Session 8: Protein Kinases

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

00:00:05.18 In the first lecture I tried to articulate for you
00:00:08.02 why protein phosphorylation is so fundamental for biology.
00:00:11.25 What I'd like to do here is to delve more deeply into the protein kinase structure and function.
00:00:19.17 And if we look at PKA, PKA is, again, a prototype kinase we understand best.
00:00:28.18 And it is activated by hormone neurotransmitter binding to the outside of a cell
00:00:37.00 in this case adrenalin. Every time...adrenalin works the same as glucagon.
00:00:40.25 And every time you secrete adrenalin into your bloodstream
00:00:43.21 you are activating PKA and you're binding to a G protein coupled receptor.
00:00:48.11 You're going through a G alpha subunit and you're hooking up to adenylate cyclase,
00:00:53.08 which leads to the generation of cyclic AMP.
00:00:56.12 And in this case, PKA, again, is dormant and it's activated when
00:01:03.22 cyclic AMP binds to the regulatory subunits and then you unleash the catalytic activity.
00:01:09.02 And there are many substrates.
00:01:10.10 So I'm going to focus here on the catalytic subunit. How does that work as a catalyst?
00:01:16.03 And next time we'll talk about how it's kept in this dormant state by the regulatory subunits
00:01:21.25 and how it's targeted to specific sites by scaffold proteins.
00:01:25.25 But, today I want to focus on the kinase.
00:01:28.07 So if we go back to this kinase history, we began to find...
00:01:35.17 there are many many protein kinases and this came from cloning many different proteins.
00:01:41.25 So you could very quickly get the sequence.
00:01:43.24 Instead of doing this sequence the hard way, by chemically doing that sequence.
00:01:49.15 And so the first protein kinase sequence was PKA. It was solved by Tatoni in 1981.
00:02:00.11 And it served then as a template, as a framework.
00:02:05.13 We had...I told you Src was cloned in 1979 but it wasn't until PKA was sequenced
00:02:11.03 that Margaret Dayhoff realized that Src was related to PKA. The two sequences were related.
00:02:18.12 So that's what put them on the same tree here.
00:02:21.02 And then in 1991, we did the first structure of a protein kinase.
00:02:25.10 And that allowed us to see the 3-dimensional features of this enzyme family.
00:02:32.07 So, let me tell you the kinds of information we gleaned from each of those findings.
00:02:40.04 So here's the kinome. The kinome is based on sequence analysis only.
00:02:46.03 And most genomes now have about 2% of their genome is coding for protein kinases.
00:02:54.11 And plants are the winners. They have 4%. They have the most protein kinases.
00:02:59.04 So it's one of the largest gene families in every eukaryotic organisms.
00:03:05.26 If we look at the sequence. So what does the sequence tell us?
00:03:10.07 It tells us that...this is a chemical description of your molecule.
00:03:15.10 You can see how from that sequence,
00:03:18.00 we could then immediately know that the cloned Src is a homolog of PKA.
00:03:23.11 And then we could do chemical things; modification with an ATP analog, here modified lysine 72.
00:03:31.08 That told us this lysine was close to the active site, close to the ATP binding site.
00:03:36.06 And it also...we later showed that you could
00:03:40.25 cross-link it to two different carboxylic acid residues, acidic residues.
00:03:47.07 So that all three of these residues, which are far apart in the linear sequence,
00:03:52.00 must be close together and near the active site of the folded protein.
00:03:57.07 And then the other piece of information here is the phosphorylation site,
00:04:02.01 which is essential for activity and that was associated with this particular threonine.
00:04:10.02 So those are the little bits of information we had.
00:04:12.09 You could take those two kinases,
00:04:16.02 once you knew that Src and PKA were related and other kinases became cloned.
00:04:20.04 The initial finding was from Margaret Dayhoff that Src and PKA were related
00:04:25.10 and had many conserved residues.
00:04:28.02 And then Tony Hunter...Hanks and Hunter did an analysis of the subdomains
00:04:35.13 and elucidated these various subdomains, each having a specific motif.
