Bio 2B03 Module 6 Lecture 2 Script PDF

BIO2B03 scripts Module 6, lecture 2 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we continue our Module 6 on Cell Signaling, and in particular, in this Lecture 2, we will focus on...

BIO2B03 scripts Module 6, lecture 2 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we continue our Module 6 on Cell Signaling, and in particular, in this Lecture 2, we will focus on Phosphorylation in signaling pathways. So let’s get started! Slide 2 The objectives of this lecture are: to describe key steps in two signal transduction pathways. These are the cytokine receptor and JAK-STAT pathway and the receptor tyrosine kinase (RTK) and Ras pathway. In particular, we will identify similarities and differences between these pathways, and we will also interpret experimental results that define components of these signal transduction pathways. Let’s start first, with the cytokine receptors and JAK- STAT pathway. Slide 3 About 2 million new erythrocytes (or red blood cells) are produced per second in the adult human body. These cells develop in the bone marrow and circulate for about 4 months in the body, before they are digested and recycled by phagocytic white blood cells called macrophages. Typically, red blood cells are replaced when mitotically-proliferating pluripotent stem cells (or progenitor cells) stop dividing and instead start to differentiate, or commit into a specific cell type. The signal for maturation of the erythrocytes is the signaling cytokine protein, erythropoietin (or Epo). Expression of this erythropoietin is regulated by an oxygen-binding transcription factor in our kidney cells. Once produced, this erythropoietin is released from our kidneys into the circulatory system. While this is a very public signal, note however, that it is only the erythrocyte progenitor cells that carry the erythropoietin receptor (or EpoR). Since erythropoietin is a cytokine, then the erythropoietin receptor is a cytokine receptor. This receptor is linked to the JAK-STAT signal transduction pathway. Overall, when activated, the resulting cellular responses in the target cell include inhibition of cell death, changes in patterns of gene expression, and differentiation. Let’s look closer at the components of this cell signaling pathway. Slide 4 Following the principles of cell signaling, within this pathway, there is a signal (the Epo protein), a receptor (the erythropoietin receptor), an intracellular signal transduction pathway (JAK-STAT), and a responding change in target cell behavior. Now normally, the erythropoietin receptor is inactive as a monomeric, single-pass transmembrane protein. B u t , w h e n erythropoietin is available, the erythropoietin signal interacts with two Epo receptors, initiating dimerization. Shown here are the cytosolic domains of two cytokine receptors associated indirectly through a single Epo molecule. Slide 5 Now what’s special about the erythropoietin receptor, is that it has three functional domains: the cytosolic domain, the transmembrane alpha-helix domain, and the extracellular domain. Functionally, each erythropoietin receptor is associated with a JAK kinase protein on its cytosolic domain. A JAK kinase in the unphosphorylated state is inactive, with very weak kinase activity. However, when erythropoietin binds, this leads to dimerization of the erythropoietin receptors, and the two JAK kinases that are associated with the receptors are brought close together. In fact, so close together, that their weak kinase activity is sufficient to phosphorylate a neighbouring JAK kinase. This is called autophosphorylation. In particular, phosphorylation of the activation lip on JAK kinases is what activates kinase activity in these JAK proteins. JAK kinases have many targets of phosphorylation, including tyrosine residues on the intracellular domain of the receptor.Overall, the JAK kinase is specifically a tyrosine kinase, meaning that only tyrosine residues are phosphorylated. So what happens after this receptor activation, and how does this lead to the cellular response in erythrocyte progenitor cells? Slide 6 Activation of the erythropoietin receptor initiates a cascade of intracellular events. To start, the phosphorylated docking sites, once phorsphorylated, become available for protein-protein interactions. Importantly, this includes binding with the STAT transcription factors. These STAT proteins go from inactive to active, based on their interactions with these receptors. Specifically, monomers are inactive, however, once they dimerize they become activated. This activation is dependent on STAT protein