The Very Last Bio23 Lecture – Cell cycle and oncogenes

August 21, 2001

Figure 27.1 – a typical cell cycle

S phase – DNA synthesis takes place.

There is a specific decision point during G1 called the RESTRICTION POINT (START, in yeast). This is the time when the cell makes the commitment to another cycle of division (e.g. the cell will check for DNA damage during this time). There is a slight lag phase before going into S phase.

G2-M transition – The cell monitors whether there was complete DNA replication, DNA damage, or whether the cell is large enough for division. If there are repairs needed, the cell hangs out in this stage for a while.

Figure 27.3 – in the top half, each arrow = molecular changes in regulatory molecules (e.g. kinase)

Bottom half, red lines = additional superimposed check points that check to see if the cell is ready for the next step (assesses success of the previous step)

e. g. if you did not successfully segregate chromosomes, you don’t want to enter G1; this

is prevented by monitoring in a check point relay system.

1). Biochemical experiments

2). Yeast genetics

It turned out that both were working on the same molecules!!!

Xenopus cell – good for biochemical experiments because the female produces a huge oocyte/eggs (a single cell = 1mm in diameter!); the cell is full of stuff needed for development and you can isolate it!

Looking at meiotic cells (some changes that take place at meiosis), transition from:

S phase

Pre-meiosis I

-oocyte arrest at this stage

say it is n G2 phase

so that chromosomes can condense,

build spindle apparatus

meiosis I is completed

M phase

Metaphase of meiosis II and arrests again

Sperm enters and releases arrest

(no gap phase) S>M>S>M>…etc……

these divisions are synchronous

if you have four cells in M, all 8 subsequent cells will be in S phase at the same time

-take cytoplasm from arrested eggs at metaphase

-inject it into G2 oocyte

-this drives the G2 cell into M phase, and you get nuclear envelope breakdown, chromosome condensation, and build up of spindles

-therefore, there is an M-phase promoting factor in the cytoplasm known as MPF (M phase promoting factor)

-(if you had injected G2 cell with cytoplasm from interphase egg, the cell would not enter M-phase)

It turns out that, during the cell cycles, MPF fluctuates. (Graph) It is low during interphase, and high during M phase.

So, they purified this activity, and find out that at a biochemical level, MPF is a KINASE, which can phosphorylate other proteins.

One substrate is H1 (histone) – therefore if MPF is radioactively labeled and mixed with H1, then H1 will be radioactively labeled because the MPF transfers a phosphate to H1.

1). Catalytic subunit – cyclin dependent kinase (cdk) – this is a kinase whose activity depends on cyclin, which is the second subunit!

[catalytic subunit] remains fairly constant.

2). Regulatory subunit – cyclin

[cyclin] changes during the cell cycle.

High [cyclin] at M phase.

Low [cyclin] at interphase.

MPF’s concentration cycles, but its catalytic subunit’s concentration does not; therefore, the cyclin regulatory subunit must bind to the catalytic subunit to activate it during M phase, in order to produce the general MPF cycles.

2). Genetic experiments – they were looking for mutations that screwed up the cell cycle, so that you can identify the gene that was mutated, so that you could identify the gene products needed for a normal cell cycle.

Figure 27.8

2 types of yeast: S. pombe (Fission) –makes two equal sized daughter cells

Want to isolate mutations that prevent yeast from dividing, but they they’re not going to live! So we create conditional lethals which exhibit a phenotype at a restriction temperature (36C) but have no phenotype at another permissive temperature (23C). Look for mutations that screw up the protein product, which is made at both conditions, but does not work only at the restrictive temperature.

Possible to get mutations in genes that are not cell cycle regulators-

e.g. DNA polymerase, but this isn’t regulator of cell cycle, it’s a structural product needed for all DNA synthesis

What do you get?

Regulatory molecules – cdc (cell division cycle) mutants --->different kinds of phenotypes->some are extra big, or extra small, have 4N chromosomes (replication), or 2N chromosomes (no replication).

Fig 27.9 – wt – stained wall with dye

cdc-25 mutant – bigger than normal, couldn’t divide normally

Concentrate on cdc-2 mutant in pombe– found in above temperature shift experiment

To function properly at START and G2-M phase, it required the permissive temperature.

They cloned the gene, and realized that they had the same protein that corresponded to the cdk kinase as the oocyte people running the biochemical experiments!!!

They also cloned the human gene, which, if introduced into mutant yeast, would rescue the phenotype. Therefore, the function and sequence were conserved in evolution to drive the transition from G2-M (and at START in yeast cells).

SO…..

