CPPS 325 FINAL EXAM QUESTIONS WITH 100% CORRECT ANSWERS Latest Updates 2024, Exams of Nursing

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CPPS 325 FINAL EXAM QUESTIONS

WITH 100% CORRECT ANSWERS

Latest Updates 2024

What are the 5 well-known enzyme-linked cell surface receptors? - Answer 1. Receptor Guanylyl Cyclase: produces cGMP

2. RTK: phosphorylate tyrosine residues leading to association of signalling molecules and cascade
3. TK Associated Receptors: associate with proteins having TK activity
4. Receptor Tyrosine Phosphates: Removes phosphate on tyrosine residues
5. Receptor Serine/Threonine Kinases: phosphorylate specific Serine/Threonine residues on IC signalling molecules How are RTK and TK Associated Receptors different? - Answer RTK phosphorylate tyrosine while associated receptors bind/find proteins with TK activity to initiate the cascade of signalling. What is the reciprocal of RTK? - Answer Receptor Tyrosine Phosphatases. They take the phosphate off the tyrosine residues to inactivate signalling cascades. Ex. Transforming GF Beta superfamily of receptors, cell type dependent. What is the major mechanism for receptor signal transduction? - Answer Tyrosine kinase phosphorylation RTK vs. Serine/Threonine Phosphorylation? - Answer Tyrosine phosphorylation is rare (1%) relative to serine/threonine residue phosphorylation. Tyrosine phosphorylation is the major mechanism of receptor signal transduction. What is the net effect of activated TKs? - Answer Tyrosine phosphorylation on target proteins. What do TK pathways mediate? - Answer Cell growth (growth factors), differentiation, host defense, metabolic regulation, cell survival pathways (survival factors). What are amplifiers of the TK pathways? - Answer PI3-K - > PIP PLC - > DAG and InsP3&Ca2+ What are RTK mediated actions in the cell? - Answer regulation of cell proliferation, differentiation, and cell motility, promotion of cell survival and modulation of cellular metabolism.

TKs exist in either the cytosol or as transmembrane receptors, what are the differences in the two types? - Answer Cytosol receptors have an src N-terminal region. Cytosol has no membrane spanning domain. TM receptor has a single membrane spanning hydrophobic TM domain. Ex) Epidermal GF RTK. What is src? - Answer Src is a non-receptor TK, so it does not span a membrane. It is a tyrosine kinase. And although it is cytoplasmic TK it can still bind to activated RTKs. What is contact inhibition confluency? - Answer Stops Src domains signalling and therefore cells stop dividing. If there is an issue then cancer can result and mutation causes an inhibition of the stop signal so the cells keep growing and dividing. What are SH domains? - Answer Src homology domains. Src and other proteins that have src-homology domains can bing to activated RTKs. What are the different SH domains in proteins? - Answer There are SH1, SH2 and SH domains. What isSH1 domain characterized by? - Answer Catalytic domain of a protein, for example the receptor. and it has the kinase activity. It is responsible for phosphorylating tyrosine residues. What is SH2 domain characterized by? - Answer Binds peptides with consensus (positional information on C-terminal side of the phosphorylated tyrosine). Very specific. Mediates protein-protein interactions in cellular signalling cascades. Very common in proteins outside the src family. What do SH2 and SH3 domains have as a common funciton? - Answer They mediate protein-protein interactions in cellular signalling cascades. Very common in proteins outside the src family. What is SH3 domain characterized by? - Answer Interacts with proline-rich peptide targets (minimal consensus). Mediate protien-protein interatctions in the cellular signalling cascade. Very common in proteins outside the src family. What are PTB domains? - Answer (Phosphotyrosine binding domain) Bind to phosphorylated tyrosine. Functional equivalent of the SH2 domain, except for the positional informational. Is not the C-terminus like SH2, but the N-terminus side of the phosphorylated tyrosine. shc is a PTB protein.