phosphorylation. How does this happen? Well STAT has a protein-protein interaction domain called SH2 that specifically recognizes phosphorylated tyrosine residues. In this way, STAT proteins can accumulate on the erythropoietin receptor docking sites, and the STAT protein will be proximal to the JAK kinase. Because of this, STAT itself becomes a target of phosphorylation by JAK kinase. With phosphorylation of STAT, this then allows STAT dimerization and changes the  conformation such that a nuclear localization sequence is unmasked or made visible. With that, now, the STAT dimer can be transported through the nuclear pore complex into the nucleus to activate transcription of target genes. Let’s take a closer look at how STAT is able to recognize phosphorylated tyrosine residues on this receptor. Slide 7 The SH2 domain is a protein-protein interaction domain that is essential to the function of the cytokine signaling pathway. The domain has no enzymatic function, it simply allows a protein to bind to specific target substrates. This may have the effect of re-localization of a protein within the cytoplasm. This is the case as we saw the STAT protein. Alternatively, an SH2 domain can also play a role by linking together proteins in a pathway. In this case, the SH2 domain itself did not change, but its target did: in the case of the cytokine receptor, either the docking site tyrosine was phosphorylated or unphosphorylated. We see here a more detailed representation of the SH2 domain. Specifically, the SH2 domain of a protein is represented in red. Above that is the target peptide, which fits perfectly within the SH2 binding pocket. Here, the target peptide backbone is shown in yellow and the R-groups are shown in green. The target peptide sequence is: Pro-Asn-pTyr-Glu-Glu-Ile- Pro. So, when the target protein has this sequence, SH2 domains will bind with high affinity and specificity to this sequence when the tyrosine is phosphorylated (shown here in blue), but binds with low affinity when the tyrosine is unphosphorylated. You can see how the P-tyrosine and isoleucine fit precisely within the SH2 binding pocket. This allows for reversible binding of the SH2 domain to the target peptide. Slide 8 But the SH2 domain and its interactions is only one example of a protein to protein interaction domain. In reality, there are many examples of protein- protein interaction domains. Interestingly, all are responsible for linking together two proteins. In some cases, binding is dependent upon reversible modifications to the target peptide. The SH2, PTB, and 14-3-3 domains bind peptides containing phosphorylated tyrosine with high affinity, but not the corresponding unphosphorylated peptide. This allows protein- protein binding to be reversible. Other domains bind to sequences that are not modified, for example PDZ domains bind hydrophobic residues at the C-terminus of a protein, and SH3 and WW domains bind proline-rich domains on proteins. Such binding would not be reversible. Slide 9 Now let’s get back to the model cytokine receptor that we have been looking at. Red blood cell production, often referred to as erythrogenesis is associated with activation of a specific STAT protein, that is the STAT5 transcription factor. Once the erythropoietin receptor is activated, there are many genes that are regulated by the activated STAT5 transcription factor that are necessary for the differentiation of erythroid progenitor cells into mature red blood cells. One example is the gene, Bcl-xL. This gene codes for the Bcl-XL protein that is an inhibitor of apoptosis. By inhibiting apoptosis, the erythroid progenitor cells persist and eventually differentiate. Keep in mind though, that while bone marrow is the primary source of erythrogenesis, red blood cells are also formed in the liver. This is particularly evident during development when all red blood cells are formed in the fetal liver. This provides a visual assay or technique for detecting the activation of the cytokine/JAK-STAT pathway. Let’s take a look at this example here. On the left, we see a wildtype mouse embryo. As you can see, the abdomen is bright red because the fetal liver is creating red blood cells. In contrast, on the right, we see a mouse embryo that lacks the erythropoietin receptor. In this case, the mouse is homozygous for a loss of function allele of the erythropoietin gene. So while the liver is still intact, we do not see the bright red colouration because no red blood cells are being made. As you can expect, other mutations in genes that code for proteins that are required at different steps in the cytokine