Lecture 4 Oncogenes and Signalling Flashcards
How did Skolnick change his assay to determine if Ras was a kinase which generated a positive result
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
Wild type Ras requires a phosphate from the hydrolysis of GTP T or F
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
<p>What domains in Ras target proteins does it interact with</p>
<p> Ras binding domain (RBD)</p>
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
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.
Describe how Ras causes changes in cell morphology and movement
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
Give some examples of oncogenes
HRas, KRas
What is confusing about the nomenclature of cdc25
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
What are the three arms would see changes required for cell proliferation
Changes in cell growth changes in gene expression and changes in cell morphology and movement
How did McCormack identify RasGAP
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).
How did Skolnick first investigate whether Ras was a kinase
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
How was it shown that Ras binds both GTP and GDP
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
<p>What region of the Ras protein changes conformation in the GTP-bound active conformation</p>
<p>Effector loop</p>
GTP hydrolysis by Ras activates the protein T or F
F – GTP hydrolysis inactivates the protein and switches it off
Use the data below to describe how Gross showed that Ras was a GTPase
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.
How was the mammalian homologue of SEM-5 found
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
How was it determined where Ras fits in in terms of growth factor signalling
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
What can be said about the binding of effector proteins to Ras is its different functional states
Signalling proteins bind to the GTP bound form of Ras that don’t bind the GDP bound form
What kind of molecule is Ras
Ras is a small GTPase
Give some examples of proto-oncogenes
cRas, EGFR, Src
How was FRET used to investigate the binding of effector proteins to Ras
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
Describe how Ras causes changes in gene expression
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.
What is the significance of threonine 59 that is present in some Ras mutants
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
What did the crystal structure of Ras reveal about the key residues involved in coordinating its function
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
Give some examples of oncogenes
HRas, KRas
Below is a table of a list of EGFR signalling genes from mammals Drosophila and C. elegans. Match up the genes that are homologous
See completed table below
