• Skip to primary navigation
  • Skip to main content
  • Skip to footer

Session 8: Protein Kinases

Transcript of Part 1: Protein Phosphorylation in Biology

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

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

© 2022 - 2006 iBiology · All content under CC BY-NC-ND 3.0 license · Privacy Policy · Terms of Use · Usage Policy
 

Power by iBiology