Session 9: Cell Cycle

00:00:03.02 Hello, my name is Dave Morgan. I'm from the University of California in San Francisco.
00:00:07.07 And today we're going to talk about mechanisms of eukaryotic cell cycle control.
00:00:11.12 In this first introductory lecture I'm going to give you a basic overview
00:00:15.10 of the cell reproduction process and a little bit of information about
00:00:18.13 the regulatory system that guides the cell through that process.
00:00:22.09 And then in my second and third lectures, we'll go into some details about
00:00:25.23 experiments from my own laboratory.
00:00:29.09 So it's been known since the early 1800's
00:00:31.16 that all living things are composed of individual units called cells.
00:00:34.25 And so the growth, development and survival of all living things
00:00:38.19 depends on the reproduction of those cells.
00:00:40.22 So cell reproduction is clearly and fundamentally an important biological process.
00:00:46.17 Cell reproduction is also important for human disease.
00:00:49.07 One of the major diseases of the Western world, cancer,
00:00:51.23 is essentially a disease of excess cell reproduction.
00:00:55.12 This disease occurs when a cell in a tissue acquires a mutation
00:00:59.03 that allows it to proliferate more rapidly than its neighboring cells.
00:01:02.12 And through the acquisition of additional mutations, the progeny of that cell
00:01:06.08 can form into a tumor that can eventually escape the tissue,
00:01:09.06 as shown down here, and that results in malignant cancer spread throughout the body.
00:01:13.19 So an understanding of cancer absolutely requires an understanding of
00:01:17.26 the mechanisms of cell reproduction.
00:01:20.19 So the question is: How do cells reproduce?
00:01:23.01 And that's the questions we're going to address in my lecture today
00:01:25.01 and in my other two lectures. Where do new cells come from?
00:01:28.25 Well, this question has occupied scientists ever since those early days
00:01:32.11 when it was first realized that all tissues are made up of individual cells.
00:01:36.14 And in those early days, there were a number of competing theories
00:01:39.15 about how cells reproduce. In one hand, a number of prominent psychologists
00:01:44.13 of the early 1800's suggested that cells formed by so called "free cell formation",
00:01:48.26 whereby they essentially crystallized out of the intercellular fluid.
00:01:52.11 Another theory was that all cells are derived by the division of pre-existing cells.
00:01:58.08 And through 20 or 30 years of intense microscopy in a wide range of cells and tissues and organisms,
00:02:03.05 it became clear that this second theory was the correct one
00:02:05.27 and that, indeed, all cells are derived by the division of pre-existing cells.
00:02:10.08 In other words, cell reproduction is essentially a process
00:02:13.20 by which this remarkably complex machine, the cell, duplicates its contents
00:02:18.05 and then distributes those contents into a pair of genetically identical daughter cells.
00:02:24.00 Now this idea has profound implications because it means that all cells in existence today
00:02:29.12 are likely to be derived from a single, ancestral cell that divided perhaps 3.5 billion years ago.
00:02:36.29 So in the hundred years since the discovery that
00:02:39.04 all cells are derived from other cells by division, microscopists and, to a lesser extent, geneticists
00:02:44.09 dedicated their efforts to understanding the basic mechanics of the cell reproduction process.
00:02:49.07 And that led to our current view of what we call the cell division cycle,
00:02:53.03 which is illustrated in the next slide. So we now believe that
00:02:56.13 the cell cycle, or the series of events by which the cell duplicates itself,
00:02:59.22 is divided into a series of distinct phases that are typically
00:03:03.04 defined on the basis of chromosome duplication.
00:03:05.25 S phase is the period during which the chromosomes duplicate.
00:03:09.07 And M phase is the period during which those duplicated chromosomes,
00:03:12.20 or sister chromatids as they're called, are segregated to opposite poles of the cell
00:03:16.29 and then packaged into individual, genetically identical daughter cells.
00:03:20.14 Now, throughout this process, it's not just the chromosomes that duplicate,
00:03:24.19 but all the other components of the cell also duplicate.
00:03:27.12 All the proteins, organelles, RNAs and all the other macromolecular complexes of the cell
00:03:32.04 are all duplicated throughout...continuously throughout the entire cell cycle
00:03:36.07 so that by the time the cell divides at the end of M phase the resulting daughter cells
00:03:40.29 are essentially about the same size as the cell that first began that cell division cycle.
00:03:46.18 Now, M phase is a particularly spectacular phase of the cell cycle
00:03:49.19 that is typically defined as involving two distinct events-mitosis and cytokinesis.
