Session 8: Protein Kinases
Transcript of Part 1: Protein Phosphorylation in Biology
00:00:03.08 My name is Susan Taylor. I'm a professor of chemistry and biochemistry 00:00:08.04 and also of pharmacology at the University of California, San Diego. 00:00:12.14 I'm also an investigator of the Howard Hughes Medical Institute. 00:00:16.28 And what I'd like to do today is to go through three lectures 00:00:25.04 and hopefully leave you with three general concepts. 00:00:29.11 First, I'd like to point out why protein phosphorylation is so important in biology. 00:00:36.03 Then, in the second lecture, I'd like to introduce you to 00:00:40.16 the protein kinase molecular and how that functions. 00:00:43.25 And in the third lecture, I'd like to focus on how protein kinases are regulated and localized. 00:00:51.05 So if we go to the central dogma of biology, where DNA makes RNA makes proteins 00:01:01.15 This is quite an extraordinary era that we live in now; 00:01:04.15 this genomic era of science where we have so much information available to us. 00:01:13.02 So if we look at DNA: DNA is a linear template of four bases. 00:01:17.27 And the speed with which we can sequence DNA is just beyond anyone's comprehension, 00:01:24.16 even a decade ago, that we could have whole genomes so rapidly. 00:01:29.21 So it's a linear template and the information is in this linear template. 00:01:33.11 And the same is true for RNA. It's linear template of four bases 00:01:37.20 and the information is encoded in that linear template. 00:01:42.09 But if we look at proteins, proteins are a little bit different. 00:01:45.21 They're made up of 20 different amino acids 00:01:48.16 and the chemical properties of those amino acids are quite different. 00:01:52.23 And in addition, they get modified once they're made. 00:01:58.09 And that modification can make a critical difference in how they work. 00:02:02.15 So, understanding how a protein works is more complicated. 00:02:10.18 So with DNA and RNA, transcription transcribes the DNA into RNA. 00:02:16.27 They're both linear templates. 00:02:19.22 If you look at proteins and translation now: RNA is translated into proteins, 00:02:25.05 where you have all these diverse amino acids and if we look at a protein's sequence, 00:02:30.23 it, of course, defines the chemical composition of that structure of that protein molecule. 00:02:37.22 But it doesn't really tell you how it works. 00:02:40.24 And to really understand how a protein works, you need to have structure so that you 00:02:46.11 know where those amino acids are, you know how they work together to create an active and functional protein. 00:02:54.16 And understanding this is much more complicated than 00:02:57.26 just reading out the template from the DNA or the RNA. 00:03:01.15 And so I like to think of our present era of science not as the genomic era of science but 00:03:08.03 the proteo-genomic era of science. 00:03:10.09 And ultimately we're going to have to understand this erite gamut going from DNA to RNA to proteins. 00:03:17.17 And it's going to be much more challenging to do the proteins but 00:03:20.13 we're already making enormous progress there. 00:03:25.14 So what are the building blocks, the atoms, that make up proteins? 00:03:30.06 So we look at carbons, nitrogens, oxygens, hydrogens. 00:03:38.18 OK, those are all there. There's also a little bit of sulfur there. 00:03:42.00 But then, what about phosphates? 00:03:43.20 So where do phosphates come in and why are they important? 00:03:47.14 So this is the phosphate. It's 80 kiloDaltons. 00:03:52.01 It's a little, small moiety that you add onto a very very large protein. 00:03:57.18 And I like to go back to a review that Frank Westheimer did back in 1988. 00:04:04.11 So he was one of the chemists who studied phosphoryl-transfer, 00:04:09.04 one of the major pioneers in this area. 00:04:13.18 And he elucidated the importance of phosphates for biology. 00:04:17.19 So he made two major points. 00:04:19.03 One is, its importance for DNA and RNA, for genetic information. and for transfer of information. 