What is the normal action that stops signalling for cell division in the src pathway? - Answer Contact Inhibition Confluency How do PTB and SH2 domains differ? - Answer SH2 domains binds consensus on the C-terminus. PTB domains binds the N-terminus. They both mediate cellular signalling. What is shc? - Answer PTB protein that docks at the N-terminus consensus docking site. What activates a RTK? - Answer Ligand/agonist binding Upon ligand binding the two activation pathways are? - Answer 1. Conformational change

2. Dimerization What happens with conformational change activation? - Answer A conformational change that is sufficient enough to activate SH1/catalytic domain results in phosphorylation. What is the only example of conformational change activation? - Answer Insulin receptor kinase. All other RTKs are activated through forms of dimerization. What are the two forms of dimerization? - Answer i) Receptors exist as unactivated dimers until ligand binds (Twist theory) ii) exist as monomers that are brought together and activated by ligand binding. What is the most common method of dimerization activation? - Answer Receptors existing as monomers. What is the Twist Theory? - Answer RTKs exist as formed dimers and when a ligand binds the dimer undergoes a twist/shift in shape so the catalytic domains can become fully active and therefore allow for autophosphorylation in key tyrosine residues outside the catalytic domain. What is the monomer dimerization activation pathway? - Answer The monomers need to dimerize to fully activate the TK/SH1 domains. Ligand must bind external domains of two monomer RTKs so they dimerize. This results in increased enzymatic activity of the IC catalytic SH1/TK domains by phosphorylating key tyrosine residues outside the catalytic domain. Transphosphorylation of tyrosines occurs so the kinase activation can occur, and transphosphorylation of regions that create the docking site. The phosphorylated tyrosine residues serve as docking sites for cytoplasmic signalling molecules.

What does RTK activation require? - Answer Dimerization and transphosphorylation!! Where are tyrosines phosphorylated - Answer They are first phosphorylated within the kinase/catalytic domains to increase the activity of enzyme and this triggers phosphorylation outside the catalytic/kinase domain that produces a docking site. What does phosphorylation of tyrosines within the kinase/catalytic domain do? - Answer Increases the kinase activity of the enzyme. What does phosphorylation of tyrosines outside the kinase/catalytic domain do? - Answer It creates high-affinity binding sites for a number of IC signalling properties. What are some IC signalling proteins that bind to the docking sites created by phosphorylation outside the kinase/catalytic domain? - Answer - PLC (amplifiers) which leads to release of Ca2+

• SRC TK (non-receptor TK)
• Adaptor proteins like Grb-2. (activation of Grb-2 leads to recruitment of SOS and activation of Ras) What is Ras? - Answer A small monomeric G-protein anchored to the inner PM involved in many signalling responses when activated to trimeric G-protein. What is Ras regulate by? - Answer Ras is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs). Ras switches between active and inactive states. Ras G protein inactive bound to GDP. GDP is exchanged for GTP by GEFs and Ras is activated. GAPs accelerate OFF by enhancing the hydrolysis of GTP to GDP. What happens when Ras proteins are mutated? - Answer Mutated Ras proteins are unable to dissociate GTP, so they are stuck in the ON or proliferative state. Can lead to cancer. What happens when Ras-GAPs when they are mutated? - Answer Mutations lead to disease because the Ras-GAPs proteins can't hydrolyze GTP back to GDP efficiently so Ras stays activated longer than it should. What is SOS? - Answer Is a guanine nucleotide exchange factor. How is Ras linked RTKs? - Answer An adaptor protein (Grb-2) and a GEF (SOS) link activated RTKs to Ras (downstrea signalling proteins). What is Grb-2? - Answer Grb-2 is a adaptor (linker) protein that couples activated RTKs to downstream signalling proteins like Ras. What is Grb- 2 composed of? - Answer Composed of SH2 and SH3 domains.

What is the function of SH2 domain of Grb-2? - Answer SH2 (Src-homology 2) domain of Gr- 2 binds to specific phosphotyrosines on activated RTK. Mediates activity. What is the function of SH3 domain of Grb-2? - Answer SH3 is involved in protein interaction and binds to proline rich regions of SOS. SOS is a GEF so it regulates Ras activity. How are RTKs linked to G-proteins? - Answer RTKs are activated and then they are coupled to downstream signalling proteins such as Ras (G-protein). RTK signalling activates G-protein signalling. RTK activation leads to association with SH2 domain of the linker Grb-2 protein. and the SH3 domain links with SOS that is a GEF that exchanges GDP for GTP on Ras. How are Ras and RTK different? - Answer Ras is a G-protein that is activated through RTK signalling. What is the linkage pathway between RTK and G-protein Ras? - Answer RTK-(SH2- SH3)-SOS-Ras-Raf (SH2-SH3) domains constitute the Grb-2 linkage protein. SH3 domain binding to the proline-rich region of SOS brings the GEF from the cytosol to the membrane. The Grb- 2 linker protein does not actually link with the signalling protein, but the exchange factor that activates the G-protein. What is Raf? - Answer Is a MAPKKK. Ras-Associating Factor. Serine/threonine kinase What is the activation of Raf associated with? - Answer Activation is associated with several proteins:

