit cellcycle progression, thus highlighting the functional relevance of our findings. Furthermore, our investigation reveals that reactive cysteine residues are highlyconserved across the Kinase Inhibitory Domain (KID) sequences of p21 proteins from higher eukaryotes, and the p27 and p57 human paralogs. We propose that thepresence of conserved regulatory cysteines within the KIDs of p21 family members from multiple taxa provides those proteins with the capability for directly sensingROS, enabling the direct regulation of cyclin kinase activity by ROS levels.