pathway can cause the same phenotype. This includes mutations in the genes coding for the erythropoietin signal itself, the JAK kinase, the STAT protein, or even the Bcl-xL apoptosis inhibitor protein. Slide 10 Just as important as turning on the signal is turning off the signal. As you can imagine, disabling erythrogenesis or red blood cell formation by eliminating the erythropoietin receptor is lethal. But…failing to turn off the signal can also be lethal. This is because continual activation of the cytokine pathway leads to overproduction of red blood cells. This results in an elevated haematocrit, also known as an elevated red blood cell count. So what’s the problem with this? Wouldn’t this be a good thing for animals? Being able to make the most red blood cells possible? Actually no. It’s not a good thing! This is because increasing the concentration of red blood cells in the blood increases the viscosity of the blood. The result of this could lead to a blockage of the narrow capillaries of the circulatory system. With any blockage in a blood vessel, this can result in a stroke or heart attack. Now keep in mind that sometimes, increasing haematocrit can be used in advantageous way as well. For example, some athletes intentionally increase their haematocrit by taking exogenous erythropoietin (that is erythropoietin from an external source to their body). This practice is known as “Epo doping”. But why would an athlete do this? Well, in this case, the increased red blood cell count leads to increases in the capacity of the athlete to carry oxygen and increases endurance. Especially during aerobic activities. Y e t while this may seem like a reasonable approach to take, this is actually a very dangerous practice that can be quite lethal. But overall, how is the cytokine/JAK-STAT pathway typically turned off? Well, two mechanisms for disabling the pathway have been identified. The first is by reversing phosphorylation. In this case, a phosphatase protein will dephosphorylate modified amino acid residues. For example, the SHP1 phosphatasehas two SH2 domains that allow it to dock at the same docking sites as the STAT transcription factor. Phosphatase activity itself is actually stimulated by binding of the SH2 domains to its phosphorylated ligands. As a result, localization of SHP1 to the docking sites mediates dephosphorylation of JAK kinase, turning off the signaling pathway. This is a short-term inactivation because removal of SHP1 allows for the fast reactivation of JAK kinase. Slide 11 The second way of turning off erythrogenesis is through the SOCS protein. SOCS is also able to bind to the phosphorylated docking sites via an SH2 domain. SOCS is expressed in response to high oxygen levels in the body. By using SOCS proteins, many of these will bind to the docking sites, resulting in blocking access to these sites by the STAT protein. In addition, SOCS is an E3 ubiquitin ligase that targets JAK kinase for ubiquitinlation and degradation through the proteasome. The removal of the JAK kinases turns off the signaling pathway. Following this degradation of JAK kinase, reactivation is slow as it requires the expression of new JAK kinase proteins. Experimentally, mutations have been identified in the erythropoietin gene that creates truncated versions of the erythropoietin receptor. These proteins have shorter docking sites. These mutant versions of the protein have been associated with a decreased sensitivity to the negative regulators of the JAK-STAT pathway, SHP-1 and SOCS. The result is a higher haematocrit. Interestingly, individuals carrying this variation in their own genome, will show red blood cell levels that are comparable to a person who is taking exogenous erythropoietin,but yet they are not participating in any Epo-doping at all. Finally, another long-term inactivation of the pathway would include receptor recycling and signal release. Many signaling pathways can be turned off when the receptor is internalized through endocytosis and the ligand dissociates. With this, while the receptor can be recycled back to the surface of the cell when it is needed, if systemic erythropoietin levels have gone down, the receptor will not be reactivated. Slide 12 So far, we have looked at the cytokine receptor and JAK-STAT signal transduction pathways. A second model signaling system is the Receptor Tyrosine Kinases (or RTKs) which are associated with Ras G- protein activation. RTKs are involved in many signaling pathways that induce cell differentiation, cell survival or apoptosis, cell division and proliferation, or changes in cell metabolism. One example illustrated