• serine/threonine phosphatase P2A
• Hsp90 chaperone heat shock protein
• Scaffold protein 14 - 3 - 3 What is the Hsp90 function in activation of Raf? - Answer Is a chaperone heat shock protein. It helps to stabilize Raf and is involved in proper cell localization. What is the scaffold protein 14 - 3 - 3 function in Raf activation? - Answer Is a phosphoserine adaptor/chaperone protein. It locks Raf in its inactive formation. It interacts with the RBD N-terminal region bound at two phosphoserine residues on Raf to inhibit Raf activation. 14 - 3 - 3 is autoinhibitory. What is an essential feature of Raf? - Answer The N-terminal region hinders the activity of the catalytic domain of Raf. There are a number of modifications that need to occur in order to remove the restraint on Raf to enable its activation.

Scaffold protein 14- 3 - 3 interacts with the N-terminal region to lock Raf in the inactive form. What is the importance of RBD in Raf activation? - Answer It is the N-terminal regulatory domain of the Raf protein. The RBD needs to interact with RasGTP to enable activaiton of Raf. Interaction of RBD with RasGTP at the membrane destabilizes Raf interaction with 14- 3 - 3. This allows for PP2A to dephosphorylate Raf phosphoserines (the locks on the inactive state). What is RBD? - Answer Ras Binding Domain. It is the Raf N-terminal domain of the Raf protein. How is Raf maintained in an inactivate state? - Answer Scaffold protein 14- 3 - 3 is bound to two phosphoserine in the N terminus on the Raf protein and locks it in the inactive form. What is the function of serine/threonine phosphatase PP2A in the activation of Raf? - Answer Once RBD has bound RasGTP the interaction between Ras and 14- 3 - 3 destabilizes. PP2A comes and dephosphorylates the phosphoserines on the Ras. Allowing for other phosphorylations on Raf activating it and causing dimerization. How does Raf associate with RasGTP to become activated? - Answer RBD binds to the activated Ras (RasGTP) and causes the destabilization of the inhibitory protein 14- 3 - 3 which allows the PP2A to dephosphorylate Raf and allow for other phosphorylations on Raf that result in activation and dimerization. How does Ras activation lead to Raf dimerization in the Raf/MEK/ERK pathway? - Answer Ras forms nanoclusters when activated and promotes Raf dimerization in the Raf/MEK/ERK pathway. Monomeric Raf is autoinhibited in cytosol. RBD domain of Raf binding to Ras is a high affinity interaction and releases autoinhibition. Ras-RBD interaction leads to Raf activation through side-by-side dimerization. What is Raf? - Answer Raf (Ras-associating factor) is a MAPKKK = Mitogen activated protein kinase-kinase-kinase. It is a serine/threonine kinase. What does activated Ras do? - Answer Activated Ras recruits MAPKKK (Raf) to the cell membrane and induces a conformational change in MAPKKK that activates its ser/thr kinase activity. What is another term for MAPKK? - Answer MEK How is MAPKK activated by MAPKKK? - Answer MAPKK binds to the c-terminal catalytic domain of Raf (MAPKKK). It is phosphorylated on two serine residues = activation.

