Lecture 5 Flashcards
(22 cards)
Explain how the tyrosine kinase catalytic domain works.
It has autophosphorylation activity. 2 neighboring monomer receptors of the tyrosine kinase come together after ligand binding to the extracellular receptor domain. This allows for the recruitment of specific proteins to those phosphosites, leading to the propagation of the response.
Does phosphorylation of a protein by PKA activate or inhibit the protein?
It depends on the protein. Phosphorylation of a protein by PKA (downstream in a pathway) can either activate or inhibit it.
Phosphorylation of glycogen synthase by PKA inhibits it, whereas phosphorylation of HSL activates this enzyme.
Explain how the activation of the tyrosine kinase domain of the insulin receptor occurs.
Insulin must bind to the dimeric receptor. This triggers the tyrosine kinase domains to dimerize and autophosphorylate each other on 3 specific tyrosine residues on their activation loops. This causes the activation loop to swing out towards the aqueous environment, opening up the active site. After the activation loop moves, the c-helix will move as well. This movement of the c-helix is going to help anchor the substrate in the active site.
Explain how the formation of IP3 and DAG lead to the activation of protein kinase C (PKC.) What enzyme is responsible for the formation of IP3 and DAG and what is the molecule which is cleaved (via phosphorylase action) to form these two molecules? What is the role of PKC after its activation?
A hormone first binds to receptor region of a specific GPCR. This allows the alpha subunit to exchange its bound GDP for a GTP and the alpha-GTP dissociates from the beta/gamma G-protein subunits. It will bind to phospholipase C, leading to the activation of the phospholipase C enzyme, which begins to break down phosphatidylinositol 4, 5-bisphosphate (PIP2) into DAG and IP3.
IP3 is responsible for binding to a gated Ca2+ channel, opening the channel and releasing Ca2+ into the cytosol.
PKC will then bind to the diacylglycerol found on the inner surface of the plasma membrane and the calcium that was released from the endoplasmic reticulum will bind to the PKC.
PKC then phosphorylates other proteins found in the cell.
Many membrane receptors consist of 2 different domains (one extracellular and one intracellular.) What are these domains and their purpose?
Extracellular ligand binding domain: binds ligand in order to activate/inhibit the membrane receptor
Intracellular catalytic domain: carries out the purpose of the ligand binding
Protein kinases all share a common catalytic core. Explain how this catalytic core functions and the important structures which allow for activation of these protein kinases.
Protein kinases have 2 loops enclosing the active site: the P loop and the activation loop. Only after the ATP is bound to the protein kinase and the activation loop is moved out of the way can the protein kinase function.
What are the SH2 and SH3 domains and the function?
SH2 domains: conserved domains which can bind to phosphotyrosines. When you have a tyrosine kinase, you have a phosphorylated tyrosine and another protein into the SH2 domain. The proteins that bind to the phosphorylated tyrosine are going to be binding through the SH2 domain.
SH3 domains bind to proline rich regions.
What do serine, threonine, and tyrosine have in common?
They are all phosphorylated by kinases.
What is modularity?
different signaling complexes that can be interchanged to achieve a different effect. (like legos)
What is the role of calmodulin? How is it activated and what happens upon activation of it?
Calmodulin is an enzyme that senses and binds intracellular calcium, allowing the calmodulin to change its shape, so it can bind to other proteins to activate them. Calmodulin binds calcium at the EF hand domain, which allows for significant conformational change to occur.
What other secondary messenger molecules can GPCRs use (other than cAMP) and how are they formed?
IP3 (inositol 1, 4, 5-trisphosphate) can activate downstream targets (important for calcium release)
A modified phospholipid becomes phosphorylated twice and is then cleaved with phospholipase c to form DAG (1, 2-diacrylglycerol) and IP3.
Why is it hard to identify which ligands bind to different GPCRs? What kind of ligands bind to GPCRs?
Ligand association/dissociation to the receptors happens super quickly. Trying to identify which ligands bind takes a different approach (crystallization, UV application, etc) The classes of ligands that bind GPCRs are hormones, neurotransmitters, growth factors, etc.
Why is protein kinase A (PKA) referred to as a promiscuous kinase?
PKA doesn’t need to recognize a specific sequence in order to bind and phosphorylate a serine/threonine. There are only a few similar amino acids within the different peptide sequences that are similar which allows PKA to bind and do its job. This means that PKA is not very specific when it comes to the sequences that it recognizes.
Binding of insulin to the receptor leads to activation of the tyrosine kinase/intracellular catalytic domain. This leads to the activation of IRS-1 mediated activation of PKB, leading to phosphorylation of the c-terminal end of beta-adrenergic receptors. This also leads to SHC mediated binding/activation of MAP kinase cascade through the signaling pathway. What happens when the C-terminal end of beta-adrenergic receptors are phosphorylated? In addition, what are the effects of SHC mediated activation of the MAP kinase cascade? Why might this cross talk between the tyrosine kinase receptor and the beta-adrenergic receptor occur? Why is it important?