00:04:25.03 And each of those linear templates for DNA and RNA are linked by phosphates. 00:04:30.07 So clearly, all of this template is critically dependent on phosphate. 00:04:35.11 And the other thing he recognized was the importance of phosphate for energy. 00:04:42.09 And so in this case we need to go to another molecule. 00:04:47.10 And this is an organelle, a mitochondria which is the powerhouse of the cell. 00:04:51.19 And what the mitochondria does is to make ATP. 00:04:54.21 And what ATP does is to drive all of the biological processes that take place in every one of our cells. 00:05:02.15 So here's ATP and it's the gamma phosphate at the end that turns over 00:05:08.05 and provides our cells with energy. 00:05:10.20 And just to emphasize how important this is, 00:05:14.13 the average 70 kilogram person turns over 40 kilograms of ATP a day. 00:05:20.19 So, I always find this number astounding. 00:05:23.05 So this is critically important, that phosphate for energy in our cells. 00:05:29.24 What Westheimer did not address at all, and this field was just beginning to 00:05:35.16 really emerge in terms of its huge importance, at that time, 00:05:40.11 protein phosphorylation as a mechanism for regulating biology. 00:05:46.05 And that's what I want to try and focus on now. 00:05:50.05 And we have to go back, again, to some of the history 00:05:53.14 and in this arena it was Ed Krebs and Eddie Fischer who were the first to demonstrate, 00:05:58.27 in the late 1950s, that phosphorylation was important for regulation of proteins. 00:06:05.19 And they received the Nobel Prize for that in 1992. 00:06:09.26 So, they were looking at glycogen metabolism in the liver. 00:06:16.21 And this is a liver cell. The dense particles are the granules. 00:06:23.00 There are also mitochondria there; you need a lot of energy for anything that a cell does. 00:06:27.18 There are a lot of mitochondria. 00:06:29.08 And if we look at the glycogen particles, what all of us do 00:06:35.13 when we have a carbohydrate rich meal, 00:06:37.12 the liver takes up that glucose and you make glycogen. You store it there. 00:06:42.17 And even after a short fast, like sleeping overnight, when you wake up in the morning, 00:06:47.11 you have mobilized some of that glycogen into your bloodstream 00:06:51.04 so that your brain and the rest of your body still gets glucose. 00:06:54.24 So you make and break down glycogen as a fundamental part of metabolism. 00:07:00.14 And the enzyme that does that is called glycogen phosphorylase. 00:07:04.08 It breaks down glycogen into glucose. 00:07:06.20 And that's the enzyme that Krebs and Fischer worked on. 00:07:10.27 And what they discovered was this enzyme--now we know its structure. 00:07:15.10 It's very large. Each chain (it's a dimer, there are two subunits) each has over 800 amino acids. 00:07:23.22 And what they found is, if you...this exists in two different states. 00:07:28.23 It can be phosphorylated, one phosphate on each chain. 00:07:33.07 It can be phosphorylated or not phosphorylated. 00:07:36.05 And when it is phosphorylated, it is turned on. It is an active enzyme. 00:07:41.24 And when it is not phosphorylated, it is not active. 00:07:46.14 And so this fundamental concept is really the essence of 00:07:51.06 the importance of protein phosphorylation for regulation. 00:07:55.22 So, how does it get added? How does that phosphate get added? 00:08:00.22 It gets added by a protein kinase. 00:08:03.11 So protein kinase uses ATP, transfers that gamma phosphate to a protein. 00:08:09.01 So now you have many proteins, more than half the proteins in our bodies 00:08:13.13 exist either as a dephosphorylated molecule or as a phosphorylated molecule. 00:08:19.21 So they can be turned on and turned off. 00:08:22.24 And the phosphatases are enzymes that take the phosphate off. 00:08:26.26 So phosphates are going on and off of your proteins all the time. 00:08:31.03 They're switches, they're molecular switches 00:08:35.09 that either give a go signal or stop signal. 00:08:38.24 They are essential molecular switches for all of biology. 