here is the neural growth factor, NGF. On the left is an illustration of how NGF is able to rapidly induce differentiation of neural cells including the formation of extensive axon growth. Other hormones that act through RTK pathways include platelet derived growth factor (PDGF), epidermal growth factor (EGF), and insulin. In all cases the hormone will interact with a  transmembrane receptor tyrosine kinase, and will activate what we refer to as the intrinsic kinase activity of that receptor. Slide 13 While the RTK receptor also acts through phosphorylation of target proteins, it differs from the cytokine receptors in structure. RTKs have an extracellular signal binding domain, a single-pass transmembrane domain, and intrinsic kinase activity on the cytoplasmic domain of the protein. Similar to the cytokine receptors it is ligand binding that leads to dimerization of the receptor and autophosphorylation of the kinase domain. However, the intracellular RTK signal transduction pathways are much longer than in the cytokine pathway and involves activation of an intracellular, membrane- anchored protein called Ras. Regulation of Ras requires proteins that link it to the activated RTK including adaptor proteins (GRB2) and the Ras effectors, GEF and GAP. Ras G-protein activation then leads to a kinase cascade that culminates in activation of MAP kinase. In the end, MAP kinase modulates cell behavior by phosphorylating transcription factors and changing patterns of gene expression. Slide 14 RTK activation is similar to activation of cytokine receptors. Specifically, ligand binding leads to dimerization of the transmembrane receptors. In this example, the EGF hormone binds to each of the receptors, changing the conformation of the extracellular domain, and induces dimerization. Dimerization of the extracellular domains is seen in the ribbon diagram at the bottom. Slide 15 Now, the monomeric RTK receptor has very poor intrinsic kinase activity. But, dimerization has the effect of bringing two intrinsic kinase domains very close together, so that even the weak kinase activity can lead to phosphorylation or autophosphorylation of the neighbouring activation lip of the protein tyrosine kinase. Phosphorylation of the activation lip increases the intrinsic kinase activity of these receptors, allowing the phosphorylation of more target proteins. Tyrosine residues on the intracellular docking sites of the RTK receptor are then targets of kinase activity. These phosphorylated docking sites are then potential binding sites for protein-protein interaction domains such as SH2 and PTB. Slide 16 Adaptor proteins carry two or more protein interaction domains that allow the proteins to act as linkers between other proteins. These are two examples of adaptor proteins that bind to both an activated RTK receptor at docking sites, and also to other proteins in the signaling pathway. This has the effect of indirectly linking proteins in the signaling  pathway to the RTK receptor. In addition to the monomeric adaptor proteins that we see here, there are also adaptor proteins called scaffold proteins that have multiple protein-protein interaction domains, and can assemble proteins in an ordered series. Slide 17 GRB2 is an adaptor protein with three protein- protein interaction domains. These are one SH2 domain and two SH3 domains. The SH2 domain recognizes phosphorylated tyrosine residues on the RTK docking site, while the SH3 domains bind to the next protein in the pathway. The ribbon diagram on the right shows how protein folding brings together the two SH3 domains at each terminus. Binding of the SH2 domain is dependent upon the reversible phosphorylation of the docking sites. In contrast, the SH3 domains always bind to proline-rich domains on partner proteins, such as SOS. Slide 18 The SH3 domain is represented by the half-sphere at the left and the peptide-binding domain is shown by the clefts in the surface of the proline-rich regions of a target protein. This diagram illustrates the molecular complementarity that defines binding between the SH3 domain and the proline-rich target peptide. The same interaction is shown in a space- filling diagram at the right. In red we see the SH3 domain of an adaptor protein, in yellow is the backbone of the target peptide, and in green are the proline amino acid residues of the peptide. Again, the highly specific interaction is revealed by the way in which the shapes of the two surfaces complement one another. Slide 19 With RTK receptor activation, this will also lead to activation of a G- protein, called Ras. G-proteins, or GTPase switch proteins, are