18 - Intro to RTK Signalling

Question 1

Why is the study of the SRC oncogene important?

Answer 1

The study of the transforming v-src gene (these cause transformation or cancer generation in animal models) of Rous Sarcoma virus has led to:
o The important insights into the mechanisms of cellular transformation by oncogenes
o General features of protein-protein interactions in signal transduction pathways that control cell growth and proliferation

Question 2

What can the Ross Sarcoma Virus do?

Answer 2

The Rous Sarcoma Virus can produce tumours as a consequence of expression of pp60v-src

Question 3

What is the counterpart to the Rous Sarcoma virus?

Answer 3

This viral protein has a cellular counterpart, pp60c-src, that is believed to play a role in the normal control of cell growth

Question 4

What was an important advance in the discovery of pp60c-src?

Answer 4

An important advance was the demonstation that pp60c-src had associated protein kinase activity
* In fact, both pp60v-src and pp60c-src are protein kinases and detailed analysis of the phosphoamino acids in phosphorylated IgG-heavy-chain immunoprecipitates of pp60v-src showed that TYROSINE was the amino acid phosphorylated
* Shows that src itself has tyrosine activity and it can phosphorylate itself on tyrosine

Question 5

Describe an experiment involving the Rous sarcoma virus

Answer 5

• in this experient, src was introduced into cells by infecting them with Rous Sarcoma virus, in this case the cells have been labelled with radioactive phosphate
• In the non-infected cells, there are a number of spots that correspond to phosphorylated serine and threonine, with a very small spot of tyrosine
• In the infected cells, the amount of serine and threonine remains the same, however the phosphorylated tyrosine increases enormously
• In addition, cells transformed by Rous Sarcoma Virus contained significantly more phosphorylated tyrosine in the protein than control cells

Question 6

What has the research on Rous Sarcoma Virus lead to

Answer 6

• It is now known that the tyrosine kinase activity of pp60v-src is crucial to its transforming activity
• Until this study as carried out, it has been believed that cellular protein kinases only phosphorylated either serine or threonine residues in substate proteins
• This view was a consequence of cells containing relatively few phosphotyrosine residues

Question 7

What did the observation of tyrosine phosphorylation of pp60v-src lead to?

Answer 7

• The observation of tyrosine phosphorylation of pp60v-src was quickly followed by the demonstration that the plasma membrane receptor for epidermal growth factor (EGF) has an associated protein kinase activity with also phosphorylate tyrosine
• It was found that the protein sequence of the EGF receptor was closely related to the protein encoded by the transforming oncogene v-ERB-B of the avian erythroblastosis virus
• This suggests that the erythroblastosis virus has hijacked part of the EGF receptor involved in signal transduction after growth factor binding

Question 8

Structure of the EGF (epidermal growth factor) receptor

Answer 8

The EGF receptor contains two cysteine-rich extracellular domains and a cytoplasmic tyrosine kinase domain
o Cytoplasmic tyrosine kinase domain leads to phosphorylation of key tyrosine residues on the cytoplasmic tail of the receptor, which are important in generating intracellular signalling in the receptor
- the vErbB protein lacks most of the extracellular portion of the

EGF receptor required for EGF binding but possesses all but the final 32 C-terminal cytoplasmic amino-acids
o V-Erb-B is a truncated version of the receptor

Question 9

What was discovered about the EGF receptor?

Answer 9

Subsequently it was found that the EGF receptor itself is a tyrosine kinase

Question 10

What classes of tyrosine-specific protein kinases are there?

Answer 10

Many other tyrosine-specific protein kinases have now been identified and these fall into two classes:
o The cytoplasmic tyrosine kinases like pp60c-src
o The plasma membrane receptors with intrinsic tyrosine kinase activity, such as the EGF and insulin receptors

Question 11

What are receptor tyrosine kinases?

Answer 11

The receptor tyrosine kinases (RTKs) are a family of more than 50 different transmembrane polypeptides with a protein kinase domain in their intracellular portion

Question 12

What happens when RTKs bind to their corresponding growth factors?

Answer 12

Upon binding to their corresponding growth factors, these receptors initiate a complex series of intracellular processes leading to:
o cell proliferation
o cell differentiation
o cell motility
o changes in cell shape
o production of extracellular matrix
o transcription of specific genes

Question 13

How many different protein kinases are there?

Answer 13

• There are about 500 different protein kinases and roughly 400 of them are serine/threonine kinases and these are subclassified into different types
  
  • e.g., AGC contains protein kinase A, G, and C
• Only about a fifth of protein kinases are tyrosine kinases

Question 14

What kind of process is phosphorylation of proteins?

Answer 14

Phosphorylation of proteins and kinases is a reversible process, so in addition to protein kinases, we need protein phosphatases to dephosphorylate target proteins

Question 15

How many phosphatases are there?

Answer 15

• Thers a smaller number of phosphatases, around 140
• 28 are pSer/Thr which are split into subclasses

Question 16

How many pTyr are there in relation to pSer/Thr?

Answer 16

Because tyrosine kinase is important for cell growth, there is a much greater number of pTyr phosphatases
o These ae active in the cell to make sure the level of tyrosine phosphorylation in the cell is very low
o Subclassified into cysteine based and asparagine based
o In class I there is classical PTPs, and dual specificity phosphatases which actually phosphorylate tyrosine but they also dephosphorylate serine/threonine

Question 17

Structure of the EGF receptor

Answer 17

EGF receptor (v-Erb) with cysteine rich extracellular domains, a tyrosine kinase domain on the intracellular portion

Question 18

Structure of the insulin receptor

Answer 18

The insulin receptor has 2 cysteine rich domains, exists as a dimer linked together by disulfide bonds making it unique, is the only RTK which exists as a preformed dimer, two intracellular tyrosine kinase domains

Question 19

Structure of the PDGF receptor

Answer 19

PDGF receptor is a potent RTK, has immunoglobulin like extrtacellualr domain, has 2 intracellular kinase domains meaning it exhibits more of a growth response, involved in wound healing

Question 20

Role of VEGF (vascular endothelial growth factor)

Answer 20

VEGF is involved in angiogenesis, important in the generation of cancers as it provides the blood supply