00:03:55.21 Mitosis is the process by which the chromosomes
00:03:58.22 are segregated and packaged into individual nuclei.
00:04:01.12 And then cytokinesis is the process by which those daughter nuclei are then distributed
00:04:06.19 by cell division into a pair of genetically identical daughter cells.
00:04:10.14 The process of mitosis depends extensively on
00:04:13.07 this remarkable molecular machine called the mitotic spindle,
00:04:16.15 which is essentially a bipolar array of microtubule polymers
00:04:19.22 that forms as the cell enters mitosis
00:04:22.05 and then becomes attached to the sister chromatid pairs,
00:04:24.10 as I've shown here. The basic idea is that a mitotic spindle has these two poles
00:04:29.01 that radiate microtubules, some of which become attached
00:04:32.06 to the sister chromatid pairs, as I've shown here.
00:04:34.03 The attachment site on those chromosomes
00:04:36.28 is called the kinetochore, which is a large protein complex
00:04:39.23 that basically provides a microtubule binding site on the surface of the chromosome.
00:04:44.11 And so, by the middle of mitosis the chromosomes are aligned
00:04:49.14 along the middle of the mitotic spindle, as shown here,
00:04:52.01 with one sister chromatid attached to one spindle pole
00:04:54.23 and the other sister attached to the other spindle pole.
00:04:57.04 And so what happens next is that the glue, the protein glue, that holds
00:05:00.18 the sister chromatids together is then dissolved,
00:05:03.20 allowing those sister chromatids to be pulled apart by the spindle
00:05:06.15 in anaphase, here, and then packaged into individual nuclei
00:05:10.21 and then distributed by cytokinesis into the daughter cells.
00:05:17.15 This next slide gives us a little bit more detail about the process of mitosis,
00:05:21.00 the spectacular phase during which the chromosomes are segregated.
00:05:24.21 Mitosis typically begins with a stage called prophase which is shown in the upper left here.
00:05:29.10 Prophase is distinguished by the fact that chromosomes inside the cell,
00:05:34.05 inside the cell nucleus, begin to condense from their normally disperse state
00:05:37.23 into this much more compact rod-like structure that is more easily manipulated during mitosis.
00:05:43.10 In addition, prophase is also typified by changes
00:05:46.10 in the organization of the microtubule cytoskeleton
00:05:48.28 which is labeled here in green.
00:05:51.05 And so you can see that in prophase there are two microtubule organizing centers
00:05:54.22 and these two centers move apart from one another along the surface of the nucleus
00:05:58.20 to begin the formation of a mitotic spindle.
00:06:01.03 And then at the end of prophase, the nuclear envelope dissolves
00:06:05.10 allowing the microtubules of those organizing centers to gain access to
00:06:08.28 the sister chromatid pairs inside the nucleus until eventually the cell reaches metaphase
00:06:13.11 when those sister chromatid pairs are attached to the mitotic spindle,
00:06:16.11 as I said in the previous slide, in a bi-oriented fashion whereby
00:06:19.22 one sister is attached to one pole and the other is attached to the other pole.
00:06:24.13 And then the big event of mitosis occurs, which is when
00:06:27.01 the chromosome cohesion mechanisms
00:06:29.15 that hold those sister chromatids together are removed resulting in the separation of
00:06:33.18 the sister chromatid pairs and their movement to opposite poles of the mitotic spindle.
00:06:37.17 After which, in telophase, those separated chromosome sets
00:06:41.19 are then packaged into individual daughter nuclei.
00:06:44.11 After which, cytokinesis takes care of dividing the cell itself.
00:06:49.10 So the process of mitosis is really best appreciated by looking at movies of this process.
00:06:54.07 So this movie will show you the key event of mitosis
00:06:58.22 which is the metaphase to anaphase transition,
00:07:00.24 this point at which the chromosomes are pulled apart by the anaphase spindle.
00:07:05.08 And so this happens to be a vertebrate cell,
00:07:07.29 in which the microtubules are labeled in reddish-orange here
00:07:11.15 and then these green dots in the middle of the spindle
00:07:13.14 represent those kinetochore protein complexes
00:07:15.15 that join the sister chromatids to the microtubules in the middle of the spindle.
00:07:20.14 And so you can barely see these sister chromatids
00:07:23.06 as dark shadows in the middle of the spindle back here.
