RTK Signalling Flashcards
What are the two ways that signal transduction can be modular?
- The same protein fold/domain can be used for different downstream effects, binding to different signalling proteins
- The same principle, e.g. an increase in Ca2+ can be used for different outcomes, e.g. PKC and CAM kinase activation
Modularity is present in most signalling pathways. True or False?
True
What benefit is modularity to cell signalling pathways?
Modularity allows for flexible evolution and essential cross-talk between pathways
What are the four subfamilies of Enzyme-Linked Receptors?
- Tyrosine Kinase
- Guanylate Cyclase
- Tyrosine Phosphatase
- Serine/Threonine Kinase
Describe the transduction of a signal from a ligand to the cytosol through an Enzyme-Linked Receptor
Ligands induce receptor dimerisation which activates the enzymatic activity of the receptor on the cytosolic side.
This can be through mutual trans-phosphorylation of the two subunits, or recruitment of a catalytic subunit from the cytosol which is then activated.
The active enzyme will in most cases phosphorylate and thereby activate or inhibit targets to initiate signalling cascades
Give some examples of ligands and their specific enzyme-linked receptors
TGF-B and BMP -> TGFB receptors
Cytokines, Erythropoietin, Prolactin -> Cytokine receptors
EDG, PDGF, FGF, insulin, IGF-1 -> RTKs
Describe the conformational changes that occur when a ligand binds to an RTK
Ligand binding to extracellular domains brings the intracellular domains together; homodimerisation, this allows the kinase activity of one of the cytosolic domain to phosphorylate a tyrosine on the other cytosolic domain; transautophosphorylation, with causes phosphorylation of the other cytosolic domain at a tyrosine residue. *ATP
What structure connects the intracellular and extracellular domains of an RTK?
A transmembrane alpha-helix
What is IRS-1?
Insulin Receptor Substrate 1; a multi-docking scaffolding protein recruited upon activation of an RTK.
How is IRS-1 activated?
Through phosphorylation by an activated RTK after its recruitment to the cytosolic domain of the receptor
Explain how IRS-1 is a multi-docking/scaffolding protein
Phosphorylated IRS-1 recruits multiple factors that would individually be inactive but form an active complex in each others presence.
How does IRS-1 bind to an RTK?
The PTB domain, which binds to phosphotyrosine of the cytosolic domain of the receptor, is a platform for IRS-1 to bind to RTK
Which protein domains can bind phosphotyrosine on the cytosolic RTK?
PTB and SH2 domains
Which domains are able to bind phosphoinositides in membranes?
PH and PX domains
How does phosphorylation of tyrosine on RTKs regulate activity of SH2 and PTB domains?
Phosphorylation of tyrosine on cytosolic RTK enables SH2 and PTB binding while dephosphorylation prevents it.
How is the assembly of signalling platforms self-propagating?
RTK can phosphorylate itself many times
The first principle of domain classification is by sequence and 3D structure homology rather than function. True or False?
True.
Function, i.e. binding specificity, is therefore largely correct but can deviate from the classic functional classification for some domains.
What is the function of the SH2 domain?
SH2; Sarcoma/Src. Homology 2 Domain binds phosphotyrosine
What is the function of the SH3 domain?
To bind peptide sequences enriched in proline residues
What is the function of the PTB domain?
Phosphotyrosine Binding Domain
What is the function of the PH domain?
PH; Pleckstrin Homology Domain, binds phosphoinositides in membrane
What is the function of the PX domain?
PX; Plox Homology Domain, binds phosphoinositides in membrane
What is the function of the C2 domain?
2nd domain of PKC, to bind membranes in presence of Ca2+
What is the function of the PDZ domain?
To bind to ~5-7 AAs at the C-terminus of a target protein
Domains can be stringed together in a protein to form a scaffold on which signalling complexes can be found. True or False?
True.
The PTB domain of IRS-1 binds specifically to Tyr-P of which specific receptors?
Insulin Receptor (IR) and interleukin-4 receptor (IL-4R)
How is binding specificity of the PTB domain determined?
The amino acid preceding the Tyr
Which amino acid is best at determining binding specificity in IL-4R?
Alanine (best in IL-4R and good in IR)
The glutamate at pY960 - 1 (preceding the phosphotyrosine) In IR reduces the affinity 50-fold compared to alanine at this positive. True or False?
True
Which amino acids show very weak/no binding in the neurotrophin receptor TrkA and the EGF receptor?
Aspartate and Glutamine
How is specificity determined for SH2 domains?
By the amino acids surrounding pTyr
Which amino acids specify binding of SH2 domains in the SHC adaptor protein?
A Leu or a Val at the 3rd position after pTyr
What is SHC?
An adaptor protein with SH2 and PTB domains
Which amino acids specify binding of SH2 domains in Lck protein (a scr-related kinase)?
An Ile at the 3rd position after pTyr
Which amino acids specify binding of SH2 domains in PLC-gamma-1?
A hydrophobic residue, e.g. Ile at the 2nd position after pTyr
Which amino acids specify binding of SH2 domains in Syp (pTyr phosphatase)?
