Abstract
The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high cyclin-dependent kinases (CDK) inhibitor (CKI) levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK (Cln1,2-Cdk1) and S-phase CDK (Clb5,6-Cdk1) complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double-negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex. In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.
Highlights
The major cell cycle transitions are triggered by phosphorylation or dephosphorylation of the targets of cyclin-dependent kinases (CDKs)(Morgan, 2007)
Cln-Cdk1 complexes alone were not able to cause the degradation of Sic1 in vivo
To quantitatively measure this inhibition we performed an in vitro phosphorylation and inhibition assay with purified Sic1 and cyclin-Cdk1 complexes
Summary
The major cell cycle transitions are triggered by phosphorylation or dephosphorylation of the targets of cyclin-dependent kinases (CDKs)(Morgan, 2007). The CKI acting at the G1/S transition, the protein Sic, is simultaneously both an inhibitor and a substrate of Cdk. At the onset of S-phase Sic is phosphorylated by Cdk, thereby generating two “diphosphodegrons” containing two properly spaced phosphates that are recognized by the Cdc4-SCF ubiquitin ligase (Hao et al, 2007). The signal for the degradation was proposed to be the multisite phosphorylation of Sic by G1-specific Cln1,2-Cdk complexes. This is possible because, in contrast to S- and M-phase specific Clb-Cdk1s, Cln-Cdk1s are not inhibited by Sic (Verma et al, 1997a,b; Nash et al, 2001). The destruction of Sic was shown to release the Clb5,6-Cdk complexes required to initiate DNA replication (Schwob and Nasmyth, 1993; Schwob et al, 1994; Schneider et al, 1996)
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