Abstract
The cell division cycle consists of a series of temporally ordered events. Cell cycle kinases and phosphatases provide key regulatory input, but how the correct substrate phosphorylation and dephosphorylation timing is achieved is incompletely understood. Here we identify a PxL substrate recognition motif that instructs dephosphorylation by the budding yeast Cdc14 phosphatase during mitotic exit. The PxL motif was prevalent in Cdc14-binding peptides enriched in a phage display screen of native disordered protein regions. PxL motif removal from the Cdc14 substrate Cbk1 delays its dephosphorylation, whereas addition of the motif advances dephosphorylation of otherwise late Cdc14 substrates. Crystal structures of Cdc14 bound to three PxL motif substrate peptides provide a molecular explanation for PxL motif recognition on the phosphatase surface. Our results illustrate the sophistication of phosphatase-substrate interactions and identify them as an important determinant of ordered cell cycle progression.
Highlights
The division of cells into two daughters is a process fundamental to all forms of life
The PxL motif peptide did not alter the rate at which Cdc[14] hydrolyzes para-nitrophenyl phosphate (p-NPP) in the absence of Net[1]. These results suggest that Net[1] shares its binding site on Cdc[14] with PxL motif substrates and has characteristics of a pseudosubstrate inhibitor of the Cdc[14] phosphatase
Whether a PxL motif allows phosphothreonine dephosphorylation by Cdc[14] in vivo remains to be tested. These results demonstrate that a PxL motif enhances peptide substrate dephosphorylation in vitro, especially of otherwise suboptimal target sites
Summary
The division of cells into two daughters is a process fundamental to all forms of life. The cell division cycle involves numerous cell biological processes, entailing regulated changes to chromatin, cytoskeleton, membranes and metabolism, to name but a few. This enormous regulatory complexity has been entrusted to a ‘master regulator’, the cyclindependent kinases (Cdk) and their counteracting phosphatases. Their concerted action steers the cell division cycle through biochemical switches in the form of reversible phosphorylation of serine and threonine residues. In particular, is a period of dynamic changes in the phosphorylation states of hundreds of proteins that direct them to a multitude of molecular fates.
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