M267 Lectures 1-5

M267 Lectures 1-5
Harvey Herschman
The Cell Cycle (2006)
Tissues in mammals grow in two ways: (i) by increasing in cell size, as is the case for the size of the
rooster comb in response to androgens (hypertrophy), or (ii) by increasing in cell number, as
happens to the size of a deer's antlers in spring (hyperplasia).
Within the body, we have three types of cells with respect to the cell cycle: (i) cells that are
continuously dividing, such as intestinal crypt cells and erythroid stem cells, (ii) post-mitotic cells
such as neurons and the nuclei of fused muscle cells that never divide and (iii) those cells that can be
induced to divide (hepatocytes in response to hepatectomy, antigen-responsive lymphocytes,
mammary cells during pregnancy, glial cells after wounding). Tissues have all three types of cells.
The number of cell divisions per minute per individual is 20x10 6 , just for renewing body tissues (not
including spermatozoa, antigen responses, etc). There are two “musts” to cell division: In order to
divide cells must (i) replicate their DNA and (ii) segregate the DNA to the progeny cells in mitosis.
Mitosis is the most easily observable cell cycle microscopic phenomenon. Mitotic cells appear as
round, shiny balls. The first concept of a "cell cycle" was M-------M. Lots of articles appeared
about this briefest of cell cycle periods. There are no distinguishing morphological features between
mitoses, i.e., during “interphase”, in the light microscope. The cell cycle was thought to be a
continuum of events; i.e., no qualitatively distinct events were thought to occur between mitoses.
WHAT HAPPENS BETWEEN THE TWO MITOSES; i.e. DURING INTERPHASE? Is this
time really occupied by a continuum or are there discrete phenomena, both qualitatively and
temporally, that occur in the cycle? We will discuss (i) the sequential cyclic events between mitoses
and (ii) the regulation of the process.
The first cell cycle experiment [Howard and Pelc, Exptl Cell Res 51: 2: 178 (1951)]. (1) grow
root tips of vicia faba, do a pulse of 32 P, do
INTERPHASE
DNA Synthesis Gap
M M
Gap DNA Synthesis Gap
M M
Gap DNA Synthesis Gap
M M
S
G 1
G 2
radioautographs, and examine incorporation of
isotope into the nucleus. M takes one hour, as
determined by microscopy. They found no
incorporation into the M phase cells, but did see
incorporation into some, but not all, interphase cells.
(2) Grow a series of the root tips in colchicine, which
blocks mitosis, plus 32 P. Label for various times, (2, 4,
6 hours) and examine mitotic figures for label.
Mitotic cells accumulate, because of the presence of
the drug. For the first 6 hours or so no mitotic cells
are labeled! The data imply that there exists a period
prior to cell division in which no DNA synthesis
occurs. After six hours, some mitotic cells are
labeled; they must have synthesized DNA, gone
though a period where no DNA synthesis occurred,
then entered mitosis.
(3) If 32 P is removed after 6-8 hours, some of the cells
go into mitosis much later, and are not labeled. This
implies there is a period between mitosis and the
beginning of DNA synthesis when no DNA synthesis
occurs.
1

Let’s do a more modern experiment; pulse-label cultured cells with radioactive thymidine, a DNA
precursor. Then wash, put the cells in colchicine, and do autoradiographs after this pulse label.
(4) If we plot the
data obtained for
labeled mitoses in the
presence of colchicine
we get these results:
:
G 2
M
S
G 1
G 2
S
Total number of
mitotic figures
Total number of labeled
mitotic figures
M G 1 S G 2 M
Putting these data together, we get a STANDARD CELL CYCLE
REPRESENTATION. From other data, which I won’t describe, we
can make several generalizations: (i) the S, G2 and M periods of the
cycle are pretty constant. (ii) Differences in cycle time are usually
due to alterations in G1. (iiii) in non-dividing tissues most cells are
frozen in G1, or a distinct cell cycle phase, called "Go".
AS A RESULT OF THIS EXPERIMENT, WE NOW KNOW INITIATION AND
TERMINATION OF DNA SYNTHESIS ARE DISCRETE CYCLE EVENTS.
DETERMINING THE CELL CYCLE PARAMETERS; G1, S, G2 and M:
Number of
labelled
mitoses
G 2 S G 2 + M + G 1
Time
The second procedure for determining cell cycle
parameters is the use of flow micro-fluorimetry
(FMF). One stains cells with a dye that causes DNA
to become fluorescent. The amount of dye taken up
by any cell is proportional to the DNA content of that
cell. One then determines the amount of binding of
dye per cell, using a laser. Individual cells go through
the laser beam, and the data are analyzed by a
computer.
The classic method is known as labeled
mitoses. One counts labeled mitoses after a
pulse with a DNA label such as [ 3 H]thymidine.
The generation time, GT = G1 + S
+ G2 + M. M can be determined as the
percent of cells that appear as mitotic figures
in a randomly growing population of cells.
The two major events common to all eukaryotic cell cycles are the replication of
DNA (i.e, the replication of chromosomes during S phase) and chromosome
segregation during mitosis (i.e, the segregation of DNA to the sibling cells).
If extended, the human genome has seven feet of DNA. Faithfully replicating all this only once per
cycle, and accurately packaging it for condensation and separation in individual chromosomes is a
major problem. Moreover, this DNA has to be replicated and packaged during a specific portion of
the cell cycle (S), and segregated exactly to two sibling cells during another cell cycle portion (M).
2

A number of eukaryotic systems are used to study the cell cycle, including fly embryos, clam eggs,
sea urchin eggs, yeast --where genetics can be brought into play--, and mammalian cells in culture.
While I will be emphasizing mammalian somatic cells, I will draw on data from the other systems –
particularly yeast. There are differences in localization, activity, and function of cell cycle
components in these various systems -- not all cell cycles in all organisms are alike. However, data
have shown that the cell cycle is so similar from organism to organism in eucaryotes that many
basic principles can be extrapolated among all eucaryotes.
Tissues in the organs of a mammal (like us!) are mixed populations of Go, randomly spaced G1, S,
and G2 cells, with some cells in cycle, and others having exited the cycle. We want to study the
biochemistry of the cell division process; to identify temporal points in the cell cycle. In most
somatic tissues the greatest portion of cells are not dividing. We want to use populations of
homogeneous, growing (i.e., dividing) cells. In the lab, to study the cell cycle of mammalian cells,
we study mostly immortal cell culture cell lines derived from single cells -- BHK, CHO, 3T3, etc.
We will talk about cell cycle mutants, and how these studies have contributed to our understanding
of the cell cycle.
SYNCHRONIZING CELLS: To study most of the major landmarks in the cell cycle we need to
have homogeneous populations of cells traversing the cycle at the same time; i.e., we must have
synchronous cells, whether we are studying yeast, fly, mouse or human cell cycles. What we do to
study many of the events in the cycle is to (1) synchronize a randomly dividing population of cells
and (2) study a given parameter as a function of cell cycle position, after release from the synchrony
point. There are three major methods used to synchronize cultured cells.
[1]. WE CAN SYNCHRONIZE CELLS BY BLOCKING AT SOME POINT IN THE
CELL CYCLE AND ALLOWING CELLS TO ACCUMULATE AT THE BLOCK.
(1) Cells can be starved for isoleucine, for PO4, or for serum. These starvation paradigms will
block many cell lines in G1 [Ley and Toby, J. Cell Biol 47: 453 (1970), Xeros, Nature 194:
682 (1962)], at a point referred to as the “restriction point”.
(2) One can block DNA synthesis, by using drugs such as hydroxyurea, which interferes with
dATP production, or amethopterin, FUdR, or aphidicolin. One of the most popular methods
is the use of a high concentration of thymidine, which feedback-inhibits the production of
dCTP synthesis. These drugs, in many cases, can be reversed and the cycle can be
continued. One can wash out HOU; or reverse high thymidine with a wash.
USING A SINGLE BLOCK TO SYNCHRONIZE CELLS:
DNA synthesis
inhibitor
M G 1
M
G 1
G 2 S (time greater G 2 S
than G 2 + M
+ G 1 )
Release these cells
from inhibition, and
count cell number
3
Cell Number
G 2 + M S

Note: all agents that inhibit DNA synthesis block the cells in very early S, and not at the G1/S
transition, as is often claimed. By using this procedure we can determine [G2 + M] and S. One can
determine G1 by subtraction, since we know the generation time [GT] = G1 + S + G2 + M.
USING A DOUBLE BLOCK TO SYNCHRONIZE CELLS:
Release single blocked cells for a period greater
than S, but less than the sum of G2 + M + G1.
Add back an inhibitor of DNA synthesis. All
cells will be synchronized at "the G1/S transition".
G 1 G1
G 1
M M M
G 2 G G 2
2
S -DNA S +DNA S
inhib
inhib
[2]. WE CAN SYNCHRONIZE CELLS BY MITOTIC SELECTION:
Cell Cell number number
Release these cells
from inhibition, and
count cell number
S + G 2 + M
Many cells grow attached to the culture dish, because the microtubules hold them in an extended
shape. At mitosis, the cells round up and are only loosely attached to the dish, because the
microtubules are dissociated and used to form the mitotic spindle to separate the chromosomes.
One can shake off the mitotic cells, or wash them off the surface of the dish with a stream of
medium. You can then replate these cells and have synchronized M/G1 cells [Robbins and Marcus,
Science 144: 1152 (1964)].
The advantage of mitotic selection is that there is no biochemical manipulation. The disadvantage
of mitotic selection is that, since only ~5% of the cells will be in M at any given time; one gets only
5% of the cells in synchrony with the wash. One can increase the yield by blocking cells at M with
a drug like nocodazole (a drug which reversibly prevents microtubule polymerization). One then
collects the mitotic cells, washes, and gets synchronized M/G1 cells to replate.
SO-- MITOTIC SELECTION GIVES US CELLS AT M/G1. DNA INHIBITION
PROCEDURES GIVE US CELLS EITHER DISTRIBUTED THROUGHOUT S, OR AT
G1/S.
[3]. WE CAN SYNCHRONIZE CELLS BY CENTRIFUGAL ELUTRIATION. Cells
increase in size as they go through the cycle; from M to G1 to S to G2. The centrifugal elutriation
rotor isolates cells that are increasingly larger in size. One can then check the position of the cells in
the cycle by using flow microflourimetry (FMF) to determine DNA content [Meistrich et al, Exptl.
Cell Res. 105: 169 (1979)].
Cell
number
G 1
S
G 2
Control early mid-E mid-L late
fluorescence intensity
4