00:07:26.07 And so when I start this movie what we'll see is this metaphase cell
00:07:29.17 will move into anaphase through the separation of these sister chromatid pairs.
00:07:35.09 And so right about that instant right there, the sister chromatids are separated
00:07:40.20 and then pulled apart by the anaphase spindle. And we'll see it one more time.
00:07:48.17 OK, so that is mitosis in the context of a cultured cell on a plastic dish.
00:07:53.08 What this movie will show you is mitosis in the context of a living organism,
00:07:58.08 in this case, the early embryo of the zebra fish.
00:08:00.14 And in this case the zebra fish embryo has been filmed using a modern microscopy technique
00:08:05.18 that allows the identification of all the individual nuclei in that embryo.
00:08:09.18 And so in this image right here, for example, this first image,
00:08:12.17 we're looking at a zebra fish embryo early in development where it has
00:08:15.22 about 20 or 30 nuclei in it, which have obviously undergone a number of divisions.
00:08:19.25 And each of those nuclei is visible as one of these little, white blobs
00:08:23.10 that essentially represents the fluorescently labeled DNA in the nucleus.
00:08:26.23 And so when I start this movie you'll see how these little DNA blobs, or nuclei
00:08:32.07 rapidly divide and subdivide again and again to populate this embryo with the large numbers of cells
00:08:37.27 that are needed for embryogenesis.
00:08:41.12 And so, we can see this incredible division process occurring over and over and over again
00:08:45.21 in this embryo. Resulting in the transformation of those 20 or 30 cells
00:08:49.11 into the thousands of cells that are required to populate this embryo.
00:08:52.29 And as time goes by, over the next few minutes
00:08:56.11 we begin to see the migration of these cells to form the basic pattern of the early embryo.
00:09:03.15 So clearly, this movie emphasizes the key point that cell division and reproduction
00:09:08.08 is a crucial process in the development of all living things.
00:09:13.05 OK, so that gives you a basic overview of the basic mechanics of cell division
00:09:17.29 and what I want to do for the rest of this lecture and for my other lectures as well
00:09:21.09 is focus on a more recent problem that has occupied scientists for the last 20 or 30 years or so.
00:09:26.01 And that is the question or the problem of how these remarkable cell cycle events are controlled
00:09:30.15 such that they occur always in the correct order
00:09:33.16 and with the appropriate timing and coordination.
00:09:35.27 Why, for example, S phase always occurs before M phase and so on.
00:09:39.15 And the answer to this question has turned out to be that the cell contains this remarkably
00:09:43.12 complex regulatory system that guides the cell through the stages of cell division.
00:09:47.28 And this complex regulatory system has occupied scientists, as I said,
00:09:52.24 for about 20 or 30 years at this point.
00:09:55.21 Studies of this regulatory system have come from a wide range of different model organisms
00:10:00.02 and, in fact, most of the major discoveries about this system
00:10:02.21 have come from very simple eukaryotic organisms such as the budding yeast
00:10:06.27 and fission yeast, as I'll show you in the next slide.
00:10:10.07 So this slide illustrates a few of the really important model organisms systems
00:10:14.16 for studies of cell cycle control. In the upper left, is the budding yeast Saccharomyces cerevisiae
00:10:20.01 and below that the fission yeast Schizosaccharomyces pombe.
00:10:23.13 And these two yeasts have turned out to be extremely important
00:10:26.07 model systems for studying the basic features of cell division
00:10:28.29 for many reasons. First of all, their regulatory mechanisms for cell cycle control
00:10:33.19 are very similar to the same mechanisms that are found in human cells.
00:10:37.18 They also have a number of extremely important experimental advantages.
00:10:41.19 They have short generation times, completely sequenced genomes for quite some time.
00:10:45.18 It's possible to do a wide range of genetic manipulations in these organisms
00:10:50.02 and most importantly, it's possible to apply the methods of classical genetics
00:10:53.13 to these organisms to isolate mutant genes that allow
00:10:57.00 the analysis of the principles and the components involved in cell cycle control.
00:11:01.21 And so the budding yeast and the fission yeast have been really important
00:11:05.23 tools in our study of cell cycle control.
00:11:09.05 Another important organism is shown along the bottom here,
00:11:11.08 and this is the early embryonic divisions of the frog.
00:11:14.20 Early embryonic cells both in frogs, and in vertebrates as well,
00:11:18.27 have been extremely important in studies of cell cycle control
00:11:22.11 because these early embryonic divisions represent simplified,
00:11:26.05 stripped down versions of the cell cycle, essentially,
00:11:28.06 that can be analyzed very easily biochemically for a variety of reasons.
