Chapter 4: Growth factor signaling and oncogenes (Book 4.1) Flashcards
There are four types of proteins involved in the transduction of a growth factor signal. Which are they?
Growth factors, growth factor receptors, intracellular signal transducers and nuclear transcription factors.
What happens when a phosphate is added to a target protein?
It induces a conformational change in the protein, resulting in the activation of inactivation of an enzymatic activity and/or serve as a recognition site for new protein-protein interactions.
There are two (or actually three) types of kinases. What type of kinase receptor is EGFR?
A tyrosine kinase receptor.
HER1-HER4 contain several domains. What are these?
- Extracellular ligand-binding domain - Single transmembrane domain - Cytoplasmic protein tyrosine kinase domain
So we just concluded that HER1-HER4 share the same domains. But HER2 and HER3 have exceptions. What are these exceptions?
HER2 does not bind to a known ligand but acts as a co-receptor for the other members of the family. HER3 has only weak kinase activity.
Name 6 steps that are required to get the signal from a growth factor outside the cell to inside the nucleus where gene expression is regulated.
- Binding of the growth factor to the receptor
- Receptor dimerization
- Autophosphorylation
- Activation of intracellular transducers (including RAS)
- Activation of a cascade of serine/threonine kinases (Raf, MEK, MAPK)
- Regulation of transcription factors for gene expression
The first step in the EGF signal transduction pathway is the binding of EGF to its receptor, EGFR. What extracellular domains of EGFR form a binding pocket for the ligand?
Extracellular domains I and III.
Describe in short dimerization of EGFRs.
It is the process of two EGFR monomers interacting to form a dimer. Binding of one EGF causes conformational change which induces the reveal of an extracellular receptor dimerization domain. This facilitates the binding to a similar domain in another EGF-bound receptor monomer, resulting in a receptor dimer.
What leads to autophosphorylation?
The conformational change upon ligand binding also disrupts intramolecular autoinhibitory interactions, leading to kinase activation.
Does autophosphorylation happen at multiple tyrosine residues of the EGFR? Why (not)?
Yes it does occur at multiple tyrosine residues. This is crucial for the recruitment of cytoplasmic substrate proteins that will pass the signal from the receptor to the signal transducers.
What are mechanisms for termination of kinase activity?
- Additional phosphorylation triggering a conformational change that inhibits extracellular ligand binding and kinase activity. - Dephosphorylation of regulatory phosphorylated tyrosine residues by tyrosine phosphatases. - Binding of negative regulators to the kinase domain - Receptor endocytosis and degradation.
Some phosphorylated tyrosine residues resulting from autophosphorylation create high-affinity binding sites for proteins that contain…
…Src homology 2 (SH2) domains.
What is the function of SH2 and SH3 domains (Src homogology 2 and 3)?
They mediate protein-protein interactions in pathways activated by tyrosine kinases.
What is the main difference between SH2 and SH3?
SH2 domains recognize and bind to amino acid sequences C-terminal to phosphorylated tyrosine residues. SH domains recognize and bind to proline and hydrophobic amino acid residues on partner proteins.
Why is this difference between SH2 and SH3 important to know?
Because Grb2 (intracellular protein with SH2 and SH3 domains) is able to recognize phosphorylated EGFR via its SH2 domain and facilitates the recruitment of specific porteins to the membrane via its SH3 domains.
What protein is recruited through the SH3 domains of Grb2? What does this protein do?
The exchange protein SOS, which will facilitate the activation of the intracellular transducer RAS.
Why are RAS proteins star players in regulating cell growth?
Because of their position in the signal transduction pathway; they act as a pivotal point for the integration of a growth factor signal initiating from the membrane with a number of crucial signaling pathways that carry the signal through the cytoplasm and into the nucleus.
RAS proteins are: N– H- and K-RAS. They are GTP-binding proteins. What does this mean?
This means that when they’re bound to GDP, they are inactive. When they are bound to GTP, they are active.
How do guanine nucleotide exchange factors (such as SOS) mediate the exchange of GDP for GTP?
By catalyzing the release of GDP from the guanine nucleotide binding pocket on RAS. Now GTP is able to bind to RAS.
How is RAS activity terminated?
Through the use of GTPase activating proteins (GAPs), they catalyze the hydrolysis of GTP to GDP to terminate the signal.
What is farnesylation (I think that it’s important to know WHY it’s important, but not necessarily what farnesylation is)?
A post-translational modification of the RAS protein which is required for localizing RAS to the cell membrane. It’s addition of the C15 farnesyl isoprenoid lipid to the C-terminal CAAX motif (C=cysteine, A=aliphatic amino acid, X=any amino acid).
What will active RAS(-GTP) activate?
Serine/threonine kinase Raf.
