Search

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinases (CDKs) are the major cell-cycle regulators that phosphorylate hundreds of substrates, controlling the onset of S phase and M phase [ 1–3 ]. However, the patterns of substrate phosphorylation increase are not uniform, as different substrates become phosphorylated at different times as cells proceed through the cell cycle [ 4 , 5 ]. In fission yeast, the correct ordering of CDK substrate phosphorylation can be established by the activity of a single mitotic cyclin-CDK complex [ 6 , 7 ]. Here, we investigate the substrate-docking region, the hydrophobic patch, on the fission yeast mitotic cyclin Cdc13 as a potential mechanism to correctly order CDK substrate phosphorylation. We show that the hydrophobic patch targets Cdc13 to the yeast centrosome equivalent, the spindle pole body (SPB), and disruption of this motif prevents both centrosomal localization of Cdc13 and the onset of mitosis but does not prevent S phase. CDK phosphorylation in mitosis is compromised for approximately half of all mitotic CDK substrates, with substrates affected generally being those that require the highest levels of CDK activity to become phosphorylated and those that are located at the SPB. Our experiments suggest that the hydrophobic patch of mitotic cyclins contributes to CDK substrate selection by directing the localization of Cdc13-CDK to centrosomes and that this localization of CDK contributes to the CDK substrate phosphorylation necessary to ensure proper entry into mitosis. Finally, we show that mutation of the hydrophobic patch prevents cyclin B1 localization to centrosomes in human cells, suggesting that this mechanism of cyclin-CDK spatial regulation may be conserved across eukaryotes. • The hydrophobic patch of human and yeast cyclin B directs it to the centrosome • Loss of the yeast cyclin B hydrophobic patch allows S phase but prevents mitosis • Compartmentalized mitotic CDK phosphorylation relies on the hydrophobic patch Cyclin-CDK accumulates on mitotic microtubule-organizing centers. Basu et al. find that the substrate-docking region of cyclin B, the hydrophobic patch, governs this localization in both human cells and fission yeast. Without the cyclin B hydrophobic patch, CDK phosphorylation is impaired at mitosis but only in certain subcellular compartments. [ABSTRACT FROM AUTHOR]