What does activated Raf/MAPKKK do? - Answer Phosphorylates MAP kinase-kinase at two serine residues to activate it. What is MAPKK? - Answer Is a dual specificity kinase: Has both ser/thr and tyrosine kinase catalytic domains. What is the substrate of MAPKK? - Answer MAPK is the only substrate of MAPKK. It is a tightly regulated response. What is the function of MAPKK? - Answer MAPKK/MEK catalyzes a phosphorylation event on threonine and one on tyrosine to make MAP-kinase (MAPK) fully active. How is MAPK activated? - Answer Only when both threonine and tyrosine are phosphorylated by MEK/MAPKK. It dimerizes. What is the pathway of Ras activation to activated MAPK? - Answer RTK-(SH2-SH3)- SOS-Ras-(RBD-Raf/MAPKKK)-MAPKK/MEK-MAPK What are the six steps of Ras activation to activated MAPK? - Answer 1. Ras activated by exchange of GDP for GTP

2. Active Ras recruits, binds, and activates Raf
3. GTP hydrolysis leads to dissociation of Ras from Raf
4. Raf activates MEK
5. MEK activates MAPK
6. Dimeric form of active MAPK translocates to nucleus; activates many transcription factors. What is the function of MAPK? - Answer Activates many transcription factors. It induces transcription of c-fos by modifying two transcription factors (TCF and SRF). What is TCF? - Answer A transcription factor. Ternary Complex factor. It resides in the nucleus. What is the function of TCF? - Answer TCF is a transcription factor activated by phosphorylation by MAPK. It binds enhancer element SRE regulating c-fos and other transcriptional events What is SRF? - Answer Serum response element. SRF is a transcription factor. What is the function of SRF? - Answer MAPK phosphorylates and activates the kinase p90Rsk (in cytosol) which translocates to the nucleus and phosphorylates nuclear SRF (transcription factor). This increases the binding rate and affinity of SRF binding to SRE = increased transcriptional frequency.

What is the pathway of regulation of transcription activated by MAPK? - Answer MAPK phosphorylates p90Rsk which phosphorylates SRF and MAPK also phosphorylates TCF which both are trasncription factors that bind to the SRE enhancer element to regulate c-fos and other target gene transcriptional events. What is the function of SRE? - Answer Enhancer element that regulates cFos and other target gene transcriptional events. When transcription factors bind it induces transcription. What is SRE? - Answer Serum response elements. They allow for activated transcription following growth factor (mitogen) stimulation. They are found in genes involved in cellular proliferation. What is c-fos? - Answer An early response gene/transcription factor that is regulated by the RTK/Ras pathway. c-fos is an SRE. It is required for the induction of delayed response genes including cyclin D What regulates c-fos? - Answer Is regulated by MAPK. How is the RTK-Ras-Raf-MAPK pathway terminated? - Answer RTK signalling is downregulated by:

• Protein phosphatatses - PTP1B, SHP1/2 and DEP
• Activated MAPK/ERK1/2 is part of negative feedbac loop. It can phosphorylate SOS which decreases SOS' affinity for GRb- 2
• Endocytosis, RTK ubiquination What is Her2/neu/ErbB-2? - Answer RTK of EGF class. What is EGF? - Answer Epidermal growth factor. It stimulates proliferation of many cell types. Is Her-2/neu/ErbB-2 an oncogene? - Answer Yes. It has the potential to mutate and contribute to uninhibited proliferation and can induce cell transformation. It is frequently amplified/over-expressed in human tumours of epithelial origin. What happens when Her2/neu/ErbB-2(GF) is mutated? - Answer Mutations within the TM region can cause constitutive dimerization in an activated state and this, in the case of the ErbB2 receptor can induce cell transformation. It is an oncogene. Frequently amplified/over-expressed in human tumours of epithelial origin. What is the amplification or overexpression of Her-2 recognized as? - Answer Amplification of Her-2 is a recognized prognostic marker that is associated with poor survival for patients with node-positive breast cancer.