00:08:44.02 And I like to give just one example that is one of the most dynamic events that 00:08:49.24 a cell does, it is to go through cell division. 00:08:52.06 And this is a lily cell dividing. 00:08:55.08 And you can see as this lily cell goes through the different steps of mitosis, how dynamic this is; 00:09:03.22 organizing these chromosomes, then having the cell actually divide. 00:09:09.09 This process is mediated, primarily, by kinases and phosphatases that get 00:09:16.29 turned on and turned off and that allow mitosis to start, this phase to end, start the next phase. 00:09:23.25 It's critically regulated by kinases and phosphatases. 00:09:28.16 No cell could divide without that critical, highly correlated regulation. 00:09:38.12 So, let's go back to the history. 00:09:39.29 So, phosphorylase kinase is the kinase that phosphorylated glycogen phosphorylase and 00:09:46.00 then the second one to be discovered is called PKA or cyclic AMP dependent protein kinase. 00:09:52.16 And I'm going to tell you about those two and show you how, 00:09:56.24 in this case, they work together as a team to regulate this biological event. 00:10:02.27 So here's glycogen phosphorylase when you've just had a carbohydrate rich meal; 00:10:09.07 glucose is high, insulin is high, glucagon is low. 00:10:13.22 Insulin and glucagon are two metabolic hormones that 00:10:17.13 tell the body "are we in an energy rich stage, with glucose, or are we in more of a fasting state." 00:10:25.27 So it's turned off. 00:10:27.05 Then you look at glycogen phosphorylase, it's turned off. 00:10:31.15 You have lots of glucose. You want to be making glycogen, not breaking it down. 00:10:36.08 OK, now you look at when glucose levels are low. You have high glucagon, low insulin. 00:10:42.17 In this case you want to mobilize that glycogen that is stored in the liver 00:10:45.27 and then this enzyme is turned on. 00:10:48.13 And it's turned on by the addition of that one phosphate to 00:10:52.09 each of the chains that's in the glycogen phosphorylase dimer. 00:10:59.20 OK, so let's see how that works. 00:11:01.25 So here's glucagon. Glucagon is a hormone. 00:11:04.09 It doesn't ever get into the cell. It binds to a receptor on the surface of the liver cell. 00:11:10.22 And in this case, this is a GPCR (G protein coupled receptor), the largest gene family in our human genome. 00:11:19.29 It binds, that couples to a heterotrimeric G protein, 00:11:25.10 which becomes activated and that in turn 00:11:29.09 leads to the activation of adenylate cyclase which makes cyclic AMP. 00:11:33.28 So this concept is: cyclic AMP is second messenger. 00:11:39.08 It allows some extracellular signal to be translated into a biological response. 00:11:44.06 This was discovered by Earl Southerland earlier in the 1950s, 00:11:48.05 this second messenger concept for cyclic AMP. 00:11:52.17 It is conserved as a second messenger in all of biology even in bacteria. 00:11:58.02 So let's see what...this is summarizing what I just told you. 00:12:03.00 Your extracellular signal, in this case glucagon, a hormone from the pancreas, 00:12:07.20 binds the glucagon receptor, activates the G alpha subunit, 00:12:11.01 that activates adenylate cyclase and that makes cyclic AMP. 00:12:15.00 OK, what does cyclic AMP do? 00:12:18.16 OK, so let's look now at this biological response. 00:12:22.07 So, here we go to PKA. And PKA, like most protein kinases, 00:12:29.03 I told you they're switches, is kept in an off state here 00:12:34.03 and in this case it's got regulatory R subunits and catalytic subunits, C subunits 00:12:41.11 and when they're together and there's no cyclic AMP around, it is inactive, it's turned off. 00:12:46.26 And cyclic AMP...this is the main target for cyclic AMP: these regulatory subunits of PKA. 00:12:54.02 It binds with very high affinity to the regulatory subunit 00:12:57.29 and that then unleashes the catalytic activity. 00:13:01.03 And depending on the cell type, there are many things it can do. 00:13:05.08 PKA has many substrates. It regulates many aspects of biology. 00:13:10.13 It can also go into the nucleus and turn on gene transcription. 00:13:14.12 So, turning on one kinase can have many consequences. 