MBios 401/501: Lecture 12.1 Signaling IV Slide 1 Pathways that require regulated proteolysis 1. Notch and Delta 2. Wnt/ b-catenin 3. Hedgehog 4. NFk-B Our last topic on cell signaling are pathways that involve regulated proteolysis. We ve already discusses some situations where proteolysis plays a role in signaling. For example, we briefly discussed the degradation of cycling proteins in the lecture on mitosis, and we ve looked at examples where degradation of receptors is important for desensitizing receptors in cells. However, there are a wide variety of signaling pathways where the degradation of signaling components or inhibitors of signaling components plays a central role in the functioning of the signaling system. Many of these are involved in early development, where rapid changes in gene expression are produced by the degradation of proteins that inhibit the function of key transcription factors. What this does is it allows a cell to have many transcription factors expressed in the cytoplasm ready to go and then activate a select group of them in response to a particular signaling event. A number of examples of the expression or activation of gene regulators in drosophila larvae are shown on this slide, and many of the components of developmental signaling pathways were first discovered and named from studies using the drosophila model system. We ll be looking at 4 well studies model signaling pathways that involve degradation and listed at the bottom of this slide. These are the ones I want you to know really well. Slide 2 The Notch Receptor The notch receptor and its ligand Delta play a major role in development of most tissues. Nerve cell development in Drosophila: When a cell in epithelium begins to develop as a neural cell, it inhibits neighbor cells. Nerve cells expresses Delta signal on surface that binds to Notch receptor on adjacent cell. The first of the signaling pathways that we are going to discuss is the notch/delta receptor system. The Notch receptor and its ligand delta play a major role in many developmental processes. Delta signaling is an example of a contact dependent signaling event and it is used by one cell to tell it s neighboring what to do. In drosophila, a sheet of epithelial cells develops into a combination of nerve and adult epithelial cells. As an individual nerve cells begins developing, but then expressed delta on its surface. Neighboring cells are signaled through the Notch receptor that their neighbor has become a neuron and this 1
prevents them from also becoming neurons. In this way the correct proportion of neuronal and epithelial cells are maintained. If there is a mutation in either delta or notch the result is that too many cells will become neurons and not enough will become epithelial cells. The image at the bottom left shows the developing drosophila eye at high magnification. The center of each cluster of cells is a set of neuronal cells and the surrounding ring of cells are epithelial cells. On the right of image is the same tissue in an embryo with defective notch signaling and you can see the profound loss of epithelial cell development in that tissue. Slide 3 No title The notch receptor is the protein that actually under goes proteolysis in the signaling system. It undergoes proteolysis in three different places, but only two of which are dependent on delta signaling. The diagram on this slide shows the first stages of interaction between Notch and delta, which causes cleavage of the protein by first an extracellular protease followed by an intracellular one. The cleavage event on the inside of the cell membrane is accomplished by a proteolytic enzyme complex called gamma secretase. One the regulatory subunits, called presensilin, is mutated in some forms of early Alzheimer s disease. Slide 4 No Title Notch tail moves into nucleus to activate Notch-responsive genes by converting a transcriptional repressor to an activator. In flies, gene product blocks expression of proteins required for formation of neurons. Cleavage of the internal domain of notch produces a cytosolic protein tail fragment that enters the nucleus and activates a transcription factor, which is labeled CSL in this diagram, which is already found in the nucleus. It converts it from an inactivator of transcription to an activator of transcription. Your book notes that unlike most receptors, notch cannot be recycled. Once it is activated by proteolysis you can t rejoin the pieces back together to form the inactive form. Slide 5 Wnt regulates proteolysis of Beta-catenin Wnt is a signaling protein that is secreted by cells and detected by other cells having an appropriate receptor. This signaling pathway is involved in so many different developmentally regulated events that it has been discovered several times in different 2
model systems. Unfortunately, the legacy of this history is the naming conventions of the elements of signaling pathway is kind of complicated. I am going to restrict the discussion of the pathway to what s written on this slide mot of which comes from studies of drosophila, but there are other names for individual proteins. The basic mechanism of Wnt signaling is to stabilize a transcription enhancer called beta catenin. In the absence of Wnt signals, beta catenin is phosphorylated by something called a degradation complex and the phosphorylated beta catenin is target for proteolytic degradation by the general machinery for protein degradation and recycling in cells. In drosophila the receptors for Wnt are frizzled and the LDL receptor related protein or LRP which bind to a fatty acid linked to the Wnt signaling peptide. Frizzled is a seven pass trans membrane protein, but it s not a n actual G protein. Instead the binding of Wnt to frizzled and LRP causes the recruitment of an intermediate called disheveled. As well as the kinase that are part of the degradation complex. In turn this causes phosphorylation of the protein. Axin is a scaffolding component of the degradation complex and after its phosphorylated the degradation complex falls apart as shown on the right hand side of this slide. As a consequence, beta catenin is no longer phosphorylated which this allows beta catenin to be present long enough to enter the nucleus and regulate the transcription of Wnt responsive genes. This pathway is kind of interesting in that the active