00:11:33.03 And then finally, some other organisms that have proven to be important are
00:11:36.13 the early embryo of the fruit fly Drosophila melanogaster, which has been particularly
00:11:41.14 crucial for studies of cell cycle control in early embryonic development.
00:11:44.24 And then here of course, is a mammalian cell growing in culture
00:11:48.15 which is also a very important tool in which to translate some of
00:11:52.11 the findings that have been found in some of these other organisms into the context of
00:11:55.20 a human cell. This next slide goes into a little bit more detail about the budding yeast
00:12:02.04 and its cell cycle because this particular organism has been
00:12:05.15 so instrumental in cell cycle control studies and also because
00:12:07.22 it is the organism we study in my lab and you'll need to know some
00:12:10.15 of this for my second and third lectures.
00:12:13.22 So budding yeast, as the name implies, divides by budding.
00:12:16.02 Which means that at the end of G1, when this cell enters the cell cycle,
00:12:20.02 it starts growing a little bud on the side of the mother cell
00:12:23.01 and as the cell progresses through the cell cycle, the size of that bud increases
00:12:27.13 until by the end of mitosis the bud is almost the same size as the mother cell.
00:12:32.04 And so the size of the bud in budding yeast gives a very good indication
00:12:35.23 of the position of that cell in the cell cycle.
00:12:37.29 And this turned out to be a very useful tool because it then allowed the isolation of
00:12:42.20 mutants that were arrested at specific cell cycle stages.
00:12:45.13 And so Lee Hartwell and his colleagues back in the late 1960s and early 1970s
00:12:50.12 isolated a wide range of mutants in various genes that resulted in
00:12:55.09 cell cycle arrest at specific stages that were determined on the basis of bud morphology.
00:13:01.07 These mutants were conditional temperature sensitive mutants
00:13:03.23 which means that at normal room temperature these mutant genes were
00:13:07.14 totally functional and the cell divides,
00:13:09.07 but at high temperature, 37 degrees, these mutant genes,
00:13:12.25 or rather the gene products, become inactivated and as a result
00:13:16.01 the cell then arrests at the cell cycle stage where that gene product is required.
00:13:20.08 And a couple examples of those sort of mutants are shown in the next slide.
00:13:24.06 These were the so called cell division cycle or cdc mutants and two of them are shown here.
00:13:30.01 On the left is cdc16, a mutant that causes at the high temperature an arrest in mitosis.
00:13:36.13 As you can see by the size of this bud, this cell is arrested in mitosis.
00:13:39.16 However, you can see from DNA staining here, that the cell
00:13:43.17 has not yet segregated its chromosomes.
00:13:45.10 So this cell is arrested before anaphase.
00:13:47.29 And then looking at the spindle gives us another clue that
00:13:50.17 this cell actually contains a short metaphase spindle.
00:13:53.20 And so together, these various phenotypes indicate that cdc16 mutants
00:13:57.11 arrest in metaphase prior to the onset of anaphase.
00:14:00.24 And indeed, it turned out since the discovery of this mutation
00:14:03.18 that cdc16 encodes a component of an important protein complex
00:14:08.05 that is required for progression through the metaphase to anaphase transition.
00:14:13.08 And so cdc15, in contrast, is a little different. It's also a mitotic arrest
00:14:18.00 but in this case the DNA has segregated into two distinct masses.
00:14:21.11 And so...and by looking at the spindle in these cells it's clear that these cells
00:14:25.23 have reached anaphase but have not progressed any farther than that.
00:14:28.29 They contain the long anaphase spindle, segregated DNA, but
00:14:33.06 they've arrested at the end of anaphase. And this turns out to be because
00:14:37.21 cdc15 encodes a component of a regulatory network that drives the cell
00:14:42.04 out of anaphase and into the following G1.
00:14:45.14 And so by isolating and characterizing large numbers of these cdc mutants,
00:14:49.04 yeast geneticists working in budding yeast and in fission yeast,
00:14:53.01 with the work of Paul Nurse and colleagues,
00:14:54.23 led to the isolation of a large number of these cdc mutants that
00:14:58.12 have essentially built the foundation for our present understanding of cell cycle control.
00:15:06.05 The other major organism that we need to discuss in a little detail is the frog, Xenopus laevis,
00:15:11.16 whose early embryos have turned out to be an extremely important
00:15:13.25 tool for studies of cell cycle control as well.