Why is mutation not necessary, but overexpression of Her-2 sufficient to become tumourigenic? - Answer If there is overexpression than it leads to ligand independent activation What is Herceptin? - Answer Designer Cancer therapy. Humanized monoclonal antibody. What is another name for Herceptin? - Answer Trastuzumab. mab because it is a humanized monoclonal antibody. How is Herceptin administered? - Answer Herceptin is given IV. Is Herceptin only used for breast cancer? - Answer No. It is also in trial for late stage pancreatic cancers that over-express Her2/neu/ErbB. It is given in combination with the chemotherapeutic drug Gemzar. How does Herceptin function? - Answer It binds to Her- 2 and blocks its activation. How can mutated Ras result in cancer? - Answer Mutated Ras can make Ras insentitive to GAP binding, so GTP can't be hydrolyzed to GDP. This results in permanent activation. Resulting in unihibited cellular proliferation. What types of cancer is Ras mutation associated with? - Answer Of the 30% of human tumours that have Ras mutation, pancreatic cancer has the most common incidence (90%), followed by colon (50%), thyroid (50%), lung (30%), ovarian (15%), bladder (6%), breast, skin, liver, kidney and some leukemias. How does Herceptin act on mutated Ras that causes cancer? - Answer What is the function of farnesyl isoprenoid? - Answer It is an unusual lipid that is involved in attachment of Ras proteins to the membrane. Farensyl isoprenoid is a post- translational modification done to Ras proteins to enable them to attach to the membrane. What is farnesyl transferase? - Answer FTase. It is the enzyme responsible for the post translational modification attachment of farenesyl isoprenoid to Ras. What is farnesylation? - Answer Protein prenylation. It is critcal for Ras localization to Inner Membrane. Why is farnesyl transferase a target for anticancer chemotherapeutic agents? - Answer It is the enzyme that attaches farnesyl isoprenoid to Ras, which is the lipid that attaches Ras to the membrane. So the rational is that if you inhibit the enzyme that allows for Ras to associate with the membrane then this will block signalling associated with abnormal cell division.

What is another target therapy, other than Her2 inhibition? - Answer FTase inhibitors. It also blocks cellular proliferation. What is Lonafarnib and Tipifarnib? - Answer They are farnesyl transferase inhibitors. What is Rigosertib? - Answer It is a drug that prevents Raf/Ras interactions to block the MARK signalling pathway to inhibit tumourigenic proliferation. What is the function of Rigosertib? - Answer It is a small molecule that binds to the RBD N terminal of Raf and prevents its association with Ras. What are three therapeutic targets in the RTK/MAPK pathway? - Answer HER2 - Herceptin binds to the RTK and prevents cellular proliferation FTase Inhibitors - Lonafarnib/Tipifarnib inhibits Ras from binding to the inner membrane and prevents proliferation RBD of Raf - Rigosertib is a molecule that binds the N terminal and prevents RAS from binding to the RBD of Raf. How is PI3-K activated? - Answer They are activated by binding to specific phospho- tyrosines on activated RTKs. What is the ultimate goal of PI3-K? - Answer It initiates the activation of a major intracellular signalling pathway that promotes cell survival. PI3-K is responsible for the activation of Akt. What does PI stand for? - Answer Phosphatidylinositol What is Atk also known as? - Answer Protein Kinase B. PKB What are the components of PI3-K? - Answer There are 2 subunits; a regulatory subunit (p85, with SH2 domain) and a catalytic subunit (p110). What is the characteristics of p85 subunit of PI3-K? - Answer There are two SH domains, an N-terminal and a C-terminal domain that recognize phosphorylated tyrosine motifs. There is consesnus. What is Akt? - Answer It is a serine/threonine kinase. What is the direct action of PI3-K? - Answer Due to the close proximity of the RTK associated PI3-K to the PIP2, PI3-K is easily able to phosphorylate PIP2, yielding the lipid 2nd messenger PIP3. What is PIP2? - Answer It is phosphatidylinositol diphosphate. It is embedded in the inner membrane awaiting phoshporylation by PI3-K so that it becomes the second messenger PIP3.

How does PIP3 function in the Akt pathway? - Answer PIP3 is able to interact with target proteins via pleckstrin homology (PH) domains found in target proteins. PIP3 is able to traslocate Akt and PDK1 to the membrane. What are the PIP3 target proteins with PH domains? - Answer Akt and Phosphatidylinositide dependent Protein Kinase 1 (PDK1) are colocalized to the plasma membrane. What is the function of PDK1? - Answer PDK1 phosphorylates colocalized Akt on threonine308, leading to partial activation of Akt. Are there multiple steps to Akt activation? - Answer Yes. It is partially activated by PDK phosphorylating its Thr308, and fully activated by the mTOR complex phosphorylating Ser473. What is the mTOR complex composed of? - Answer aka mTORC2. Contains the kinase mTOR protein kinase. Phosphoryltes ser473 on Akt. What is the function of the mTOR complex in Atk signalling pathway? - Answer mTOR complex fully activates the partially activated Akt by phosphorylating serine 437. Is it known how mTOR is regulated? - Answer No. But somehow by growth factors. What is the result of Akt activation? - Answer Phosphorylation of a number of target proteins, including mTORC1, GSK3, p53/NK-kB, FOXO, and Bad. Proteins involved in the survival pathway. What is FOXO? - Answer It is the target gene, a member of the Forkhead transcription factors that regulate transcription of death genes. How is FOXO activated? - Answer Akt enters the nucelus and phosphorylates FOXO on threonine/serine residues. What is the role of 14- 3 - 3 in the Akt pathway? - Answer 14- 3 - 3 facilitates the transport of phosphorylated FOXO from the nucleus to the cuytoplasm. Interaction with 14- 3 - 3 blocks NLS (nuclear localizing signal) and nuclear import. What is the function of phosphorylated FOXO? - Answer Phosphorylated FOXO binds 14 - 3 - 3 and translocates to the cytoplasm. This is good becuase this is an anti-apoptotic mechanism that supports cell survival. Dephosphorylated monoubiquinated FOXO enters nucleus to regulate expression of stress-associate and pro-apoptotic genes.

