In this lecture, I have given an overview of protein kinase structure and function using cyclic AMP dependent kinase (PKA) as a prototype for this enzyme superfamily. I have demonstrated what we have learned from the overall structural kinome which allows us to compare many protein kinases and also to appreciate how the highly regulated eukaryotic protein kinase has evolved. By comparing many protein kinase structures, we are beginning to elucidate general rules of architecture. In addition, I have attempted to illustrate how PKA is regulated by cAMP and how it is localized to specific macromolecular complexes through scaffold proteins.
All Course Materials for this Session (Educators only) – Created by Susan Taylor
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.
Scientists have discovered a new drug that inhibits an overactive kinase implicated in cancer. How do the scientists determine the drug’s mechanism of action?
To help you prepare for this, answer the following sub-questions:
- What are the key structural components of a kinase?
- How do kinases regulate signaling pathways inside the cell?
- Why is it important for the cell to have an on/off switch?
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.
Dr. Taylor received her BA in Chemistry from the University of Wisconsin-Madison and her PhD in Physiological Chemistry from Johns Hopkins University. After completing a fellowship at the MRC in Cambridge, she moved to the University of California, San Diego where she soon secured a faculty position in the chemistry department. Shortly after joining UCSD,… Continue Reading