3 H TdR
HL LL
Density
Chicken cells
BIOCHEMICAL CHANGES IN THE CELL CYCLE: DNA SYNTHESIS.
cpm
G 1
RATE
S
G 2
cpm
G 1
RATE
AMOUNT
There are a large number of questions to be addressed: What initiates DNA synthesis; i.e., is the
signal positive for S, or is DNA synthesis repressed in G1 and G2? Does DNA synthesis depend on
RNA synthesis? Does DNA synthesis depend on protein synthesis? How is DNA replication
restricted to once per cell cycle for each gene? How does chromatin structure change during the cell
cycle? How is chromatin assembled following DNA replication? Is DNA synthesized in a random
or an orderly fashion -- does DNA synthesized early in one cycle always get synthesized early in a
second cycle? When are specific genes replicated? When are enzymes involved in DNA replication
synthesized? Does DNA damage modulate DNA synthesis?
QUESTIONS CONCERNING DNA SYNTHESIS:
Is DNA synthesized sequentially or randomly? [Mueller and Kajiwara, BBA 114: 108 (1966)]
Cytochemical evidence suggest there exist "early replicating" and "late replicating" DNAs. Is DNA
that is synthesized early in one cell cycle again synthesized early the next cell cycle?
Optical
density
(1) Synchronize HeLa cells with a double-thymidine block at G1/S (S = 6hr).
(2) Release from block, add [ 3 H]-TdR for 3 hours (ie, during the first half of S). Then
switch the cells to medium containing non-radioactive, "cold" thymidine.
(3) Grow the [ 3 H]-TdR labeled cells for a few generations. (They will loose synchrony).
(4) Resynchronize the cells with a second double-TdR block at G1/S. Release in the
presence of BudR for 3 hours.
(5) Isolate DNA, shear the DNA, and centrifuge on CsCl gradient.
(6). Follow radioactivity and density. If early replicating DNA is resynthesized early,
then [ 3 H] will always go with the BUdR density label. If DNA synthesis is random,
then [ 3 H] will be present in all fractions. RESULT: [ 3 H] AND BUdR TRAVEL
TOGETHER. CONCLUSION: DNA SYNTHESIZED EARLY IN ONE
REPLICATION CYCLE IS SYNTHESIZED EARLY IN THE NEXT CYCLE.
CELL FUSION EXPERIMENTS SUGGEST MECHANISMS FOR
CELL CYLE TRANSITIONS.
Is initiation of DNA synthesis due to (1) a positive signal acting on/in S phase nuclei or (2)
release from a negative signal acting on G1 or G2 cells? To put this another way, are there G1
and G2 phase products which inhibit DNA synthesis, or S phase products which initiate DNA
synthesis? [Rao and Johnson, Nature 225: l59 (1970)].
Human cells
Flu virus or
polyethylene glycol
Use cells that have morphologically distinct nuclei. They fused together, using
inactivated sendai virus, a G1 phase cell and an S phase cell from different species,
to make a "heterokaryon" – a cell with two nuclei in a common cytoplasm. Then
they added [ 3 H]-TdR, and prepared autoradiographs, to identify labeled nuclei. The
S phase nucleus makes DNA. In addition, the G1 nucleus is induced to make DNA.
Fuse a G2 and an S cell. G2 nucleus makes no DNA; S nucleus makes DNA.
Fuse a G2 phase and a G1 phase cell. No DNA synthesis in either nucleus.
5
S
G 2
M
2X
1X

THESE FUSION EXPERIMENTS SUGGEST [1] THAT DNA SYNTHESIS OCCURS IN
RESPONSE TO A POSITIVE, NONSPECIES SPECIFIC INDUCING FACTOR PRESENT
IN S PHASE CELLS AND [2] THAT G2 CELLS –WHICH HAVE DUPLICATED THEIR
DNA -- HAVE INFORMATION THAT TELLS THEM TO IGNORE THE SIGNAL.
Is there a "dominant factor" for chromatin condensation, or is there an inhibitor of THIS
mitotic activity in interphase cells? [Johnson and Rao, Nature 226: 717 (1970)]. Fuse a mitotic
cell to any interphase cell (in G1, S or G2), and the interphase cell undergoes premature
chromosome condensation, or PCC. The mitotic cell is "dominant"; something present in mitotic
cells can initiate chromosome condensation in interphase cells! What might this be?
HISTONE SYNTHESIS
C
B
Fraction number
try ptophan
arginine
lysine
A3
A2
A1
Histones are synthesized in S phase. A large percent of
the cell protein is histone; the histones must be
duplicated, get into the nucleus, and associate with the
DNA for each cell cycle. We will define histones as
chromosomal (nuclear) proteins that are high in arginine
and lysine, low in tryptophan and are acid extractable.
The first experiment was done by Robbins and Borun
[PNAS 57: 409 (1969)]. They labeled cells with [ 3 H]lys,
[ 3 H]-arg or [ 3 H]-tryp, then prepared nuclei, extracted
other proteins first with NaCl, then extracted histones
with 0.25N HCl, ran on SDS PAGE gels, and
determined radioactivity to identify histones.
To determine when in the cell cycle histones are synthesized, they synchronized cells at M/G1 (how
might they do this?), released, pulsed with radioactive [ 3 H]-lys/arg at various times (shown in the
figure below as 1, 2 and 3), and measured histone synthesis. Histones are only synthesized in S
phase. How is transcription of the histone genes restricted to S-phase??
M/G 1
1
2
3
Fraction number
If one adds cytosine arabinoside, an inhibitor of DNA synthesis, histone synthesis ceases!! Tight
coupling exists between DNA synthesis and histone synthesis.
HISTONE MODIFICATIONS DURING THE CELL CYCLE. Histones undergo a variety of
modifications; including acetylation, phosphorylation, ADP ribosylation, ubiquitination, sumolation,
and methylation. These modifications are primarily on the flexible, basic domains in the N and C
termini of the histones.
Histone phosphorylation: There is an ordered pattern of histone phosphorylation during the cell
cycle. The data for histone H1 is best described. (1) H1 is not phosphorylated in resting cells. (2)
Two hours prior to DNA synthesis, H1 begins to be phosphorylated. Thus "old" G1 histone (i.e,
histone synthesized in previous cell cycles) --for that cell cycle-- is phosphorylated. (3) H1
phosphorylation increases somewhat in S. (4) H1 phosphorylation continues to increase in G2 and
in the first part of M. (5) In mitosis, H1 is rapidly and dramatically dephosphorylated.
6

Histone Kinase: Zellig and Langan [BBRC 95: 1372 (1980)] showed that histone H1 kinase
activity, measured in extracts of synchronized cells, increased 4-6 fold from late G2 to mid
metaphase. The activity disappears at the end of M!! The H1 kinase enzyme activity had a half-life
of 30 minutes. Is this due to enzyme activation? synthesis? Degradation? Remember this paper; it
was "before its time".
THE DISCOVERY OF CYCLIN, ANOTHER PROTEIN SYNTHESIZED CYCLICALLY
DURING THE CELL CYCLE: In l983 Evans et al [Cell 33: 389 (1983)], using gel
electrophoresis, observed that a protein builds up in fertilized sea urchin eggs during interphase,
reaches a maximum just before/during mitosis, and is rapidly degraded in mitosis. They named this
protein "cyclin". We will not talk much about embryonic systems, since I want to emphasize
somatic cells. We now know that all cells contain a homologue of this sea urchin cyclin, cyclinB.
This is the experiment that lead to the Nobel Prize for Tim Hunt in 2001.
G 1 + S + G 2 G 1 + S + G 2 G 1 + S + G 2
M M M
RESCUING YEAST CDC MUTANTS WITH A HUMAN GENE: THE cdc28/cdc2 STORY.
Lee Hartwell isolated temperature sensitive (ts) mutants of the yeast S. cerevisiae that – when
grown at the “permissive temperature” (23 o C) – grow well. But, when shifted to the “nonpermissive”
temperature (36 o C), these mutants arrest at specific points in the cell cycle –
presumably the point at which the function of the gene in question is needed; what Hartwell termed
the “execution point” for the function of that gene. One can easily tell where in the cycle the cdc
cells are, when they arrest, by the size of the bud. These are the experiments that lead to the Nobel
Prize for Lee Hartwell in 2001. Hartwell called these “cell division cycle” or cdc mutants.
When shifted to the restrictive temperature, the S. cerevisiae cdc28 mutant arrests at a point in G1
called START. This is the point where the cell makes its decision to initiate DNA synthesis. Paul
Nurse isolated similar mutants in the fission yeast, S. pombe. cdc2 is a ts cell cycle mutant in S.
pombe. S. pombe cdc2 mutants arrest predominantly at G2/M at the non-permissive temperature.
What was surprising was that the S. cerevisiae cdc28 mutant and the S. pombe cdc2 mutant
complement one another, even though there is great evolutionary distance between these two strains
and that the former arrests at START and the latter at G2/M. However, closer analysis showed that
the cdc28/cdc2 gene product actually controls both functions in both yeast strains. Because of
difference in other aspects of their biology, they arrest predominantly at different points, but the
cdc28/cdc2 gene is actually responsible for both functions in both strains.
S. pombe can (amazingly) use a promoter from the SV40 monkey virus to express foreign genes.
Lee and Nurse [Nature 327:31 (1987)] transfected a human cDNA expression library, with cDNAs
driven by the SV40 promoter, into a cdc2 ts/leu - S. pombe strain. They cotransfected with a leu+
plasmid and let the cells grow for a while at the permissive temperature, to first select for leu+
transformants. They then plated at the restrictive temperature, and selected for growth. From one
million leu+ transfectants they got 5 that could grow at the restrictive temperature. From 2 of these
they could recover plasmids containing human expression cDNAs that could correct a cdc2
mutation! Thus this human gene complements the yeast cell cycle mutant. The yeast cdc2 gene
product is a 34 kd phosphoprotein, known at that time to have protein kinase activity in vitro. What
are the properties of the human cdc28/cdc2 homologue? It is also 34 kd, is phosphorylated.
Moreover the p34 cdc human homologue – like the products of the cdc2/cdc28 genes, is a protein
kinase. This is the experiment that lead to the Nobel Prize for Paul Nurse.
7

Draetta and Beach [Cell 54: 17 (1988)], using [35]S-labelled HeLa cells, found that the protein
product of the p34 gene, the cdc2 protein kinase, complexes with a protein called p62.
Percent
cells in
each phase
G 1 S G 2
2 4 6 8 10 12 Fraction
p34
p62
p34
p62
casein
[35]S-labelled HeLa cells were fractionated by
elutriation. The p34 level [determined by western
blots (row 1 of the figure)] does not change much
during the cycle. However, p34 associates with a 62
kd protein, p62, during G2, as shown by coimmunoprecipitation
of [35]S-labeled proteins with
antisera to p34 (row 2). Moreover, p34 kinase
activity is greatly elevated in G2/M, using the p34IP
for casein kinase activity (row 3). In addition, p62 is
a substrate for p34 kinase (row 3)! The kinase activity
– but not the p34 protein itself – is eliminated as a
part of the M to G1 transition. The data suggest that
p34 associates with p62 as a function of cell cycle
position, and that p62 association is necessary for
activation of p34 kinase activity on casein and on
p62.
The p62 protein is a cyclin. Several experiments, between 1989 and 1990, identified the molecular
nature of the p62 protein.
1. The yeast cdc13 gene encodes a 62 kilodalton, phosphorylated protein, p62, that complexes with
yeast p34. The yeast cdc13/p62 gene was cloned [Bodner and Beach, EMBO J. 7:2321 (1988),
Solomon, Cell 54: 738 (1988)] and sequenced. The product of the yeast cdc13 gene, the p62
protein, is yeast cyclin!
2. Is the p62 protein that associates with p34 in mammalian cells a cyclin? In experiments carried
out at about the same time, Langan et al [MCB 9: 3860 (1989)] (remember that paper that "was
before its time", published 8 years earlier?) showed that the mammalian histone kinase active at the
G2/M time period also contains p34cdc2 and cyclin. Thus it appears that a complex of p34 kinase
and cyclin (p62) is the active G2/M histone kinase; cyclin converts a relatively inactive p34 kinase
to a histone kinase. A cell cycle dependent heterodimer of the p34cdc2 protein kinase and cyclin is
formed to activate histone kinase activity. Loss of cyclin as the cell goes from M into G1 eliminates
the M-phase specific histone kinase activity. We now call this cdc2 p34 protein kinase enzyme a
cyclin-dependent kinase, or cdk.
Cdc2
p34
Cdc2
p34
Inactive kinase
+ cyclin + cyclin
Cdc2 cyclin cyclin
p34
Cdc2
p34
cyclin cyclin
active cyclin-dependent kinase
HISTONE + ATP HISTONE-P + ADP
8