00:15:16.18 Following fertilization, the egg...the fertilized egg of the frog
00:15:20.13 divides rapidly in these so called cleavage divisions which
00:15:23.29 transform that large fertilized egg into thousands of cells in a matter of a few hours essentially.
00:15:30.12 And it goes through these very simplified versions of the cell cycle
00:15:33.18 that have only S and M phases and no gap phases.
00:15:36.15 And these occur over a period of about 30 minutes each.
00:15:39.14 And so this very rapid, simple cell cycle has turned out to be an important
00:15:43.15 tool for these studies of the basic features of cell cycle control.
00:15:47.18 One big advantage of frogs and their embryos is that these eggs are so large
00:15:51.23 that they can be injected very easily with test substances
00:15:54.15 or for that matter it's possible to isolate large amounts of the cytoplasm
00:15:57.29 from these fertilized eggs and recreate the events of the cell cycle in a test tube.
00:16:05.08 And so it was these early studies of yeast genetics on one hand and frog embryonic cells on the other
00:16:10.04 that led to a couple of competing or alternative views of how cell cycle control is achieved.
00:16:14.26 And those are illustrated in this slide.
00:16:18.02 On the left is the view that resulted from studies in yeast and
00:16:20.25 particularly from various studies of cdc mutants like the ones I showed you.
00:16:24.12 Those studies suggested that many cdc mutants arrest...when they arrested
00:16:28.21 the cells in early cell cycle stages also blocked progression through later cell cycle stages.
00:16:33.15 And so, for example, a cdc mutant that arrests cells prior to DNA replication
00:16:37.23 also caused a block to the onset of mitosis.
00:16:42.04 And so this suggested that there were these dependency relationships
00:16:44.09 among the events of the cell division cycle such that DNA replication, for example,
00:16:49.02 has to occur before mitosis can occur.
00:16:52.14 And so the idea was that one event, such as event A or DNA replication,
00:16:56.06 must be completed in order for event B to begin.
00:16:59.23 And so these events might some how regulate each other in some way.
00:17:04.06 Now, the alternative view, on the right here,
00:17:06.23 came from studies in the early embryos of the frog which
00:17:09.23 clearly suggested that these frog eggs, these early frog embryos, I should say
00:17:14.04 contain an intrinsic biochemical timer that turns on cell cycle events
00:17:19.08 at specific times and in a specific order.
00:17:21.16 In other words, the events of the cell cycle and their order and timing
00:17:25.10 are determined by a programmed timer or a clock that essentially flips switches
00:17:29.28 at specific times to initiate the cell cycle events.
00:17:32.25 And so that timer is independent of the events that it controls.
00:17:36.11 And in fact, in frog embryos it's possible to block DNA replication or
00:17:40.00 completely remove the nucleus and still see evidence
00:17:42.05 that this timer is operating normally and oscillating.
00:17:46.19 So how do we reconcile these two different models?
00:17:48.25 Well, the answer turned out to be that both were at least partially correct.
00:17:51.28 There is, indeed, an intrinsic biochemical timer in all eukaryotic cells
00:17:55.20 that guides the cell through the cell division cycle, but in many cells
00:17:58.27 the events of the cell cycle can feed back
00:18:01.08 to that timer and adjust the timing of later events
00:18:03.28 if early events fail for one reason or another.
00:18:06.13 And that's summarized in this next slide.
00:18:10.22 So, once again, the basic idea is that the eukaryotic cell cycle is
00:18:14.20 governed by this intrinsic biochemical timer or control system
00:18:17.29 that essentially, is a series of biochemical switches that turn on specific cell cycle events
00:18:23.19 in a specific order and in a specific time.
00:18:26.28 However, that timer can be regulated in such a way that those cell cycle events
00:18:30.23 can feed back and send information to the timer to arrest progression through certain stages
00:18:35.05 if necessary. And so there are three major transitions or so called checkpoints in the cell cycle
00:18:40.21 where this timer can be arrested if conditions are not appropriate.
00:18:45.29 And so for example, if the timer initiates S phase as shown here,
00:18:49.09 but for some reason DNA synthesis fails and a chromosome fails to duplicate,
00:18:53.03 then that will send a message back to the controller that will block progression through
00:18:57.11 the G2/M checkpoint over here. So the timer will basically arrest
00:19:00.08 at this point here, thereby preventing
00:19:02.28 entry into mitosis if the chromosomes are not fully duplicated.
