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General Phosphorylation Characteristics
A simplified understanding of the regulation of Sic1 degradation involves the phosphorylation of multiple CDK sites, which consist of optimal and suboptimal consensus phosphorylation motifs. Recent studies conducted by Koivomagi et al. have revealed the many intricacies of the multi-phosphorylation reaction between the cyclin-CDK complex and the Sic1 protein. These studies unveil the important characteristics of the Sic1 CDK phosphorylation sites, which include priming sites, binding sites, degron pairs, distancing of phosphorylation sites, and relative site location. In addition, the studies also emphasize the influence of other factors on Sic1 phosphorylation, including the Cks1 phospho-binding pocket, cyclin docking motifs, and Cdk1 active site specificity. All of these mechanisms contribute to the dynamics of the sequence of events leading to Sic1 degradation and initiation of S-phase.

Cks1 Role
Apart from being an often-overlooked component of the Cdk1 cyclin complex, Cks1 is critical for Sic1 multi-phosphorylation and degradation. The phospho-binding pocket of Cks1 is capable of binding independently to phosphorylated CDK sites on Sic1. Additionally, the binding affinity of Cks1 for phosphoserines is extremely weak, essentially making Cks1 binding dependent on the presence of phosphothreonines only. Thus, in Sic1 mutants with one Cdk1 phophorylation site or only phosphoserines present, Cks1 is unable to properly bind to the substrate and promote Sic1 multi-phosphorylation. This provides a strong argument for a processive phosphorylation mechanism instead of the previous theory of a random distributive phosphorylation model. In addition to requiring threonine, Cks1 binding to Sic1 can be enhanced with the introduction of a proline residue at the -2 position relative to the threonine residue.

Site positioning of Cks1-Dependent Phosphorylation
Sic1 is a molecule with disordered regions, which aids in the manipulation of phosphorylation site distances. For the following findings, Koivomagi et al. utilized a Sic1 construct with a T33 optimal consensus motif, acting as the primary phosphorylation site, and a suboptimal motif, acting as a secondary site.

When limiting observations to only double-phosphorylated Sic1 constructs, a two-step phosphorylation process was observed, where the first step was primary site phosphorylation. However, the secondary site must be located towards the C-terminus of the protein relative to the primary site for phosphorylation to occur. Secondary site phosphorylation is also sensitive to positioning. Peak phosphorylation rates were found between the +12 to +16 amino acid distances, with a distinct increase around the +10 to +12 range and gradual decrease across the +20-30 range. Interestingly, the introduction of the -2 proline residue enhances phosphorylation both in vitro by expanding the peak phosphorylation range, but does not increase phosphorylation activity at distances less than +10. This expansion of the peak phosphorylation range could possibly be attributed to enhanced binding of the priming site to Cks1.

A simple Sic1 construct containing 5 phosphorylation residues (1 priming site and two phosphodegron pairs) revealed that any slight movement of the priming site can have significant effects on cell cycle progression. The priming site should be within the +12 to +16 range of both residues in the phosphodegron pair to maximize phosphorylation.

Directionality of the Clb5 and Cln2 Phosphorylation
Sic1 phosphorylation is initiated by the G1 cyclins, Cln1,2, and then completed by S-phase cyclins, Clb5,6 (Fig. 1). The distant docking motifs of the cyclin participate in Sic1 phosphorylation dynaimcis. S-phase cyclins use RXL docking while G1 cyclins use LLPP docking. Sic1 phosphorylation increases when the RXL motif of Clb5 is +16 to +20 positions relative to the optimal CDK motif. RXL positioning located N-terminal to the motif led to negligible amounts of phosphorylation. In contrast, moving the LLPP motif away from the priming site increases Cln2 phosphorylation, regardless of directionality.

Phosphorylation Processivity
Cks1-dependent multi-phosphorylation occurs in a processive or semi-processive manner, evidenced by the lack of intermediate Sic1 phosphorylation states in normal cells. This processivity is also dependent on the presence of the cyclin docking site since increasing the numbers of mutations in this site decreases the net phosphorylation rate. Processive phosphorylation has two plausible mechanisms where a single binding event leads to the phosphorylation of two or more sites. The first mechanism proposes that, without dissociating from the enzyme complex, the primary site is phosphorylated and immediately shifted from the active site to the Cks1 binding pocket to allow for the additional phosphorylation of other CDK sites. The second mechanism proposes that the phosphorylated primary site binds to another location and is continuously bound while other CDK sites bind to the active site in a sequential manner for multi-phosphorylation. Simulations predict that the probability of a second phosphorylation event after the first, without dissociation, is 40% and 20-40% for the first and second mechanism, respectively.

Sic1 is targeted for degradation by SCF (Cdc4), which recognizes Sic1 phosphodegron pairs, which are closely positioned paired phosphorylation residues that each have strong affinities for Cdc4. In a Sic1 construct with the S69/S76/S80 cluster, processive phosphorylation of these phosphodegron pairs are reliant on Cdk1 sites. Clb5 processivity is dependent on the T5 and T33 sites, while Cln2 processivity is dependent on T5. Reintroduction of various residues led to the discovery of the T33 residue serving as a docking site for the T45/T48 phosphodegron pair, which is able to promote Sic1 degradation to a certain extent in the absence of other phosphodegron pairs.

Proposed In vivo Mechanism
The following is a proposed mechanism by Koivomagi et al. of the in vivo cascade to promote Sic1 phosphorylation and degradation.

In late G1, Sic1 is inhibiting the Clb5-Cdk1 complex, simultaneously inhibiting its own degradation. The phosphorylation cascade proceeds by Cln2-Cdk1 phosphorylation of the T5 priming site. Following this, the T33, T45, and S76 residues are phosphorylated by Cln2-Cdk1, but no degron pairs are phosphorylated. However, these phosphorylated sites enhance Clb5-Cdk1 docking, leading to increased Sic1 phosphorylation at suboptimal sites and a positive feedback loop where Clb5-Cdk1 inhibition is continually decreased while Sic1 degradation is increased.