Chem*3560
Lecture 10: Cyclins, cyclin kinases and cell division
The eukaryotic cell cycle
Actively growing mammalian cells divide
roughly every 24 hours, and follow a precise
sequence of events know as the cell cycle.
For simpler organisms like yeast, the cycle is
similar, but is compressed to as little as 2
hours.
The cell cycle was first described by
cytologists, who described two distinct
phases of the cycle, based on events that
were directly observable through a
microscope (Lehninger p.469).
M phase (Mitosis) starts by dissolution of
the nuclear envelope and condensation of
chromosomes, which are initially in the form
of replicate pairs. A series of dramatic and
very visible events results in the division of
the chromosome pairs , followed by orderly segregation carried out by the mitotic apparatus, so that
the daughter nuclei each receive one copy of each chromosome. Finally two nuclei re-form around the
chromosomes and the cell divides in two.
During S phase (Synthesis) the DNA content of the cell doubles. The length of S phase depends to
some extent on the size of the organism's genome
Since no obvious change is visible in the microscope, the intervals from M to S and from S to the next
M were labelled as Gap phases G1 and G2. At the biochemical level, the two gap phases are actually
filled with activities such as expression of proteins, metabolism and synthesis of other structural
components of the cell. Events in G2 are more specialized towards preparing for the coming mitosis.
Cells that have ceased dividing exit the regular cell cycle, and enter a state called G0.
G1 can be variable in duration, and is the phase that gets longer as cell growth slows down. A distinct
point exists towards the end of G1 called Start or the restriction point. Once cells pass start, they
proceed around the remaining stages of the cell cycle in a fairly orderly progression. However at
various critical stages, there a checkpoints that only allow a cell to progress if they have satsfactorily
completed a previous stage, so for example entry to G2 phase is stalled if DNA damage is detected,
allowing time for repair.
Specific protein kinases determine the timing of the cell cycle .
Progress through the cell cycle is monitored and controlled by set of protein kinases whose activity
rises and falls at different cycle phases. These operate through two distinct components, an
activating protein called a cyclin, and the protein kinase catalytic subunit called a cyclin dependent
kinase.
Cyclins were first identified as
proteins of unknown function whose
concentration varied as a
function of stage of the cell
cycle, and go through a period of
gradual build up followed by rapid
protein destruction. The graph
shows how cyclin B peaks at M
phase, and is then destroyed.
Genetic studies in fission yeast Schizosaccharomyces pombe provided the most important information
from a set of mutations called cell division compromised or cdc. Mutations are a powerful way to
study biological processes. Each mutant is observed so as to determine which biological function is
lost, and then biochemical studies can identify which proteins are involved. In the case of cell division
mutations, cell growth is arrested, so the mutations are derived as conditional or temperature
sensitive (ts) mutants. The mutant functions perfectly normally allowing cell growth at what is called
the permissive temperature , but becomes defective at the restrictive temperature , usually higher
because thermal instability is the triggering factor.
A yeast mutant first identified as cdc2 was found to be unable to progress through the cell cycle at
the restrictive temperature. The cdc2 gene expressed a protein first identified as p34 (a protein
recorded as 34 kDa on polyacrylamide gels. The p34 protein was found to function as a protein
kinase that was only active in combination with a protein expressed by the cdc13 gene , which
turned out to be a cyclin B homolog.
Since the cdc2 protein is a protein kinase that requires a cyclin for activation, it was called Cyclin
dependent kinase (CDK). Before long it was found that all eukaryotes express families of
homologs of both CDK and cyclins. Different combinations of cyclins and CDKs control passage
through different phases of the cell cycle.
In mammals:
Stage
Targets
Cyclin D-Cdk4 or 6
Progression through G1 phase
Cell proliferation regulators
Cyclin E-Cdk2
Entry to S phase
DNA Replication proteins
Cyclin A-Cdk2
Progression through S
Cyclin A-Cdk1
Progression through G2
Cyclin B, CDK phosphatase
Cyclin B-Cdk1
M-phase
Chromosomal proteins
Nuclear envelope proteins
There are several other proteins that are cyclins or CDKs based on
sequence similarities, but which have nothing to do with cell cycle
regulation, e.g. Cyclin H and Cdk7 are part of the RNA polymerase
II complex. Having found a regulatory mechanism that works, Nature
uses it in many different ways.
