Cell Cycle 2 - Signalling mechanisms of growth and division
Anil Chopra
1. Explain how ligands which activate tyrosine kinase receptors signal through the
small G protein, Ras, to activate the extracellular signal-regulated kinase (ERK)
cascade.
2. Describe how the ERK cascade pathway regulates gene expression and leads to
progression through G1 of the cell cycle.
3. Outline the principle of the molecular timing process which regulates the cell
cycle through oscillating amounts or activities of cyclins, their kinases and
inhibitor proteins
Modulating Cellular Function
Cells can be told to do different things in response to extracellular signalling
molecules:
These can include telling the cell to grow and divide:
Growth Process:
1. Hormones act on cell surface receptors resulting in the stimulation of pathways.
2. Active second messengers are produced resulting in amplification. (single
receptor results in production of many other molecules)
3. Other factors are modulated and the produced molecules eventually result in many
responses by the target cell.
Key Components of Signalling Pathways
1. Protein Phosphorylation – transfer of terminal phosphate group
from ATP to a hydroxyl group to form a phosphoester.
a. Both kinase and phosphatase are regulated within the cell.
Can turn proteins “on” or “off”.
b. Effector proteins are also affected by phosphorylation.
(works both ways).
c. Kinase cascades exist: in which kinases activate kinases
leading to signal amplification.
2. GTP Binding Proteins – molecular switches
a. In its inactive state, it has a binding site that binds the nucleotide GDP
b. When pathway activated GTP is made from GDP which changes the
confirmation of the G-protein to its active form.
Exchange
Factors
GTPase
Activating
Proteins
(not kinases!)
3. Adapter Proteins
a. Involved in binding other proteins together forming links between them
b. Grb 2 - has different
Src Homology
domains
SH3
Proline-rich
regions
(constitutive)
regions
SH2
Phosphorylated
tyrosines
(inducible)
SH3
Growth Factor Stimulation
Dividing cells are going around the cell cycle however most cells are in G0  when
growth pathways are inactivated. No growth factors around.
Cells in G0 enter the cell cycle e.g. in the process of wounding which results in the
release of many growth factors. This causes cells to enter the cell cycle at G1.
Mitogenic growth factor
(i.e. growth signals from other cells)
Receptor protein tyrosine kinase
Small G protein (Ras)
Kinase cascade
Immediate early genes
(c-jun, c-fos, c-myc transcription factors) – stimulate
the expression of other genes
(1) “myc” is a very important transcription factor and its level changes with
differing levels of growth factor. Different growth factors include:
- epidermal growth factor (skin)
- platelet derived growth factor
(2) When the receptors are in their inactive state they are “monomers”. The
ligands are DIMERS which dimerise the receptors resulting in the transphosphorylation of the two monomers.
- These phosphates are docking sites for signalling molecules.
- Receptor has tyrosine kinase activity – Receptor
Protein Tyrosine Kinase (RPTK)
(3) The receptor becomes trans-phosphorylated on tyrosine (Tyr) residues and then
adapter and signaling proteins are recruited. (e.g. Grb 2)
(4) Receptor Protein Tyrosine Kinases (RPTKs) signal to Ras
Grb
2
Exchange factor
Sos
(5) Ras
- activates protein kinase
cascade.
- Known as MAPK cascade
generically, however this
particular one is the ERK cascade.
- All this results in the changes in the target
proteins.
The amount of GTP-loaded Ras can be activated by
oncogenes. This can result in mutations which
eventually lead to an increased risk of tumour
development.
Specific
Raf
Generic
MAPKKK
(MKKK)
MEK MAPKK
(MKK)
ERK MAPK
How it Affects the Cell Cycle
The cell cycle requires strict regulation:
- complex checkpoints at different stages of the cycle
Based on “Cyclin dependent kinases” (Cdks) in the proliferating
cells.
- Cyclins bind to these molecules.
- These are expressed at certain points in the cell cycle.
(by stimulation of genes)
- Synthesised then degraded.
Different cyclin Cdk complexes trigger different events in the cell cycle such as S
phase (from the interaction of Cdk2 and Cyclin A) mitosis (Cdc2 + cyclin B) G1
progression (Cdk2 + Cyclin E). The complex formed by the binding of Cdk1 to
mitotic cyclin is Mitosis Promoting Factor (MPF).
In order for the MPF (Mitosis
CdkCdk1
promoting factor) to become
active, it needs two molecules
CAK
- Cdk Activating Kinase
(CAK): a kinase that
phosphorylates the
activating kinase.
- Wee1: an inhibitory kinase which removes the inhibitory
phosphate.
When these two molecules are present, the active MPF can form and
result either in another Cdk being activated or for the next stage of the
cell cycle to proceed. At different points in the cycle, different Cdks and
cyclins are required to form complexes and trigger the next
stage in the cell cycle. Cyclins are
Requires activating phosphorylation
sequentially synthesised so that
AND removal of inactivating
they trigger the expression of the
phosphorylation
genes necessary for the next phase of the cycle.
P
The complexes formed from Cdks and cyclins are used to phosphorylate proteins that
are needed in the cell cycle e.g.
- Nuclear lamins are proteins that breakdown the nuclear envelope but are not
activated until Cdk1 binds to cyclin B.
- Retinoblastoma proteins are used to inactivate transcription factors, however,
when a complex formed by Cdk2 and cyclin E phosphorylates the retinoblastoma
protein (Rb), then it becomes inactive and releases transcription factors:
Rbs are TUMOUR SUPPRESSORS.
E2
F
Cdk4/6cyclinD
Cdk2-cyclinE
Inactive
Transcription factor
(e.g. cyclin E)
Cdks are also regulated by Cdk inhibitors (CdkIs) e.g.:
- INK 4 family
- CIP/KIP family
Active Transcription
factor
E2
F
(e.g. cyclin E)