[MUSIC] Welcome to lecture three. In this lecture, I will continue to talk about bottom-up approaches to develop and understanding how pathways become networks using the MAP-kinase pathways/network, if you want to call it that. You might remember that in the previous lecture, I talked to you about the cyclic AMP pathway and how components within that pathway connect to one another to transmit information from the outside of the cell, information arriving in the form of a, a hormone or neurotransmitter or such as adrenaline to produce physiological events such as glucose production or activation of calcium channels or, or other such events. Here I will talk, in this lecture, I will talk some more about how the molecular interactions of the various pathways allow one to connect to a different pathway. I'm going to use a series of cartoons taken from some very nice reviews to sort of illustrate these points and I have further drawn into them as I thought I needed to, to sort of illustrate these points. So pay close attention to the pictures, so that this will allow you to sort of frame these interactions and connections. So pathways become networks when components of one pathway interact with and regulate the components of another pathway. So, here we have the MAP-kinase pathway and by way of introduction the MAP-kinase pathway is another prototypic signalling pathway that is used by many growth factors and other signals to and other extra cellular signals to transmit information to the inside of the cell to control cellular proliferation, status of cellular health etc. So the, the growth factor pathway goes to these proteins MAP-Kinases or Nitrogen activated protein Kinases and in this diagram, in this picture you can see that signal come from the epidermal growth factor receptor to the intermediary kinase called MEK to the MAP-kinase which in this pathway is called ERK. That in turn then goes ERK goes to transcription factors and to regulate gene expression and so on. The MAP-kinase pathway is actually connected and regulated by the cyclic AMP pathway and in two different ways, both stimulatory and inhibitory. The stimulatory effect comes to the cyclic AMP binding of the GEF EPAC, which we talked about last time, and this, or called cyclic AMP GEFs, and EPAC can activate the small g protein draft that allows signal to flow to the, to MEC and the map kinase pathway from Raf. Raf can also or Rap sorry, Rap can also be activated by the growth factor receptor through this, through an exchange factor called C3G please don't ask why these are named the way they are. That's a whole another story. But just remember them as proper names. Through C3G that also activates Rap. So that's the activating signal from cyclic AMP connecting into the growth factor pathway. There is also an inhibitory signal where protein kinase A can phosphorylate and inhibit the protein Raf1 and Raf1 Is important for the signal flow from the [UNKNOWN] from the growth factor receptor through Ras to Mek and by inhibiting the regulation of Raf1, the signal flow from RA, the small g protein RAF to to Mek and ERK is inhibited and just, so this cyclic AMP can in, inhibit signal flow through the ERK or MAP-kinase pathways. So you can see, depending on which components are present, the one cyclic AMP pathway can be flexibly connected to the MAP-kinase pathway. Now let's focus a little bit on the MAP-kinase pathway itself and in by focusing on the growth factor MAP-kinase pathway, I want to introduce a concept or a class of proteins that is very important for signalling. These are called adaptor proteins such as the protein Grb2 or Crk. So these adaptor proteins connect activated growth factor receptors to the exchange factors, which, in turn, can then activate the small g proteins. So in this case, again with the EGF receptor the presence of the adapter proteins, such as Grb2 and Crk, allows for the activated receptor to to use the exchange factor SOS for this, for the GTPase Ras or C3G for the GTPase Raf, and, and regulate the activation of both Rap or Ras. When Rap is activated, that in turn activates the isoform of Raf, which is a protein kinase called which is a protein kinase and this isoform is called braf. When SOS is activated, it activates the g protein, the small GTP is Ras. And Ras in turn activates another isoform of Raf called Raf 1. And both the Raf 1 and braf activate Mek, which then activates ERK, which then goes on to transcription factors. So the adaptors are very important for connecting the activated receptor to the signaling pathways, so that signal can flow from the cell surface membrane to the receptor. These adaptors and, and some GEFs use specific domains called SH2 and SH3 domains, these domains are part of these proteins and allow them to interact with one another in a highly specific manner. So now one has a really complex pathway. Please don't be sort of scared when you look at all these arrows running all over, but it's really sort of important to look at this picture carefully through each of these different areas to understand that the same receptor can activate multiple pathways, and of course, these pathways are connected to each other. Here again is this picture taken from the writ, a review written by Lemmon and Schlessinger shows the different pathways that can be connected to that are connected to the EGFR receptor. Of course as I just mentioned to you there is the grab SOS pathway that goes to Ras and to Rap Raf and to Mek, which is which is called the MAP kinase, kinase and down to MAP kinase, which is ERK, This this is ERK to to the transcription factor Fos and AP1. In addition, the EGFR can also activate, the EGF receptor can also activate the enzyme PI3 kinase that produces PIP, that produces PIP2 a lipid, that in turn activates another protein kinase AKT and that goes on to signal to cell death pathways or and it can through the exchange factor Wav activate the small g protein Cdc42 that can through through again through the various Meks and through the transcription factor CREB. Remember CREB that was activated by protein kinase A, it can also be activated by the MAP kinase pathway and control the cell cycle, and control cell cycle. In addition EGFR can activate, EGFR can activate the Enzyme Phospholipase C, Gamma Isoform that produces IP3 and Diacilglicerol remember I told you Phospholipase beta, the