all regulated in the same way. These are GTP-binding proteins. When bound to GTP, the protein is active, but when bound to GDP, the protein is inactive. The G-protein also has intrinsic GTPase activity; that GTPase activity is always active, though its activity can be modulated by other factors. At the left is the GTP bound G-protein (here, in the ON state). As you can see, in the GTP-bound state the arms, or switches, interact specifically with the terminal phosphate on the GTP in the nucleotide binding site. The GTP here is represented as GDP plus that terminal third phosphate. Here, GTP fits specifically within the binding pocket of the G- protein, and the negative charge on that terminal phosphate interacts with glycine and threonine residues on each switch. This interaction causes the pulling of the switches together. We further see this “ON” conformation illustrated in the ribbon diagram below.  The intrinsic enzymatic activity of the G-protein is distinct from the ON and OFF states of the protein. It is not the GTPase activity that is turned on and off. However, this GTPase activity is required for turning the protein “OFF”. On the right is the conformation of the G-protein in the “OFF” state. With this G-protein, GTPase hydrolyzes the terminal phosphate of GTP, releasing inorganic phosphate (Pi). GDP now remains in the nucleotide-binding pocket. In the absence of that terminal phosphate, the switches are no longer held inwards,and instead fold out. In this conformation, the G- protein is “OFF”. The ribbon diagram illustrates how the glycine and threonine residues cannot interact with GDP and so the switches fold out. Finally, GDP has a very low affinity for the nucleotide- binding pocket, so GDP will leave. Because of this, the G protein will remain in its “OFF” state, until a GTP comes in the binding pocket and activates the protein again. This cycling between GTP hydrolysis, GDP release, and GTP binding regulates G-protein activity of any G-protein, including Ras. Slide 20 We see overlaid in this image, the “ON” and “OFF” states for the G-protein. Here, the ON state is represented in red, and the OFF state is represented in green. This figure illustrates the difference in conformation of the switch proteins that modulate G-protein activity. Slide 21 Though monomeric G-proteins have intrinsic GTPase activity, there are other proteins that assist in the regulation of G-protein inactivation and activation. GEF is a guanine nucleotide exchange factor. Any GEF promotes dissociation of GDP and allows GTP to enter the nucleotide-binding pocket. In this way, GEF acts as an activator of G-protein activity. In contrast, a GTPase activating protein, or GAP, promotes inactivation of the G-protein by enhancing the intrinsic GTPase activity. GAP proteins are able to accelerate the intrinsic GTPase activity by 100 fold or more and rapidly inactivates the G-protein. A third regulator is a guanine nucleotide dissociation inhibitor, or GDI. This protein increases the affinity of the nucleotide-binding pocket for GDP, keeping the G-protein inactive or “OFF”. Slide 22 The cycle of Ras G-protein activation and inactivation in the RTK pathway is summarized in this diagram. To start, the G-protein is represented in the inactivate conformation by the turquoise rectangle. The “OFF” state is maintained when GDI promotes GDP binding. Counteracting this,a GEF protein promotes the dissociation of GDP. When GDP leaves the binding pocket, GTP readily comes in. GTP binding happens quickly because there is a high concentration of GTP in the cell and the nucleotide binding pocket has a  high affinity for GTP. Once the protein is bound to GTP it is “ON”. While the G-protein is on, it can interact with its target protein. From there, GAP promotes the inherent GTPase activity of the G- protein, which hydrolyzes GTP to GDP. Now the G- protein is “OFF” and can no longer interact with the target protein. This diagram also illustrates the idea of a cellular clock. That is, that a G-protein remains ON for a fixed amount of time, depending upon the presences of the modulator protein, GAP. With that, the length of time that the G-protein is ON determines how much of the target protein is activated and for how long it is activated. Elimination of GAP would result in an increased duration of the active G-protein. In the case of a signaling pathway, this would mean that signaling would persist for longer. Slide 23 So let’s bring this all back to the Ras G-protein and its GEF and GAP proteins. The Ras G-protein interacts with a GEF called SOS. The SOS protein is a protein that is relocated to the