00:04:39.09 And those subdomains have really held up with time as the definition of the protein kinase core.
00:04:47.20 But then when we had the structure, now you could begin to understand
00:04:52.15 how those motifs fold together to form an active kinase.
00:04:57.09 So now you can begin to map that sequence
00:05:03.22 more comprehensively onto a structural framework.
00:05:07.18 So here we show the two major domains that are conserved in the kinase family.
00:05:12.17 One, the small lobe, is mostly associated with ATP binding.
00:05:16.12 The large one has a lot of residues associated with peptide binding and catalysis.
00:05:21.28 Those are the conserved residues and you can see they're spread out over the whole kinase core.
00:05:27.02 It also has this phosphorylation site here. This is essential for its activity.
00:05:33.01 And another phosphorylation site out here at the C-terminus, outside the core.
00:05:37.18 OK, so this is the N-lobe and this is the C-lobe.
00:05:41.26 OK, so now we can have a more in depth understanding of those motifs.
00:05:48.09 And this is what we now know is the conserved core that is shared by all of those proteins on that kinome tree.
00:05:56.25 They all have this common element to them.
00:06:02.09 OK, so if we look at different examples,
00:06:05.27 I've showed you PKA, I earlier talked about Src down here with its SH3 and SH2 domains.
00:06:13.15 These are just some other kinases. And you can see PKA is actually kind of unusual
00:06:19.06 in that it has a relatively small...it has little tails at each end but it's mostly
00:06:26.04 the kinase domain and its regulatory part is a separate subunit--the regulatory subunit.
00:06:33.00 Whereas other kinases have fused domains: C1 and C2 attached to PKC.
00:06:37.21 They bind to calcium and diacyl-glycerol.
00:06:41.01 Cyclic GMP protein kinase, very similar to PKA, only it's cyclic GMP binding domains are fused to the kinase core.
00:06:48.12 So there's all of those have in common the kinase core but each has these variations.
00:06:54.18 So now, again, if you come back to the core,
00:06:58.23 you can actually map those subdomains onto the kinase core.
00:07:03.22 And so I'm going to show you, again, how these now correlate with the N-lobe and C-lobe.
00:07:10.20 And tell you a little more about the two lobes as well.
00:07:13.14 Here's now the sequence of PKA where we can map the secondary structure;
00:07:20.00 a helix versus a beta strand. You can map it onto to those subdomains
00:07:24.22 and have a 3-dimensional context to those subdomains.
00:07:30.12 So here's those subdomains mapped onto the core of PKA and color-coded again.
00:07:38.14 So you can see the subdomains that comprise each of these lobes, the entire conserved core.
00:07:47.06 And, I'm going to walk you through these really quickly
00:07:50.09 just to show you some information about these.
00:07:54.25 So, this is the first on. Subdomain I is what we call the gylcine-rich loop.
00:08:00.03 And here you can see the gylcine-rich loop here.
00:08:03.21 You can see the 3 gylcines that are conserved in most kinases up at the top.
00:08:08.21 And the features you can begin to sort out what are the features that this motif does.
00:08:15.01 And then that's followed by subdomain II.
00:08:18.10 Here's that lysine, that K. That's the lysine that gets modified by ATP analog.
00:08:24.02 This is the C-helix. This is only helical element that is in the small lobe.
00:08:32.08 And then you have another beta strand.
00:08:37.07 And then you have this subdomain V that actually links the two domains.
00:08:43.12 This beta strand is in the N-lobe and this helix is in the C-lobe.
00:08:49.25 And this little piece joining them is the linker that joins the two lobes.
00:08:55.09 And then the E-helix. This is a very hydrophobic helix.
00:09:00.01 And the catalytic loop.
00:09:02.11 This is going to be essential for catalysis. It's a little beta sheet that's in the C-lobe.
00:09:09.04 And this is another part of that beta sheet. This is the activation loop.
00:09:15.14 And so I'll talk about these in more detail.
00:09:18.03 This is the F-helix--very, very important. This entire molecule is organized around this F-helix.
00:09:25.05 It's unusual in that it goes right through the middle of the C-lobe.
