Lecture 4 Oncogenes and Signalling

Question 1

How did Skolnick change his assay to determine if Ras was a kinase which generated a positive result

Answer 1

Instead of using radioactive ATP as before he used radioactive GTP (32P-GTP) and incubated the Ras protein he had extracted with this nucleotide instead. He then repeated the ion exchange chromatography and this generated a band that corresponded with the size of the Ras protein. Hence he hypothesised that Ras was autophosphorylating with GTP rather than ATP but could indeed be a kinase

Question 2

Wild type Ras requires a phosphate from the hydrolysis of GTP T or F

Answer 2

F – wild type Ras is a GTPase and doesn’t require this phosphate released from its GTP hydrolysis. Interestingly Ras mutants with a threonine at position 59 do require this phosphate hence having a gain of function

Question 3

<p>What domains in Ras target proteins does it interact with</p>

Answer 3

<p> Ras binding domain (RBD)</p>

Question 4

Wigler recognised that Ras existed in 2 distinct forms GTP bound and GDP bound. He then carried out a genetic screen in yeast. What component of Ras signalling was identified from this work and how does it fit into Ras signalling

Answer 4

Wigler identified the yeast gene cdc25 which seemingly promoted cell growth in the same way that Ras did. It was later found that this gene Cdc25 was in fact a guanine nucleotide exchange factor (GEF) involved in promoting the displacement of GDP by GTP which activates the protein.

Question 5

Describe how Ras causes changes in cell morphology and movement

Answer 5

Ras activates RalGEF which ultimately leads to the activity of cdc42 which is involved in the formation of filopodia which are important in cell migration. In addition Rac is also activated which leads to the formation of lamellipodia

Question 6

Give some examples of oncogenes

Answer 6

HRas, KRas

Question 7

What is confusing about the nomenclature of cdc25

Answer 7

The cdc25 GEF is expressed by S. cerevisiae. Another yeast strain S.pombe expresses a completely different gene product called cdc25 which is a phosphatase

Question 8

What are the three arms would see changes required for cell proliferation

Answer 8

Changes in cell growth changes in gene expression and changes in cell morphology and movement

Question 9

How did McCormack identify RasGAP

Answer 9

He incubated cRas with the lysates from a cell line (MCF7) to investigate the effects on GDP production when the cells were incubated with tritiated-GTP. This was compared to cells not incubated with the MCF7 lysate. He noticed that cells that were incubated with the MCF7 lysates saw a much greater increased in radioactive GDP levels compared to control. This corresponded to a massive decrease in the levels of radioactive GTP present. Hence something in the lysates was promoting the hydrolysis of GTP to GDP by the Ras protein. From this he was able to extract the individual protein responsible for this activity which was termed a GTPase activating protein (RasGAP).

Question 10

How did Skolnick first investigate whether Ras was a kinase

Answer 10

Skolnick carried out similar experiments to that of Erikson and Collett used to determine that src was a kinase. He extracted Ras from cells using an immunoprecipitation reaction. The extracted Ras protein was then incubated with radioactive ATP. He then used ion exchange chromatography to separate the protein based on its size and exposed this membrane to film to try and see if there was a band of radioactivity in the region where the Ras protein would be expected. This would indicate that Ras has incorporated the radioactive phosphate from the ATP and hence is autophosphorylation something which is seen in the src tyrosine kinase. Surprisingly there was no band of radioactivity which corresponded with the Ras protein

Question 11

How was it shown that Ras binds both GTP and GDP

Answer 11

Competition experiments were carried out where the wells of a 96 well plate were coated with antibodies against the Ras protein. Cell lysates were then washed over the plate allowing for Ras protein to bind to the anti-Ras antibodies. Once the Ras was bound in the wells radiolabelled GDP was added in the form of tritiated-GDP (3H-GDP). This would bind to the Ras protein allowing you to quantify how much Ras protein was present in each well by the amount of radioactivity. Then a number of different non-radioactively labelled guanine nucleotides were add to see if they were capable of displacing the tritiated-GDP. This would be indicated by a drop in the degree of radiation emitted from the well as a result of addition of the new guanine nucleotide. It was subsequently found that radioactivity was alleviated upon the addition of GTP GDP and dGTP. This determined that Ras must be a guanine nucleotide binding protein capable of binding to both GTP and GDP

Question 12

<p>What region of the Ras protein changes conformation in the GTP-bound active conformation</p>

Answer 12

<p>Effector loop</p>

Question 13

GTP hydrolysis by Ras activates the protein T or F

Answer 13

F – GTP hydrolysis inactivates the protein and switches it off

Question 14

Use the data below to describe how Gross showed that Ras was a GTPase

Answer 14

Gross expressed and purified recombinant Ras in bacteria. He used two different forms of the Ras protein proto-oncogenic cellular Ras (cRas) and the viral oncogenic form Harvey Ras (HRas) that possess the G12V amino acid substitution. He then incubated HRas and cRas with radioactive 3H-GTP and analysed the guanine nucleotides present in the sample by chromatography over a period of 120mins. The cRas lanes began to show production of GDP that could only be from the GTP that was initially incubated as no other reagents were added. A band of radioactivity was beginning to be visible at 20 mins in the cRas lane which increased in intensity up until 120 mins (lane 1). Interestingly HRas didn’t lead to any production of GDP over the entire time course of the experiments. This means that the G12V change in HRas seemed to prevent its ability to produce GDP.