What happens when the PI3-K/Akt signalling pathway is switched off? - Answer Phosphorylated FOXO in the cytopasm can be dephosphorylated, enabling it to return to the nucleus. Once in the nucelus and ubiquinated, FOXO to regulate expression of stress-associated and pro-apoptotic genes. What happens when FOXO is dephosphorylated in the cytoplasm? - Answer During stress, FOXO4 isoform is bound by a uqiquitin molecule and this facilitates its entry into the nucleus. What does ubiquitin associated to FOXO facilitate? - Answer The entry of FOXO to the nucleus. Also, the interaction with various transcriptional co-activators such as p300 (HAT). How is stress-induced activation reversed in FOXO activation? - Answer There is a ubiquitin specific protease that removes ubiquitin from FOXO so that it stops stress- associated and pro-apoptotic genes. What is Usp/HAUSP? - Answer It is a ubiquitin specific protease that removes ubiquitin from FOXO so that its pro-apoptotic activity is inactivated. When ismanipulation of the PI3-K pathway used as a cancer treatment? - Answer It is a hallmark of endocrine therapy-resistance, hormone receptor-positive breast cancer and Her2 negative. When other interventions are not possible. PI3-K inhibitors are used to induce apoptosis in tumours. What is BKM120? - Answer A PI3-K inhibitor. It targets and antagonizes the PI3-K survival pathwat of the cancer cells. What are cytokines? - Answer Soluble proteins, mainly glycoproteins, or peptides that mediate interactions between cells directly and modulate the individual cells and tissues. What are cytokines involved in? - Answer They regulate hematopoiesis, immunity and development (of many different cell types), and repair of the nervous system. What types of signalling modalities are cytokines involved in? - Answer Autocrine, paracrine and endocrine What is Epo? - Answer Erythropoieitin. It is a cytokine involved in new formation of RBC. What are Epo receptors? - Answer Cytokine receptors What is CFU-E? - Answer Colony forming units erythroid

What is the mechanism of cytokine signalling? - Answer Cytokines bind to non receptor tyrosine kinases NRTK (cytosol tyrosine kinases). And the NRTKs phosphorylate variety of intracellular proteins on tyrosine following cytokine R activation. How does cytokine signalling NRTKs differ from MAPK pathway? - Answer NRTKs have a more direct link to transcriptional regulation in the nucleus. What is a key cytokine signalling pathway? - Answer JAK/STAT pathway What are JAKs? - Answer Janus Kinases. They are non-receptor tyrosine kinases. What do JAKs contain? - Answer A carboxyl catalytic kinase domain (SH1 domain, just like RTK). What is the function of the SH1 domain of JAK? - Answer SH1 domains of JAKs are phosphorylated on tyrosine residues and this increases their catalytic activity and makes phosphorylation of key tyrosine residues on the receptor possible. What is STAT? - Answer Signal trasducer and activator of transcription. It is a transcription factor. Where is STAT present in its inactive form? - Answer Cytosol What is the functional domains of STAT? - Answer An SH2 domain. The SH2 domain recognizes phospho-tyrosines and flanking sequences in the activated JAK in the receptor kinase complex. What is required for STAT activation? - Answer Phosphorylation of tyrosine by JAK. SH2 of STAT flanks the JAK which has a SH1 catalytic domain. How are STAT-DNA complexes formed? - Answer Serine phosphorylation is required for the formation of STAT-DNA complexes What does Erythropoietin require for development of erythrocytes? - Answer EpoR and JAK Does cytokine signalling share features with RTK signalling? - Answer Yes. Ligands(cytokines) bind receptors and the receptors then dimerize. Dimerization of receptors with JAK associated transphosphorylates activation lip tyrosines. Then there is phosphorylation of additional tyrosine residues. The activation lip in cytokine NRTKs is aka the blocking loop in RTKs. What is the Cytokine NRTK activation pathway? - Answer A ligand/cytokine binds to receptor and the receptors dimerize, which bring the receptor associated JAKs close to each other. JAKs becomes activated by transphosphorylation of each other's blocking/activation lip.