00:09:29.20 It's very hydrophobic. Just analyzing it you would think it was a membrane-spanning helix.
00:09:35.04 Extremely hydrophobic.
00:09:37.14 And then this G-helix serves as a docking site for proteins and H-helix.
00:09:42.10 And here just to show you an example comparing the subdomains of PKA and Src,
00:09:48.10 you can see there are little differences but overall, those subdomains are conserved.
00:09:53.10 The fold is conserved in all of those protein kinases, all of those 500.
00:09:58.28 So, I'm going to tell you about these lobes first
00:10:02.06 because I think it will give you an understanding of how those subdomains work together
00:10:07.02 synergistically to create an active kinase.
00:10:10.00 And so this is the N-lobe and it contains this glycine rich loop
00:10:15.02 which I told you about. Those are the three glycines.
00:10:17.16 And then the C-helix is the other really conserved feature here.
00:10:22.09 And you can see the lysine 72 and Glu91.
00:10:26.03 Those are the residues that were linked together by our chemical studies.
00:10:30.14 So when we first saw this structure, my first thing was to say,
00:10:34.07 "Where's that lysine? And where are those residues that cross-link to it?"
00:10:37.19 And it was...they were all right next to each other just as we thought from the chemical studies.
00:10:43.17 But we couldn't understand how they related to each other until we saw that structure.
00:10:49.04 OK, so the glycine loop is the most mobile part of the kinase.
00:10:58.18 And it has to open and close.
00:11:01.08 And when it closes it fits down on top of the gamma phosphate of ATP
00:11:05.28 and positions it to be transferred to a protein substrate.
00:11:11.08 So this opening and closing of the glycine loop is essential for catalysis.
00:11:16.01 And that hydrogen bond there between the glycine loop and the gamma phosphate of ATP is critical.
00:11:26.22 Now we go to the large lobe.
00:11:28.17 So it's mostly helical but it has this beta sheet that's right at the active site cleft.
00:11:33.20 And some of those conserved residues lie here in these two loops
00:11:39.18 and those are very essential for transferring that phosphate.
00:11:42.29 And that lies on top of this very stable helical subdomain.
00:11:48.03 The N-lobe is very malleable. This lobe is very, very stable.
00:11:53.00 And some key features that the phosphate that's important for activating the kinase
00:11:59.16 is here, and when you phosphorylate that site, the active site is maximally active.
00:12:10.10 Without that phosphate it's not active.
00:12:12.09 So even though it's 20 angstroms away from the active site, it is essential to create the active kinase.
00:12:18.15 And without that phosphorylation, the enzyme really unfolds very easily
00:12:23.16 and its chemical properties are quiet different.
00:12:27.02 One phosphate, just one phosphate.
00:12:30.02 This is the catalytic loop. Many of the catalytic residues that are important.
00:12:34.07 This P+1 loop is important from docking peptide substrates.
00:12:40.11 So, if we look at a mimic of what we think might be a transition state for transferring the phosphate,
00:12:47.06 this is the...aluminum fluoride serves as a mimic for the gamma phosphate of ATP
00:12:54.19 and you have the residues from the catalytic loop in the C-lobe,
00:12:58.17 the residues from the glycine rich loop in the N-lobe.
00:13:01.15 They converge on that gamma phosphate and transfer it to the peptide substrate.
00:13:06.17 So, very beautiful chemistry of how these come together at the active site.
00:13:12.05 So let's just look, for a moment, at catalysis.
00:13:15.03 So, the kinase must open and close as part of its catalytic cycle.
00:13:20.19 So, when it's open, it's actually quiet malleable.
00:13:24.28 Then, it binds ATP and its peptide substrates and here, for that transient moment of catalysis,
00:13:32.05 it's in a closed conformation.
00:13:34.08 So, if we look at how these look in the molecule, this is the open conformation on the left
00:13:44.20 where you have a hole there, that's where the ATP is going to fit into that big hole.
00:13:48.19 And then the closed conformation, you can see how that active site cleft really comes down and closes.
00:13:55.09 And now, in this...this is just going to give you an image of how this kinase opens and closes.