Question 15

How was the mammalian homologue of SEM-5 found

Answer 15

A library of bacteria were created each expressing a mammalian gene. A sheet of nitrocellulose was placed on top of agar plate causing the bacteria expressing proteins on the surface to stick to the membrane. The bacteria on the nitrocellulose membrane were then lysed on the membrane. The DNA encoding the intracellular TKD of a receptor tyrosine kinase was used as a probe by attaching a fluorophore to it. This fluorescent TKD probe was washed over the cells expressing the library of mammalian genes. If the bacteria are expressing a gene which binds to the TKD probe then the fluorescence will become restricted to that colony. It was known where on the nitrocellulose membrane each colony were and what gene they were expressing which allowed the identification of colonies that bound to the TKD probe. This lead to the discovery of growth-factor receptor binding protein 2 (Grb2) which was later found to be homologous to SEM-5

Question 16

How was it determined where Ras fits in in terms of growth factor signalling

Answer 16

Genetic screens in Drosophila and C. elegans revealed genes with high degrees of homology with Ras and other signalling components of growth factors. It was noticed that Drosophila with mutations in the seveneless gene developed ommatidium lacking the 7th rhabdomere cell. This gene was later identified to be the invertebrate EGFR homologue

Question 17

What can be said about the binding of effector proteins to Ras is its different functional states

Answer 17

Signalling proteins bind to the GTP bound form of Ras that don’t bind the GDP bound form

Question 18

What kind of molecule is Ras

Answer 18

Ras is a small GTPase

Question 19

Give some examples of proto-oncogenes

Answer 19

cRas, EGFR, Src

Question 20

How was FRET used to investigate the binding of effector proteins to Ras

Answer 20

A FRET donor was fused to the Ras binding domain protein (protein that recognises GTP-bound Ras). This was CFP and was fused to Raf. Then the FRET acceptor (YFP) was fused to the Ras protein. Association of Ras with RasBD protein leads to FRET signal that when blue light was shone onto the cell the yellow fluorescence increased and the cyan fluorescence decreased. This only occurred in the GTP-bound form of Ras hence effector proteins recognise the specific GTP-bound form of the protein

Question 21

Describe how Ras causes changes in gene expression

Answer 21

Ras activates Raf or MAP3K. This leads to the activation of the MAPK pathway which culminates in the activation of transcription factors that translocate to the nucleus and upregulate gene transcription.

Question 22

What is the significance of threonine 59 that is present in some Ras mutants

Answer 22

This mutant form of Ras means that upon hydrolysis of GTP the phosphate that is released ends up binding covalently to threonine 59 where it remains stuck. Thus this would result in the mutant form of Ras possessing an additional autophosphorylation ability which the wild type Ras would not

Question 23

What did the crystal structure of Ras reveal about the key residues involved in coordinating its function

Answer 23

This identified to key residues glycine 12 and glutamine 61. These residues coordinate the process by which the terminal phosphate of GTP is accommodated by the Ras and hydrolysed. If glycine 12 or glutamine 61 are mutated GTP isn’t hydrolysed and Ras adopts a conformation in which it is permanently on

Question 24

Give some examples of oncogenes

Answer 24

HRas, KRas

Question 25

<p>Below is a table of a list of EGFR signalling genes from mammals Drosophila and C. elegans. Match up the genes that are homologous</p>

Answer 25

<p>See completed table below</p>

Question 26

Describe how Ras causes changes in cell growth

Answer 26

Ras activates the lipid kinase PI3K which in turn leads to the activation of PIP3 and subsequently Akt and RhoGEFs. This ultimately lead to an inhibition of apoptosis by Bad stimulation of the mTOR pathway which increases protein synthesis and additional GSK3β which is specifically involved in the stimulation of cell proliferation

Question 27

Skolnicks hypothesis that Ras was a kinase was in fact wrong explain how he got the results that he did

Answer 27

Skolnick used a cancer cell line to immunoprecipitate the Ras protein. It turns out that this cancer cell line was expressing an oncogenic form of Ras with a threonine resided at position 59 of the protein. This mutant form of Ras means that upon hydrolysis of GTP via its GTPase activity the phosphate that is released ends up binding covalently to threonine 59 where it remains stuck. This would give the impression that Ras is a kinase and is capable of autophosphorylation when in fact this is only due to the specific mutation in the oncogenic form

Question 29

Give some examples of proto-oncogenes

Answer 29

cRas, EGFR, Src

Question 30

Gross’ experiments showed that HRas was unable to produce GDP when incubated with tritiated-GTP. Explain what the actual cause of the result was and how it relates to activating proliferation

Answer 30

Cellular Ras is a smallGTPase that hydrolyses GTP to GDP. However oncogenic HRas has the G12V amino acid substitution. This means that HRas can’t hydrolyse GTP and is constitutively active. So oncogenic HRas doesn’t have its GTPase activity which normally switches it into the off state. This means that this form of Ras is stuck in the on form where it can continually activate downstream effectors.

Question 31

How did Macara’s experiments lead to the identification of the mammalian homologue of cdc25

Answer 31

Macara took purified Ras and pre-bound it to radioactive GDP (3H-GDP). He then measured the radiation over time to act as a representation of the dissociation of GDP. As GDP dissociated from the Ras protein the amount of radioactivity would decrease. He carried out these experiments with the addition of brain extract cytosol and compared the effects of this to cells not exposed to brain cytosol to see if the addition of this extract effected the dissociation of GDP. Indeed the brain extract cytosol massively increases the dissociation of GDP from Ras in a concentration-dependent manner. He later purified the single protein from this extract capable of causing this stimulation of GDP dissociation which turned out to be homologous to cdc25. This was a mammalian GEF.