signaling component is always being produced, that s the beta catanin, but is then inactivated by destroying it, unless an appropriate activation signal is present. This seems kind of wasteful, but it allows the cell to respond rapidly to signaling events. Slide 6 Wnt signaling regulates bristle patterning in drosophila Pictures So I mentioned that the Wnt signaling pathway is involved in a large number of developmental events, including events that regulate then orientation and distribution of epidermal hair forming cells in drosophila. The components of this pathway were initially identified by screening fruit flies for mutations that altered hair cell patterning. Two of these mutations generated flies with bristles that were randomly oriented, leading to the appearance of being frizzled or disheveled, as shown in these images from the original paper that was published in 1993. Slide 7 Wild type and hedgehog mutant drosophila larvae Pictures The next signaling pathway we are going to discuss is the hedgehog signaling pathway. The hedgehog signaling pathway was also first discovered by screening drosophila looking for unusual looking mutants. The images on this page show a normal larva on the left and a mutant that lacks hedgehog signaling activity on the right. The short body and prickly appearance of the mutant reminded the discover of a hedgehog. I ll discuss precisely how signaling systems alter the morphology of the embryo in an upcoming 3
lecture, but for now, just concentrate on the mechanics of how the signaling system works. Slide 8 Hedgehog Signaling The diagram on this page shows the hedgehog signaling pathway in its inactivating state. In the absence of an activating signal, a protein called cubitus interruptus or CI, is targeted for degradation. However the degradation is only partial and results in a small negative regulator of gene expression being produced. This negative regulator enters the nucleus of cells and inhibits the production of hedgehog dependent genes. The processing of CI is accomplished using a degradation complex much like the Wnt signaling. Interestingly this degradation complex is linked to microtubules by one of its components called Costal. This appears to help keep unprocessed Ci from accidentally entering the nucleus and initiating transcription. The signaling system also involves three trans membrane proteins. ihog is the receptor for the hedgehog signal. Two other trans membrane proteins are also involved called patched and smoothened. From their names, you can guess that mutations in these proteins also produced mutant flies that had odd looking bristles. Smoothened is interesting because it is present on intracellular vesicles instead of plasma membrane and it is maintained there by the function of inactivated patched. Slide 9 No Title This slide shows what happens when the hedgehog ligand binds to ihog, which is the receptor for hedgehog, and patched. The first thing that happens is smoothened becomes localized to the plasma membrane. It then recruits the degradation complex and inactivates it. So once again this looks a lot like Wnt signal. The inactivation of the degradation complex allows unprocessed Ci to be produced, which enters the nucleus and now activates gene expression of hedgehog dependent genes. So once again we see an example of an active regulator of transcription that is constitutively degraded. Some of the interesting features of the hedgehog pathway are the inactive intermediate found on intracellular vesicles, and the linkage of the degradation complex to the microtubules. Slide 10 Stress and inflammation signaling through NFkB Well, we ve make it the last slide of the series of signaling lectures. Our last pathway is the NFkB signaling system. NFkB stands for nuclear factor kappa light chain enhancer of 4
activated B cells, which basically means that it is a factor that enhances antibody production by B cells. NFkB is another latent regulator of gene expression meaning it is always around waiting to be activated in cells. In the absence of an activating signal, NFkB is bound to an inhibitor called ikb, which stands for inhibitor of NFkB. A number of signaling elements can activate a regulatory complex that phosphorylates ikb and is therefore called the inhibitor of NFkV kinase or IKK. One of the most important examples of receptors that can activate IKk is the tumor necrosis factor alpha ligand binding receptor or TNF alpha. The activation of the IKK complex, which phosphorylates ikb, targeting it for degradation. This then liberates NFkB, which enters the nucleus and alters gene expression in combination with cofactors that are already present. So this system is unique among the four we ve discussed in that the protein that gets degraded is not a transcription factor itself, but instead is a protein that is inactivating the active transcription factor. Slide 11 Major Points in Signaling Systems Notch and Delta Delta is the signal. Common in epithelial cells and neurons. Intracellular domain of Notch is degraded by gamma secretase\ Cleavage product is active regulator of gene expression Wnt/Beta-catening Wnt is the signal, LRp and frizzled are the receptor Receptor activation activates disheveled, which inhibits a degradation complex\ Beta-catenin is no longer degraded, enters the nucleus and initiates gene expression Hedgehog Hedge is the signaling ligand ihog and patched are the receptor Binding of hedgehog deactivates repressor activity of patched Smoothened arrives at membrane and deactivates a complex that normally binds to microtubules and degrades Ci. (The degradation product is a functional inhibitor of gene expression) Full length Ci enters the nucleus and activates gene expression NFk-B Many inflammation and stress signals and receptors, one important one is TNFalpha NFkB is normally inhibited by binding to ikb Receptor activation leads to activation of a complex that phosphorylates ikb and causes it to be degraded. The complex is ikb kinase (IKK) Degradation of the inhibitor (IIKB) frees NFkB and allows it be up regulate expression of target genes. This is an outline of the major points of each signaling system discussed in this lecture. If you know what s on this slide and can answer the summary questions. You should 5
have an understanding of everything I d like you to know about these pathways. 6