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Protein Kinases: Structure, Function, and Regulation

Part 1: Protein Phosphorylation in Biology

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

Part 2: Architecture of a Protein Kinase

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

Part 3: Protein Kinase Regulation and Localization

00:00:06.01 So in the first lecture I stressed the importance of protein phosphorylation for biology
00:00:13.15 and in the second lecture I tired to articulate for you
00:00:19.08 the protein kinase molecule, the three-dimensional molecule and how it works.
00:00:23.08 What I'd like to do here is focus on how that kinase is regulated and how it is localized.
00:00:31.07 And so again, I'm going to focus on PKA as the model system
00:00:36.02 in part because it's the kinase we really understand the most about in biochemical terms
00:00:41.07 and structural terms.
00:00:43.21 So, again, it is activated by a hormone binding to a GPCR,
00:00:49.18 which then leads to the generation of cyclicAMP
00:00:53.17 and then that unleashes the catalytic activity of PKA, the kinase.
00:01:00.19 What I'm going to focus on today is this other side where it is in its inactive state
00:01:06.03 and also targeted to specific sites in the cell by A Kinase Anchoring Proteins.
00:01:14.18 This is really an essential feature of specificity and biological function for PKA.
00:01:21.20 And so we're going to look at the holoenzymes.
00:01:26.08 I showed you previously what we learned from the structure of the catalytic subunit by itself.
00:01:33.06 What I'm going to tell you here is how much more information we obtained from
00:01:39.28 having a structure of the holoenzyme complex.
00:01:42.13 And then I'm going to focus on the A Kinase Anchoring Proteins
00:01:47.10 and tell you how they target PKA to specific sites
00:01:50.22 and again really leave you with the global challenge for all of us
00:01:57.22 to understand how these kinases exist as part of larger, often very large, macromolecular complexes.
00:02:06.25 So, if we look at that kinase that I told you about before,
00:02:12.10 we have the structure of PKA bound to an inhibitor peptide
00:02:21.15 and I told you about the active site residues and what I want to look at now...
00:02:28.00 Here's the kinase with the two lobes with ATP bound at the cleft between the two lobes
00:02:34.04 and I've told you about those subdomains. I think you have a good concept of those subdomains now.
00:02:40.08 What we're going to focus on now is the surface of that kinase
00:02:44.08 because it's the surface that the kinase uses to interact with other proteins.
00:02:49.25 And these kinases are protein interaction modules.
00:02:53.19 They communicate with other proteins and they use their surface to do that.
00:02:57.24 So, here's the PKA with its conserved subdomains
00:03:01.22 and now if we look at that core of PKA you can see its crevices, the charged features of that surface.
00:03:10.05 Each kinase is quite different on its surface and that dictates what proteins it can interact with.
00:03:16.06 So one of the major proteins that binds to the catalytic subunit
00:03:20.10 are the regulatory subunits and they are multi-functional, modular and highly dynamic.
00:03:25.27 It has two cyclic AMP binding domains at the C terminus
00:03:30.20 and then it has an inhibitor sequence that binds to the active site cleft of the catalytic subunit
00:03:37.21 and then at the N-terminus it has a dimerization domain.
00:03:40.07 This linker region, in the absence of cyclic AMP or catalytic subunit,
00:03:49.26 is a very dynamic entity, this linker region.
00:03:54.00 And so it's hard to do structure of the whole thing.
00:03:56.20 We could do structures of the monomeric forms
00:03:59.20 and they're perfectly functional in terms of binding cyclic AMP and binding catalytic subunit
00:04:04.01 with very high affinity, sub-nanomolar affinity.
00:04:07.03 And so we did structures of this one. We've also done a structure of the monomeric form.
00:04:13.00 This first structure in 1995 showed us the cyclic AMP binding domain
00:04:21.28 and told us how this regulatory subunit functions as a receptor for cyclic AMP.
00:04:28.28 And cyclic AMP is a major signaling molecule in all of biology.
00:04:36.15 It's a second messenger signal of external stress in bacteria as well as in man.
00:04:43.05 And the domain that it binds to, this Cyclic Nucleotide Binding domain, is also conserved.
00:04:49.12 So it's binding to this domain that now translates that cyclic AMP signal into a biological response.
00:04:57.29 So the general features of this domain were
00:05:01.24 really elucidated by this structure of the cyclic AMP bound R subunit
00:05:05.26 and you can see the same domain is conserved in a number of other proteins.
00:05:10.01 So, looking more closely at that motif there is what we call a phosphate binding cassette,
00:05:17.05 this PBC which anchors the phosphate of cyclic AMP--another very important phosphate in biology.
00:05:25.06 And then in addition you have as part of that conserved motif are these B and C helices