Activation of the insulin receptor leads to the activation of IRS-1, leading to the activation of PKB and the phosphorylation of the c-terminal end of the beta-adrenergic receptors, which is important for the recruitment of beta-arrestin, such that endocytosis of beta-adrenergic receptors is able to occur.
Simultaneously, the insulin receptor leads to phosphorylation events, thus allowing SHC to bind to phosphotyrosines utilizing their SH2 domains. SHC binding to phosphotyrosines allows for the MAP kinase cascade to occur.
By engulfing beta-adrenergic receptors and activating the MAP kinase cascade, we are ensuring that one pathway occurs and the other does not. Since insulin and epinephrine are “opposing,” this cross talk between tyrosine kinase receptors and beta-adrenergic receptors ensures that we don’t have simultaneous activation of opposing pathways.
Explain the entire insulin signaling cascade and how the binding of insulin to the receptor leads to the stimulation/expression of GLUT4.
1) Insulin binds to the extracellular receptor region of the insulin receptor, causing dimerization of the tyrosine kinase to occur. This allows for the catalytic domain to autophosphorylate each other.
2) Autophosphorylation of the catalytic domain on tyrosine residues causes the activation loop of each subunit to swing open, exposing the active site of the tyrosine kinase and activating it.
3) This will then recruit an insulin receptor (IRS-1) to bind to the tyrosine kinase receptor and that will phosphoyrlate IRS-1.
4) The phosphorylated tyrosine residues on IRS-1 creates a binding site for proteins with SH2 domains. The protein that binds is Grb-2. Sos then binds to Grb-2 and Ras is recruited to Sos, causing the release of GDP from Ras and the binding of GTP to Ras.
5) The activated Ras will bind Raf-1 to activate it. It then phosphorylate MEK on 2 serine residues to activate it. MEK will then phosphorylate ERK on threonine and tyrosine residues to activate it.
6) Activated ERK will move into the nucleus of the cell and phosphorylate SRF/Elk 1 dimer that binds to DNA and change the expression of genes.
Explain the erythropoietin tyrosine kinase pathway. What is the purpose of this pathway?
In the erythropoietin tyrosine kinase pathway (JAK-STAT,) erythropoietin will bind to a different tyrosine kinase and will cause the catalytic domain to autophosphorylate.
Dimerization/autophosphorylation of the intracellular domain allows for the recruitment of STAT to the phosphorylation sites. STAT will then get phosphorylated and have an open SH2 domain. Grb 2 gets recruited through a coupling protein which will then activate the MAP kinase cascade.
MAP kinase will enter the nucleus and STAT will homodimerize and those will enter the nucleus to bind to DNA. When the homodimerized STATs are in the nucleus, MAP kinase will phosphorylate some of the transcriptional machinery and allow for changes in gene expression. These 2 pathways end up coming together to activate the specific gene expression of Erythropoietin (EPO). The purpose of EPO pathway is used to increase red blood cells such that you are able to carry more oxygen.
How do gated ion channels work?
Through changes in membrane potential and binding of specific ligands to the receptor sites of these gated ion channels, opening of the gated ion channels allows ions to pass through a membrane.
How does the acetylcholine receptor work?
When acetylcholine binds to the acetylcholine receptor, it will rotate the subunits of the receptor, causing hydrophobic groups of the M2 helices to face away from the interior. Now, polar residues line the inside of the channel, allowing sodium and calcium to enter the neuron.
Membranes are electrically polarized. What is typically the membrane potential of most cells? Is the inside of a cell positively charged or negatively charged? How are membrane potentials maintained?
The membrane potential of (-50 to -70 mV) is maintained through the transport of sodium out of the cell and the transport of potassium into the cell. This process sends 3 Na+ out and 2 K+ in and uses 1 ATP. Since more positive charge is leaving the cell, the inside is more negative than the outside.
What does the flow of ions across a cell membrane depend upon?
It depends on the concentration of the ion and the charge of the membrane vs the ion. If there is more negative inside the cell, it’ll be hard to bring more negative ions in. However, if there’s little Cl- in the cell, the concentration gradient makes it easier for Cl- to go in.
What is GLUT4 and why might the insulin signaling cascade result in higher expression of GLUT4 genes and an increase in GLUT4 transcription/translation?
GLUT4 is a glucose transporter (membrane protein) which allows glucose to move from the blood into the cytosol of cells. The insulin signaling cascade results in higher expression of GLUT4 genes since the end goal of the release of insulin is to lower blood sugar.
What is the role of receptor guanylyl cyclases?
They are receptors that produce cGMP. The binding of a ligand to the extracellular receptor region of the guanylyl cyclase allows for the intracellular domain to produce cGMP from GTP. The effects of these guanylyl cyclases work through the activation of protein kinase.