The human cyclin B gene: Pines and Hunter [Cell 58: 833 (1989)] isolated a cDNA for a HeLa cell
cyclin, cyclin B, expressed the protein and made antibodies to it. They synchronized HeLa cells at
the G1/S transition by sequential thymidine and aphidicolin blocks. Following release, cyclin B
RNA increased four fold from S to G2, and was relatively low in other phases of the cell cycle.
[This may be the first mRNA to clearly be shown to be induced in G2.] When cells were collected
in mitosis with nocodazole, then released at the M/G1 interface, the level of p62/cyclin mRNA
plunged dramatically in the M to G1 transition. Northern analyses are shown below:
0 2 4 6 8 10 12 14 16 18 20 22 24
G 2 /M peak
Hours after release from G G1/S 1/S
Cyclin
Histone 2A
0 2 4 8 16
Hours after release from mitosis
The cyclin protein also was low in S, and increased in G2 (western blots).
0 2 4 6 8 10 12 14 16 18 20 22 24
G 2 /M peak
Hours after release from G G1/S 1/S 1/S
Intensity of cyclin,
abitrary units
10 15 20 30
Hours after release from G 1 /S
Cyclin protein
Cyclin protein
Intensity of cyclin,
abitrary units
0 0.5 1.0 1.5 2 4 5
Hours after release from mitosis
0 0.5 1.0 1.5 2 4 5
Hours after release from mitosis
Cyclin
The cyclin of Hela cells is destroyed in the transition from M to G1, just as it is in frog eggs,
clam eggs, sea urchin eggs and yeast. Pines and Hunter also showed that the Hela cell p34/cyclin
complex is an H1 kinase, with highest activity in mitotic cells. To repeat: active H1 kinase is a
heterodimer composed of a stable p34 subunit with latent kinase activity and cyclin -- a protein
made and destroyed cyclically.
MULTIPLE CYCLIN GENES ARE PRESENT IN CELLS
Multiple S and G2 cyclins in mammalian cells. Pines and Hunter cloned a second cyclin, cyclin
A, from Hela cells. [Nature 346: 760 (1990)]. Cyclin A is expressed slightly before before cyclin B
(in S, G2, and M), and is degraded before cyclin B in mitosis. Yeast have now been shown to have
six B-type cyclins; Clb1-Clb6.
G1 "cyclins" in yeast. Recall that p34 cdc2/cdc28 is required at START and at G2/M in budding
yeast. Three yeast "cyclins" required in G1 were isolated [Hadwiger, PNAS 86: 6255 (1989)]. They
are referred to as “cln” cyclins. In addition to p34cdc28 kinase, these cln proteins are required to
pass through START. Co-immunoprecipitation experiments demonstrated that the p34cdc28 protein
forms complexes, active as protein kinases, with G1 cln cyclins [Wittenberg, Cell 62: 225 (1990)].
9

Overlapping "G1 cyclins", "S cyclins", and "mitotic cyclins". A great deal of genetic and
biochemical work has now characterized the cyclins necessary for cell cycle transitions in yeast.
The Cln1-3 cyclins [which we can term “G1-cyclins”] activate p34/cdc2 kinase for G1 progression
through START. The B-type cyclins Clb5 and Clb6 [which we can term “S-cyclins”] are required
for S phase initiation. Clb5 and Clb6 are synthesized late in G1, overlapping Clns 1-3. Clns (G1
cyclins) are first required at START, then the Clb5/6 proteins are required for S phase progression.
The Clb1-4 cyclins [which we can term “M-cyclins”] are required for the completion of mitosis.
Cyclins specific for various phases exist for the yeast cell cycle.
The yeast p34/cdc2/cdc28 cyclin-dependent kinase can interact with different cyclins, at
different phases of the yeast cell cycle, and phosphorylate alternative substrates necessary for
various functions.
Substrate a
Substrate b
Substrate c
Cdc2 Cdc2 Cdc2
Substrate d
Substrate e
Substrate f
Substrate g
Substrate h
Substrate i
G1 cyclins in mammalian cells. Using yeast mutants defective in cln1, cln2 and cln3 (the yeast G1
cyclin genes), human "cyclin" genes that can complement this defect have been isolated [Lew et al,
Cell 66: 1197 (1991)]-- using procedures much like the original cloning (by Lee and Nurse) of the
human p34 cdc2 homologue. These genes are called cyclins C, D, and E. There are, in fact, three
different D-type cyclins; D1, D2 and D3. The C, D and E cyclins are synthesized in G1 in
mammalian cells! The same concept of G1-cyclins, S-cyclins and M-cyclins is also true for
mammalian cells.
10

DISTINCT P34CDC2-LIKE GENES AND PROTEINS IN MAMMALIAN CELLS.
Pines and Hunter showed that cyclin A in human tissue culture cells can also associate with a 33kd
protein that "is related to but distinct from p34cdc2." This protein complex also has H1 kinase
activity, suggesting there may be a family of p34cdc2-like proteins. This protein became known as
cdk2.
A family of mammalian cyclin-dependent protein kinases, or CDKs, was subsequently cloned.
Cdk2 protein associates with cyclin A [Tsai et al, Nature 353: 174 (1991)]. Subsequently a family
of at least ten proteins related to p34cdc2 was cloned [Meyerson et al, EMBO J. 11: 2809 (1992)].
Thus, although in yeastcdc2/cdc28 appears to control both G1/S and G2/M transitions, in
mammalian cells a family of related genes are responsible for these (and possibly other) transitions.
Mammalian cells have distinct cyclin dependent kinases and cyclins that catalyze the
phosphorylation of alternative substrates required for completion of various cell cycle
functions.
WHAT ARE THE ROLES OF THE VARIOUS CYCLIN DEPENDENT KINASES, OR
CDKs, AND THE VARIOUS CYCLINS IN MAMMALIAN CELLS?
HOW DO THESE CATALYTIC KINASE SUBUNITS AND CYCLICALLY PRODUCED
REGULATORY SUBUNITS REGULATE CELL CYCLE PROGRESSION AND
TRANSITIONS?
WHAT ARE THE SUBSTRATES PHOSPORYLATED BY THE CDKS?
WHAT’S A CYCLIN? Anything with a “cyclin box” – a site for interacting with a CDK, and
demonstration of an interaction with a CDK to increase its kinase activity. Here is an illustration
of the value of having a whole genome sequence: by these molecular definitions, in budding
yeast there are 22 cyclins and 5 CDKs. Nine cyclins can activate CDC28. CDC28 is the only
CDK known to have an essential role in cell cycle progression in yeast [Andrews and
Measday, Trends in Genetics 14: 66 (1998)].
11

A summary of major cyclin and Cdk
interactions in the mammalian cell cycle. We
now know that, in mammalian cells, cyclin B
and cyclin A, along with p34cdc2, are involved
in the G2/M transition. Cyclin A:Cdk2 also
seems to be involved in control of DNA
synthesis. We also know that cyclins A and E,
and cdk2, are involved in initiation of
replication of DNA.
The three D-type cyclins vary between
cell types; they may carry out different
functions when complexed with various cyclindependent
kinases. It appears that complexes
of cyclin E and/or cyclin D with Cdk2 and
Cdk4 are involved in entry into G1.
cdc2
M
cdc2 CDK? Cyclin
cdc2 CDK? cdc2 CDK? ???
Cyclin
???
Cyclin
A&B
G 2
Cyclin
B
p cdc2
p
G 1
CDK
2,4& 5
Cyclin
D1,D2
D3
PCNA
CDK2
Cyclin
E p107 &
E2F
S CDK2
Cyclin
A p107 &
Cyclin
E2F
B
Since the experiment of Lee and Nurse (page 7 of the lecture notes) in 1987, we have gone from
descriptive biology to mechanistic studies of the cell cycle. This is why I referred to the Lee and
Nurse experiment as a “landmark experiment,” leading to the Nobel Prize.
.
12
cdc2

WHAT ARE THE SUBSTRATES OF THE CYCLIN-DEPENDENT KINASES?
A CDK reaction is required for human cells to traverse the G1/S boundary. In traversing the
cell cycle, human cells decide at the "restriction point" (similar to the S. cerevisiae START
decision) whether to exit the cell cycle and enter Go, or to commit to another cell cycle. If cells
commit to progression through the cell cycle, they activate a transcription factor, E2F, necessary
to transcribe genes whose products are needed for DNA synthesis. In early G1, E2F is bound to
another protein --we will call it the restriction boundary protein, or RB protein for now-- and
E2F is not active as a transcription factor. RB protein is not phosphorylated in non-dividing, Goarrested
cells, or in cells emerging into G1 from M. Cell cycle-dependent phosphorylation of RB
releases E2F from the RB/E2F complex, and permits E2F to activate transcription of genes
necessary to enter S. The CDK4/cyclin D1 complex, which forms as cells synthesize cyclin D1 in
G1, phosphorylates RB protein, leading to release of active E2F transcription factor.
RB must subsequently be dephosphorylated before cells complete mitosis, so that it can
again monitor the decision to remain in the cell cycle or to exit the cell cycle and enter the Go
phase, the next time the cell progresses to the restriction point.
RB RB RB
Cyclin
c
D1
c Cyclin
D1
CDK
4
E2F E2F
G o , Early G 1
Cell cycle progression
Late G 1 , Early S
Among the genes transcriptionally regulated by E2F are c-myc, cyclin E (necessary to initiate DNA
synthesis), B-myb, p107, pRB, E2F-1, E2F-2, cyclin A, p34cdk2, DNA polymerase alpha,
HsORC1, dihydrofolate reductase, thmidine kinase, thymidylate sythase, PCNA, RRM2 and
Histone 2A. [Lavia and Jansen-Durr, Bioessays 23: 221 (1999)].
Recall that histones are transcribed only during the S phase of the cell cycle. How is this
controlled? Each mammalian histone had a distinct promoter, with different cis-acting
regulatory sequences that bind different transcription factors. Cyclin E expression is induced in
response to E2F activation. Cyclin E forms a heterodimer with Cdk2. Zhao et al [Genes & Dev.
12, 456-461(1998] identified NPAT (nuclear protein mapped to the AT locus) as a protein that
interacts with, and is phosphorylated by, Cdk2/cyclin E. Using antibodies to NPAT, Zhao et al
[Genes & Dev.. 14, 2283-2297 (2000)] found that NPAT localizes to histone gene complexes
(you should read how this is done). Cyclin E also co-localizes to these sites [Ma et al, Genes &
Dev.. 12, 2298-2313 (2000)]. Localization occurs in S phase, when histone transcription occurs.
The idea is that cyclin E is expressed at G1/S, interacts with Cdk2, phosphorylates NPAT at the
histone gene locus and that phosphorylated NPAT co-ordinates the transcription of the histone
gene family, either by acting as a chromatin-rearranging machine, a co-activator, etc.
NPAT NPAT NPAT NPAT
pp
pp
NPAT NPAT NPAT NPAT
NPAT NPAT NPAT NPAT X X X
H1 H2A H2B
Fold Induction Induction
H4 H4-1 Control
Cyclin E Cdk2
NPAT NPAT NPAT NPAT
pp
pp
pp
pp
NPAT NPAT NPAT NPAT NPAT NPAT
pp
pp
pp
pp
pp
pp
RB RB
RB
NPAT NPAT NPAT NPAT NPAT NPAT
pp
pp
pp
pp
pp
pp
p
H1 H2A H2B
If one transfects cells with a [histone
promoter][luciferase] reporter gene, cotransfection
with an NPAT expression vector
increases transcription 10 fold. NPAT must be
phosphorylated at a Cdk2/cyclinE site for this
increase in transcription to occur.
13