same enzyme, but capital G protein capital receptor. This is another isoform, and through these PKC and targets can go to a variety of pathways. So for instance, PKC can also activate Sark and Ras, and so on. And if of course activates and I won't even talk about this, activates the Transcription factors of the class STAT, which turns on different target genes, it can activate another tyrosine kinase called FAK, which can then regulate cytoskeleton. So that, just by, just by binding one receptor, this growth factor EGF in many cells can, can activate many pathways providing a rich, complex network to produce a variety of effects. You should remember that not all of these pathways are present in all cell types, but many of them are, so, the effects of EGF on a pa-, on a cell are often complex in the sense that EGF provides, uh,kinds of cell survival, it can cause proliferation and it can cause movement of the cell by regulating the cytoskeleton. Sometimes is can also cause cell death because as I, as I just told you, it can go to the apoptosis pathway. An even more complex picture here and what I want to sort of emphasize in this is there is not a single map kinase, but three different map kinase pathways that can sort of control a variety of transcription factors. All the way on the left side is a standard architecture of this pathways that there is a receptor that goes to a sort of an exchange factor that goes to a GTPAs, that goes to the first ki-, of the protein kinases called map kinase kinase kinase that goes to a map kinase kinase that goes to a map kinase that in turn can go to different substrates that may be that my include transcription factors. Using the same architecture, there are at least three different map kinase pathways and while I won't go through all the components you can see that there is a good mix and match approach where the different kinds of map kinase kinase kinase such as Raf can talk to different kinds of map kinase kinase either raf talks to Mek1 and Mek2, while MEKK2 and 3 talk to Mek 4 and Mek 7. And these in turn activate different kinds of G protein map kinases, and different kinds of transcription factors. So you can see that there is sort of a both, separation of signals to the different pathways, and the mixing of matching of signals at the top and the bottom. You can see here at the bottom, there is the, the overlap of signals, where signals from one protein kinase can activate multiple transcription factors and multiple, each transcription factor often can be activated by multiple protein kinase. This is a sort of a pattern called a by-fan, and I will deal with these by-fans, I will explain how by-fans are useful in a later lecture. You can see that by regulating these various transcription factors one can produce a real a variety of cellular and organismal effect and these include cellular effects such as proliferation, apoptosis, or inflammation and organismal effects that are tumorigenesis, or differentiation. One point I do want to make is, you might be wondering what these little tubes of the sides are. These are scaffolds or, adaptor proteins that can bind to these different map kinase and hold them together as a complex. So in this kind of account, in this picture you can notice the considerable interconnectedness. I would suggest that, don't worry too much about actually remembering the names, but look through these pictures carefully to understand how the connectivity at the upstream and the downstream level works when there is a sort of a relatively selective pathway in the middle. So now, I am providing you with a much more simplified version of this picture, so that you can sort of get your get to, get us understanding of the core organization. So this is again the same signalling pathway. There is Grb and SOS and then there is RAS. SOS is the exchange factor or the GEF, for Ras, the Ras then activates Raf, the MEKK the map kinase kinase kinase, that in turn activates Mek, the map kinase, kinase and Erk which is the map kinase. And by doing this, it allows signal to flow from the membrane to the nucleus. The flow of information from from the membrane to the nucleus involves three components. There is the plasma membrane component which that allows signal to flow from the receptor to Ras, to Raf. Then there is a cytoplasmic component that allows the signal to flow from Raf to erk. And the trans-location of Raf, erk into the nucleus, to phosphorylate a transcription factor, allows signal to go from the cytoplasm to the nucleus. So, mathematical models are often needed to deal with the specification of where biochemical reactions occur and, these can be represented as coupled ordinary differential equations, taking into account the three component and the mo-, and specific mo-, and movement of specific components between these components. So how is space represented in mathematical models? There are two ways of doing this. One can use compartmental models, where biochemical reactions within a compartment are represented as groups of ODEs, Ordinary Differential Equations and some components move between compartments and have a rate associated with the movement and there is mass conservation between the compartments. So, for instance, when ERK moves from the cytoplasm to the nucleus, there, there's a decrease of the amount of ERK in the cytoplasm and an increase in the amount of ERK in the membre-, in the nucleus. And the, in the compartmental model, there would be for the, for the map kinase path with three compartments, the plasma membrane compartment, the cytoplasm compartment, and the nucleic compartment. A more precise way of representing space is through partial differential equations, which is based on Fick's law of diffusion that can explicit recom compute both changes in concentration and changes in locations of reactants and products. This is a much more complicated way of calculations, and it sometimes useful for understanding live cell imaging, but often one can get away with sort of compartmental models. And so I will, I will discuss both of these models in due course and how they can be utilized for various types of information flow in, in biological and cellular systems. So this concludes the first part of Lecture 3. And in, in the next part I will deal more with how networking arises from interconnectedness of pathways. Thank you. >> [MUSIC]