cell membrane through indirect association with the activated RTK receptor. Here, the SH3 domains of the GRB2 adaptor protein hold SOS close to the membrane. This has the effect of bringing SOS proximal to the membrane anchored Ras G-protein. The interaction of SOS with Ras promotes the release of GDP and the subsequent binding of GTP, thus activating Ras. Slide 24 Three conformation states for the Ras protein are shown here in these ribbon diagrams. At the left is the inactive Ras-GDP, in the middle is SOS binding that displaces GDP, and at the right is the active Ras- GTP. Slide 25 So while SOS is the GEF protein for Ras, the GAP protein, NF1, enhances the intrinsic GTPase activity of Ras, and accelerates the rate of hydrolysis of GTP. This in the end, inactivates Ras. In particular, the presence of NF1 will shorten the length of time that the G protein is active. We know this, because experimentally, a loss of function mutation in the NF1 gene, will lead to an elimination of GAP protein activity in this pathway and results in an increased length of time that Ras is active. This allows signaling to persist longer than it should. In pathways that regulate mitosis, this can mean increased rates of cell division. This is where the NF1 gene, or neurofibromatosis 1 gene, gets its name. Specifically, because loss of function mutations are associated with tumorigenesis in neural systems Slide 26 Summarized here is the activation of the Ras protein by a series of proteins that link the activated RTK receptor dimer to Ras. So far, we have described the initial part of the signaling pathway at the cell membrane. But how does the signal get transduced to the nucleus to alter patterns of gene expression? Slide 27 Well, we have seen that Ras activation is downstream of RTK receptor activation. This means that Ras activation occurs after RTK receptor activation and that Ras activation is dependent upon prior RTK receptor activation. An experiment that first revealed this association between RTK receptors and Ras is illustrated here. Both of Ras and RTK receptors were identified as proteins that can signal cell division. We can hypothesize that this could occur in different ways 1) RTK activates Ras (that is RTK is upstream of Ras); 2) Ras activates RTK (that is RTK is downstream of Ras); 3) or RTK and Ras are in parallel, independent pathways, and that either pathway can lead to cell division; or 4)RTK and Ras are in parallel pathways, and both are needed for cell division. Those were the four models for RTK signaling at the time that these experiments were carried out. From there, researchers carried out these experiments on cell cultures grown in petri dishes. The cells are shown here in this cartoon as the red ovals that are shown within each petri dish. Now, if we add EGF, the signal binds to the EGF- RTK receptor on the surface of the cell, and this typically induces cell division, and there is a corresponding increase in red ovals. This is the control that we see on the left - showing that with the right signal, and with cells that contain the right receptor, we can induce cell division. In the middle condition, the researchers removes Ras function by adding an antibody to Ras to the dish. Adding an antibody blocks the ability of Ras to interact with its target substrate and eliminates Ras activity. This is done BEFORE EGF is added. So, with this antibody treatment, when EGF is added, there is no cell division. This suggests that despite activation of the RTK receptor by the EGF signal, by blocking a downstream step (Ras, with the antibody ), we have eliminated signaling. At the right , the researchers replaced Ras with a constitutively active form of the Ras protein, Ras-D. The D stands for a dominant mutation. This protein variant lacks GTPase activity and so the G-protein is always active. This time, no EGF is added and the RTK is not activated. Despite this, the cells are dividing rapidly. This suggests that Ras is downstream of the RTK because we have bypassed the effect of an  inactive RTK by having a constitutively active downstream component. This response looks a lot like the over-proliferation of cell division that we see in tumorigenesis. In fact, in human cells, a constitutive, dominant mutation in Ras is associated with the formation of tumours in many different cell types. Slide 28 Mutations that cause constitutively active Ras protein are associated with many human cancers. One example is a mutation that leads to the elimination of a single glycine residue in Ras that blocks binding of the GTPase accelerating protein. Mutations in genes coding for other