okay the second half of this chapter we're going to talk about is regulation of cell cycle which really has a lot to do with protein expression and regulation of protein concentrations so this image shows the basic cell cycle starting with g1 where the cell is growing s phase where DNA synthesis is happening g2 where the cell grows some more and then M phase which is mitosis and cytokinesis and the three red blocks shown are the three big checkpoints where the cell actually pauses and goes through and makes sure there's plenty of nutrients the cell size has enlarged checking for DNA damage before it allows the cell to go to the next phase of the cell cycle so we're going to talk about some of the ways this is controlled or regulated at the molecular or the protein level the biggest players are Cyclones and cyclin-dependent kinases CDKs and these work together to regulate cell cycle and protein expression and so what I want you to see from this slide is that to be active the cdk binds a specific cyclin the cdk is phosphorylated and remember that the cdk is a kinase so its function is to phosphorylate other proteins what's interesting about the regulation of Cyclones and cdks is that the cdks are always around it's the cyclin that cycles through being present and not and that's how they got their name I'll show you a figure about a minute so what happens to the Penguins is they get something called ubiquitin put on there and this marker tags the protein for destruction so when there's no cyclin present the cdk is inactive when cyclin is present oops the cdk can become active so the cyclin is either being destroyed or expressed the cdk is expressed continuously and you can see that here this is just showing the Cyclones during cell cycle so they're cyclin a and B and E and D 1 and D 2 and B is the one that the book also code calls the mitotic cyclin-cdk us two or cycles are expressed at different times so d2 is expressed during g1 Peaks somewhere in S phase and then as a cyclin level concentration is going down that means that it's being degraded cyclin b is important you can see at peak in mitosis cyclin a is important for g2 phase so where it Peaks is telling you where it's functioning do you want in an interesting one right because it's all it's expressed at high level throughout most of the cell cycle and you see E is important in g1 so as that slope comes down it's because the cyclin is being degraded as the slope goes up it means the concentration is being increased and this is just another figure to kind of emphasize that point that it's this cycling of these Cyclones so they're expressed they activate their cdk and then they're degraded and they're expressed and activated cdk integrated that the cdk remains constantly expressed mitotic cyclin-cdk after is really describing the cyclin CDK interaction so the way you interpret this graph is that in in g1 s g2 mitotic cyclins I : B increases in concentration reaches a peak in FAS which is also the peak of this complex activity and then you can see the cyclin is degraded and the activity of the mitosis promoting factor goes down and then cyclin is made again and so you can nicely see how the cyclin cycles throughout the cell cycle repeating its expression Peaks and decrease Peaks and decrease another way that Cyclones and cdks are regulated and this really should be well that's alright what I want you to focus here is the cdk regulation great so you have your mitotic cyclins and be expressed you've got your CDK these come together but what's really interesting is you actually have to have two phosphorylations that actually inhibit the cyclin-cdk complex and then you get an activating kinase that puts the phosphate on here and then to make it active you got to get rid of these two phosphate phosphate remember phosphatases are enzymes that remove phosphates tiny cesare enzymes that add phosphates so your final mitotic cdk-cyclin complex is active with one specific phosphorylation but there are cases that phosphorylate dot c DK and actually inhibit so phosphorylation can be inhibiting or it can be activating depending on the site of the protein that is phosphorylated so it's a complicated regulation and one of the things I just want you to appreciate is that this level of complexity allows us to tightly regulate such an important thing like cell cycles all right anaphase promoting complex so we're actually going to kind of work backwards through cell cycles I went back and forth trying to figure out what's the best way to present this but we're just going to kind of go with the order of the book anaphase promoting complex I have this thing in yellow here is a ubiquitin ligase which is explained right here so I showed you back here that when the cyclin gets ubiquitin or ubiquitinated ubiquitinated if i give it it's targeted for destruction so that's one of the things that anaphase promoting complex does is it targets the mitotic cyclins for destruction and when you destroy the cyclin the cdk is no longer active so anaphase promoting complex wants to help make sure everything is ready for an assay is to happen which is the separating of the sister chromatids the way anaphase promoting complex works is one it targets secure in for destruction so there's a flute sorry it'll be distant there's these two proteins separate and secure in and when they're bound the separate is inactive when anaphase promoting complex puts ubiquitin on here and secure and is degraded separates comes and what separates does is it starts breaking