00:13:18.26 We're going to focus here on this liver cell and what are the consequences for glycogen metabolism. 00:13:25.27 So let's look at this cyclic AMP. It gets made in response to glucagon. 00:13:32.19 It binds to PKA and it converts it from an inactive state to an active state. 00:13:39.01 OK, what does that do now with respect to glycogen metabolism? 00:13:44.15 Well, glycogen phosphorylase kinase that was the first kinase that Krebs and Fischer characterized. 00:13:52.02 That's the kinase that phosphorylates glycogen phosphorylase. 00:13:57.20 And PKA turns it from an off state to an on state. 00:14:03.13 So we now have one kinase, PKA, turning on another kinase, glycogen phosphorylase kinase. 00:14:09.27 And then, that in turn acts on glycogen phosphorylase 00:14:13.27 and again, that's converting it from an inactive state to an active state. 00:14:18.26 So these on-off switches are happening all the time in our cells. 00:14:26.14 So...and in these cases it's just one phosphate. 00:14:29.24 One single phosphate can make an enormous difference 00:14:32.26 for a very large protein whether it's active or whether it's inactive. 00:14:38.13 So let's go back to the history now and look at this curve a little bit more. 00:14:43.20 So in the 1980s this really expanded exponentially. 00:14:48.12 And that's because we developed the technology to clone and to sequence DNA. 00:14:52.17 So from that it became clear that there were many kinases and that their sequences were all related. 00:14:59.19 And we now fast-forward to the genomic era of today and we have whole kinomes from organisms. 00:15:11.15 And the human kinome is about 2% of the human genome codes from protein kinases. 00:15:19.19 It's one of the largest gene families. 00:15:24.08 PKA belongs to this little branch down here. 00:15:27.09 And the other one that I told you about is phosphorylase kinase. 00:15:31.02 It belongs to this other branch. 00:15:33.22 Those are both very important, classical, metabolically important kinases. 00:15:44.15 So, let's go back to this now and look at another event that was really important around 1980. 00:15:49.27 And this is the discovery that Src was also a protein kinase. 00:15:55.22 And let me tell you about Src. 00:15:58.02 So the history of Src: it was first discovered as an oncogene 00:16:04.09 in chickens from Rous Sarcoma Virus. 00:16:07.25 So the Rous Sarcoma Virus causes cancer in chickens. 00:16:15.27 And so Src is responsible for that transformation of a normal chicken cell 00:16:22.21 into malignant cancer cell and Src was the oncogene that was responsible for that. 00:16:28.06 So that was discovered back in the 70s. 00:16:30.08 1978, it was shown that this Src also had kinase activity, protein kinase activity. 00:16:38.14 And then Src was cloned. So then you had the sequence of Src. 00:16:43.10 And then Tony Hunter and Bart Stefton showed that phosphotyrosine 00:16:52.07 was also an important biological site for phosphorylation that we have. 00:17:01.19 Coming back to here, these are all serine thronine kinases. 00:17:06.27 And now we have this whole tree of tyrosine kinases. 00:17:11.19 And they are related by sequence. They all belong to the same family. 00:17:16.20 If we look at serine and threonine, 00:17:18.27 most of those kinases on the yellow line are serine threonine kinases. 00:17:22.28 They phosphorylate serine threonine and they're much more abundant. 00:17:26.16 But then you have tyrosine as another amino acid that can be phosphorylated 00:17:32.26 and tyrosine is very, very important. 00:17:37.00 Although not as abundant, critically important for biology and for disease. 00:17:41.27 So we look at the kinome now and it's this branch at the top that corresponds to 00:17:48.11 those tyrosine kinases and all the rest of these are phosphorylating serine and threonine. 00:17:55.11 So, we have a branch, a very large kinome 00:17:59.10 that includes both serine and threonine kinases and tyrosine kinases. 00:18:03.22 And so I want, at the end here, to tell you the importance again of adding one phosphate. 