There are 2 critical amino acids on cdk’s catalytic subunit: Tyr 15 and Thr 161

• The association of cyclin and cdk drives the phosphorylation of both (wee-1 phosphorylates Tyr 15), but the kinase is still inactive!
• To be active, the phosphate on Tyr 15 must be removed by cdc 25, so now the kinase is activated (this is also the rate limiting step).
• Enters G2-M
• It must be these two specific aa. e.g. if you mutated Thr161 to Ala, its phophorylation would not produce an active kinase!
• Going back to Fig27.9:
• cdc25 mutant – the phosphate on Tyr 15 is never removed, so it cannot enter G2-M and just grows without being able to do cell division!
• wee-1 mutant – there is no inhibitory phosphorylation of Tyr 15, so the cdk is always active and you enter mitosis too early, and produce very short cells!

What happens if you have an over expression of wild type cdc25?

Ubiquitin mediated proteolysis:

-important for degradation of cyclin and other anaphase regulators (M ‡ A)

APC = anaphase promoting complex

The following is a slide [….

Requires ubiquitin mediated degradation of cyclin

Ubiquitin = 76aa protein

Addition of ubiquitin to protein targets it for degradation.

Different enzymes are required to attach ubiquitin.

STEPS:

1. Ubiquitin gets linked to E1
2. Ubiquitin transferred to E2
3. E3 binds substrate (protein that will be ubiquinated)
4. Transfer of ubiquitin from E2 to substrate

……] end of slide

Anaphase promoting complex (APC) = E3 <--specificity

It turns out that mitotic cyclin is a target for APC, which is cell cycle regulated and turned on partway through mitosis.

Cyclin-U-U-U-U-U (ubiquitin=U) -----> proteosome (things go into it and come out in pieces)

This activity is required so that it can exit M phase.

APC also promotes degradation of other anaphase regulators, so that mutations in APC components (its made of 8 subunits) causes the cell to be stuck in metaphase.

Yeast cdc2 mutants also have problems in START

-Yeast have one cdk (pombe. – cdc2; cerv. – cdc28) which operates in both the G1 and G2->M transitions by associating with different cyclins

e. g. START (kinase needed here)

G1----------> ---------------------------> S phase [cig2 – G1 cyclin]

This is in contrast to the animal cells where there are many different kinases and many different cyclins e.g. mitotic kinase -----> phosphorylates substrates A, B, C, D while…

BUT IN YEAST, there is only ONE KINASE for different cyclins!!!!

Mammals….

Fig. 27.20 – different cdk/cyclin complexes control different transitions, they are G1 and M specific (multiple cyclins, multiple cdk’s)

Cdk1=mitotic in mammals/frogs; cdk 2, 4, 6=G1

REMEMBER FIGURE 27.22!!!

• G0/G1 (passing through start) – cell needs cdk 2, 4, 6, and cyclin D (which lets cell leave G0)
• cdk2/cyclin E – needed for initiation of S phase (G1->S transition)
• cdk2/cyclin A – needed for finishing S phase

- Growth factors --> induces transcription of cyclin D and causes cdk/D complex to form

• once you’re past the restriction point, you’re committed, no turning back
• One substrate for cdk/D is RB
• in resting G0 cells, RB is UNphosphorylated
• RB binds another protein (a tx factor) called E2F which has a binding site in many S phase specific genes, and allows tx activation.
• E2F is inactive as a tx factor until RB dissociates from it
• When the cdk/D complex phosphorylates RB
• RB then releases E2F which can go an activate S phase specific genes

cdk inhibitors (cdi’s) are important for regulating G1 and S cdks

Fig 27.25 cdi’s regulate G1 and S phase cdk/cyclin complexes

e. g. inhibits kinase phosphorylation of cdk Thr 161 or it can interfere with the

phosphorylation of cyclin K/cdkD complex

There are cases of cancer where both copies of the RB gene are mutated (RB-/RB-) and leads to tumor growth on retina. If you’re born with mutated forms, its familial.

But if you started with RB-/RB+ initially, then underwent a somatic mutation to become RB-/RB-, you can also get tumor growth on the retina.

Therefore RB = tumor suppressor gene, and its normal function is to restrict growth.

If there is no RB protein, there is no break in the system and E2F can just run and activate genes continuously.

Tumor suppressor genes are different from oncogenes. Oncogenes are gain of function mutations which give you altered gene products or over expression of products, and therefore have dominant effects.

i.e. Ras mutation Ras-/Ras+ will still make an aberrant protein that makes uncontrolled growth

whereas with RB, you have to knock out both copies to get uncontrolled growth