components of the pathway are also associate with cancer. Her2 is a receptor tyrosine kinase that has been linked to hereditary forms of breast cancer. Wildtype Her2 is a receptor for the EGF signal. The mutant variant of Her2 does not respond to the signal and instead is always activated. This is because the mutant Her2 is always dimerized, even in the absence of that signal ligand. Constitutive dimerization of Her2 receptor leads to uncontrolled cell division. NF1 is another component that leads to uncontrolled cell division when absent. Think now, about other components of this signaling pathway could lead to tumorigenesis? Slide 29 So we have seen that Ras can be activated and inactivated. What then? Well, Ras activation leads to activation of its own target protein, Raf. Phosphorylated residues on Raf are bound by the 14- 3-3 adaptor protein. This holds Raf in an inhibited conformation. However, binding of Raf to Ras, leads to the release of Raf from the binding 14-3-3 adaptor protein, and activates Raf. Slide 30 Raf is a serine/threonine kinase protein that is at the top of a kinase cascade in the RTK pathway. This is a kinase that will phosphorylate serine and threonine amino acids on target proteins. Once activated, Raf (also called MAP kinase kinase kinase) leads to phosphorylation of its target protein MEK (also known as MAP kinase kinase). From there, MEK phosphorylates MAP Kinase at two residues, tyrosine and threonine, activating this protein at the end of the cascade. MAP kinase is a serine/threonine kinase that dimerizes upon activation and is translocated to the nucleus where target transcription factors are phosphorylated and activated. A scaffold protein acts to hold the proteins in this cascade close together in an ordered series. Slide 31 Activation of MAP kinase is illustrated in these ribbon diagrams. The activation lip contains the threonine and tyrosine residues that are targets of the dual- specificity MEK kinase. The change in conformation of the activation lip reveals the ATP and substrate binding pockets. This is similar the mechanism that  we have seen in various kinase proteins. In the end, MAP kinase is a downstream target of all Ras-linked RTK signaling pathways. Slide 32 But what is MAP kinase doing? Well, two targets of the active MAP kinase dimer are shown here. P90 RSK kinase is phosphorylated in the cytoplasm, allowing it to be translocated into the nucleus. In addition, MAP kinase is itself translocated into the nucleus. Once inside the nucleus, these two kinase types each phosphorylate a target transcription factor. One, is the ternary complex factor, or TCF that is directly phosphorylated by MAP kinase, the other is the serum response factor, or SRF that is directly phosphorylated by P90 RSK. Together, these transcription factors bind to a DNA sequence called the serum response element. The serum response element or SRE, is an enhancer sequence upstream of a collection of genes. When the two transcription factors bind to the SRE and form this complex, they promote the assembly of RNA polymerase and transcription of the target gene. Many target genes contain the upstream SRE including C-fos. C-fos is a gene that codes for another transcription factor that enhances rates of transcription of genes that are required for regulating and turning on the cell cycle. Slide 33 Ultimately, the change in cell behavior at the end of the RTK signaling pathway is transcriptional activation and the induction of cell division, differentiation, and other cell behaviours. The size of the protein shapes in each step of this pathway illustrates an important concept. Each kinase enzyme can target multiple proteins. This allows amplification of the signal at each step. One EGF signal could lead to millions of activated ERK1/2 MAP kinase proteins and in turn millions of copies of the target proteins required for cell division. In this way, the signaling pathway in our cells can be very sensitive to low concentrations of hormones that are secreted into the circulatory system and present at even just nanomolar (or 10-9 M) concentrations. Slide 34 And that’s all for today’s lecture. In summary, we have looked at two signaling pathways: 1) Cytokine receptors are signal receptors that are associated with kinase activity. As we saw today, the JAK-STAT pathway is an example of a cytokine pathway that responds to erythropoietin signal by increasing red blood cell production. And finally, 2) Receptor tyrosine kinases (RTKs) bind to hormones and act through the Ras G-protein to initiate a kinase cascade. The end result is the regulation of gene expression.