Specific residues up to 5AAs away.
What is Phospholipase C?
A class of membrane-associated enzymes that cleaves phospholipids just before the phosphate group.
Phospholipase C can only transduce RTK initiated signals. True or False?
False. PLC can transduce both RTK and GPCR signals.
What is the function of PLC in the GPCR pathway?
PLC-beta binds with G-alpha, forming a complex which catalyses hydrolysis of PIP2 -> DAG + IP3
What is the function of PLC in the RTK pathway?
RTK forms complex with PLC-gamma at intracellular domains, which catalyses hydrolysis of PIP2 -> DAG + IP3
Which protein kinase is a common downstream target of DAG and IP3?
PKC
Explain how IP3 increases cytosolic Ca2+ concentration.
IP3 binds to IP3-gated Ca2+ channel in ER causing local and transient Ca2+ release into cytosol.
How is IP3 degraded/inactivated?
Phosphatases either remove the 4’ or 5’ phosphate or a kinase adds a 3’ phosphate.
How are Ca2+ levels regulated in the cytosol?
Ca2+ channels in the plasma membrane, CRAC and P2X, and in the ER, IP3R, when activated provide a local increase in cytosolic Ca2+.
The SERCA (sarcoplasmic-endoplasmic reticulum calcium) pump constantly removes excess cytosolic Ca2+ into the ER.
What is the cytosolic resting [Ca2+] relative to the concentrations found in ER?
Cytosolic is ~10,000 fold lower than in ER at resting.
Only a 5-fold increase in cytosolic Ca2+ is needed for Ca2+ to be active as a second messenger. True or False?
False. Its about 10-fold but still small relative to difference in concentrations between ER and resting cytosol
What is the calcium binding motif of PKC?
C2 domain
What is the calcium binding motif of Calmodulin?
EF hands
What is the result of Ca2+ binding to its motifs on calcium-binding proteins?
As is a highly charged species, induces a large conformational change, changing the way AAs interact with each other in the protein
What is the function of Ca2+-bound Calmodulin?
Active Calmodulin binds to the helix of a kinase, resulting in autophosphorylation (of the kinase) by ATP.
E.g. CAM kinase.
This activates the kinase.
Removal of Ca2+ deactivates the calmodulin and therefore the kinase.
What is PKC?
Protein Kinase C: A group of serine/threonine kinases containing a regulatory domain attached to the catalytic domain.
What are the three historical classifications of PKC?
- Conventional (alpha, beta, gamma) - activated by DAG and Ca2+
- Novel (delta, epsilon, eta, theta) - activated by DAG only.
- Atypical (zeta) - activated by ceramide
Describe the structure of conventional PKCs.
At the N-terminus, A C1 Zinc finger binding domain in regulatory subunit binding DAG/phorbol esters.
A C2 Ca2+ binding domain in regulatory subunit.
A C3 ATP binding domain in catalytic subunit.
A C4 substrate binding domain in catalytic subunit, at C-terminus.
Describe the structure of novel PKCs.
C2-LIKE domain in regulatory subunit binding DAG only (not calcium) at the N-terminus.
Zinc finger also binding DAG in regulatory subunit.
C3 ATP binding domain with C4 substrate binding domain at C-terminal in catalytic subunit
Describe the structure of atypical PKCs.
C2-LIKE domain binding ceramide, in regulatory subunit.
C3 ATP binding domain and C4 substrate binding domain at the C-terminus, in catalytic subunit.
What structural features are common between the three classifications of PKCs?
All have a regulatory and catalytic subunit, with regulatory subunits at N-terminus and catalytic at C-terminus.
All have C3 ATP binding domain upstream from the C4 substrate binding domain, which is at the C-terminus.
What is the kinome?
The human protein kinase tree constructed based on sequence homology.
E.g. RTKs, MAPKs, PKA/B/C…. etc.
~500 different human kinases in human genome!!
Similarities between RTKs?
All transmembrane proteins
All receptors
All phosphorylate tyrosine
Part of enzyme-linked receptor family
Similarities of MAPKs?
Similar targets and function
Similar in sequence
Similarities of PKA/B/Cs?
Catalytic domains and target domains can be similar but can have different regulatory domains (e.g. the classifications of PKCs)
What are the cellular effects of PKC-alpha?
Role in autoimmunity and transplant rejection
Promotes IL2 production
Promotes IgG switching
What are the cellular effects of PKC-beta?
Cell migration
IL2 production
What are the cellular effects of PKC-delta
Promote apoptosis
Inhibit IL2 production
Role in autoimmunity and transplant rejection
What are the cellular effects of PKC-zeta?
Cell migration IL2 production Inhibits cell adhesion Inhibits apoptosis Inhibits effector cell differentiation
What are the cellular effects of PKC-epsilon?
Inhibits apoptosis
IL2 production
What are the cellular effects of PKC-theta?
Promotes cell adhesion, IL2 production, effector cell differentiation, and roles in autoimmunity and transplant rejection.