w.t. hpm CyclinAHA
cdk2
Histone H1
p107
E2F
How do cyclin-dependent kinases choose their substrates? Mammalian
The crystal structure of cyclin A-cdk2 has been solved (Jeffery et al,
Nature 376: 313, 1998). A hydrophobic patch on the surface of cyclin A,
in which the sequence is conserved in many cyclins, is apparent.
Schulman et al [PNAS 95: 10453, (1998)] thought this patch might direct
substrate recognition. Many of the substrates of cyclin A-cdk2 have the
sequence RXLFG. Mutations were made in the cyclin A hydrophobic
patch (mutant htm), complexes were purified by immunoprecipitation,
and the ability of the cyclin A-cdk2 complexes to interact with
and phosphorylate substrates was examined. The wild-type and mutated
complexes could both phosphorylate H1. However, the mutant complex could not phosphorylate
RB as well as the complex containing the wild type protein, and has no activity on several other
[cyclin A][cdk2] substrates. These data suggest that, as predicted from the structural data, the
substrate is chosen and presented to the kinase by interaction with the specific cyclin of the
cdk/cyclin heterodimer.
The big questions are:
1. Which cyclins interact with which Cdk catalytic subunits?
2. How are the activities of the various cyclin-dependent kinases regulated?
3. What are the substrates for the various cyclin-dependent kinases?
4. What cell functions are regulated by these CDK phosphorylations?
5. What phosphatases reverse the cyclin-dependent kinase-mediated events (e.g., Rb,
H1)?
6. How are cyclins degraded?
7. By what other mechanisms are the various cyclin-dependent kinase complexes
activated and inactivated?
THE CYCLINS AND THE CYCLIN-DEPENDENT CATALYTIC KINASE
SUBUNITS ARE ALSO SUBJECT TO CYCLIC PHOSPHORYLATION.
CyclinB (a G2/M-cyclin) accumulates in late S and in G2, and complexes with the p34cdc2 protein.
Why, then, do we not see H1 kinase activity in G2, when cyclin B and p34cdc2 are complexed?
Instead, we find the kinase activity shooting up in mitosis. (1) During early G2 --when the
p34cdc2/cyclin complex exists, but its H1 kinase activity is not yet high-- the p34cdc2 protein is
phosphorylated, on both tyrosine and threonine [Draetta and Beach, CELL 54: 17 (1988)]. The
tyrosine/threonine phosphorylation occurs in the ATP binding site of p34, and inactivates the kinase
--even though it is complexed with cyclin B. (2) When cells enter mitosis, the threonine and tyrosine
of the p34 kinase in the complex is dephosphorylated, and kinase activity increases dramatically. We
conclude that Tyr/thr phosphorylation negatively regulates p34/cyclin B M-phase kinase activity.
Other studies suggest a distinct threonine (thr161) site on the p34cdc2 protein must be
phosphorylated to activate M-phase kinase [Gould et al EMBO J. 10: 3297 (1991)].
The amounts of cyclin B and p34cdc2 do not go up dramatically during the later part of G2
and entry into M, but the kinase activity of the complex goes up dramatically at G2/M, as a
result of dephosphorylation of the catalytic p34cdc2 subunit.
14

HERE IS A SCHEMATIC OF THE PHOSPHORYLATIONS OF THE M-PHASE KINASE.
WHAT ENZYMES ARE RESPONSIBLE FOR THE PHOSPHORYLATION AND
DEPHOSPHORYLATION OF THE p34cdc2 PROTEIN IN THE PROGRESSION OF THE
CELL CYCLE?
Cycin B
is
degraded
Active
CDK!!
T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2
T161
T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2
Cyclin
B
T161
T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2
T161
inactive p
ATP
p
G1
G1
M G2
PPase
S
T14 dephosphorylation
Y15 dephosphorylation
Cyclin
B
T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2
p p
Cyclin T161
B p
T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2 T14Y15
cdc2
p p
Cyclin T161
B
1. What kinase phosphorylates p34cdc2 on threonine and tyrosine (T14Y15) in the active
site to suppress the activity of this enzyme?
2. What kinase phosphorylates p34cdc2 on threonine 161?
3. What phosphatases dephosphorylate p34cdc2 on these residues as the cells traverse
the cell cycle?
15
p
(inactive)
(still inactive)

Genetic studies in yeast demonstrate that the products of other genes influence the mitotic
p34/cyclin complex activity.
Mik 1
wee1
Cdc2
p34
cdc 25
cdc 13
(Cyclin)
Interphase mitosis
G 2
M
Three genes – wee1, mik1, and cdc25 – appear to regulate Cdc2
kinase activity at G2/M. A schematic from the known genetics is
shown. Gene products are shown as stimulating (by the arrows) or
inhibiting (by the blunt bars) the activity of other gene products. The
cdc2 gene product is the p34cdc2 kinase, the cdc13 gene product is
cyclin. These two proteins, p34cdc2 kinase and cyclin, are shown
acting in concert to induce mitosis. In the absence of both wee1 and
mik1 cells can enter mitosis prematurely, without completing DNA
synthesis, because they have too much CDK activity.
Protein kinases that regulate M-phase [p34/cyclin B] kinase. The product of the yeast wee1
gene is a both a tyrosine and a threonine/serine kinase. Using recombinant, bacculovirus-produced
proteins, Parker et al [PNAS 89: 2917 (1992)] demonstrated that p34cdc2 by itself is not a substrate,
but that the p34cdc2 component of the p34cdc2/cyclin B heterodimer is a substrate. Phosphorylation
of p34cdc2/cyclin B blocked H1 kinase activity of an in vitro assembled H1 kinase (p34cdc2 and
cyclin B complex).
The human wee1 gene was cloned by rescuing a yeast wee1 mutant with a human cDNA expression
library [Igarashi et al, Nature 353: 80 (1991)] (the Lee and Nurse experiment all over again).
Overexpression of human wee1 in HeLa cells prevents cell cycle progression [McGowan and
Russell EMBO J 12:75 (1993)]. [How would you do this experiment? What would the results look
like, in a graph or table?] These data suggest that Wee1 must be inactivated for a cell to make a
transition into M. Watanabe et al [EMBO J 14:1878 (1995)] demonstrated hyperphosphorylation
and subsequent degradation of Wee1 in HeLa cells.
wee1 cooperates with another gene, mik1 [Lundgren et al, Cell 64:1111 (1991)]. Mik1 is also a
protein tyrosine kinase! Both genes are necessary for p34cdc2 tyr phosphorylation in vivo. wee1 and
mik1 are essential for p34cdc2 phosphorylation.
PROTEIN PHOSPHATASES THAT REGULATE THE M-PHASE KINASE.
The yeast cdc25 gene product is a protein tyrosine and threonine phosphatase [Strausfeld et al,
Nature 351: 242 (1991); Gautier et al, Cell 67; 197 (1991)]. The enzyme shows remarkable
substrate specificity for tyrosine-phosphorylated p34cdc2. The enzyme can also dephosphorylate the
adjacent threonine residue; it a both a ser/thr and a tyrosine phosphatase of remarkable specificity.
Dephosphorylation of p34cdc2 protein by cdc25 protein phosphatase activates the kinase activity of
the p34/cyclin complex.
.
Cdc 25
p
p
p34 cyclin
Wee 1
p34 cyclin
Histone Histone p
Histone Histone p
16

% G 2 + M
The cdc25 gene product, a cdc2 phosphatase, appears to be expressed primarily in the G2 phase of
the cell cycle, both in S. pombe [Moreno, Nature 344: 549 (1990)] and in Hela cells [Sadhu, PNAS
87: 5139 (1990)].
Hela Cells
cdc 25 mRNA
cdc 25 mRNA
cdc 25 mRNA
Hela cells Yeast cells
% Septated
% Septated
M
M
Time (20 min intervals)
Yeast cells
Several mammalian homologues of Cdc25 have now been cloned. Cdc25C can dephosphorylate
p34cdc2, the G2 Cdk. What phosphatases act on Cdk2, Cdk4, Cdk6, etc?
cdc2
Cyclin
B
PPase
p
p
cdc2
Wee 1 Cyclin
B
Cdc 25
Cdc 25
Histone
p
+ ATP
p
cdc2
Cyclin
B
mitosis
Histone p Histone p
Cdc 25 message
Phosphorylation activates the
phosphatase activity of the Cdc25
protein. The Cdc2 protein component
of the Cdc2/cyclin B complex (the
“histone kinase”) must be
dephosphorylated at residues 14 and
15. Active Cdc2/cyclin B histone
kinase can also phosphorylate the
Cdc25 phosphatase; moreover,
phosphorylation of Cdc25 activates
this protein phosphatase. Thus a "selfamplification"
is set up, in which a
small amount of active cdc2/cyclinB
can activate the cdc25 phosphatase,
which then activates more kinase.
[Hoffmann et al, EMBO J. 12:53
(1993)]. This sets up a cycle in which
very rapid activation of p34cdc2/
cyclin B histone kinase can occur.
A Cyclin-dependent kinase phosphorylates and regulates cyclin-dependent kinases; cyclindependent
kinase cascades. In order to be activated, p34 cdc2/28 must be phosphorylated on
Thr161. The enzyme that carries out this phosphorylation is called Cdc2-activating Kinase, or CAK.
Fisher and Morgan [Cell 78: 713 (1994)] demonstrated that CAK is composed of a cyclindependent
protein kinase Cdk catalytic sub-unit called MO15 and a new G1 cyclin, called cyclin H.
CAK can activate both Cdc2 (a G2/M Cdk) and Cdk2 (a G1 Cdk).
17

