Immunology Leitenberg January 22, 2014 2:00-3:00 PM Notetaker #31 Signal Transduction and Lymphocyte Activation Slide 2: Every

year this lecture gets bogged down with molecular details, but I want you guys to focus on the big picture. Slide 3: One thing that I want you guys to think about a little bit is the complexity of the system. So when lymphocyte receptors interact with an antigen, theres a variety of different outcomes that can result. Lymphocytes dont just do one thing, and Ive listed them here. These two lists arent mutually exclusive. Slide 4: Related to the previous slide, we can think about this in a very simplistic way, where the cells have a receptor and bang something magic happens (left) or you can think about it in a slightly more complicated way (right.) Ive blacked out the names because we dont really care about all the names of all of these molecules, but were going to learn the name of some of these molecules today. Slide 5: I want you guys to think about the changing outcomes of T cell activation as it correlates with different signal strengths. We talked about positive and negative signal strengths next lecture. Well touch on how changes in signaling may affect survival. And also how changes in the signaling may alter the types of cytokines the T cells make. And sometimes the strength and type of signal turns off antigen signaling. Slide 6: Were going to talk about some general principles of how signal transduction pathways are initiated and regulated. And then were going to talk about some of the things that happen after the TCR and BCR interact with the ligand. Slide 7: Read slide verbatim. Slide 8: Weve talked about the structure of these antigen receptors. This is the top of the molecule; this is the part that engages with ligand. What weve skipped so far is how that ligand recognition event is translated into things that go beside the cell, and one of the things were going to talk about today are these associated molecules. In order to send a signal these TCRs need to associate with a CD3 complex, and for the BCR, they need to associate with what is called IgBeta and IgAlpha. Slide 9: So another things that we learned about these receptors is that theyre structurally similar to each other, although they recognize different forms of antigen. So TCRs recognize MHC peptide complexes whereas BCRs recognize conformational epitopes, but even though they recognize different forms of antigen they really signal in a very similar way. So here, you notice the cytoplasmic tail

theres not much of a tail at all. The receptor has no intrinsic enzymatic activity. So in order for these receptors to transfer a signal inside the cell, they need to associate with other molecules that have a longer cytoplasmic tail that can influence what is going on inside the cell. Slide 10: This is what I just said, so in order to signal they need to associate with invariant protein complexes. For the T cell, thats called the CD3 complex. And for the B cell its IgAlpha and IgBeta. But even these molecules dont have an intrinsic enzymatic activity. They need to recruit additional molecules to the site of receptor ligation in order for them to work. Slide 11: And one of the ways that this works is that these molecules have these little motifs in the cytoplasmic tail that are shown as little yellow bars on the slide, these are referred to as ITAM motifs. Immunoreceptor Tyrosine-based Activation Motifs. And all that means is that they have conserved sequences of AAs that have tyrosines in them. And these tyrosine residues get phosphorylated by enzymes that come to the site of receptor ligation. Those enzymes are called tyrosine kinases. In order for this receptor to be expressed on the surface of the cell in the first place, all these proteins need to assemble first. Theyre assembled in the Golgi and need to be shipped to the cell surface. So if youre missing or have a mutation in the CD3Epsilon gene, it prevents them from expressing the CD3Epsilon protein. That means they wont express the TCR at all, because you need the whole thing to come together in order to be expressed on the cell surface. Slide 12: Again the same thing is true for the B cell receptor. It associates with the IgAlpha and IgBeta proteins. These proteins also have these ITAM motifs in that cytoplasmic tail that are phosphorylated by tyrosine kinases. The BCR engages with ligand, this picture shows a ligand having two repetitive epitopes that allows the BCR to get cross-linked. And sometimes this cross-linking event is thought to facilitate the recruitment of tyrosine kinases to the site of receptor ligation. Slide 13: And this is just the sequence of AAs that defines the ITAM motif. Each ITAM motif contains 2 tyrosine residues, and that is what defines these molecules. And so this is found on invariant proteins for BCRs/TCRs. This sequence is also on other immune receptors to activate them. And so the phosphorylation of ITAMS is mediated by a particular family of tyrosine kinases that is known as the SRC family. And when the ITAMS get phosphorylated, that phosphorylation even can in turn recruit other tyrosine kinase and other adaptor proteins to the site of receptor ligation. Slide 14: This is just an example of some other receptors in addition to the TCRs/BCRs that associate with these proteins that have ITAM motifs. The names are not so important, but the way they signal is really very similar. Slide 15: And so we talk about this way of signaling by which receptors themselves have no intrinsic enzymatic activity, but they rely on recruiting enzymes to the site