00:19:06.12 And likewise there are similar mechanisms
00:19:09.07 in operation at the metaphase-anaphase transition
00:19:11.08 and at the start or G1/S checkpoint at the beginning of the cell cycle.
00:19:14.23 At all of these checkpoints or transition points it's possible to arrest
00:19:18.00 the timer under certain conditions if certain previous events are not carried out successfully.
00:19:24.29 OK, so with this basic idea of cell cycle control in mind
00:19:28.18 the big question that has occupied many of us for the last 10 or 20 years
00:19:31.22 has been understanding what the molecular composition of this timer is.
00:19:35.17 What are the proteins that make up this timer and
00:19:38.19 are assembled together into this remarkable regulatory system?
00:19:42.04 And here again, the major breakthroughs in this field
00:19:44.28 arose out of a combination of both yeast genetics and
00:19:47.22 biochemical studies primarily in early embryonic cell types.
00:19:52.02 And that all collided essentially, in the late 1980's when it was realized that
00:19:56.03 people working in yeast genetics and people working in early embryos
00:19:59.25 of frogs and other systems
00:20:01.04 were actually studying essentially the same regulatory molecules.
00:20:05.06 In budding yeast for example, a protein kinase called Cdc28 was found to be required for
00:20:11.12 entry into the cell cycle. In fission yeast, a highly related protein kinase called Cdc2
00:20:16.25 was identified that was required for progression into mitosis.
00:20:20.21 And these two protein kinases, Cdc2 and Cdc28 appeared to be the homologues of one another
00:20:25.20 in these two species and were clearly crucial regulators of cell cycle progression in these yeasts.
00:20:31.11 Meanwhile, in the frog, Masui and others had identified a Maturation Promoting Factor
00:20:36.09 or MPF activity which appeared to be capable of driving mitotic eggs
00:20:42.01 of the frog into mitosis. And so this so called M-phase promoting factor was capable of
00:20:47.04 initiating mitosis when injected into non-mitotic cells. So that clearly represented
00:20:53.25 some sort of important biochemical activity that might be important for cell cycle control.
00:20:57.20 And then finally, Tim Hunt and colleagues identified a protein
00:21:01.01 called Cyclin whose levels oscillate during
00:21:03.09 the cell cycle and rise in mitosis and fall there after suggesting that
00:21:06.24 it might be some component of an important regulatory molecule.
00:21:10.28 And so as I said, in the late 1980's all these fields collided when it was realized
00:21:15.01 that all of these people were actually working on the same thing
00:21:19.10 and that MPF for example, this Maturation or M-phase Promoting Factor was
00:21:22.26 in fact, a complex of a small protein kinase that was related to Cdc28 and Cdc2 and Cyclin.
00:21:28.28 And so a complex of Cdc28 or Cdc2 and a Cyclin molecule forms a protein kinase
00:21:34.21 complex that is responsible for driving progression through the major stages of the cell cycle.
00:21:40.00 And that's illustrated in the next slide.
00:21:41.23 The basic idea is that the heart of the control system that guides
00:21:45.25 the cell through cell division is a protein kinase called a Cyclin-dependent kinase or Cdk.
00:21:51.23 And Cdks are inactive until they associate with their Cyclin regulatory partners,
00:21:55.28 resulting in the activation of a Cdk-cyclin complex.
00:22:00.11 Those Cdk-cyclin complexes then phosphorylate large numbers of substrates in the cell
00:22:05.08 resulting in the onset of various specific cell cycle events.
00:22:11.22 And so our current view of this system is that there aren't simply
00:22:16.05 one or two Cdks in the cell but in fact there are series of Cyclin-Cdk complexes
00:22:20.26 each of which is responsible for driving the specific events of the cell division cycle.
00:22:25.04 And so for example, a complex of Cdk with an S-phase Cyclin forms in late G1 and
00:22:30.09 is activated at that point to trigger the onset of DNA replication.
00:22:34.27 And then likewise, later in the cell cycle, an M-phase cyclin-Cdk complex forms and
00:22:38.29 is activated, resulting in the initiation of mitotic entry.
00:22:43.07 And so through this series of Cdk-cyclin complexes, the events of the cell cycle are
00:22:47.27 triggered in the appropriate order and with the appropriate timing.
00:22:52.13 Of course, since the initial discovery of Cdks it's become clear that they're
00:22:56.14 activities are controlled by far more that just the Cyclin regulatory subunit.
