Cell cycle control

Mitogens

Factors (e.g. epithelial growth factor) that bind to receptors on a cell and trigger mitosis

What is the restriction point

The point at which the cell has been exposed to the right combination of factors for a sufficient length of time, to trigger its entry into the cell cycle. After this point it is now committed to cell division, and the growth factors can be removed and the cell will complete a full cycle (DNA replication, growth and cell division).

How does the transduction of growth signals occur

After the receptor has bound to a mitogen, a cascade of mitogen activated protein kinases is activated (MAPK pathway). Protein kinases use the hydrolysis of ATP to phosphorylate proteins to activate them - often transcription factors. The transcription factors enter the nucleus and cause the expression of genes encoding cyclins D and E, which bind to CDKs which phosphorylate proteins needed for cell cycle entry, growth and DNA replication

Which residues are phosphorylated in proteins to control their activity

Serine, threonine or tyrosine (they all have available hydroxyl groups)

Positive and negative control of the cell cycle

• Mitogens (including growth factors) and integrins (cell contact) trigger cell division

• Growth inhibitory signals and stress (oxidative or low nutrient) inhibit cell division

• DNA damage pauses the cell cycle until the damage is repaired (but cannot stop the cycle once it has begun)

When does mitosis occur in terms of cyclin levels

When cyclin levels are the highest W

When does DNA replicate in terms of cyclin levels

When cyclin levels are rising W

When does chromosome segregation and cell division occur in terms of cyclin levels

When cyclin levels fall

Two types of yeast

Budding yeast and fission yeast

What residues do CDK-cyclin complexes phosphorylate

Serine and threonine residues that are upstream of a proline residue that is followed by a basic residue (e.g. lysine)

What do Cdks do

Cdks (cyclin dependent kinases), when they are bound to cyclins, phosphorylate proteins involved in the cell cycle - this can either activate them or deactivate them.

Link between cyclin, CDKs and CDCs

CDKs (metazoans) and CDCs (yeast) can only function as kinases when they are bound to cyclin

Roles of different cyclins

• Cyclin D - promotes the cell to move from G1 to S (G1-Cdk)

• Cyclin E (and cyclin A) - DNA replication and growth (G1/S-Cdk)

• Cyclin A - growth (S-Cdk)

• Cyclin B - promotes movement from G2 to mitosis, and chromosome segregation and cell division (M-Cdk)

Three major regulatory transitions in the cell cycle

• Start / restriction point - occurs during late G1, when the cell commits to entering the cell cycle and chromosome duplication

• G2/M transition - early mitotic events, leading to chromosome alignment on the spindle, are triggered

• Metaphase-to-anaphase transition - sister chromatid separation stimulated

How are cyclins involved in positive feedback

Previous CDK-cyclins phosphorylate transcription factors that enable the expression of later phase cyclins

Role of protein phosphatases

Reverse the effects of CDKs, by dephosphorylating proteins - this is crucial to reset the cell cycle, and to dephosphorylate proteins that prevent the next stage of the cell cycle from occurring

How does ubiqiutin work

• Ubiquitin acts as a tag that marks proteins for destruction

• Ubiquitin E3-ligase enzymes recognise degrons on their target proteins (in this case cyclin), and identify the lysine residues on the substrate

• They covalently attach ubiquitin to this lysine by an isopeptide bond from the ubiquitin’s C terminus

• Ubiquitin can then form isopeptide bonds with other ubiquitin proteins, forming an ubiquitin chain

How does the proteasome destroy proteins

• Proteasome recognises ubiquitinated proteins - the ubiquitin chains can bind to the caps of the proteasome

• Proteasome cleaves off ubiquitin chain so that it can be reused

• Proteasome destroys the protein in an ATP dependent manner

Two main cell cycle ubiquitin ligases

• SKP1/Cullin/F-box (SCF) - recognises phosphodegrons (degron sequences marked by phosphorylation - this is a phosphorylation-dependent switch - the enzyme will then add ubiquitin)

• Anaphase promoting complex/cyclosome (APC/C) - recognises a small linear sequence - the destruction box (Dbox)

What does APC/C destroy

• Securin (in metaphase) - destruction of securin activates a protease which separates the sister chromatid pairs, so triggers anaphase

• S- and M-cyclins (cyclins A and B) - this inactivates most of the CDKs in the cell, which means that many proteins that the CDKs phosphorylated in early mitosis are dephosphorylated by phosphatases

• APC/C triggers the metaphase-to-anaphase transition

When do SCF and APC/C act?

• SCF is active early on in the cell cycle

• APC/C becomes active later on, when it has been phosphorylated by CDKs

How are cyclins involved in negative feedback

Later cyclins (e.g. cyclins A and B) phosphorylate the degrons of earlier cyclins (D and E), which promotes their ubiquitination by SCF, so they are destroyed. Cyclins D and E are only really stable when mitogens are causing their synthesis.

Functions of cyclin B

Cyclin B associates with its Cdk to form M-Cdk. This phosphorylates proteins, activating them, that are trigger the formation of the mitotic spindle, chromosome condensation and the attachment of sister chromatids to the mitotic spindle, and in animal cells, the breakdown of the nuclear membrane.

Why must cyclins be destroyed

So that the proteins they phosphorylate can be dephosphorylated by phosphatases, so that the next stage of the cell cycle can proceed, or so that cyclin levels, and therefore Cdk levels, fall to the levels required for G1 to begin again.

M cyclin (cyclin B in vertebrates) and positive feedback

• Accumulation of M-cyclin in G2 leads to more M-Cdk complexes

• The Cdk subunit is phosphorylated by CAK (Cdk activating kinase), but inhibitory phosphorylation at two neighbouring sites by Wee1 means that it is inactive

• The activation of the phosphatase Cdc25 removes the inhibitory phosphates

• The activity of Wee1 is suppressed

• M-Cdk is able to activate its own activator (CAK) and inhibit its inhibitor (Wee1) - this leads to positive feedback, leading to all the M-Cdk complexes being quickly activated, “leading to rapid phosphorylation of many proteins that drive the early events of mitosis”