00:05:31.16 which are functionally conserved in all of those domains
00:05:35.02 and each cyclic nucleotide binding domain has a hydrophobic capping residue.
00:05:39.07 So you can see that the adenine ring is capped between two hydrophobic residues.
00:05:44.03 And this can come from different part of the molecule
00:05:49.17 but the PBC, the B and C helices are conserved.
00:05:53.28 And so, if we now look at what we have learned from the individual proteins,
00:06:00.11 from cyclic AMP bound structure of the regulatory subunit and from the free catalytic subunit,
00:06:06.00 we've learned a lot. How does this function as a receptor?
00:06:09.07 How do we understand that cyclic AMP binding motif?
00:06:11.24 How does it work as a catalyst? So we've learned a lot.
00:06:16.01 But we didn't really understand how is the catalytic subunit actually inhibited by the regulatory subunit
00:06:23.15 nor did we understand how the holoenzyme is activated by cyclic AMP.
00:06:28.26 And these were things that we really have learned just in the last few years
00:06:33.00 and for me it has certainly demonstrated to me the importance of
00:06:37.15 having larger complex to be able to really understand function
00:06:41.19 and certainly we have understood so much more about the function of PKA
00:06:45.16 having a structure of a holoenzyme complex compared to what we knew
00:06:49.17 with just the free catalytic subunits and regulatory subunits.
00:06:53.28 So this is the small cyclic AMP binding domain protein bound to the catalytic subunit
00:06:59.15 and you can see several things from this structure.
00:07:03.03 First, the red part is the inhibitor peptide which was always disordered before.
00:07:07.28 Now it becomes ordered at the active site cleft.
00:07:11.04 The major interactions here of this surface are with the large lobe.
00:07:14.23 So the large lobe is an important docking site for many proteins to bind to protein kinases.
00:07:23.20 OK, so let's look more closely at what was really surprising to us about this structure.
00:07:30.00 We really didn't anticipate this at all.
00:07:32.17 So, on the left we have the structure of the cyclic AMP bound domain
00:07:37.23 and then on the right I'm going to show you what happens when you release cyclic AMP
00:07:45.05 and you bind to the catalytic subunit and you can see a major conformational change.
00:07:52.10 The helical subdomain changes and this inhibitor peptide becomes ordered,
00:07:57.02 that is, ordered at the active site cleft.
00:07:59.03 And in this structure that inhibitor region was there but it was always disordered.
00:08:03.29 I told you that linker is very disordered when it's a free regulatory subunit.
00:08:09.25 So let's look at first this ordering of the inhibitor site.
00:08:16.06 So I told you in the last lecture how PKI, a small inhibitor peptide,
00:08:19.26 is bound to the active site cleft and they have a common mechanism,
00:08:26.21 this region that's circled here is the common inhibitor region.
00:08:30.20 It also mimics a substrate. So that binds directly to the active site cleft
00:08:35.01 and they do it in the same way.
00:08:36.18 PKI on the other hand bound with that amphipathic helix.
00:08:40.10 That achieved, again, sub-nanomolar affinity but on a different surface.
00:08:44.14 Whereas here you can see that the regulatory subunit
00:08:46.15 is using a completely different surface to achieve high affinity.
00:08:50.27 And I'm going to show you this again so you can appreciate the dynamic features,
00:08:55.25 especially this B/C helix, how that B/C helix goes from a kinked structure to an extended structure.
00:09:02.12 So, a very, very dynamic module
00:09:05.02 and we didn't anticipate such a conformational change at all before seeing the structure.
00:09:11.02 So here you can see again that we've docked the inhibitor peptide
00:09:14.16 and then you can see as your morph from the cyclic AMP bound conformation into
00:09:18.22 the unbound conformation you can see that C helix, how, as it extends, it binds onto the surface of the large lobe.
00:09:28.07 So, it makes a major contact with the large lobe
00:09:31.22 So this is the activation loop that I told you about before.
00:09:34.28 Here's that phosphate. This is an active kinase.
00:09:36.27 The regulatory subunit doesn't bind very well if it's not an active and phosphorylated kinase.
00:09:42.17 And so, I also told you that this threonine feeds back
00:09:45.19 to all those different parts of the molecule, including the active site.
00:09:48.26 It makes an active kinase.
00:09:50.13 So, if we look where that helix from the regulatory subunit is docking,
00:09:56.13 it's docking right up against that activation loop and this is a critical interface.
00:10:03.13 So, we now have a further understanding of what you achieve by phosphorylating this kinase.
00:10:12.04 You create an active kinase. So you stabilize the active site so it's optimized for catalysis.
00:10:18.18 But the other thing that you do based on the holoenzyme structure,
00:10:22.21 is that you also create a surface here that is now providing a docking site for the regulatory subunit.