00:23:01.03 Cdks, both the mitotic and S-phase sort are also regulated by phosphorylation
00:23:06.26 on the Cdk subunit and also by inhibitory proteins that can associate with Cdks
00:23:10.23 at certain cell cycle stages to restrain their activity.
00:23:13.28 And so, Cdk activity and the up-regulation and down-regulation of Cdk activity
00:23:20.03 during the cell cycle depends on a wide range of different regulatory mechanisms.
00:23:26.21 So, the next question following the discovery of Cdks
00:23:29.23 dealt with this interesting observation over here
00:23:33.12 which is that Cyclin levels drop precipitously during mitosis.
00:23:37.14 And in fact studies, a wide range of studies, suggested that the destruction of
00:23:41.08 Cyclins in mitosis is actually required for progression out of mitosis.
00:23:45.19 So for example, if you make a version of Cyclin that cannot be degraded, that is highly
00:23:48.25 stabilized and remains high throughout mitosis, cells expressing that stabilized Cyclin
00:23:54.21 fail to exit. They fail to get out of anaphase and exit into the following G1,
00:23:59.10 indicating that Cyclin destruction is in fact required for the exit from mitosis.
00:24:04.11 And so a lot of effort was placed on identifying the mechanisms
00:24:07.14 that determined this Cyclin destruction.
00:24:09.04 And that led, primarily through biochemistry with a little bit of yeast genetics on the side,
00:24:12.26 led to the identification of a large protein complex that is responsible
00:24:17.00 for the destruction of cyclins. And that protein complex is called
00:24:20.28 the Anaphase-Promoting Complex or Cyclosome, otherwise known as the APC.
00:24:25.04 And its activity rises in mid-mitosis and is responsible for destroying cyclins.
00:24:31.22 The APC is not a protein kinase but a ubiquitin-protein ligase or E3 which means that its job
00:24:37.18 is to catalyze the attachment of a small protein called ubiquitin onto its targets.
00:24:42.04 And by attaching large numbers of these ubiquitins onto its targets
00:24:45.18 that sends those targets to a protease in the cell called the proteasome
00:24:49.02 where they are destroyed. And so the APC is essentially capable of triggering
00:24:52.26 the destruction of its target proteins, including the cyclins.
00:24:57.15 The next slide illustrates in a little bit more detail what it is that the APC does.
00:25:01.10 Now, as I mentioned, the major target of the APC is the Cyclin of this Cdk regulatory subunit.
00:25:08.26 And so the destruction of cyclins results in the inactivation of Cdks in late mitosis.
00:25:13.02 The other major target of the APC is a protein called securin.
00:25:17.06 And securin is a tight binding inhibitor of a protease called separase.
00:25:22.14 And so when securin is destroyed in mitosis by the APC that results in
00:25:27.02 the liberation of separase in the cell and separase then goes to the sister chromatids
00:25:31.10 and cleaves a single subunit of a protein complex called cohesin
00:25:35.04 that holds those sister chromatids together.
00:25:38.01 And by cleaving that subunit, separase induces the separation of the sister chromatids
00:25:42.04 which can then be segregated by the microtubules of the mitotic spindle.
00:25:46.08 And so the APC, through this mechanism, directly triggers the initiation of anaphase.
00:25:52.11 Now, as I said, it also triggers the destruction of cyclin and therefore
00:25:55.17 the inactivation of the associated Cdks, and this turns out to be very important, as I mentioned
00:26:00.06 because this allows the dephosphorylation of substrates of the Cdks.
00:26:05.12 And this dephosphorylation of Cdk substrates is required for
00:26:08.22 normal progression out of mitosis.
00:26:10.27 Normal anaphase and normal telophase and cytokinesis and mitotic exit all depend on
00:26:16.12 the dephosphorylation of Cdk substrates.
00:26:19.08 And so, cyclin destruction is therefore crucial, one of the crucial jobs of the APC in late mitosis.
00:26:26.28 And so we're left with this general scheme of cell cycle control
00:26:30.08 whereby the cell cycle control system is essentially a series of biochemical switches
00:26:35.05 made up of cyclin-dependent kinases of various sorts
00:26:38.00 that turn on the various events of the cell cycle until the cell reaches metaphase
00:26:42.05 when the chromosomes are aligned on the mitotic spindle. At which point the APC
00:26:46.05 triggers the destruction of securin and cyclins to take the cell out of mitosis
00:26:50.09 and complete the cell cycle.