JAKs (SH1) then phosphorylate several tyrosine residues on cytosolic domain. Inactive monomeric STAT binds to phosphotyrosine via its SH2 domain. The pSTATs dissociate from the receptors and spontaneously dimerize. The pSTAT dimers translocate to the nucleus to regulate transciption. What genes to STATs regulate? - Answer They transcribe Suppressors of Cytokine Signalling (SOCS). Negative feedback. What are the four ways of negative regulation of the JAK/STAT pathway? - Answer 1. Short-term Regulation: phosphatases (SHPs)

2. Longterm regulation: Suppressors of Cytokine Signalling (SOCS) family. They are transcribed by STAT.
3. Degradation: ubiquitin-proteasome systems
4. Dominant Negative STATs What is short-term regulation of JAK/STAT signalling? - Answer SHP1 Phosphatase. JAK2 deactivation induced by SHP1 Phosphatase. SHP1 phosphatase can also dephosphorylate nuclear STAT proteins. What is SHP1 Phosphatase? - Answer It dephosphorylates JAK2. It has both an SH domain and a phosphatase domain. In its inactive state the SH2 domain is covering the phosphatase domain. What is the short-term regulation of JAK/STAT signalling pathway - Answer The SH domain on inactive SHP1 associates with the tyrosine that was phosphorylated by the activated JAK2. This association results in release of the phosphatase domain so that it can dephosphorylate the tyrosine on the activation/blocking lip, thereby inactivating JAK2. What is longterm regulation of JAK/STAT signalling? - Answer SOCS = Suppressors of Cytokine Signalling They are part of a regulatory feedback loop because their transcription is regulated by the JAK/STAT pathway. Signal blocking and protein degradation is induced by SOCS proteins. What is the longterm regulation of JAK/STAT signalling pathway? - Answer Dimerized pSTATs translocate to nucles where they regulate gene expression, including SOCs (negative feedback). Binding of SOCs blocks catalytic domain and/or binding of signalling molecules. Receptors are also tagged with ubiquitin and targeted for degradation. What is Primary Familial and Congenital Polycythemia (PFCP)? - Answer When your hematocrit is elevated. causing an increase in RBC mass and Hb due to a mutation in the erythropoeoietin receptor (EpoR) gene. Increases up to 50% in the oxygen carrying capacity of the bood.

Could bind STAT5 but couldn't bind SHP-1 phosphatase which normally assists in terminating the signal in short term. How is the JAK/STAT pathway involved in melanoma brain metastasis? - Answer STAT3 is upregulated in human brain melanoma cells. JAK2 is also upregulated, while expression of a negative regulator SOCS1 is decreased when compared between metastatic to levels in primary melanoma tissues. What is the critical event linked to the promotion of brain metastasis in melanoma cells?

