SIC1 & MULTISITE PHOSPHORYLATION

What is Sic1?

A CDK inhibitor (CKI) that prevents S phase entry in budding yeast

Which kinase does Sic1 inhibit?

CDK1–Clb complexes

What phase does Sic1 regulate?

G1 to S transition

Why must Sic1 be degraded?

To allow CDK1–Clb activation and initiation of DNA replication

How is Sic1 destroyed?

By phosphorylation followed by ubiquitination and proteasomal degradation

How many phosphorylation events are required to target Sic1?

Multiple — Sic1 undergoes multisite phosphorylation

What does multisite phosphorylation mean?

Phosphorylation at several distinct residues is required to trigger a response

Why does Sic1 require multiple phosphates?

Prevents accidental degradation from a single spurious phosphorylation

What phosphorylates Sic1?

Cyclin-dependent kinases (primarily CDK1–Clb complexes)

What is the initial site called?

The priming phosphorylation site

What is a priming site?

A site that must be phosphorylated first to promote binding to Cks1

Why are priming sites important?

They change Sic1 conformation and recruit Cks1 for additional phosphorylation

What is a secondary site?

A suboptimal phosphorylation site dependent on a priming phosphorylation

What determines whether secondary sites are phosphorylated?

Their spacing relative to the priming site (14–18 residues)

Why 14–18 residues?

Allows Cks1 to bind the primed phosphate and present the next site to the CDK

Which accessory protein promotes Sic1 multisite phosphorylation?

Cks1

What does Cks1 recognise?

Phospho-serine/threonine residues already on Sic1

How does Cks1 affect phosphorylation speed?

It dramatically increases the rate of secondary site phosphorylation

What happens when Cks1 is mutated?

Cks1mut cannot bind phospho-sites and multisite phosphorylation collapses

What is the consequence of lost multisite phosphorylation?

Sic1 remains stable

What happens to Sic1 mutants lacking key phosphorylation sites?

They are not degraded and cells arrest in G1

What is the phenotype of active Sic1?

Strong G1 arrest

What is the phenotype of inactive Sic1?

Cells continue cell cycle normally & pass START

What does Sic1 level reflect?

CDK1 activity — as CDK1 rises

What is Sic1’s role in commitment?

It provides a threshold; only high sustained CDK1 activity overcomes it

Why is Sic1 considered a “timer”?

It requires cumulative phosphorylation events spread over time

What happens if CDK activity fluctuates?

Threshold is not met and Sic1 persists

How does Sic1 enforce directionality?

Once degraded

What concept does Sic1 illustrate?

Ultrasensitivity — a highly switch-like response to gradual kinase increases

Why do cells evolve multisite control?

To ensure noise-resistant

Which ubiquitin ligase targets Sic1?

SCF complex (Skp/Cullin/F-box)

What does SCF recognise?

Phosphorylated Sic1

Why does SCF require phosphorylation?

Ensures only fully tagged proteins are degraded

What happens if SCF components are absent?

Sic1 accumulates and cells arrest in G1

Which cyclin drives Sic1 degradation most strongly?

Clb5/6 CDK1 complexes at the G1/S transition

Why is Clb5/6 activation initially blocked?

Sic1 binds them tightly to prevent premature replication

How is inhibition released?

CDK1–Cln phosphorylation gradually inactivates Sic1

What type of feedback loop removes Sic1?

Positive feedback — CDK1 activity destroys Sic1

What transition does Sic1 destruction mark?

Commitment to S-phase and initiation of DNA replication

Which process begins once Sic1 is gone?

Origin firing and replication licensing in S phase

How does Sic1 protect genome stability?

Prevents premature or accidental replication

What experiment demonstrated multisite spacing importance?

Mutant Sic1 variants altered spacing and stalled phosphorylation

What tool visualises Sic1 phosphorylation patterns?

Phos-tag SDS-PAGE separating differently phosphorylated forms

What determines the order of phosphate addition?

Sequence context + docking affinity + presence of Cks1

How does Sic1 relate to mammalian CKIs?

It is analogous to p27/p21 which restrain CDK2 until mitogen signaling

Why is Sic1 critical to START fidelity?

Ensures only fully prepared cells enter S phase

How does Sic1 tie metabolism to division?

Cyclin/CDK levels reflect nutrient conditions before Sic1 removal

What would happen if Sic1 were degraded by accident?

Cells would replicate DNA without sufficient mass or nutrients

What happens to Sic1 after degradation?

Proteasome breaks it down into peptides; it must be resynthesised for the next cycle

When is Sic1 resynthesised?

Late mitosis to early G1

Why is Sic1 resynthesis important?

Ensures cells do not re-enter replication immediately after finishing mitosis

What determines Sic1 half-life?

Balance of phosphorylation

How does Sic1 illustrate modular control?

Integration of cycling

What does Sic1 teach about cell cycle networks?

Decision points depend on accumulation of many small molecular events