00:10:30.22 So, again, it impresses, I think on you and all of us, the importance of
00:10:36.22 one single phosphate on the functioning of this protein.
00:10:42.18 This is now both domains and the questions was: does that helix extend in both domains?
00:10:49.01 And of course it does. It's even longer.
00:10:51.15 And so the consequences of this are really very important in looking at this entire structure.
00:10:58.00 Again, it's a very large, interface that you have here
00:11:01.11 for the A domain and the B domain binding to the catalytic subunit.
00:11:06.06 And looking at it as a static difference, this is the cyclic AMP bound state
00:11:14.20 where this inhibitor site is present but disordered. You don't see it.
00:11:20.25 And then, now when you look at releasing cyclic AMP and binding to the catalytic subunit,
00:11:26.11 you now splay apart the two domains.
00:11:28.14 You have a very long extended helix that separates the two domains.
00:11:33.21 And you have the inhibitor peptide docked to the active site cleft.
00:11:37.11 So it's a major conformational change that you induce in the regulatory subunit
00:11:42.05 as a consequence of releasing cyclic AMP and binding to the catalytic subunit.
00:11:46.17 And the catalytic subunit does not change very much in its conformation.
00:11:51.03 And so this is just letting you see this again and flipping back and forth
00:11:56.16 the change in the proximity and the organization of these two domains.
00:12:01.19 And so you can look, just going back and forth, at how
00:12:06.16 that B domain is really just hooked onto that helix and so when that helix extends,
00:12:12.00 it flips away the B domain and really dissociates the two domains from one another.
00:12:18.17
00:12:20.16 OK, so we've looked at the consequences of binding the regulatory subunit,
00:12:27.10 how the C subunit is inhibited, how it gets activated by cyclic AMP.
00:12:31.08 That came from having a holoenzyme structure.
00:12:33.13 Let's look at this other feature: the kinase...PKA is not typically just floating around in solution.
00:12:41.10 It's often targeted to specific sites.
00:12:45.00 And if you want to look at the dynamics of time and space as part of your signaling equation,
00:12:50.14 then you have to look at how is PKA localized.
00:12:54.07 And in large part, this is achieved by A Kinase Anchoring Proteins,
00:12:59.02 scaffold proteins that bind with high affinity to the regulatory subunit.
00:13:05.02 So let me explain that a little bit more.
00:13:07.13 These are just some of examples that John Scott gave me
00:13:11.14 of different places in which the regulatory subunits can be targeted;
00:13:17.08 to the plasma membrane, to mitochondria,
00:13:21.04 to all different sites so that you have localized communities of signaling.
00:13:27.19 And if we look at what I've told you so far, it's this part of the molecule
00:13:31.24 that's important for binding to the catalytic subunit and inhibiting the catalytic activity.
00:13:38.06 It's this part, the dimerization domain, that is important for targeting to AKAPs.
00:13:44.29 We've looked in particular at Dual Specific AKAPs.
00:13:49.03 Most AKAPs bind to the RII subunits.
00:13:51.25 They bind with very high affinity, in the nanomolar range, to RII subunits.
00:13:55.18 There are some that bind to both RI and RII subunits
00:13:59.12 and those we call Dual Specific AKAPs or D-AKAPs.
00:14:02.03 So, just to again give you a sense of the complexity of this signaling system:
00:14:09.26 This is the L-type calcium channel where you have
00:14:13.11 one of its tails regulated by a PKA phosphorylation site.
00:14:17.19 We've had these extraordinary advances in getting structures of channels and membrane proteins
00:14:25.13 and understanding for the first time, at atomic level resolution,
00:14:28.22 how an ion passes through a channel.
00:14:31.07 But, it's often the tail and the loops that are disordered on the cytoplasmic side
00:14:36.01 that provide the mechanism for regulating, whether that channel is open or not.
00:14:40.02 And this is the case for this L-type calcium channel. There's a PKA phosphorylation site there
00:14:46.15 and PKA is targeted to this site by this AKAP179 which has many binding partners
00:14:54.00 but it targets to PKA by this little mechanism which is an amphipathic helix in the AKAP.
00:15:02.03 And so you bring the kinase there.
00:15:03.19 You have PP2A as a phosphatase, Calcineurin as a phosphatase.
00:15:07.07 Very often, you have in this system, kinases and phosphatases that work together in a synergistic way.
00:15:14.27 And it's a synergy which is another thing that I hope to convey to you.
00:15:19.06 How these molecules work together, how different parts of one molecule,
00:15:23.26 like the kinase work together to create an active enzyme,
00:15:27.29 and how different molecules come together to create a signaling unit.
00:15:33.02
00:15:34.28 So, one example, D-AKAP1, targets PKA to the outer mitochondrial membrane.
00:15:41.28 And if we look at D-AKAP1, these AKAPs have many binding modules
