Creating Switch-Like Responses
Recent work in our lab into mechanisms by which phosphorylation events can be utilized to regulate complex biological events has shown that multi-site phosphorylation of the CDK inhibitor, Sic1, sets a threshold for the onset of DNA replication and creates an ultrasensitive biological switch. (Reference: Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F., Mendenhall, M.D., Sicher, F., Pawson, T., and Tyers, M. (2001) Nature 414, 516-523.)
Creating Switch-Like Responses
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SCF complexes are multi-subunit E3 protein ubiquitin ligases, comprised of a scaffolding protein (Cullin), which interacts with an E2 enzyme, a RING-H2 protein, and an adaptor, Skp1. Skp1 in turn recognizes the N-terminal F-box of a targeting subunit which binds through a variable C-terminal domain, often composed of WD40 repeats or leucine rich repeats, to the substrate for ubiquitination. The binding of the substrate to the C-terminus of the F-box protein is frequently dependent on the phosphorylation of the target on serine or threonine, and the ubiquitination and subsequent degradation of such a protein is therefore regulated by its serine/threonine phosphorylation. We have recently analyzed the phospho-dependent degradation of the Sic1 cyclin-dependent kinase (CDK) inhibitor in the yeast S. cerevisiae, using a combination of biochemical and genetic tools. Sic1 is a specific inhibitor of the S phase CDK, composed of the Cdc28 kinase associated with Clb-type cyclins. In the G1 phase of the yeast cell cycle, the G1 CDK (Cdc28 associated with Cln-type cyclins) phosphorylates Sic1 at 9 Ser/Thr-Pro motifs, and the phosphorylated form of Sic1 is then recognized by the WD40 repeat domain of an F box protein termed Cdc4, which is part of a larger SCF E3 ubiquitin ligase

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The recruitment of phospho-Sic1 to the SCFCdc4 complex leads to its ubiquitination and destruction, and this relieves the inhibition of the S phase Cdc28-Clb CDK, allowing the initiation of DNA replication. Thus Sic1 is the key substrate of the G1 CDK, because Sic1 phosphorylation, and consequent recognition by the Cdc4 F-box protein, acts as a switch to promote transition for the G1 to the S phase of the cell cycle. A mutant form of Sic1 that lacks all Cdc28-Cln phosphorylation sites is stable, and therefore toxic because cells cannot replicate their DNA.

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In trying to understand how Sic1 serine/threonine phosphorylation mediates its recognition by the Cdc4 WD40 domain, we found that no single Sic1 phosphorylation site is sufficient to mediate stable Cdc4-binding, Sic1 ubiquitination, or progresion into S phase. Indeed, Sic1 must be phosphorylated on at least 6 sites to associate with Cdc4 and allow passage through G1. Although the physiological Sic1 phosphorylation sites do not bind Cdc4 with high affinity, we observed that phosphopeptides from human Cyclin E1 or yeast Gcn4 (a transcriptional regulator that is also targeted by the SCFCdc4 complex), bound to the Cdc4 WD40 domain with a Kd of ~1 mM, and a Hill coefficient of 1, suggestive of a single class of binding site. Analysis of peptide SPOTS arrays yielded a consensus binding motif for the Cdc4 WD40 repeats of Ile/Leu-Ile/Leu/Pro-pThr-Pro, with a selection against basic residues at the +2 to +5 positions. These results explain why no single Sic1 phosphorylation site binds well to Cdc4, since these sites are all sub-optimal with respect to the preferred consensus. We found that insertion of the optimal recognition motif into a Sic1 variant lacking all endogenous phosphorylation sites was sufficient for stable binding to Cdc4, and for elimination of the Sic1 CDK inhibitor. However, this variant form of Sic1 was not able to fully replace the endogenous protein, as it was degraded prematurely, leading to precocious entry into S phase, and genome instability likely due to premature firing of DNA replication origins. These and related results have suggested that the Cdc4 F-box protein has a single phosphothreonine-binding pocket, containing three essential arginine residues, which binds in equilibrium to multiple low affinity sites on Sic1. This requirement for multisite phosphorylation of Sic1 establishes a threshold of G1 CDK activity which must be surpassed for stable Cdc4 binding, and thus for Sic1 degradation and activation of the S phase CDK. Thus, Sic1 multisite phosphorylation and consequent Cdc4 recognition in essence provide a timing device for transit through the G1 phase of the cell cycle, and guard against adventitious entry into S phase in response to low level Cdc28-Cln activity, as well as providing for a switch-like degradation of Sic1 once the appropriate level of G1 CDK activity has been reached.

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References

Pawson T., Raina M., Nash P. Interaction Domains: From Simple Binding Events to Complex Cellular Behaviour (in Press) FEBS Letts.