lecture 6 - Signalling by Secreted Peptides

Question 1

Describe how protein phosphorylation acts as a molecular switch

Answer 1

•Activities of many proteins in signalling pathways are regulated
by the removal or addition of phosphate groups.
•Addition or removal of phosphate groups changes the
conformation of the proteins
•Phosphorylation can either activate or inhibit protein function
•One third of eukaryotic proteins are phosphorylated

Question 2

What are protein kinases?

Answer 2

•Enzymes that transfer the terminal phosphate group from ATP to
the hydroxyl group of tyrosine, serine or threonine residues in
protein chains.
•2% of genes in the human genome encode kinases
•Each cell may contains hundreds of different protein kinases.
•The amino acid sequence of each kinase determines its target specificity
•Each kinase has many different protein targets

Question 3

What are protein phosphatases?

Answer 3

• Phosphatases catalyse the reverse reaction of removing phosphate groups from tyrosine, serine of threonine residues in phosphorylated proteins (phosphoproteins).
• Kinases and phosphatases can either activate of inhibit protein function

Question 4

What are GTP binding proteins?

Answer 4

•GTP (guanosine triphosphate) binding proteins are an important
class of molecular switch
•GTP binding proteins exist in two states
1) An inactive GDP (guanosine diphosphate) bound form
2) An active GTP (guanosine triphosphate) bound form

Question 5

What are Small monomeric GTP binding proteins?

Answer 5

•Small proteins involved in relaying growth factor signalling and regulating many cellular processes
i. cytoskeletal behaviour
ii. growth and cell division
iii. differentiation
•In active state are able to hydrolyse bound GTP back to GDP
•This GTPase activity means that they can inhibit their own activity

Question 6

Describe how GTP binding signalling takes place

Answer 6

• Cycling to the active GTP bound state requires another protein, guanine nucleotide exchange factor (GEF).
• Cycling to the inactive GDP state requires the GTPase activity of the GTP binding protein and a GAP protein (GTPase activating protein)

Question 7

What are fibroblast growth factors (FGFs)?

Answer 7

•Large family of peptide ligands.
•22 FGFs have been identified in the human genome.
•FGFs have multiple biological activities.
1) Regulation of cell growth and survival
2) Regulation of cell differentiation
3) Regulation of embryonic development

• FGFs are important in human disease eg skeletal
abnormalities and cancer.
• Tyrion Lannister and a Dachshund have a mutation in the gene coding for an FGF cell surface receptor

Question 8

Describe how FGF signalling takes place

Answer 8

•FGF are paracrine or autocrine signals that bind to a
family of four transmembrane receptors.
•The intracellular domain of the protein has tyrosine
kinase activity.
•FGF ligands form a complex with extracellular
proteoglycans (proteins rich in attached sugars).
•Binding of ligand to the receptor causes receptor
dimerization and activation of receptor kinase activity.
•Activated receptors phosphorylate tyrosines on various
target proteins and autophosphorylate tyrosine residues
within their own intracellular domains.
The signal has now entered the cell

Question 9

How is the receptor activated?

Answer 9

Extracellular matrix has Immunoglobulin-like domains required for ligand binding specificity.
Plasma membrane has hydrophobic domain that allows insertion into the membrane.
Cytoplasm has intracellular tyrosine kinase domain

Question 10

Describe how adaptor proteins pass the signal on

Answer 10

Phosphotyrosine residues within the receptor’s intracellular domain act as docking sites allowing other signalling proteins to interact with the receptor complex.

Question 11

Describe how Ras GTPase passes the signal on

Answer 11

•The Grb2 adaptor protein recruits a guanine nucleotide exchange factor (GEF) to the receptor complex.
•A GEF (SOS, son of sevenless) stimulates Ras to cycle to the active GTP bound form
•GAP (GTPase activating protein) stimulates the GTPase activity of Ras and its cycles back to inactive GDP bound form.
Activated Ras can pass the signal on to a number of downstream signalling pathways.
•Ras is a critical regulator of signalling by many growth factors.
•Mutations in Ras which lock into the active GTP bound form are oncogenic and are very common in human cancer.

Question 12

Describe how amplification takes place by the MAP kinase cascade

Answer 12

• The signal is passed on by a cascade of three protein kinases
• Raf (MAP kinase kinase kinase) is recruited to the inside of the cell membrane by activated Ras.
• Raf phosphorylates and activates MEK (MAP kinase kinase).
• MEK phosphorylates Mitogen Activated Protein Kinase (MAP kinase) also known as Erk(Extra-cellular signal Regulated Kinase)

Question 13

Describe how MAP Kinase acts as a mediator of FGF signalling

Answer 13

•Erk (MAP kinase) is phosphorylated and activated
within minutes of stimulating a cell with an FGF ligand.
•Erk phosphorylates and regulates the activity of multiple proteins including other kinases and regulatory proteins.
•Erk translocates to the nucleus where it phosphorylates
and modulates the activity of multiple transcription
factors.
•Stimulation of this pathway can rapidly lead to changes
in gene transcription.

•Treatment of embryonic cells with FGF rapidly
elevates levels of phosphorylated ERK
• Treatment with MAP kinase phosphatase inhibits this
phosphorylation
• Levels of total ERK are unchanged

Question 14

Describe how transcription of early response genes is activated

Answer 14

•Activation of MAP kinase and phosphorylation of
transcription factors in the nucleus leads to changes in
gene expression.
•Stimulation of MAP Kinase pathway rapidly (30-60
minutes) activates transcription of at least 100 early
response genes.