00:15:47.26 and D-AKAP1 has a little PKA binding motif there that I'll explain to you further.
00:15:53.19 And it also has a targeting region. That's what takes it to the mitochondria,
00:15:59.01 the N-terminus and if you add an extra 30 residues at the N-terminus it goes to ER.
00:16:03.15 D-AKAP1 is an example of one of these AKAPs.
00:16:10.05 And here you can just see, if you ordinarily have RI in these cells, it's cytoplasmic.
00:16:15.25 But, if you co-express RI which is labeled with the GFP in green,
00:16:22.02 with D-AKAP1, you find that it all goes to the mitochondria.
00:16:27.01 And this is very characteristic of the mitochondrial morphology in this cell.
00:16:34.09 OK, D-AKAP2 is another one that we've looked at.
00:16:38.06 And D-AKAP2 binding both to RI and RII.
00:16:41.13 It regulates ion transporters and so this is another big function of these AKAPs;
00:16:48.00 to regulate ion transporters.
00:16:49.12 And so, the ion transporter that it was first shown to regulate
00:16:54.21 is the sodium-phosphate ion transporter.
00:16:56.23 And this is regulated not by phosphorylation but by internalization.
00:17:02.20 When you don't want...this is in kidney proximal tubules,
00:17:05.20 when you don't want phosphate to be taken up, you internalize the transporter.
00:17:10.04 And D-AKAP2 is linked to the transporter though a PDZK domain protein
00:17:16.13 and these are little binding modules (PDZ domains) that now link the AKAP to the co-transporter
00:17:25.21 and PKA, through the AKAP.
00:17:30.04
00:17:32.16 So, let's look more closely at D-AKAP2, at just its binding motif.
00:17:38.17 So, the C-terminus of D-AKAP2 contains all the information that important for binding to PKA.
00:17:45.21 It's this little region in red here. It's modeled to be an amphipathic helix.
00:17:50.02 It is an amphipathic helix.
00:17:51.07 And then this little bit at the end, the three residues at the end are what target it to that PDZ domain.
00:17:57.23 But it's going to be this amphipathic helix that we took,
00:18:02.23 this peptide, and crystallized it together with the dimerization domain of RII-alpha.
00:18:09.00 You can see here this complex.
00:18:11.10 The red is the AKAP peptide. It binds with 2nM affinity, very, very tightly.
00:18:17.07 And you can see the N-terminus is a 4 helix bundle of both chains of the regulatory subunit.
00:18:25.04 And then, most recently we did the structure of the RI dimerization domain.
00:18:31.26 Here, where this has a lower affinity, 50nM
00:18:36.27 but critical for docking, particularly under stress conditions,
00:18:44.11 of targeting PKA, the RI subunit. So the RI subunit it typically more soluble than the RII subunits.
00:18:54.00 The RII subunits are mostly targeted.
00:18:56.16 The RI subunits are cytoplasmic but they can be dynamically recruited.
00:19:02.00 If you cap a T cell for example, RI is normally soluble, you can cap it with an antigen.
00:19:07.14 RI goes to the cap site.
00:19:09.19 So, RI is much more dynamic.
00:19:12.13 And it also has disulfide bonds that actually link the two protomers.
00:19:16.09 So, we have a chemical understanding of these docking motifs.
00:19:20.17 And here is just showing you, again, that RI subunit...
00:19:25.05 how this amphipathic helix is binding specifically to this surface
00:19:29.11 and seeing that as a surface, a very close packing of the small helix to this dimerization domain
00:19:40.12 and when you remove the peptide you can see this groove.
00:19:43.00 It's a very characteristic groove and quite different for RI and RII.
00:19:46.25 So that's how you achieve targeting.
00:19:51.02
00:19:53.05 So, let's look at one more feature.
00:19:56.06 I told you how important it was for us to be able to understand the holoenzyme complex,
00:20:03.05 that complex of a monomeric R subunit and C subunit.
00:20:06.18 So, in the physiological state, RI exists as a dimer.
00:20:14.02 So this is a homodimer of two chains of the regulatory subunit.
00:20:20.21 You have at the N-terminus the dimerization domain that I just told you about.
00:20:26.15 We have the cyclic AMP binding domains at the C-terminus and this very disordered linker region.
00:20:33.25 And so when we look at this linker region you can see
00:20:37.16 first of all, this is all disordered in the free R subunit
00:20:40.05 and you have the inhibitor peptide that binds to the active site cleft.
00:20:44.05 You have other motifs. There are phosphorylation sites, a putative Grb2 binding site,
00:20:49.08 There are many kinds of things that can be going on in this disordered region.
00:20:54.22 So what happens when you bind and make a holoenzyme?
00:20:58.26 Well, we've only seen heterodimer: monomeric R with C,
00:21:03.23 but we know under those conditions that this part becomes ordered.
00:21:07.12 So that becomes ordered.
00:21:09.00 So we're left with a small region that still seems to be quite disordered.
00:21:14.10 So, how do we get an handle on that?
00:21:18.12 So, one way that you can do this is to actually introduce...
00:21:24.28 so this is the part that's left, you can introduce cysteine residues.