00:14:01.19 And it's morphing the open and closed states.
00:14:03.24 And I'm just going to point out, before we see the movie, the lysine 72 and the Glu91.
00:14:09.22 Those were two of the residues that cross-linked.
00:14:11.24 So now you can get a sense of how opening and closing of the active site cleft is taking place.
00:14:21.11 And so this is just a morphing--the open conformation and the closed conformation,
00:14:25.25 essentially the breathing of a protein kinase molecule.
00:14:30.21 OK, so if we look up close now at that closed conformation where ATP is bound,
00:14:37.06 you can see most of the ATP is buried in that deep hole there.
00:14:40.22 And just the little gamma phosphate is sticking out at the edge of the interface
00:14:46.12 between the glycine rich loop from the N-lobe and the catalytic loop from the large lobe.
00:14:53.00 And then you can see here how a peptide fits into that site.
00:14:57.01 So, it docks mainly onto the large lobe.
00:15:00.27 Here's the P+1 residue. It's a nice, in this case, hydrophobic pocket
00:15:04.20 where that hydrophobic residue docks.
00:15:07.29 This is a pseudo-substrate. It's an alanine instead of a serine.
00:15:11.06 If that was a serine it would accept the phosphate and transfer the phosphate.
00:15:16.08 And then, PKA likes basic residues and so you have acidic residues on the protein in red
00:15:23.06 that recognize those basic amino acids in the peptide.
00:15:26.09 And then for the inhibitor peptide which is bound in...this is a very high affinity binding peptide.
00:15:35.17 It has a helix that docks to the active site cleft and this serves as a tethering site.
00:15:45.08 So I've told you how PKA works. It's the kinase that we understand best in terms of structure and function.
00:15:53.10 But now we have this whole kinome tree.
00:15:55.22 And, what can we learn now from a large family?
00:16:02.15 We have...because these are so important for diseases, we have many kinase structures.
00:16:07.17 Kinases have become a major target for drug discovery. So we have many kinases.
00:16:12.22 So I've shown you what we can learn from in depth analysis of one kinase.
00:16:18.13 What can we learn from this structural kinome now where you have
00:16:22.26 not only many sequences but you have many structures?
00:16:26.16 And, we can learn different kinds of things.
00:16:28.28 I'm going to tell you some global lessons we can learn about the entire family.
00:16:32.10 One can delve deeply into a single family
00:16:35.05 and find out what are the unique features of one subfamily versus another.
00:16:39.27 So this is just six of the different kinase structures, kinase cores.
00:16:47.25 And you can see that the subdomains are mostly conserved in all of these kinases.
00:16:53.22 What can we learn from that structural kinome?
00:16:59.04 One of the things that we did was to develop a method which we call Local Spatial Pattern Alignment.
00:17:07.01 And this is just rapidly comparing any two structures and looking at the spatially similar residues.
00:17:15.09 And this provides you with a pattern.
00:17:18.05 You can do this independent of any sequence alignment.
00:17:20.25 Just two structures, compare them.
00:17:23.07 And you get figures like this that shows the spatial relatedness of residues in two protein kinases.
00:17:34.00 And so if we look here you can see a couple of key residues
00:17:38.09 that these edges indicate a spatial link between two amino acids.
00:17:46.23 And the more edges that go to a particular amino acid, the higher its involvement score.
00:17:52.15 We call that an involvement score.
00:17:54.24 So we make a network of spatial related residues in two protein kinases.
00:18:01.24 And this is called the involvement score. The higher the involvement score,
00:18:05.15 the more spatially-related amino acids interact with that particular amino acid.
00:18:12.24 So we use this first to compare active and inactive kinases.
00:18:20.04 What is different?
00:18:21.06 Can you find spatially-related differences that are unique to
00:18:28.01 the active kinase and that are not there in the inactive kinase?
00:18:31.06 And from this analysis we defined what we call a spine.
00:18:38.06 We've subsequently called it a regulatory spine
00:18:42.00 It's a hydrophobic spine that is spatially conserved in every active kinase
00:18:47.12 and is broken when the kinase is inactive.