NEGATIVE REGULATION OF THE CELL CYCLE, CYCLINS AND CDKS
Eukaryotic cell progression through the cell cycle is regulated by the sequential formation,
activation and subsequent deactivation of a series of cyclin-dependent kinases. Are there ways
in which external agents can interfere with the sequential cyclin/cdk activations, and regulate
progression through the cycle?
Cdc28/Clb2 Cdc28/Cln2
pET pETSIC1 pET pETSIC1
Purified Sic1 inhibits the Clb2-associated
kinase, but not the Cln2 kinase in in vitro.
Hist H1
HisHASic1 (pETSIC1) was added to yeast extracts from
CLB2HA3 orGAL-CLN2HA3. After 30 min. incubaton at 4o HisHASic1 (pETSIC1) was added to yeast extracts from
CLB2HA3 orGAL-CLN2HA3. After 30 min. incubaton at 4 C,
Clb2 or Cln2, Sic1 and their associated protiens were
immunprecipitated with 12CA5 Antibody, and H1 kinase
activity was measured. In control lanes (pET), E. coli extracts
from cells containing the empty vector were added.
oC, Clb2 or Cln2, Sic1 and their associated protiens were
immunprecipitated with 12CA5 Antibody, and H1 kinase
activity was measured. In control lanes (pET), E. coli extracts
from cells containing the empty vector were added.
cdc28
SIC 1
p p
Clb2
cdc28
Clb2
SIC 1
Histone Histone p
p40/Sic1, regulates the G1/S transition in yeast.
p40/Sic1 was originally identified as a protein that
associates with cdc28/cyclinB complexes. It was
purified and shown to inhibit the activity of Cdc28-
Clb kinase in vitro [Mendenhall, Science 259:216
(1993)]. In yeast, cdc28 p34 protein can complex
with Clb2 (an S phase cyclin). When the p34
cdc28/Clb2 complex is associated with p40/Sic1
protein, this cdk/cyclin complex is inactive as a
kinase. p40/Sic1 inhibition is specific, inhibiting p34
cdc28 complexed to Clb5 and Clb2 (S/G2 cyclins),
but not Cln2 (G1 cyclin). [Schwob et al, Cell 79: 233
(1994)]. To enter S, p40/Sic1 protein is
phosphorylated and then degraded, releasing the
inhibition of the CDK activity. Think about the
following question: how does Sic1 get degraded?
Cyclin dependent kinase [CDK] activities are
regulated by a group of molecules known as cyclindependent
kinase inhibitors, or CKIs. Sic1 is now
termed a cyclin-dependent kinase inhibitor, or CKI. The
CKIs of yeast inactivate cyclin-dependent kinases even if
tyr15 and thr14 are dephosphorylated and thr161 is
phosphorylated.
Recall that we wanted to ask the question “Are there ways in which external agents can
interfere with the sequential cyclin/cdk activations, and regulate progression through the cycle?”
The answer is “yes”, both in yeast and in mammalian cells.
Regulation of a CKI joins the cell cycle and response to external stimuli that inhibit cell
growth, both in yeast and mammalian cells. Yeast have two mating types, a and α. In response to
α-factor, which is released by α-mating type yeast, a-mating type yeast exit
the cell cycle in G1, and prepare to mate. What makes proliferating, haploid
cells withdraw from the cell cycle at G1 in response to mating factors? The
far1 gene is required for mating-factor induced cell arrest. Expression of the
far1 gene is induced by binding of α-factor to the α-factor receptor
expressed on type a cells. Far1 protein associates with p34/cdc28
complexes to the G1 Cln1 and Cln2 proteins, and inactivates their
kinase activity, but does not inhibit the activity of p34Cdc28/ClnB
complex. Far1 is an inducible G1 cyclin-specific CKI [Peter and Herskowitz,
Science 265: 1228 (1994)].
18

We have seen that there are at least two yeast CKI molecules in yeast. p40/Sic1, a clb-specific
cyclin, is a part of the intrinsic cell cycle machinery, regulating the G1/S transition. Far1 is a cln
specific cyclin. Far 1 mutants have normal cell cycle control in proliferating cells; Far1, an "add
on" to the cell cycle machinery, controls progression through the cell cycle in response to external
stimuli.
TGF-β is a “growth factor” that stops proliferation of human keratinocytes.
p15 protein is also a CKI. TGF-β induces expression of p15 message and
protein in human keratinocytes, producing an inhibitor of G1 Cdk/cyclin
kinases [Hannon and Beach, Nature 371: 267 (1994)].
Morgan [Nature 374:131 (1995)] summarized the principles of CDK regulation:
19

PROTEOLYSIS RULES: MECHANISM OF MITOTIC CYCLIN DEGRADATION
There are two excellent earlier reviews on this topic; King et al, Science 274: 1652 (1996), and
Hoyt, Cell 91: 149 (1997).
Before beginning the discussion of proteolysis and the cell cycle, we need to review the phases
of mitosis, the transition from G2 to G1. Sister chromatid separation occurs in the transition
from metaphase to anaphase. The degradation of cyclin B is required for the exit from
telophase into the subsequent interphase.
cdc2/28
Cohesins
cyclin deg.
G 2 Prophase Metaphase Anaphase Telophase G 1
-p34cdc2 +p34cdc2 -p34cdc2 +p34cdc2 0 15 30 50 0 15 30 50
min min
interphase mitotic
Active cyclinB/p34cdc2 gets one into mitosis. How do we
get out? To get out of the mitotic state we must inactivate
the p34cdc2 kinaseWe also know that cyclin B must be
degraded for the cell to exit the mitotic state. What is the
signal for cyclin B degradation? How is cyclin B
degraded? If [35S]radio-labeled cyclin is prepared
and added to an interphase extract from frog embryo
cells it is stable. If active p34cdc2 kinase (i.e.,
[p34cdc2][cyclinB]) is also added, the [ 35 S] cyclin B is
rapidly degraded [Felix, Nature 346: 379 (1990)]. What triggers the degradation of the
radioactive cyclin in these extracts?
Cyclin B degradation uses the ubiquitin system. Glotzer et al [Nature 349: 132 (1991)] used a
system in which they generated a “stable mitotic extract” (you should read this paper and the
relevant papers, to see how this was done). They also made a hybrid protein of the N-terminal of
cyclin (containing what is now termed the "cyclin destruction box", or “D-box”; RXXLXXXXN)
fused to protein A (for ease of purity of the radiolabeled, recombinant protein). Protein A binds to
immunoglobulins, so the fusion protein can be purified on an immunoglobulin-coated bead. They
showed this cyclin-protein A fusion protein, labeled with radioactive 125 I, could be degraded by
mitotic extracts, as Felix et al showed for native cyclin (vida supra). Long exposure of the gels
suggested a "ladder" of higher molecular weight forms. By using affinity binding of the 125 I-cyclinprotein
A protein to IgG (used for both illustrations below) they showed convincingly that “stable
mitotic extracts”, which degrade cyclin, form ubiquitin intermediates of the cyclin-protein A
complex. Interphase extracts do not form the ubiquitin complexes. I suggest you read this paper.
buffer
buffer
M extract
M extract
I extract
Time 0 6 12 20 25 30 35 40 60
20

Does p34cdc2/cyclin B activate a cyclin-specific enzyme involved in ubiquitination of cyclin in
M? Does p34cdc2/cyclin kinase make cyclin a better substrate in M phase for a constitutive
ubiquitination system?
The protein complex that degrades ubiquitinated proteins in cells is called the “26S
proteasome.” It appears to always be present in cells. The major question to answer is “What
targets proteins destined for degradation to the proteasome in a cell-cycle specific manner
(e.g., cyclin B in this case)?”
cyclin B
cdc2 p
cdc2 p
p p
cyclin B
cyclin B sythesis
cdc25
p p
cdc2 p
cdc2 p
p p
cyclin B
cdc2 p
cdc2 p
cyclin B
+
cdc2
protein X
protein X
p
p
protease
Felix et al suggest that activated M-phase
kinase (p34cdc2/cyclinB) phosphorylates a
substrate, X, that leads to the activation of a
protease that will then degrade cyclinB. We
now know this degradation pathway includes
phosphorylation and ubiquitination of
cyclinB. New synthesis of cyclinB allows
reactivation of the M-phase kinase in the next
G2 period. A corollary of this hypothesis is
that the protease that degrades G2 cyclins in
M must be inactivated in the next cell cycle;
otherwise cyclinB could not be degraded, and
the next cycle could not occur.
The components in clam extracts that ligate ubiquitin to cyclin B have been identified.
[Hershko et al, JBC 269:4940 (1994)]. One component is active only from M-phase extracts.
Moreover, this component can be activated from interphase extracts by p34cdc2/cyclin B, as
suggested by Felix. The cyclin ubiquitinylation activity is part of a large protein multimer. This
result suggests a mechanism by which the destruction of the cyclin of G2/M Cdk activity can be
initiated and cells can make the transition to interphase. The enzyme complex that ubiquitinates
cyclin B in M phase is called the “cyclosome” or the Anaphase Promoting Complex or APC
(because it is necessary for the transition from G2 into anaphase). The cyclosome/APC marks
cyclin B for degradation by ubiquitination, preparing cyclin B for degradation by the proteasome.
Clearly, the cyclin B degrading system must be turned off prior to G2 of the following cell
cycle, so that cells can proceed through a subsequent cell cycle; cells must be able to make
“stable” cyclin B at a later time, for the next cell cycle.
Cyclin
B
p
Cyclin
B
proteasome
APC
Ub
Ub
p
Cyclin
B
Ub
Ub
Ub Ub Ub Ub
Ub UbUb
What turns off the cyclin B
degradation system – i.e., what
turns off the cyclosome/APC – so
cyclin B can be synthesized, and
form the p34cdc2/cyclin B
complex required for mitosis in
the next cell division?
Cyclin B proteolysis continues in G1 in yeast, until p34/cdc28 is reactivated by Cln G1 cyclins.
Once the cyclin B degradation system --the APC-to-proteasome complex-- is triggered, how long
does it stay active? Is cyclin B all degraded within the period of M, or can it keep being degraded as
cells enter interphase?
21

cyclin B RNA cyclin B protein H1 kinase activity
Cyclin B2 (ClnB2) protein cannot accumulate in cells deprived of G1 cyclins. Amon et al [Cell
77: 1037 (1994)] constructed a yeast cell mutated in the three Cln G1 cyclins, and carrying a
methionine-suppressible G1 Cln2 gene. In the presence of met, these cells arrest in G1, because they
are not expressing G1 cyclins. If these same cells carry a ClnB2 gene driven by the Gal promoter,
one can regulate the expression of the cyclin B gene. If these cells are given methionine, they have
no clns; they arrest in G1. When stimulated with galactose, they accumulate ClnB2 mRNA, but no
ClnB2 protein or, therefore, H1 kinase activity. Cyclin B protein is degraded in G1. Expression of
G1 cyclins is necessary to shut off cyclin B degradation.
If the procedure described above is repeated with cells washed free of met, and given galactose,
CLN2 expression will occur. Within 15 minutes, cyclin B protein and H1 kinase activity appear;
degradation of the cyclin B protein is abrogated. Accumulation of G1 cyclins leads to inactivation of
CLB proteolysis.
Cyclin B is degraded in G1 if the G1 CLNs are not synthesized; i.e., until the G1 CLNs are
synthesized. The APC remains active until it is (somehow) shut off by the G1-Cdk activity that
requires G1 cyclins. The APC machinery, which marks cyclin B for degradation, is turned off
when the G1 cyclins are synthesized.
A summary of CLN production, CLB production and cyclin B destruction machinery in yeast.
CLNs CLNs
CLBs
G1 S M G1 S M
Start Finish Start
Cyclin Oscillations in yeast
CLBs
APC mediated destruction
machinery for cyclin B
CLN1, CLN2 and CLN3 associated kinases are absent in early G1, reach peak levels
at the G1-S transition, and decline as cells enter G2. These kinases not only promote
budding and DNA synthesis, but also inactivate CLB cyclin proteolysis. Activation of
the mitotic kinase during G2 causes repression of CLN1, CLN2 and CLN3 synthesis
and onset of M phase and ultimately leads to the reactivation of CLB proteolysis.
Cyclin B proteolysis then persists during G1 until the reactivation of CLN cyclins
22