Cyclins are helix bundles, and share a common core of about 100
amino acids (blue/green) that make up the five helices that contact the
CDK. Outside this region (yellow/red) there can be considerable
variation, both in size and sequence. Cyclins can help determine
which substrates are targets for the cyclin-CDK combination (in
particular for yeast, where there is only one CDK for the whole cell
cycle, and only the cyclins vary for different
stages).
CDKs have an N-terminal β-sheet that forms
the ATP and substrate binding site,
followed by the PSTAIRE helix which
cyclins bind to. Hence PSTAIRE (these are
the single letter codes of the amino acids in the
helix) is a sequence signature to identify
CDKs. This is followed by the Thr 160 loop,
which blocks access to the substrate site in the
absence of bound cyclin. When cyclin binds,
the Thr 160 loop moves into contact with the
cyclin, and this realigns Glu 51 correctly for its
role in catalysis. Presence of cyclin
activates CDK by a factor of 10,000.
Finally, the C-terminal end of CDK forms
another helix bundle (Lehninger p.470).
CDK is itself regulated by phosphorylation.
Each CDK is subject to a combination modes of regulation:
by a specific pattern of phosphorylation within its own polypeptide;
the required association with the activating cyclin;
some CDKs can associate with factors called cyclin kinase inhibitors (CKI), which act by
masking the kinase catalytic site with an autoinhibitory loop. CKI must be eliminated or
inactivated for CDK to be available for catalysis.
Different phosphorylation sites in CDK have different consequences for activity. Presence of phosphate
on Tyr 15 occurs early, and keeps the CDK from being active prematurely. This phosphate is near the
ATP substrate binding site, so the negative charges of Tyr-PO4 2– and ATP repel. Phosphorylation of
Thr 160 helps shift the Thr loop out of the protein substrate binding site, and activates the CDK. This
phosphorylation may be carried out by the cyclin-CDK combination controlling the preceding phase of
the cycle.
The M-phase cyclin B-Cdk1 combination must reach peak activity very rapidly, because M is the
shortest of the four phases of the cell cycle. Cyclin B-Cdk1 phosphorylates and activates the
phosphatase that removes its
inhibitory Tyr-PO4 2-. This increases the
activity of the CDK, which increases the
activity of the phosphatase, a positive
feedback loop. Positive feedback can be
exploited to bring about very rapid
transitions between two states
(Lehninger, Fig 13-13, p.471.)
Cyclin accumulates gradually but is destroyed rapidly
Cyclin accumulates
as its polypeptide is
expressed (which
may be controlled
by the cyclin-CDK
of a previous phase
or by external
growth factors). A
common feature of
Cyclin-CDKs is that
they enable entry
or progress
through a stage of
the cell cycle, but to
complete passage
through and
terminate that
phase, the cyclin
must be
destroyed.
Targeted destruction of a protein is mediated by tagging it with a small protein called ubiquitin (ubique
= Latin for everywhere). An enzyme called ubiquitin ligase links the C-terminal carboxylate of
ubiquitin to a convenient lysine side chain of the doomed protein. Once a protein is tagged, it is picked
up by a cellular complex called the proteasome , which uses ATP hydrolysis energy to unfold the
polypeptide and feed it to a cluster of proteases.
The essence of this mechanism is the ability of the ubiquitin ligase to identify which proteins are fated for
destruction, so there are many different ubiquitin ligases in cells. Cyclin identifies itself as a target
because it contains a sequence -Arg-Thr-Ala-Leu-Gly-Asp-Ile-Gly-Asn- called the cyclin destruction
box. Each cyclin-CDK also activates an adaptor protein that is specific for linking that cyclin to its
ubiquitin ligase, e.g. so that cyclin A can be destroyed while leaving cyclin B intact.
Cyclin Kinase Inhibitors (CKI) keep cyclin kinase inactive if there's a
problem
CKI may be induced as a result of a cell not meeting the requirements of a cell cycle checkpoint.
DNA damage triggers activity of a factor called p53 (protein of 53 kDa), which controls transcription
of many genes, including CKI p21. CKI p21 binds to and inhibits Cyclin E-Cdk2, halting progress
into S phase until the DNA damage is repaired. If the damage can't be repaired, p53 goes on to trigger
a process called apoptosis, or programmed cell death.