• Answer The loss of SOCS1 negative feedback that can inhibit STAT3 activation. Increased SOCS1 expression resulted in reduced incidence of melanoma metastasizing to brain and longer lifespan. What is RNA interference? - Answer An evolutionaily conserved cellular defense mechanism for controlling the epxpression of genes. It is caused by sequence specific mRNA degradation and is mediated by double stranded RNA homologous in sequence to the target RNA. What are the practical aspects of RNAi in biological research? - Answer It is used to define a gene function through gene knockout/knowckdown. It is used to define biochemical pathways by microarray screening RNAi knockouts. What are the practical aspects of RNAi in therapeutic treatment? - Answer - Cancer
• Viral Infection
• Parasitic infections How does RNAi work? - Answer RNAi works posttransciptionally. First the dsRNA needs to be processed into 21-23 nt fragments and then once the guide strand from the fragment binds to the RISC and the complex initiates the cleavage of the complementary host mRNA. This suppresses gene expression. What is DICER? - Answer It is a specific enzyme that recognizes the dsRNA and chops it into small fragments between 21-23 bp with 2 nt 3- overhangs. These short RNA fragments are called small interfering RNA. What is siRNA? - Answer It is the product of the enzyme DICER. It is 21-23 nt fragments with 2 nt 3' overhangs. It has endonuclease activity. Small interfering RNA. What is the function of siRNA? - Answer It binds to the RNA-induced silencing complex (RISC). One of the two strands that make up the siRNA is the guide strand that is incorported into the RISC and then it pairs with complementary sequences in the cells. What is the guide strand of siRNA? - Answer It is the strand from the siRNA fragment that incorporates into the RISC. It pairs with complementary sequences in the cell. Antisense.

It targets mRNAs. What is the RISC? - Answer RNA-induced silencing complex. It is composed of ARGONAUTE and SLICER. How is the RISC activated? - Answer Activated when siRNA unwinds and the activated complex binds to complimentary target mRNA using the guide RNA. What is ARGONAUTE? - Answer It is a protein in the RISC that has a PAZ domain that binds the 3' end of the guide siRNA before they unwind. Binds the guide strand to the RISC. What is SLICER? - Answer It is part of the RISC. It cleaves the bound complementary mRNA in the middle and therefore cuase gene suppression. Once the mRNA is cleaved it can no longer be translated into functional protein and is vulnerable to degradation by RNases. What is the mechanism of action of RNAi? - Answer Double stranded RNA is introduced into a cell and gets chopped up by the enzyme DICER to form siRNA. siRNA binds to the RISC and is unwound. The antisense/guide RNA complexed with RISC binds to its corresponding mRNA which is then cleaved by the enzyme SLICER rendering it inactive. The siRNA/RISC complex can silence many copies of target mRNA. What is the mechansim of action for genome-wide RNAi screen? - Answer Screen for cell growth and viability. Each microwell contains siRNA corresponding to an individual gene in the drosophila genome. Those wells in which the cells fai to grow identify genes important for growth or viability. What is NO? - Answer Nitric Oxide It is a potent relaxant of peripheral vascular smooth muscle. Used by the body as a signalling molecule. Is NO a signalling molecule? - Answer Yes. It serves different functions depending on body system. It could be a NT or a vasodilator. It is the first gas to act as a biological messenger. What mediates local signalling by NO? - Answer cGMP. NO acts to produce cGMP which promotes smooth muscle relaxation, and increased blood flow. What is the NO signalling regulatory pathway? - Answer Ach binds to muscarinic AchRs. Ca2+ channels open causing Ca influx into the cell. Activation of calmodulin, which activates NOS (NO synthase). NO can go into muscles and act on guanylate cycase to produce cGMP.

cGMP phosphodiesterases are important in shutting down the response. What kind of concentration is NO gas normally at? - Answer Low [ ] of NO are normally present. When are drugs used to manipulate NO? - Answer To induce NO synthesis. As NOS inhibitors. Inhibitors of cGMP phosphodiesterases. What conditions are drugs used to induce NO synthesis? - Answer - Pulmonary Hypertension (nitroprusside-NO donor, open up the airways)

• Acute Respiratory Distress Syndrome (dilates pulmonary arteries, some bronchodilator activity)
• Angina Pectoris (nitroglycerin - NO donor) What conditions are drugs used as NOS inhibitors? - Answer Clinical severe septic shock induces massive drops in BP (hypotension) and so NOS inhibitors are administered as iv bolus to help increase blood pressure. What conditions are drugs used as cGMP phosphodiesterase inhibitors? - Answer Viagra is a potent and selective inhibitor of phosphodiesterase type 5 which breaks down cGMP. cGMP promotes smooth muscle relaxation, increased blood flow to the penis and inhibiting PDE5 maintains concentrations of cGMP necessary for regional smooth muscle relaxation in the corpus cavernosum. What is pains functional role? - Answer Serves a functional role in survival. What happens to people lacking pain receptors? - Answer People who are lacking pain receptors are at great