How is the APC/cyclosome activated to target cyclin B for degradation? Identification of the
cell-cycle regulated component of the complex that targets cyclin B destruction by the
proteasome. Identifying the genes that encode the protein components of the APC/cyclosome.
WT
Cdc16-123
Cdc23-304
Cse1-22
pGAL-CLB2
-GAL
+GAL
Irniger et al [Cell 81: 269 (1995)] made a ClnB2-β-gal fusion, and
expressed this gene in the Amon et al [cln1-, cln2-, cln3-] mutant.
When the cells are arrested in G1 (because G1 CLNs are not
synthesized), colonies transcribe clnB2-βgal mRNA. Parental colonies
remain white, because the fusion protein is degraded by the
cyclosome/APC complex. If a cell is mutant in the cyclosome/APC
cyclin B degrading system, colonies will be blue. They obtained three
mutants -- all previously known genes; CDC16, CDC23 and CSE1.
This question has also been approached biochemically. When one fractionates frog egg extracts for
the components necessary to ubiquitinate cyclin B, the cell cycle-determining factor is a complex
that contains the CDC27 and CDC16 proteins. [King et al, Cell 81:279 (1995)]. Immunodepletion
of these proteins prevents cyclin B ubiquitination. This is the mitotic E3 component of the cyclin B
ubiquitination complex suggested by Hersko (1994).
Antibody staining demonstrated that human CDC27 and CDC16 proteins co-localize to
centrosomes and mitotic spindles. It appears a complex of Cdc27, Cdc16 and other proteins is a
machine – the cyclosome/APC – regulated in M, that mediates cyclin B destruction.
NON-CYCLIN PROTEINS ALSO MUST BE TARGETED FOR
DEGRADATION BY THE CYCLOSOME/APC COMPLEX FOR CELLS TO
TRAVERSE MITOSIS.
If a non-degradable cyclin B is present, cells do not arrest in anaphase. Sister chromatid separation
still occurs, and the cells arrest in telophase. Therefore, cyclin B inactivation cannot be the trigger
for anaphase sister chromatid separation. However, if one blocks APC activity, either genetically or
by injecting antibodies to APC components, sister chromatid/chromosome separation is blocked;
anaphase is not completed. These findings create a paradox: entry into anaphase does not require
degradation of mitotic cyclins (e.g., cyclin B), yet anaphase remains dependent upon proteolysis
initiated by the action of the APC. APC/cyclosome directed degradation of some other proteins –
before cyclin is degraded – must be necessary for this sister chromatid separation -- i.e.,
metaphase to anaphase -- to occur.
CUT2 is a protein found in S. pombe that increases in anaphase, and accumulates in G1 if
cells are mutant in APC components. The data suggest that CUT2 must be degraded by the APC for
cell cycle progression to continue in M. Expression of a non-degradable form of CUT2 (without a
D-box) blocks cells in anaphase, i.e., before they enter telophase. [Funabiki et al, Nature 381:438
(1996)]. CUT2 degradation must occur before cyclin B degradation.. A similar story holds for a
protein known as PDS1 in S. cerevisiae. (1) PDS1 is degraded during mitosis, and its degradation
requires an active cyclosome/APC [Cohen-Fix et al, Genes and Development 10:3081 (1996)]. (2)
Over-expression of non-degradable PDS1 (PDS1 without a D box) arrests cells in metaphase (i.e.,
the cells don’t enter anaphase). (3) Degradation of PDS1 requires an intact D box and functional
APC. PDS1 is the budding yeast equivalent of CUT2 in the fission yeast.
CUT2 and PDS1 are also known as securins. Securin binds to a molecule called separase. Separase
(the Esp1 protein in budding yeast, and the CUT1 in fission yeast) is a protease that is inhibited by
securin. When CUT2/PGS1/securin is degraded by the APC/proteasome prior to anaphase, separase
can be activated by proteolytic activity. Separase then cleaves the family of molecules known as
cohesions, which hold the sister chromatids in anaphase together, allowing the sister chromatids to
separate and anaphase to proceed. [Reviewed in Peters, Mol. Cell. 9:931 (2002); Yanagida, Genes to
Cells 5:1 (2000)]].
23

So – the APC/proteasome-directed destruction of proteins such as CUT2/PDS1/securin is
necessary for cells to move from metaphase to anaphase. But mitotic cyclins (and some other
proteins that are involved in spindle formation) are not destroyed until later in the mitotic
sequence, after telophase, in order for cells to exit mitosis and enter G1.
securin
Proteolysis at metaphase, anaphase, telophase, G1 transitions [King et al, Science 274: 1652 (1996)]
How is the APC activity regulated so that distinct proteins (e.g., CUT2/PDS1/securin versus
cyclinB) are ubiquitinated by the APC, and delivered to the proteasome for degradation, at
different times – at different stages – during the G2 to G1 transition through M?
Schwab et al [Cell 90:683 (1997)] demonstrated that
yeast cells deficient for a protein called HCT1/CDH1
are impaired in their ability to degrade mitotic cyclin
B2. In the experiment shown at the right, G1 phase
wild-type yeast cells and G1 phase hct1 mutant cells
were isolated by elutriation. At various times after
isolation of G1 phase cells and continued growth,
populations of cells were analyzed by western
blotting for Clb2 protein. Hct1 mutant cells cannot
degrade Clb2.
These authors also demonstrated that HCT1 does not direct
APC/proteasome activity to the PDS1/securin protein. In this
experiment HCT1 is expressed from an inducible gal promoter.
Cells were treated with galactose to induce HCT1 expression, and
the ability of the cells to degrade both Clb2 and PDS1 was examined.
Clb2 is rapidly degraded, PDS1/securin is not degraded.
24

The data of Schwab et al suggest that HCT1 directs the activity of the APC/cyclosome
ubiquitination system to mitotic cyclins, but not to proteins such as PDS1/CUT2/securin – a protein
that must be degraded to initiate anaphase. In a similar experiment, Visintin et al [Science 278: 460-
463 (1997)] demonstrated that the CDC20 protein directs the APC/cyclosome activity to
PDS1/securin, but not to mitotic cyclins. Hoyt [Cell 91:149 (1997)] proposes the following model:
Cdc20 and Htc1 have been cloned. When recombinant Htc1 is added to interphase
cyclosome/APC preparations, the cyclin B ubiquitination ability of the complex is greatly
stimulated (Fang et al, Mol. Cell 2: 163, 1998); i.e., the APC/cyclosome activity is stimulated.
The APC cdc20 complex peaks in activity in M, and is responsible for Pds1p/securin
proteolysis and anaphase onset. APC Cdh1 cyclosome activity peaks in Anaphase/G1, and is
responsible for promoting Clb2p proteolysis and consequent G1 entry from M. [Nasmyth,
TIBS 24:98 (1999)].
1 2 3 4 5 6
Time (min) 0 15 30
0 15 30 60
What determines whether a target protein (e.g. cyclin B or
CUT2/securin) is directed to the proteasome by the E3 ligase
APC CDH1 or by the E3 ligase APC CDC20 ? Remember the cyclin B
“destruction box” [Glotzer et al, Nature 349: 132 (1991)]? Many
APC CDH1 substrates contain this sequence, RXXLXXXXN. Cdc20
is, itself, a target of APC CDH1 ; Cdc20 is rapidly degraded at the end
of mitosis. Reconstituted mitotic extracts containing CDH1
degrade Cdc20; without recombinant CDH1 the Cdc20 protein is
stable [Pfleger and Kirschner, Genes & Devel. 14:655 (2000)]. By
mutating regions of the Cdc20 protein and adding them to
APC CDH1 , they identified a KENXXXN sequence required for
APC CDH1 -dependent ubiquitination. Lanes 1, 3 and 5 are w.t.
Cdc20, lanes 2,4 and 6 are the mutant. The Cdc20 protein is
destroyed in an APC CDH1 extract, the mutant is not. APC CDH1
substrates may contain a KEN box.
Pfleger et al [Genes & Devel. 15:2396 (2001)] subsequently showed the CDH1 and Cdc20
proteins can bind directly, through their N termini, to the substrates that they direct to the
proteasome.
APC APC
cdc20 cdh1
proteasome proteasome
Substrate Substrate 11
25
Substrate Substrate 22

G1 CYCLINS (AND OTHER PROTEINS) ARE PREPARED FOR
PROTEASOME DEGRADATION BY A DISTINCT UBIQUITINATION
MECHANISM – THE SCF COMPLEX.
The cdc34 gene, which causes arrest at the G1/S transition at the non-permissive temperature,
encodes a ubiquitination enzyme. Mutants in the cdc34 gene arrest at the G1/S transition. Cloning
and sequencing the cdc34 gene showed that it has homology to ubiquitin-conjugating enzymes.
Recombinant cdc34 protein has ubiquitin-conjugating activity with histone 2A (how would you
demonstrate this experimentally?) [Goebl et al Science 241:1331 (1988)].
Cdc34p ubiquitinates Cln2 and leads to Cln2 destruction. Cln2, a G1 cyclin, is the regulatory
subunit of the G1 CDK, cdc28p34/cln2, that binds to Cdc28/p34. The Cln2 in the complex is
phosphorylated. In these in vitro reactions, an additional modified form of Cln2-P is observed. This
Cln2-P modified form can be precipitated with anti-ubiquitin. The ubiquitination occurs in extracts
of wt. cdc34 cells, but not extracts of cdc34 t.s. cells. Moreover, ubiquitination did not occur in
cdc28 t.s. extracts; phosphorylation of cln2 is required for ubiquitination of Cln2 by the cdc34
enzyme. [Deshaies et al EMBO J. 14:303 (1995)].
Cln3-beta gal remaining
Cdc34
w.t.
15 30 45 60
Chase time (min)
Cln3 is also phosphorylated by Cdc28/p34, and is then ubiquitinated by
a Cdc34-dependent step, leading to Cln3 degradation. Cln3, another G1
cyclin, is also ubiquitinated. Cln3 is also not degraded in the cdc28 t.s.
mutant. Finally, Cln3 is not degraded in a cdc34 t.s. mutant [Yaglom et al,
Mol. Cell. Biol. 15:731 (1995)]. In this experiment, a Cln3/β-gal fusion
protein was expressed in wt cdc34 cells or in a cdc34 mutant. The proteins
were labeled for five minutes with radioactive amino acids, then the cells
were chased with cold amino acids. At the times shown, Cln3-βgal was
immunoprecipitated and subjected to electrophoresis and autoradiography.
CYCLIN-DEPENDENT KINASE INHBITORS, OR CKIs, ARE ALSO
DEGRADED BY A CDC34-DEPENDENT UBIQUITINATION SYSTEM.
Sic1 degradation in yeast requires Cdc34 enzyme activity. Recall that Sic1 – a CKI inhibitor of
the CDK activity required for the G1/S transition; i.e., an inhibitor of CDKs that contain S-phase
cyclins – regulates the G1/S transition. Sic1 must be phosphorylated by a cdc28/-G1 cyclindependent
step, and Sic1 is then degraded. An essential role for the yeast G1 CDKs is to promote
the transition to S phase by the phosphorylation of Sic1, leading to its degradation. How is Sic1
degraded and removed, to allow S phase to begin? Sic1 degradation in late G1 depends on Cdc34!
[Schwob et al Cell 79:233 (1994)]. (How would you do the experiment?)
The transition from G1 to S should be a “step function” –
i.e, it should be a rapid initiation of DNA synthesis – but
it should occur only after a series of events in G1 has
taken place. Moreover, one doesn’t want this transition to
be dependent on small errors in the level of
phosphorylation of Sic1. Nature has solved this by
requiring that six out of nine phosphorylation sites on
Sic1 be phosphorylated before ubiquitination/degradation
becomes effective. This results in at least a finite minimal
G1 phase period. [Nash et al, Nature 414:514-521 (2001).
This results in a delayed, but sharp initiation of Sic1
degradation, activation of cdc2/S cyclin protein kinase
activity, and the G1/S transition.
26