of receptor ligation and something called receptor-associated tyrosine kinases. These receptors have no intrinsic kinase activity, but they recruit other molecules to the site, and the point of this is to allow flexibility of responses because it can vary the number and spread of enzymes that are recruited to the site and may vary quantity or quality of different 2nd messenger signaling pathways. This is different from other receptors we see in biology that have intrinsic enzymatic activity built into their cytoplasmic tails. Slide 16: Were going to be talking about receptor associated tyrosine kinases, were going to spend some time now talking about what regulates whether or not these proteins come together in the same place; what regulates the formation of these macromolecular signaling complexes. And well talk about 3 things: how proteins interact with each other, the role of the cytoskeleton in cells to bring all the molecules together to one location in the cell, and also the role of heterogeneity within the lipid bilayer to segregate different classes of moleculesthese things are called lipid raft membrane binding domains. Slide 17: This cartoon shows you how proteins interact with each other. And thats because these proteins have domains in their structure that mediates specific interactions with other proteins with other domains. Here you have a receptor engages with ligand, phosphorylation happens as shown by the little pink circle, and so the phosphorylation event results in the recruitment of a cytosolic protein that has multiple different protein-protein interaction domains. And one of these domains interacts with these phosphorylated tyrosines. And this protein may have other domains, that may interact with other proteins, and so you can see that upon receptor ligation, you get the recruitment and assembly of a lot of different proteins at that site. Slide 18: And so heres some examples of proteins that have multiple different protein interacting domains. Here you see adaptor proteins have SH2 and SH3 domains. SH2 domains interact with phosphorylated tyrosines, and SH3 domains interact with proline residues. You see this protein has multiple interacting domains. Its not just protein-protein interactions; some of these domains deal with protein-lipid interactions, which help target proteins to the lipid bilayers. Slide 19: So another term that you should be familiar with is a scaffolding protein. So you have one structure that allows you to hang a lot of stuff off of that structure in one place. And one way this might work is you have a scaffolding protein that is not phosphorylated, but as a consequence of signaling, it gets its tyrosine residues phosphorylated, and those residues can recruit a bunch of proteins that have SH2 domains all to one place, clustered around that scaffolding protein. Slide 20: Its not just recruiting stuff all into one place, its also amplifying signals nearby a cascade of enzymatic reactions. So here you see RAF, which is a serinethreonine kinase, its activated, and it interacts with multiple substrates, and one of the consequence of interacting with a substrate is it puts a phosphorylated group on this enzyme to make it active, and allow that enzyme to act on its own substrates.

So you have a cascade of enzymatic reactions that amplify signal transduction that may have originated from 1 or 2 antigen receptors being ligated. Slide 21: This is the concept about heterogeneous regions within the plasma membrane. There are variations within the lipid bilayer that restrict the movement of proteins that are within those variations. So for example you could have cholesterol-rich regions that make this region less fluid, and so proteins that live in this region, have a more limited ability to float around, so they may aggregate in one spot. Some proteins may move into these regions of reduced fluidity as a result of interactions with these ligands. There are some proteins show in this cartoon that dont go all the way through the lipid bilayer that have a GPI linkage that like to stay here, and some of the Src-family kinases have a lipid modification that allows them to insert into this region. Slide 22: So one of the things that may happen after receptor ligation you may alter where these integral membrane proteins go. So before receptor ligation they might be outside of these lipid rafts, but as a consequence of receptor-ligand interactions, something changes so that they get brought into these lipid rafts, and that may allow them to be exposed to proteins that theyre not normally exposed to that help propagate signaling further. Slide 23: This gets across the principle that you can get aggregation of multiple different proteins together and they can interact with each other to activate 2nd messenger signaling events that in turn regulate cell fate. Slide 24: So the other thing about the assembly of these signaling complexes is that it may change over time. And the rate at which these guys assemble/disassemble can be rate-limiting events. Slide 25: And so one of the things that I want to get across is that this whole is dynamic in the sense that it changes over time. So this movie will be posted on Bb. Slide 26: This is a frame-by-frame depiction of what happens in the movie. Youre looking at two colors. The green represents the TCRs and the red represents adhesion molecules that facilitate the interaction between the TCR and the antigenpresenting cell (APC.) So this area where the TCR and the APC interact with each other is called the immune synapse where they exchange lots and lots of information. So this is an example of proteins that are changing over time. Youll notice that at the beginning, the TCRs are engaging with the MHC complexes at roughly the periphery, and then what you notice over time, the TCR becomes more concentrated at the center. The adhesion molecules they start off at the center and they end up in the periphery. This is to show you that the assembly and location of signaling complexes is dynamically changing over time as the TCR is interacting with the APC. Slide 27: Reads slide verbatim