00:18:50.22 And it's made up of hydrophobic residues.
00:18:53.02 They come from non-contiguous residues. So one is coming from beta-4, one is from alpha-C,
00:18:59.16 one is from the DFG motif, one is from the HRD motif, two are in the N-lobe, two are in the C-lobe.
00:19:08.14 And they align to make this hydrophobic spine.
00:19:12.00 You would not ever find this by sequence comparisons
00:19:15.11 because, by sequence analysis, they're non-contiguous
00:19:17.26 but they are spatially well-defined as a conserved motif.
00:19:25.20 So we then went back and looked at all of the kinases
00:19:29.00 and found that there were actually two spines.
00:19:34.03 And so we found a second spine, which we call the catalytic spine, in addition to the regulatory spine.
00:19:40.22 And the importance of...again, it's hydrophobic residues that are from both the N-lobe and the C-lobe.
00:19:48.04 The unique thing about the catalytic spine is that it's the adenine ring of ATP that
00:19:52.19 completes the spine and links the two.
00:19:54.26 So that means once you add ATP, you now have a new coordination between the N-lobe and the C-lobe.
00:20:04.02 So the other feature that came from this analysis is this F-helix.
00:20:08.09 This a very hydrophobic helix that spans the C lobe.
00:20:12.06 This is very unusual for a globular protein like a kinase.
00:20:17.02 But, it's very highly conserved and it serves as the framework...
00:20:22.22 it's linked to both the regulatory spine and to the catalytic spine
00:20:29.05 And it nucleates...it really provides the architecture on which you assemble an active kinase.
00:20:36.27 And we call this the S2H motif or two spines and a helix.
00:20:41.19 And that is really the fundamental architecture that every protein kinase.
00:20:46.21 So, here I show you just that F-helix.
00:20:49.12 You can see how it goes right through the middle of this C lobe.
00:20:55.02 You would never predict that this was part of a globular protein by sequence analysis.
00:21:01.10 And you can see, as this rotates around, the relationship of that F helix to the rest of the protein.
00:21:13.06 It really...everything is nucleated around that F helix.
00:21:16.25 You can see some of the key residues that emerge
00:21:20.29 that are going to be important for linking to the two spines that are coming out of that F helix.
00:21:26.26 There you can see the two spines,
00:21:28.04 how those two spines are really anchored in a very fundamental way to the F helix
00:21:33.10 and the catalytic loop. All the functional elements are really linked to this F helix.
00:21:44.12 So, again, you can see here, when you open and close the kinase,
00:21:48.22 those conformations...you can see that the two spines stay intact as part of that breathing motion.
00:21:55.10 It does not interfere with the breathing motion of the kinase.
00:22:02.03 So, I'll give you an example here of the...this is the insulin receptor,
00:22:06.15 another member of that tyrosine kinase branch.
00:22:11.03 And this is the active conformation of the insulin receptor and you can see that
00:22:15.00 the two spines are intact. This is a fully active conformation of the insulin receptor kinase.
00:22:20.27 When it's not active (and it's activated by phosphorylation of its activation loop here),
00:22:28.06 when it's not phosphorylated on its activation loop,
00:22:31.13 then you can see the spine in broken.
00:22:33.25 And this is one way in which it can be broken.
00:22:37.28 Actually, a residue from the regulatory spine goes over and fills the adenine pocket
00:22:43.07 for the catalytic spine.
00:22:44.10 But there are many, many variation of how you can break that spine.
00:22:49.20 So, why is that phosphate so important?
00:22:53.00 I talked about this phosphate being really important.
00:22:58.08 It's important for the insulin receptor. It's important for PKA.
00:23:02.04 That phosphate on the activation loop...
00:23:04.22 And I'm just going to show you: Here's a phosphate in PKA
00:23:08.22 and it's making seven different either electrostatic or hydrogen bond interactions
00:23:17.10 with different side chain residues.
00:23:20.09 And it's such an integrating phosphorylation site.
00:23:24.19 It goes to this histidine which is in the C helix, in subdomain 3.