There are several other genes whose products are required for the G1/S transition; genes
which – when mutated – produce phenotypes like cdc34 (i.e., arrest at G1/S). Mutations in the
skp1, cdc53 and cdc4 genes all produce phenotypes similar to that of a cdc34 mutation.
S-phase
cyclin
cdc28
Sic 1
Skp1
S-phase
cyclin
cdc28
cdc4
Cdc53
Cyclin
cdc28
ub
cdc34
S-phase
cyclin
cdc28
Sic 1
Skp1
p p
cdc4
Cdc53
S-phase
cyclin
cdc28
Sic Sic 1
p p
ub
cdc34
ub ubububub
ub ub ubub
ubub
ubub
ub
A macromolecular complex recognizes
phosphorylated Sic1 protein and catalyzes its
ubiquitination. Skowyra et al [Cell 91:209
(1997)] expressed recombinant forms of all these
proteins in an insect cell system and used coimmunoprecipitation
procedures to demonstrate
that these four proteins – Cdc34, Cdc4, Cdc53,
and Skp1 – could form a macromolecular
complex. Feldman et al [Cell 91: 221 (1997)]
demonstrated that this complex, in the presence
of ubiquitin and the E1 and E2 enzymes, can
ubiquitinate phosphorylated Sic1. The cdc4
protein recognizes the multi-phosphorylated Sic1
protein, and causes its binding to the
ubiquitinating complex.
Ccd4 associates with Skp1 through a motif on Cdc4 that is called an F-box. The complex that
ubiquitinates Sic1 is called an SCF complex (for Skp1, Cdc53, F-box protein).
Bai et al [Cell 86:263 (1996)] identified a number of proteins that contain F-box domains. They
suggested that different F box domain proteins might interact with the SCF complex (i.e., with
Skp1, Cdc53 and Cdc34) and – by interacting with alternative target proteins – lead to the
destruction of such target proteins by ubiqitination and proteasome degradation.
Skowra et al [Cell 91:209 (1997)] then demonstrated that this is the case: Another F-box protein,
Grr1, associates with Skp1, Cdc53, and Cdc34. This complex does not bind to phospho-Sic1, but
does bind to phospho-Cln2 and phospho-Cln3 – two G1 cyclins that are degraded as cells make the
G1 to S transition. It appears that the Skp1, Cdc53, Cdc34 complex – the SCF complex – can
interact with a variety of F-box proteins, via the Skp1 recognition site. The F-box containing
subunits target the SCF ubiquitination activity to different substrates.
This should all sound familiar; just as the G2/M APC/cyclosome ubiquitinating machine
can be targeted by to distinct substrates -- Cdc20 and Hct1/Cdh1-- , the G1/S SCF complex
ubiquitinating machine can be targeted to distinct substrates by distinct F-box proteins.
27

Wee1 is also degraded by an SCF Cdc34 mechanism. Recall that wee1 (a kinase that
phosphorylates p34cdc2) in S. pombe must be inactivated for cells to move from G2 into M, and
that the wee1 protein is phosphorylated and degraded. How is this degradation accomplished?
Using an oocyte extract system, Michael and Newport [(Science 282: 1886, 1998)] demonstrated
that a dominant-negative cdc34 mutant can block wee1 degradation. Thus, in addition to
ubiquitinating G1 phase cyclins and CKIs, an SCF complex regulates entry into mitosis by
initiating wee1 protein degradation. What F-box protein targets wee1 kinase for ubiquitination?
Tome-1, a protein connecing the APC- and SCF-directed protease cell cycle mechanisms.
Percent wee1
H1 H1 kinase kinase activity activity
100
50
0 50 100 150
Time (min)
0 50 100
Time (min)
1
2
Ayad et al [Cell 113:101-113 (2003)] used a biochemical search to find
new substrates for APC CDH1 . They in vitro translated pools of cDNAs,
mixed them with [mitotic extracts + CDH1] vs [mitotic extracts without
CDH1], and looked for proteins degraded in the presence of CDH1. They
identified several substrates. One they called Tome-1. As expected for a
APC CDH1 substrate, Tome-1 is degraded in a cell cycle dependent manner
similar to cyclin B. Western blots are shown for G1/S synchronized cells.
The Tome-1 sequence contains what looks like an F-box. Tome-1 co-IPs
with Skp-1 and Cul-1. Is it an F-box protein that directs SCF-dependent
ubiquitination and degradation of some substrate? If Tome-1 is reduced in
cells by using a small interfering RNA (2), wee1 degradation is inhibited.
A control oligo (1) does not block wee1 degradation. The experiment was
done by pulse labeling cells with [ 35 S]cysteine after incubation with the
oligos, then doing immunoprecipitates at various times after labeling.
Non-mitotic extracts can be stimulated to enter mitosis, and activate CDK
activity on histones. If Tome-1 is depleted from extracts with anti-Tome-1,
and the extracts are then stimulated “to enter mitosis”; i.e, to activate
histone kinase, Tome-1 depleted extracts enter mitosis much later than
IgG depleted extracts. Tome-1 is named for trigger of mitotic entry.
Tome-1 helps to regulate the transition from G2 into M by acting as an F-box protein leading to
the degradation of wee1, shifting the wee1/cdc25 balance in the direction of cdc25. This causes
dephosphorylation of cdc2 and activation of the cdc2/cyclin B kinase activity required for
mitosis. However, Tome-1 is degraded by an APC-dependent process, so that wee1 can be built
back up in the next cycle, preventing premature mitosis. Tome-1 connects the APC and SCF
protein degradation cycles.
From the yeast data base, there are over 20 “F-box” containing proteins that are candidates for
SCF-regulatory molecules that control cell cycle transitions of various types, by promoting
ubiquitination and degradation Koepp et al [Cell 97:431 (1999)].
.
Through a combination of genetics and biochemistry many substrates for the APC/cyclosome
complex (for the G2/M/G1 transition) and many substrates for the SCF complex for
interphase transitions have now been identified. This list should grow now rapidly
.
28

SCF
APC
APC
CDH1
CDH1
CDH1
APC
Grr1
Cyclin Cyclin BB
Tome-1 Tome-1
Cln1 Cln1 Cln1
Sic1 Sic1
G 1
M
Cdc 20 separase separase
securin securin
separase
29
SCF
oo oo
o o
o
S
G 2
cdc4
wee1
A SUMMARY OF PROTEOLYSIS IN THE CELL CYCLE
SUBSTRATE (SCF) FUNCTION F BOX PROTEIN
Cln1 a G1 cyclin Grr1
Cln2 a G1 cyclin Grr1
Cln3 a G1 cyclin ?
Clb5 S phase cyclin ?
Sic1 a CDK inhibitor Cdc4
Far1 a CDK inhibitor Cdc4
Rum1 b CDK inhibitor Pop1 + Pop2
Swe1 a /Wee1 c Mitotic inhibitory Met30/?
kinase
Cdc6 a /Cdc18 b DNA replication factor
Cdc4/?
Gcn4 a Transcription factor for
Cdc4
amino acid regulation
Armadillo d /β-catenin e Wnt/Wg-activated Slimb/β-TRCP
transcription factor
Cubitus interruptus (Ci) d Hedgehog-activated Slimb
transcription factor
E2F-1 e Cell cycle transcription
Skp2
factor
Gic2 e Polarized bud growth Grr1
regulator
IKB e NF-KB inhibitor β-TRCP
CD4 e (via HIV Vpu) T H cell HIV receptor β-TRCP
Putative Substrates
Cyclin D1 e G1 cyclin
Cyclin E e G1 cyclin
p21 e CDK inhibitor
p27 e CDK inhibitor
p57 e CDK inhibitor
Superscripts refer to organism: a , S. cerevisiae; b , S. pombe; c , X. laevis; d ,
D. melanogaster; e , H. sapiens; f , S. solidissima; and g , A. nidulans.
SCF Tome-1
SUBSTRATE (APC) FUNCTION
Clb2 a Mitotic cyclin
Cyclin A e,f Mitotic cyclin
Cyclin B c,e,f Mitotic cyclin
Cdc20 a /p55Cdc e APC regulator
Cdc5 a Mitotic kinase and APC regulator
Pds1 a /Cut2 b Anaphase inhibitor
Geminin c DNA replication inhibitor
Ase1 a Spindle protein
NIMA g Mitotic kinase
Tome-1 wee 1
Superscripts refer to organism: a , S. cerevisiae; b , S. pombe;
c , X. laevis; d , D. melanogaster; e , H. sapiens; f , S. solidissima;
and g , A. nidulans.