00:21:29.11 Do cysteine scanning mutagenesis and put fluorescent probes in and just look very generally at flexibility.
00:21:35.19 How flexible is that residue when nothing is bound versus when you have a holoenzyme?
00:21:40.15 And when we did this with David Johnson at Riverside,
00:21:44.29 we found that these residues, all of these, in the absence of any ligand, cyclic AMP or C subunit,
00:21:53.13 these were very, very flexible.
00:21:55.07 When you added holoenzymes, some of them stayed flexible but others became much less flexible.
00:22:02.03 So this one at the end, for example, stay flexible.
00:22:04.15 This region here becomes very fixed. It's part of that tight holoenzyme complex here.
00:22:10.20 But some of these became more fixed in the holoenzyme complex
00:22:15.21 and that led us to think, "Maybe there are some other interactions there."
00:22:18.21 So, we made a new construct that actually extends further in this N-terminal direction
00:22:26.11 and we crystallized that to see if this communication is going between the two inker regions
00:22:33.13 that we might get some more ordering.
00:22:35.20 And so Angela, a graduate student who carried out this work and got a crystal
00:22:45.02 but it was still a heterodimer. So it had, in one asymmetric unit,
00:22:50.15 a catalytic subunit and a monomeric regulatory subunit
00:22:54.11 but this tail did become ordered and if we looked at that more closely,
00:23:00.02 it didn't order all of it. There's still 10 or so residues missing, but it ordered about 10 more residues.
00:23:06.01 And it ordered it in a way that it wasn't binding to its own cyclic nucleotide binding domain
00:23:12.08 and it wasn't binding to the catalytic subunit.
00:23:14.15 And so we had to really go to the symmetry related dimer to understand how this might be ordered.
00:23:24.05 And so you can rotate it now. We're looking at it from the perspective of
00:23:28.16 the bottom of the dimerization domain looking down on this.
00:23:31.11 And so we can see that the N-terminal part of this inhibitor site
00:23:36.20 is ordered by its interaction with the C and B domain of the symmetry related dimer.
00:23:42.24 And, again, this is the inhibitor site for this dimer
00:23:46.25 and the N-terminus of that is coming over and interacting with the C and B domain.
00:23:50.21 And so this provides an incredible opportunity for allostery
00:23:56.02 where we know the Hill coefficient for activating the heterodimers is about 1.2
00:24:02.11 whereas the Hill coefficient for activating this tetrameric holoenzyme is 2.3-2.4.
00:24:11.05 So there's an incredible about of allostery that you achieve by having the tetrameric holoenzyme.
00:24:19.07 And one can begin to see how you might achieve that.
00:24:22.08 Here you have the inhibitor peptide docking to both of the symmetry related dimers
00:24:28.11 and then cyclic AMP binding.
00:24:30.18 When it binds to this domain it's going to unleash that inhibitory peptide
00:24:36.13 from its own bound catalytic subunit.
00:24:40.09 But you can see how it also has the potential to have an allosteric effect
00:24:44.19 on releasing the inhibitor peptide that's bound to the other heterodimer.
00:24:48.09 So this is very, very exciting for us
00:24:50.24 and although we obviously need to do the structure of the full length holoenzyme and validate this,
00:24:59.08 this structure is of the tetrameric holoenzyme, the model that we create from this
00:25:04.06 is very consistent with much of the information that we have about the tetrameric holoenzyme.
00:25:12.10 So you can see here, as you rotate around, you pick up now a symmetry related dimer
00:25:16.29 which gives you the framework for the tetrameric holoenzyme.
00:25:20.24 As you go around the next time, you pick up the B domain
00:25:26.16 which is highly cooperative for this RI-alpha subunit.
00:25:30.21 You must bind cyclic AMP first to the B domain, then to the A domain
00:25:34.27 and then you release the inhibitor peptide from the active site of the catalytic subunit.
00:25:39.29 And then it's also a really nice model because the N-terminal part here is
00:25:45.09 positioned so that it can beautifully link up to the dimerization domain.
00:25:50.15 We have only about 20 residues that are missing from this structure now.
00:25:55.25 So it's a very good model for really beginning to think about a tetrameric holoenzyme
00:26:04.21 and I think as we move forward, one of the real challenges for structural biologists
00:26:11.29 who are working on these signaling proteins is to try to elucidate larger complexes
00:26:18.00 and I think from that we will really gain
00:26:21.13 a much better understanding of how this works as an integrated system.
00:26:25.13 And just an example of the RI subunit that has a class 2 PDZ motif at the C-terminus,
00:26:33.05 which is quite exposed here
00:26:34.29 and we were pretty sure there is some PDZ domain protein that's going to be binding there as well.
00:26:39.27 Building this higher assembly of molecules
00:26:43.16 is going to be a really important thing for all of us to try to achieve.
00:26:50.00