00:18:12.14 As I showed for glycogen phosphorylase, 00:18:14.09 one phosphate makes a difference between it being active and inactive. 00:18:17.22 So, let me tell you, just for kinases in general, they not only add phosphates to other proteins, 00:18:25.12 they are typically phospho-proteins themselves. 00:18:28.10 And when you just encode that protein, translate that protein from the sequence, 00:18:35.08 that has all the amino acids there but that kinase is not active. 00:18:40.04 And typically you add one phosphate to what we call the activation loop 00:18:45.24 and that converts an inactive kinase into active kinase. 00:18:50.14 So, kinases themselves are highly regulated by phosphorylation. 00:18:56.12 OK, so again, one phosphate. 00:19:01.11 So now let's look at Src and PKA and I'll get more into these domains and things in the next lecture, 00:19:08.17 but to point out that PKA has this kinase core which is important for its kinase activity. 00:19:17.12 And Src has...its sequence is related and that conserved kinase core is what builds that whole kinome tree. 00:19:28.10 All of those kinases have this core. 00:19:30.10 What was unusual about Src was that it had these other domains 00:19:35.26 that turned out also to be conserved sequences but 00:19:38.23 they were not conserved in other kinases. A small subgroup had these domains. 00:19:44.06 And these were discovered by Tony Pawson in the 1980s 00:19:51.13 and were named...he named them SH3, SH2 and SH1. 00:19:57.07 SH1 was the kinase domain. 00:19:59.29 And then, what he found was that the SH2 domains, in particular, bind to phosphotyrosine. 00:20:08.23 And so this introduced the whole concept of adaptor molecules. 00:20:14.06 That this is a domain whose main function is to bind to phosphortyrosine. So it's an adaptor. 00:20:22.29 Now, let me show you how this works for Src. 00:20:24.27 And Src is just an example of all of the tyrosine kinases there are. 00:20:31.20 Each has a different variation on this theme. 00:20:34.28 So Src...ordinarily it's turned off. 00:20:38.00 I've told you kinases are switches, you turn them off and you turn them on. 00:20:41.17 And in this case it's turned off by a phosphate that's at the C-terminus or Src. 00:20:52.09 And it binds to its own SH2 domain. 00:20:55.15 So, it's kind of its introverted mode. It's not interacting with other proteins. 00:21:01.28 It's interacting with itself and turned off. 00:21:04.18 And so the key event for activating Src is to remove that inhibitory phosphate. 00:21:10.07 So you take it off. 00:21:11.17 You take the phosphate off and then you now convert it into an active enzyme. 00:21:15.20 And the first thing it does is to phosphorylate itself. 00:21:18.27 So you have this activating phosphate here. 00:21:22.09 So now it's able to phosphorylate many other proteins. 00:21:28.14 So now it's an active kinase. 00:21:30.11 So it also has many substrates that it can phosphorylate. 00:21:33.21 But you've also done is to release the SH2 domain and the SH3 domain 00:21:41.07 from their interactions with the kinase core to now interact with other proteins. 00:21:47.00 And so, in particular, if you look at SH2 domain it's now serving as 00:21:53.14 a docking site for another phosphotyrosine that belongs to another protein. 00:21:57.11 And so in this way, by activating one kinase, and introducing several different phosphotyrosines, 00:22:05.18 you nucleate a molecular complex. 00:22:09.18 And these can be very large and many biological events radiate from that 00:22:14.20 single activation of kinase. 00:22:17.01 That then functions to integrate many other molecules into a biological response. 00:22:25.00 So those are two examples and what I would like to do in the next lecture 00:22:31.10 because these kinases are so important for disease, 00:22:35.08 they have become important structural targets. 00:22:38.17 And so in the next lecture I'd like to talk about the structure of the kinase 00:22:44.14 using PKA as an example but try to help you understand how a kinase works as a molecule. 00:22:51.11 And then after that I'll talk about how it's regulated and localized. 00:22:55.28