CKIs in mammalian cells are also degraded by a ubiquitin-dependent mechanism. p27(Kip1),
a CKI, is present in quiescent cells. In quiescent cells, p27 degrading activity is low, and the protein
is stable. Following mitogenic stimulation, p27 is ubiquitinated, p27 is more rapidly degraded, and
cells move into the cycle. If we remove growth factors, the ubiquitination reaction is suppressed,
p27 is elevated, and cells withdraw from cycle. p27 levels go down as the cells go into cycle,
without any change in mRNA level or change in rate of protein synthesis [Pagano et al Science
1995]. Spruck and Strohmaier [Cell Cycle 1: 250 (2002)] review mammalian SCF ubiquitin ligases.
Steve Reed [Nature reviews in Molecular Cell Biology 4:855 (2003)] reviews the relationship
between cyclic phosphorylation reactions, cyclic degradation reactions and the cell cycle.
DNA REPLICATION LICENSING. ONCE AND ONLY ONCE FOR DNA SYNTHESIS.
Recall (1) that replication of the DNA must occur once, and only once, for each gene in a cell
cycle, and (2) that – when G2 phase cells are fused to S phase cells – the G2 phase cell DNA
does not replicate, despite the presence of a factor that can cause G1 phase cell DNA to replicate.
Is there a factor that “licenses ” DNA replication, that is removed after replication in S phase? If
so, the DNA must be re-licensed before the next S phase.
DNA replication occurs at origins of
replication. There is a complex, called the
origin replication complex, or ORC, that
binds to replication origins. The ORC is
associated with origins of replication
throughout the life cycle (how might you
demonstrate this?) Licensing occurs as the
cells complete mitosis: Cdc18p, Cdt1p, and
the MCM proteins (a multi-subunit oligomer
called the mini-chromosome maintenance
oligomer) are loaded onto the ORC,
licensing it for replication.
The licensed replication complex is activated for DNA replication at the G1/S transition. In
yeast, this activation requires Cdc28/clb5 or Cdc28/clb6 –“the S phase cylcins”. In mammalian
cells, this activation requires Cdk2-cyclinE. The activation of the licensed replication complex
by the CDK results in additional proteins binding to the origin. The ORC is left at the origin,
while the assembled, activated replication machinery proceeds down the DNA. The functional
target(s) of the CDK required to activate a licensed origin are not yet known.
When the MCM replication complex leaves the origin after initiation of DNA synthesis, the
origin is converted to the unlicensed state. How does a cell prevent re-replication?
Genetic and biochemical studies have shown that the cdc18p is degraded by a F-box/SCF
ubiquitination mechanism. The cdc6/18 protein has to be resynthesized in G1, in order to license
the DNA for subsequent replication.
Without knowing about cell cycle mutants and proteolytic cycles in eukaryotic cells, we could not
have begun to understand this “must” of cell cycle biology. Nishitani and Lygerou [Genes to Cells
7: 523 (2002)] have reviewed DNA licensing recently.
30

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS AND
PROTEOMICS COME TO CELL CYCLE ANALYSIS.
The analysis of events in the cell cycle – particularly in yeast – have been the target of high
throughput screens, genomic and proteomic analysis.
Genomics, proteomics bioinformatics and cell cycle.
High throughput screens: do the temperature sensitive screens
of Hartwell and Nurse identify all cell cycle genes? It is
possible, and in fact likely, that the temperature sensitive screens
of Hartwell and Nurse would not identify all genes involved in cell
cycle control. (You might want to think about reasons why this is
the case). Stevenson et al [PNAS 98: 3946 (2001)] used overexpression
of yeast genomic fragments and cDNA libraries, in
which the genes are over-expressed from the GAL1 promoter, to
look for genes that would cause cell cycle arrest in S. cerevisiae
when the cells are shifted to galactose. Using high-throughput
screening, 150,000 colonies were screened and 179 genes/ORFs
were identified that modulate the cell cycle. Cells were analyzed
by flow cytometry after 6-8 hours in the presence of galactose.
Strains in the left column are shifted toward a 1C amount of DNA
relative to cells containing a control plasmid, cells in the right
column are shifted toward a 2C content of DNA. 43% of the
known genes cloned had previously been associated with cell
cycle. 19 new ORFs were identified that modulated cell cycle.
Seven of these ORFs cause accumulation in M phase, when
examined microscopically.
Spellman et al. [Mol Biol Cell. 9: 3273 (1998)] synchronized yeast cells three ways (with mating
factor, using the cdc15 ts mutant, and using an elutriation rotor). They extracted RNA at various
times after release from the cell cycle blocks, and then performed microarray analyses of mRNA
levels for the whole yeast genome, versus RNA from exponentially growing cells. They first
used “phase analysis,” in which they plotted the peak time of expression for these genes. They
found 800 cell cycle regulated genes; 300 in G1, 71 in S, 121 in G2, 195 in M and 113 “M/G1”
genes. They compared the upstream promoter sequences of the genes, and found increased
presence of appropriate transcription factor response elements.
They then used “cluster analysis”, which “sorts through all the data to find the pairs of genes that
behave most similarly in each experiment, and then progressively adds other genes to the initial
pairs to form clusters of apparently co-regulated genes.” Nine clusters were identified (3 in G1,
2 in S, 1 in M, and 3 in M/G1). About half the 800 cell cycle regulated genes are in these nine
clusters. Analysis of the 5’ regions of the genes in a cluster shows that such genes share common
promoter elements. They suggest these data “provide a foundation for understanding the
transcriptional mechanisms of cell cycle regulation”. For example, within the G1 group of genes
is the “CLB2 cluster” of 76 genes . 58% have at least one copy of ACGCGT (the target of MBF;
a heterodimer of Mbp1p and Swi6p; see below), vs 6% of control genes. 52% have a
CRCGAAA (the target of SBF; a heterodimer of Swi1p and Swi6p, see below), vs 13% of
control genes. There are several other “clusters” that peak in G1.
Can we discover anything new with this analysis? The “MET cluster” of 20 genes “was
completely unexpected”. Ten genes are known to be involved in methionine biosynthesis,
sequences of the others suggest roles in this pathway. 15 have AACTGTGG sequences.
31

All the data from Spellman et al, 400,000 data points, are freely available on the web for
investigation and manipulation by any informatics aficionados.
However, all is not necessarily well….. Shedden and Cooper [Nuc. Acids. Res. 30:2920 (2002)]
have reviewed this paper and conclude (1) “…when the degree of cyclicity for genes in different
experiments are compared, a large degree of non-reproducibility is found.” (2) “Specific genes
can show a wide range of cyclical behavior between different experiments; a gene with high
cyclicity in one experiment can show essentially no cyclicity in another experiment.”
Cell cycle gene profiling has also been extended to mammalian cells.
Cho et al [Nature Genetics 27: 48 (2001) synchronized fibroblasts by a double thymidine block
and analyzed mRNA profiles every two hours by microarray, using Affymetrix arrays, for
cyclically expressed genes. A duplicate experiment was performed. Twelve “patterns” were
identified, which included 731 transcripts. An attempt to classify these patterns into groups of
genes involved in related functions (e.g, amino acid metabolism, cell adhesion, etc). Again, the
entire data set is available on the web for analysis.
Whitfield et al [Mol. Biol. Cell 13: 1977 (2002)] synchronized Hela cells by (1) double
thymidine block, (2) high thymidine followed by nocodazole or (3) mitotic collection and
pooling on ice. Cells were released, RNA was isolated at various times, and microarray analysis
was performed. RNAs were prepared and assayed using spotted cDNA arrays. They identified
~850 cell cycle regulated genes. Extensive temporal correlations and functional classifications
are provided. Like the previously cited studies, the entire data set is available for analysis.
Whitfield et al compared the ~700 genes from the Cho et al paper with 595 genes that mapped,
in their own study, to unique gene clusters. Only 96 genes were identified as cell cycle
regulated in both studies!! They comment “We have no ready explanation of this difference in
results, except to note that there are differences in the cell lineage, the microarray technology,
and in the analysis methods…. We suspect that the most significant differences may well be in
the degree of synchrony achieved…”
Again, all is not well….. Shedden and Cooper [PNAS 99: 4379 (2002)] have also reviewed the
data from the Cho et al article. They performed a statistical analysis of the data and conclude that
the cyclic variations observed by Cho et al could be explained by chance fluctuations, and
conclude “We propose that the microarray results do not support the proposal that there are
numerous cell-cycle-specifically expressed genes in human cells.” An unpublished response to
the Shedden and Cooper article can be read at
http://www.salk.edu/docs/labs/chipdata/response.html. Dr. Cooper’s comment on the response of
Cho and Lockhart can be found at http://www-personal.umich.edu/~cooper/Replytocho.pdf.
Dr. Cooper argues that the chemical methods of synchrony result in “cyclic” gene expression
patterns that reflect responses to particular chemical treatments, and not functions of the cell
cycle. He argues that essentially all “synchrony” experiments (not just those involved in
microarray analysis) are flawed in their premises, design and interpretation. For those of you
interested in his alternative point of view regarding the cell cycle, you can consult his web site,
http://www-personal.umich.edu/%7Ecooper/, where his own personal views are presented, as
well as links to many of his papers.
32

A total genomic analysis of transcription factors and cis-acting genomic sequences
regulating cell-cycle specific gene expression:
A variety of transcription factors have been shown to bind to
the upstream regions of members of the various “clusters” of
cell-cycle regulated genes in S. cerevisiae. Many of the
genes expressed in the G1 phase of the yeast cell cycle bind
either SBF, a heterodimer of Swi4p and Swi6p, or MBF, a
heterodimer of Mbp1p and Swi6p. Swi4p and Mbp1p are
DNA binding proteins; Swi6p may have a regulatory
function. Iyer et al [Nature 409: 533 (2001)] used chromatin
immunoprecipitation assays to identify genes that bind SBF
and MBF targets. They used an array of intergenic
sequences, as well as coding sequences, from the yeast
genome to identify sequences that are coimmunoprecipitated
with Swi6, Swi4p or Mbp1. Using data
for enrichment of sequences in the ChIP assay and
expression data from Spellman et al, they identified about
200 candidate target genes. 49% of the Swi4 ChIP targets
contain CRCGAAA, the SBF target; only 10% of all
intergenic regions have this sequence. 55% of the MBF
putative targets have the ACGCGN sequence; only 20% of
all intergenic regions have this sequence.
Of particular interest: “Not every promoter bound by SBF or MBF in vivo contained a
recognizable consensus binding site. Moreover, most of the coding sequences and many of the
promoters that contain the consensus sequences show no evidence of binding to these factors in
vivo.” Using the data of Spellman et al, they found that 66% of the genes identified by ChIP
with Swi4p,Swi6 or Mbp1 are cell cycle regulated, as opposed to 13% of all yeast genes.
CHECKPOINTS AND THE CELL CYCLE
If a cell were to begin to divide DNA up into daughter cells before the DNA has been completely
replicated, the result would be catastrophic for the cell.
If there are errors in the DNA replication, then it would be disadvantageous to the cell to segregate
incorrectly replicated DNA.
Reinitiation of replication before duplicated genomes are segregated to daughter cells by mitosis
would be harmful to the cell.
To control for these processes cells have developed "checkpoints". For example, cells monitor the
extent of DNA synthesis, and do not allow mitosis to occur unless all the DNA has been replicated.
Failure of this sort of checkpoint results in premature chromosome condensation. Similarly, if DNA
is damaged the cell will not permit mitosis to occur until the damage has been corrected. Finally,
cells will not initiate a new round of synthesis of DNA until the 4c DNA has been reduced to a
diploid complement by mitosis. Each of these checkpoint controls is genetically determined;
mutations in specific genes in yeast can cause (i) PCC, (ii) replication of damaged DNA, and (iii)
reduplication of DNA prior to mitosis. If cells have damaged DNA they cannot enter mitosis, unless
the RAD9 gene is not functioning; this is a "checkpoint". Similarly, if cells have not completed
DNA synthesis, they cannot enter mitosis (recall the cell fusion experiments with M phase cells, in
which PCC occurs and kills the cells). These data are the experimental evidence for the existence of
"checkpoints" in the cell cycle, where completion of prior events is monitored before subsequent
events are allowed to occur.
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Fold Induction Fold Induction
Fold Induction
H4 H4-1 Control
H4 H4-1 Control
H4 H4-1 Control
34
Fold Fold Induction
H4 H4-1 Control