SUBSTRATE RECOGNITION: RXL, SLiMs, Cks1/2, DOCKING

What determines CDK substrate specificity?

Short recognition sequences and docking motifs on substrates

What is an RXL motif?

A conserved short linear motif that binds directly to cyclins

Where is the RXL-binding site found?

On cyclins

How do RXL motifs influence CDK activity?

They recruit specific substrates to cyclin–CDK complexes

Why is the RXL motif important?

It increases affinity and catalytic efficiency of phosphorylation

Do all cyclins recognise the same RXL motif?

No

How does cyclin identity change substrate choice?

Distinct cyclins expose different docking surfaces

What experiment revealed RXL motifs?

Adams study identifying conserved substrate docking sequences

How was binding specificity measured?

Competition between substrates for cyclin docking

Why is competition important?

Multiple substrates with different affinities fight for cyclin access

What regulates which substrate wins?

Relative abundance + affinity + motif strength

What are SLiMs?

Short Linear Motifs — minimal amino acid motifs enabling protein–protein interactions

Where are SLiMs found?

On substrates and adaptor proteins interacting with kinases and regulators

Why are SLiMs powerful?

They enable multiple weak interactions that collectively determine binding

What type of interaction do SLiMs provide?

Low-affinity

How can multiple SLiMs cooperate?

A protein with several SLiMs can dock to multiple components simultaneously

How do SLiMs integrate signalling?

They allow substrate recognition to reflect phosphorylation state

What happens if SLiMs mutate?

Substrates fail to dock

What is Cks1?

A conserved accessory protein that binds phospho-amino acids on substrates

What does Cks1 recognise?

Phosphorylated threonine/serine motifs on CDK substrates

How does Cks1 aid phosphorylation?

It binds priming phosphates and repositions substrate on CDK for additional phosphorylation

What does Cks1 form a complex with?

Cyclin + CDK to form a trimeric complex

What are the three docking interfaces in a CDK–cyclin–Cks1 complex?

Cyclin RXL site

Why are three docking sites beneficial?

They coordinate priming

What is Cks2?

A related cyclin-dependent kinase subunit that acts in cis with CDK2

What happens in Cks1-mutant proteins?

They cannot recognise priming phospho-sites

What does Cks1 mutation demonstrate?

Phospho-recognition is essential for ordered phosphorylation cascades

What technique separates phosphorylated protein forms?

Phos-tag SDS-PAGE

Why does Phos-tag SDS-PAGE help?

It separates proteins by number of phosphates to visualise multi-site events

How does substrate architecture affect phosphorylation?

Spacing between sites determines whether secondary sites can be phosphorylated

How many residues apart are priming + secondary sites for Cks1 action?

14–18 residues between phospho-sites enables sequential phosphorylation

Why is residue spacing important?

Allows Cks1 to bind one phosphate and position the next target correctly

How do different substrates create different phosphorylation signatures?

Variations in motif spacing

What happens if priming site is absent?

Secondary phosphorylation fails

Why do CDKs phosphorylate some sites first?

Priming sites have optimal sequence context for faster catalysis

What is a suboptimal site?

A secondary site only phosphorylated after priming site modification

How do RXL and Cks1 work together?

RXL motif docks substrate; Cks1 anchors phospho-site to reorient for multisite phosphorylation

Why do cyclins not share all substrates?

Each cyclin exposes docking grooves best suited to a subset of SLiMs

Give one clinical application of substrate specificity

Design of drugs that target only specific cyclins (e.g.

How do macrocyclic inhibitors exploit docking?

They mimic binding motifs and selectively occupy RXL pockets on cyclins A/B

Why is cyclin-selective inhibition valuable?

Allows blocking specific cell cycle transitions without shutting all CDKs down

What happens if CDKs lacked docking sites?

Phosphorylation would depend only on catalytic encounter

Why is modular binding evolutionarily favoured?

It expands signalling control without needing more enzymes

How does substrate docking enable feedback?

Phosphorylation alters docking affinity allowing sequential timing

Why can one CDK phosphorylate hundreds of substrates?

Docking interfaces adapt catalytic core to diverse targets

How does docking support order of events?

High-affinity substrates phosphorylate earlier; low-affinity later

Why are multi-layer interactions better than single binding?

Stronger specificity without irreversible binding interactions

How do SLiMs contribute to signalling network complexity?

Tiny motif changes drastically rewire pathway outcomes

Why can two substrates respond differently to same CDK?

Different motifs dictate binding kinetics and turnover rates

Why are docking interactions reversible?

Ensures CDKs move rapidly between many substrates

How do scaffolds affect docking?

Scaffold proteins bring kinases and substrates close to increase efficiency

What is the advantage of weak binding?

Allows fast turnover and dynamic cycling without sequestration

Why is substrate docking not just passive?

CDK–cyclin complexes are tuned to recognise motifs and generate patterned phosphorylation

How does motif diversity help disease treatment?

Cancer-causing motif changes can be targeted with selective inhibitors