down these proteins called cohesion so cohesion is part of that centromere that's actually holding the sister chromatids together and so when you break this down you can see your sister chromatids can split okay so we've got two things happening we've got anaphase promoting complex taking the mitotic cyclins or degradation and tagging securin for degradation and there's this little red component that is key to making anaphase promoting complex functional and that is protein called CDC 20 so here's a picture slightly different from your textbook but I think it shows the rules of these proteins a little bit better so here your cohesions holding those sister chromatids together here is a protein called mad and there's also a protein called bub they don't show that here and here is CDC 20 and these this protein complex binds to that kinetochore and it hangs out there until a spindle fiber is connected and once the spindle fiber is connected to the kinetochore this complex disassembled Mattu and bub go off to do their job CDC 20 now binds your anaphase promoting complex and makes it active so it's a nice way people back here here that anaphase promoting complex does not become active until all the chromosomes have spindle fibers attached because if you have a chromosome or sister chromatids without a spindle fiber attached when the cells go to split right this whole thing is going to go one way versus what we want to happen is splitting the sisters so anaphase promoting complex is not active to allow the splitting of the sisters until CDC 20 is bound and CDC 20 is not bound until the spindle fibers are connected to the kinetochore so this is a really nice system that has checks okay yes now we're ready for you to activate so you have to have enough of this active which means all the sister chromatids bound to spindle fibers the active anaphase promoting complex now breaks down the cohesion molecules and the sisters can split something else that is happening so again we're working backwards so now we've talked about some controls here in m-phase now we're going backwards to s-phase one thing we want to make sure in our eukaryotic cells is that the DNA is replicated only once until mitosis happens so we don't want our chromosomes going to this and having too many of everything right we want to keep them as nice sister chromatids with our homologous pairs so in g1 while the cell is growing proteins are put on what's what happened to that application to a place on the DNA called the ori which stands for origin of replication the ori is a site where helicase can open up the double strand of DNA and DNA primers can come in and start copying the DNA and so you only want this to happen once so in g1 phase these proteins are loaded on to the origin in S phase they're doing their job and what also happens in S phase is that these proteins can be phosphorylated so the pre-replication complex proteins some get phosphorylated so that they can tree bind there is also a protein called Geminid which blocks MCM our green thing here from rebinding once it comes off so replication licensing means in g1 we're saying okay you guys have to go ahead to replicate but once you come off in S phase we have proteins that are going to modify you so that you cannot rebind and so that you cannot replicate again because you only want your DNA replicated one time also in S phase we have a protein called retinoblastoma and retinoblastoma we will see you next chapter is called a tumor suppressor tumor-suppressor for cancers a protein that helps control cell cycle so that it doesn't go out of control and so how our B is important is that during g1 phase here our B will be phosphorylated so normally our B binds e to F and E to F is a transcription factor that helps recruit RNA polymerase to turn on or express genes and so when our B is not phosphorylated it binds e to F and the genes and proteins for S phase are not expressed when the cell gets the signal yes it's time to divide and we'll talk about growth factors in the next talk then our B is phosphorylated it now no longer binds e to s because it has a conformational change and gene transcription happens and what we'll talk about in the last chapter is that RB when mutated does not bind to e to F and so transcription for S phase proteins can happen or can be expressed all the time which means the cells are getting the signal hey we got to keep replicating our DNA and what's happening in cancer is you get DNA replication when things like DNA damage have not been checked yet and so you start to replicate damaged or mutated DNA one other important protein and this goes along with DNA mutations is p53 this we will talk about is also a tumor suppressor and important for F phase control and how this protein works is if DNA is damaged by either what we call double-stranded DNA breaks right so you've actually broken your helix or single-stranded breaks and they need to be repaired by the cell before replication can happen so if there's damaged DNA either the ATM or the ATR protein is activated it comes through and activates kindnesses that are important in checking for damaged DNA and p53 gets phosphorylated so when p53 is phosphorylated it works to turn on genes so it may turn on p21 which tells the cell stop or it may turn on or stop dividing pull up which tells the cell to undergo apoptosis or cell death and we will talk about apoptosis and cell death in the next talk so I'm going to stop there and we'll come back and talk about growth factors [Music]