The cell cycle
chromosome duplication
S
G1
G2
M
cell division
(cytokinesis)
nuclear division
(mitosis)
The cell-cycle control system
metaphaseanaphase
transition
G2/M
transition
M
G2
S
Start transition
G1
The heart of the cell cycle control system: Cyclin-dependent kinase (Cdk)
Cdk
Cyclin
Basic framework of the cell-cycle control system
Yeast:
Cln1, 2
Clb5, 6
Clb2
Vertebrate: Cyclin E
Cyclin A
Cyclin B
Order - Completeness - Irreversibility - Robustness - Regulatability
Cdk1 has hundreds of targets in the cell
Cdk1 phosphorylation sites often cluster in disordered loops and termini
Site A
Site B
Predicted disorder
Quipu
Phosphorylation is like Quipu
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
…how is the pattern generated, and how is it decoded?
Cyclin-specific Cdk1 substrates contain motifs that interact
with a docking site on the cyclin
Cyclin
Cdk
Active site
of Cdk
‘Hydrophobic patch’
on cyclin
S/T*-P-x-K/R
R-x-L
phosphorylation site
on substrate
motif
on substrate
The Cks1 subunit provides
an additional docking site for
pre-phosphorylated sites
substrate
Turning on S-Cdk in budding yeast
Start
The switch at G1/S: textbook dogma
Phosphorylation of Sic1 by Cdk1
triggers its destruction via the ubiquitin ligase SCFCdc4
Ub
Ub
Ub
Cdc4
E2
Ubiquitin
SCFCdc4
Rbx1
Cul1
Skp1
Sic1 is loaded with Cdk phosphorylation sites
that drive binding to the Cdc4 subunit of SCF
Cdc4
E2
Ubiquitin
SCFCdc4
Rbx1
Cul1
Skp1
Structure of an ideal phospho-peptide
binding to the Cdc4 subunit of SCF
The optimal Cdc4-binding site (‘CPD’): L/I-L/I/P-pT-P-[R/K]-[R/K]-[R/K]-[R/K]
disfavored
Nash et al. (2001) Nature 414:514-21.
Orlicky et al. (2003) Cell 112:243-256.
But the Cdc4-binding sites in Sic1 are pretty bad…why?
Optimal Cdc4-binding site (‘CPD’):
L/I-L/I/P-pT-P-[R/K]-[R/K]-[R/K]-[R/K]
Suboptimal Cdc4-binding sites in Sic1:
Nash et al. (2001) Nature 414:514-21.
Binding to SCFCdc4 requires six sites of phosphorylation on Sic1
Phospho-Sic1 binding
to Cdc4
Nash et al. (2001) Nature 414:514-21.
Six suboptimal sites somehow lead to high affinity?
Sic1
Cdc4
Cdc4
Cdc4
The model: distributive phosphorylation of Sic1 on six sites
leads to ultrasensitive Clb5-Cdk1 activation at G1/S
The model: distributive phosphorylation of Sic1 on six sites
leads to ultrasensitive Clb5-Cdk1 activation at G1/S
The first fly in the ointment:
a second phosphate binding site on Cdc4
Cdc4
Cyclin E
peptide
Hao et al. (2007) Mol. Cell 26:131-143.
Two or three good di-phosphodegrons in Sic1
7 mM
3 mM
Hao et al. (2007) Mol. Cell 26:131-143.
The latest complication:
processive phosphorylation of Sic1 in vitro
PhosphoSic1
(Phos-Tag gels)
Kõivomägi et al. Nature 480, 128 (2011).
Processive phosphorylation depends on the Cks1 subunit
substrate
Kõivomägi et al. Nature 480, 128 (2011).
Phosphorylation by Clb5-Cdk gets a boost from a docking site
Clb5
hydrophobic
patch
mutant
Clb5
RXL mut
substrate
Kõivomägi et al. Nature 480, 128 (2011).
Cascades of phosphorylation drive Sic1 degradation:
Cln-Cdk1 starts things out with the help of a docking site
P
P
P P
P
Kõivomägi et al. Nature 480, 128 (2011).
Clb5-Cdk1 takes it the rest of the way
P
P
P P
P P P
Kõivomägi et al. Nature 480, 128 (2011).
Cascades of phosphorylation events drive Sic1 degradation:
mystery solved!
P
P
P P
P P P
Kõivomägi et al. Nature 480, 128 (2011).
The switch at G1/S depends on positive feedback
The central problem of mitosis
?
one nucleus
containing
duplicated
chromosomes
two nuclei,
each with
one set of
chromosomes
The solution:
biorientation of sister chromatids
on a bipolar spindle
sister chromatid pair
kinetochore
spindle pole
A thing of beauty is a joy forever
Prophase
Prometaphase
Anaphase A
Anaphase B
Metaphase
Telophase
How sister chromatid pairs are attached:
the ‘search and capture’ model