Slide 28: Ok, so thats basic principle stuff so Im just going to go into the steps of antigen receptor signaling. Slide 29: The first step is the antigen receptor engaging with antigens. Here the BCR is engaging with a multivalent antigen thats cross-linking two different BCRs together. This allows the recruitment of activation of Src-family tyrosine kinases, which phosphorylate ITAM motifs within the IgAlpha and IgBeta proteins that are associated with the immunoglobulin molecule. And so here it names some of the Src-family tyrosine kinases, Ive identified the ones that are testable, and so for the BCR, the poster child for the Src-family tyrosine kinase well talk a lot about is Lyn. Slide 30: So one of the complicated questions, is how do these Src-family tyrosine kinases know that their receptor is being ligated? Its answered in part by invoking co-receptors. So for T cells weve talked about co-receptors already. So the main coreceptors for T cells are the CD4 proteins and CD8 proteins, and the MHC classes they engage with. This stabilizes the interactions of the TCR with the MHC complex. One of the things that I left out was that on the cytoplasmic panel of the CD4 coreceptor is that they associate with some of these Src-family tyrosine kinases, and the one that associates with co-receptors is called LCK. So when you have complexes like this, you can imagine that the co-receptor engaging with the MHC complex at the same time that the TCR is will bring in the LCK molecule into the area where it can engage with CD3s ITAM motifs, and phosphorylate the ITAMS and initiate signaling. BCRs also have co-receptors that can associate with Src-family tyrosine kinases like Lyn. And so this is an example of that, it includes a protein called CD19. Q: Can you go over what p56 is? A: p56 is LCK, LCK is a Src-family tyrosine kinase that phosphorylates tyrosines, and in this case its phosphorylating tyrosines on these ITAM motifs on the CD3 complex. So the B cell co-receptor works the same way, here is the BCR engaging with antigen. Sometimes antigens come in relatively complicated complexes; they can have associated compliment molecules attached to them. And so a compliment is attached to the antigen, the CD21 molecule is actually a compliment receptor, and thatll allow this complex to come into the neighborhood of this complex, and bring in one of these Src-family tyrosine kinases, which in turn can phosphorylate ITAMS. Slide 31: So heres a cartoon for T cell receptors. So here you have a TCR engaging with MHC peptide complexes, its an MHCII so this is a CD4 T cell, and so you have the CD4 molecule also binding with the MHCII molecule. And so what you notice is that after the TCR interacts with the MHC peptide is the phosphorylation of the ITAMs within the CD3 complex. And that phosphorylation is mediated by LCK, the Src family tyrosine kinase. Actually there is another one here and its called Fyn. And phosphorylation of these ITAM motifs allows the recruitment of yet another molecule, and in T cells, this other molecule is called ZAP70, which is also a tyrosine kinase.

Slide 32: So this initial ITAM phosphorylation is mediated by the Src-family tyrosine kinases. How are these kinases regulated? There are regulatory regions within these proteins; theres an activating region and an inhibitory region. And these regions are regulated depending on whether or not theyre phosphorylated. Slide 33: And so there are two proteins that are important in regulating the phosphorylation of these regulatory regions. One protein, is yet another tyrosine kinase, and its called Csk. It puts phosphates on the inhibitory site, and promotes the conformational change of the protein so that its folded up and diminishes its enzymatic activity. And its acted on by a counter enzymeits a phosphatase (CD45.) What a phosphatase is, is its an enzyme that removes phosphates from proteins, so in this case it removes a phosphate group from that inhibitory tyrosine and promotes this (left) conformation; a more active form of the Src-family tyrosine kinase. And so you have this dynamic equilibrium thats regulated by the relative activity of Csk and CD45. Slide 34: Reads slide verbatim Slide 35: Putting all of that together, you have the set up in and of itself from the point of view of the T cell. We have the role of CD45, which is important; it maintains the SFKs in an active configurationand that allows them to act on the ITAM motifs of the CD3 complex upon receptor ligation. Upon receptor ligation, the active kinases are brought into the neighborhood, they phosphorylate the ITAM motifs, the recruit this other protein, and in this case for T cells its called ZAP70. And the SFKs also phosphorylates additional molecules that result in multiple signal transduction events. Slide 36: So the BCRs do the same thing except the names are changed. So CD45 is also important for BCR activation; it primes the SFKs to be active upon receptor ligation. The SFKs phosphorylate the ITAM motifs, that leads to the recruitment of an enzyme that is analogous to ZAP70 of the T cells called Syk. And once Syk is recruited to the site of antigen receptor stimulation, Syk acts on other things that transduce other signal transduction events inside of the cell. Slide 37: Shows movie (available on Bb) Slide 38: What happens next after activation of SFK or ZAP70? Basically, it activates at least 3 different 2nd messenger signaling pathways. One of the big ones is that it causes the activation of an intracellular Ca flux. It activates this enzyme called PLC gamma that cleaves PIP2 into two pieces. One of them is IP3, and it binds to receptors on the ER that allow the ER to release Ca into the cytoplasmic space, which in turn activates other Ca channelsso you get a big Ca flux inside the cell, and that Ca flux activates other proteins that ultimately results in the activation of a transcription factor called NFAT, which relocates from the cytoplasm to the nucleus to regulate gene transcription. The other thing product of PLC gamma is DAG, which

is important for the activation of protein kinase C, which in turn, activates another transcription factor called NFkB. The other 2nd messenger signaling pathway is mediated by Ras, which activates small G proteins like those that activate MAP that in turn regulate yet another transcription factor. And so you can get the activation of multiple transcription factors that go to the nucleus and regulate patterns of gene transcription. Slide 39: Shows one other movie (available on Bb)