Videos in this Talk
  • Part 1: Protein Phosphorylation in Biology
    Part 1: Protein Phosphorylation in Biology
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Architecture of a Protein Kinase
    Part 2: Architecture of a Protein Kinase
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 3: Protein Kinase Regulation and Localization
    Part 3: Protein Kinase Regulation and Localization
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
Speaker: Susan Taylor
Total Duration: 1:18:24
Recorded: October 2009
All Talks in Biochemistry

Talk Overview

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.

Speaker Bio

Susan Taylor

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

Playlist: Signaling

Related Resources

Dynamics of cAMP-dependent protein kinase. Johnson DA, Akamine P, Radzio-Andzelm E, Madhusudan M, Taylor SS. Chem Rev. 2001 Aug;101(8):2243-70.

Regulation of protein kinases; controlling activity through activation segment conformation. Nolen B, Taylor S, Ghosh G. Mol Cell. 2004 Sep 10;15(5):661-75.

Active and inactive protein kinases: structural basis for regulation. Johnson LN, Noble ME, Owen DJ. Cell. 1996 Apr 19;85(2):149-58.

A helix scaffold for the assembly of active protein kinases. Kornev AP, Taylor SS, Ten Eyck LF. Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14377-82.

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Kornev AP, Haste NM, Taylor SS, Eyck LF. Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17783-8.

The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module. Kannan N, Haste N, Taylor SS, Neuwald AF. Proc Natl Acad Sci U S A. 2007 Jan 23;104(4):1272-7.

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