Besides contribution of active RAS, what else is needed for activation of Raf?
Relief of a Raf auto-inhibitory mechanism.
What will active Raf activate?
The kinase Raf phosphorylates mitogen-activated protein kinase kinase (MAPKK or MEK).
What is special about MAP kinases?
They have dual specificity, so MAP kinase activation requires both tyrosine and threonine phosphorylation.
Is Raf a MAPKK or MAPKKK? And what is MEK?
Raf is a MAPKKK and MEK is a MAPKK.
Why do MAPKs provide the cytoplasmic link between active RAS on the plasma membrane an regulation of gene expression?
Active MAPKs can enter the nucleus and here they can regulate the activity of many transcription factors via phosphorylation.
What is an important target of the MAPK cascade (name + function)?
AP-1 transcription factor, it regulates the expression of genes involved in growth, differentiation and death.
How can AP-1 induce cell cycle progression?
By binding and activating the cyclin D gene, which is a critical regulator of the cell cycle.
Of what gene families does AP-1 consist of?
Of jun and fos (these proteins contain basic leucine zupper domains that facilitate their binding as dimer to either the cAMP response element (CRE) or the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element in a target gene.
AP-1 activity is induced by two mechanisms. What are these?
- Direct phosphorylation of members of the Fos family by MAPK affects their DNA-binding activity. 2. MAPK phosphorylation and subsequent activation of other transcription factors increase the expression of both fos and jun genes. As a result AP-1 activity increases.
The Myc family of transcription factors (Myc, Max, Mad, Mxi) can dimerize in different ways and lead to distinct biological effect of grwoth, differentiation and death. What are gene targets of Myc?
N-ras and p53
What does Myc need in order to function?
Constitutive expression of Max.
What happens when Myc is present and Max is constitutively expressed?
Myc and Mac form heterodimers and bind to a regulatory sequence called the E-box, in their target genes.
What is crucial for the oncogenic, mitogenic and apoptotic effects of Myc?
Heterodimer formation.
What is the function of other heterodimers, such as Max and Mad/Mxi?
They are inhibitory for Myc function. They do this by binding to the E-box in gene promotors, but here they acts as repressors.
True/false: All receptor tyrosine kinases activate RAS.
True
Raf is an effector protein that leads to the activation of the MAPK cascade, but there are several other effector proteins of RAS activation. Name one.
Phosphatidylinositol 3-kinase (PI3K), which is a lipid kinase.
If you’ve studies the lecture, the function of PI3K is already known. What happens when the second messenger, PIP3, is produced?
PIP3 recruits the serine/threonine kinases PDK-1 and Akt to the membrane. PDK-1 then phosphorylates and activates Akt.
Where is Akt involved in and how?
It is involved in anti-apoptotic and survival roles by phosphorylating distinct target proteins. (Activated Akt can also be translocated into the nucleus where it phosphorylates nuclear substrates, including transcription factors).
What is the target of the drug rapamycin (m-TOR)?
A serine threonine kinase, it is a downstream target of Akt that is involved in promoting anabolic programmes such as lipid and nucleotide synthesis.
What is the function of SRC?
It is an intracellular tyrosine kinase that plays a role in cell proliferation and in regulation of cell adhesion, invasion and motility.
What is demonstrated by the intracellular tyrosine kinase SRC?
That cell signaling can have effects on cell behavior.
SRC is a phosphoprotein which contains SRC homology domains, including a …
SH2 and SH3 domain. It also contains a negative regulatory domain near the carboxy-terminus.
What is demonstrated when Tyr530 is phosphorylated in the negative regulatory domain of SRC?
That it will bind to the internal SRC SH2 domain which results in an intramolecular association which represses the kinase domain and thus keeps SRC in an inactive state.
How can the negative regulatory intramolecular conformation/association within SRC be disrupted?
Through the use of an EGF receptor (EGFR) and a growth factor. The autophosphorylated receptor can interact with the SH2 domain of SRC, which disrupts the conformation.
What is another way to disrupt the negative regulatory intramolecular conformation/association within SRC?
It can also be done through the use of focal adhesion kinase (FAK), FAK can bind to the SH2 domain of SRC. SRC-FAK complex can interact with target proteins (focal adhesion protein, adaptor proteins and transcription factors).
Fill in: The target proteins of the SRC-FAK complex are important for the regulation of….. (1) These …. (1) are important for cell shape and motility.
- cell matrix junctions (called focal adhesions).
Assembly of focal adhesions facilitates cell *motility/adherence*, while disassembly facilitates *motility/adherence*.
adherence and motility respectively
Acitvation of SRC leads to *assembly/disassemby* of focal adhesions and thereby permits increased *motility/adherence*.
disassembly, motility.