00:23:30.10 It goes to lysine 189 which is actually in beta-strand 9.
00:23:37.26 It goes to arginine 165 which right before the catalytic loop.
00:23:44.26 And it integrates this whole activation loop.
00:23:48.24 So, it's playing a critical role in integrating all of the subdomains of the protein.
00:23:56.18 So in this last part I wanted to go back to the kinases and disease
00:24:00.07 and again, relate to what I told you about these subdomains and the spines.
00:24:08.25 There are probably more now, but at least 30% of kinases are implicated in various diseases
00:24:13.28 and that is just growing all the time as we delve more deeply into the different kinases.
00:24:20.08 Many more are likely to follow as we get more and more genomic information
00:24:24.21 and disease correlations.
00:24:28.10 Kinases are tractable as drug targets because you can inhibit them and
00:24:34.00 I'm going to show you can example of that.
00:24:36.01 So this is the human kinome and I'm showing you one kinase in the tyrosine branch there.
00:24:42.28 This is Abl, a relative of Src.
00:24:46.07 And in this fatal disease chronic mylogenous leukemia
00:24:51.23 it's this Abl which is modified. It's fused to another protein and it makes it an oncogene.
00:24:56.27 So, it's constitutively active. It's turned on all the time.
00:24:59.23 That's what makes an oncogene--you can't turn it off.
00:25:03.03 So, this Gleevec was discovered as a very specific inhibitor of BCR-Abl.
00:25:12.07 And this is the real proof of principle that says that kinases are very tractable drug targets.
00:25:23.07 If we look at Abl with ATP bound to it you can see here the two spines;
00:25:28.09 the regulatory spine, the catalytic spine. This is active Abl.
00:25:32.16 And then you compare that now to the structure where Gleevec is bound
00:25:37.17 and this was done by John Kuriyan's laboratory.
00:25:42.04 It is bound to the active site cleft but you can see when Gleevec binds,
00:25:46.22 the regulatory spine is broken and so it's actually weaving through
00:25:52.07 both the catalytic spine and the regulatory spine.
00:25:57.10 So it binds to an inactive conformation
00:25:59.12 and you can see precisely the features that are being taken advantage of by Gleevec.
00:26:05.03 So, one of the sites that is frequently associated with resistance to Gleevec
00:26:11.20 is a mutation of this threonine residue in the ATP binding pocket here.
00:26:17.10 So this threonine is sometimes referred to as the gatekeeper.
00:26:20.04 And I think in the previous slide...here's the gatekeeper residue.
00:26:25.09 I show you where the gatekeeper residue is.
00:26:27.09 It's a threonine. And so the mutation that takes place often is that it's converted to methionine.
00:26:33.10 So threonine is a small residue. Methionine is a much bulkier residue.
00:26:37.24 It's so bulky that it will prevent Gleevec from binding.
00:26:41.15 It's a steric hindrance so it blocks that.
00:26:43.19 But, it does more than that and this recent study by Daley and Kuriyan shows that
00:26:51.07 when you have the methionine there instead of the threonine,
00:26:55.08 methionine is a nice hydrophobic residue and it fills a gap between
00:27:01.22 the catalytic and the regulatory spine and what it does is it now creates a very stable, active spine.
00:27:08.06 So by changing that one residue, threonine, to an isoleucine (methionine),
00:27:13.17 you not only abolish the binding of Gleevec, you also create an oncogene.
00:27:19.03 You now have stabilized this regulatory spine in a way that no longer requires phosphorylation.
00:27:26.01 So this is not only resistant to Gleevec, this protein is an oncogene.
00:27:35.06 So, I've talked about the subdomains and how they're conserved in the family
00:27:39.26 and I have given you an appreciation of how kinases work
00:27:42.24 and why they are a tractable target for drug discovery
00:27:46.18 and then what I want to talk about next time is how the kinases regulate
00:27:51.23 and to really do that we're not going to be so interested in the catalytic machinery,
00:27:56.01 we're going to be really interested in the surfaces of the kinase
00:27:59.18 and how does PKA in particular bind to other proteins.
00:28:04.04