00:26:52.20 Now obviously, as I said before, this is a highly simplified view of things
00:26:55.16 because there are countless other regulators that are sending
00:26:58.23 inputs into this system to refine the activation of Cdks and the APC in various ways.
00:27:03.27 For example, there are numerous phosphorylation events on the Cdks and on the APC itself
00:27:09.08 as well as various inhibitory proteins that bind to those proteins.
00:27:13.00 Now, recent studies have begun to focus...
00:27:15.02 now that we have the basic components of the cell cycle control system in place,
00:27:18.06 have begun to focus on how these components are assembled into a regulatory system that
00:27:22.17 achieves the behaviors that we're so interested in.
00:27:26.05 So this field is now reaching into the systems biology field
00:27:29.01 and beginning to use mathematical modeling to put these components into a regulatory system
00:27:34.03 and model that system to see how it achieves the various behaviors that it achieves.
00:27:38.09 So for example, one key question is
00:27:40.23 the kinetics of Cdk activation at the beginning of S-phase and M-phase.
00:27:45.10 It's well established that these kinases are both activated in a highly
00:27:49.06 switch-like fashion at the onset of S-phase or the onset of M-phase.
00:27:52.22 And so, in other words, their activity goes from very low activity to very high activity very rapidly.
00:27:59.21 And so there are biochemical mechanisms such as positive feedback loops involved
00:28:02.28 that ensure that these Cdks go from low to maximal activity abruptly.
00:28:07.15 And this, of course, allows the complete commitment of the cell
00:28:10.22 to a new cell cycle stage through this abrupt activation of Cdks.
00:28:15.19 And so, that gives you an overview of the basic features of the cell cycle control system
00:28:20.21 and some of the issues that are attracting scientists today
00:28:23.09 and it prepares you for my next two lectures
00:28:25.22 in which I will address some of these issues in a little bit more detail.

00:00:02.03 So, hello, my name is Dave Morgan. I'm from the University of California in San Francisco.
00:00:06.04 And in this lecture I'm going to go over some of my own work on studies of
00:00:09.23 how the cyclin-dependent kinases drive the events of the eukaryotic cell division cycle.
00:00:15.29 Now it’s well established at this point that the major regulators
00:00:19.11 of the eukaryotic cell cycle are the cyclin dependent kinases or Cdks.
00:00:23.13 And the basic idea is that a series of Cdk-cyclin complexes are activated
00:00:28.11 in a specific sequence during the cell cycle
00:00:30.08 to trigger the events of the cell cycle in the appropriate order.
00:00:33.21 And so, for example, S-phase Cdk-cyclin complex is formed in late mitosis or in late G1
00:00:39.15 and are then activated in the beginning of S-phase to initiate DNA synthesis.
00:00:43.03 And then M-phase Cdk-cyclin complexes form at the end of G2
00:00:47.06 and are activated to initiate the events of mitosis and take the cell to metaphase.
00:00:52.17 So the big question that I want to address today is how is it that these
00:00:55.26 Cdks actually drive these cell cycle events?
00:00:59.05 Now obviously, Cdks are protein kinases, which means that the most likely mechanism
00:01:02.24 by which they promote cell cycle events is through the phosphorylation of other proteins
00:01:07.10 which then bring about those events.
00:01:09.15 And so over the past 10 or 12 years or so, we've dedicated quite a lot of effort
00:01:13.18 to identifying the substrates of the cyclin-dependent kinases
00:01:16.20 in the hope that that will lead us to an answer to this question of
00:01:19.17 how the Cdks actually initiate cell cycle events.
00:01:23.23 So in this lecture I'm going to tell you about two methods that we've used
00:01:26.23 to systematically and comprehensively identify Cdk substrates
00:01:30.09 and then in the second half of the lecture we'll go into some interesting ways
00:01:33.20 in which we use those lists of substrates to address some general questions of
00:01:37.19 Cdk function and phospho-regulation.
00:01:41.07 So the first method we use to identify Cdk substrates
00:01:43.22 began about 10 or 12 years ago in a collaboration with Kevan Shokat, a chemist here at UCSF.
00:01:51.00 Now, Kevan came up with an idea whereby it would be possible to label the specific targets of
00:01:57.04 a protein kinase in a crude cell mixture and this slide attempts to explain that basic method.
00:02:02.09 On the left...let's focus on the left first.
00:02:04.08 On the left is a wild type regular protein kinase like Cdk1 with its cyclin regulatory partner.
00:02:10.08 And typically when one wants to label the targets of a protein kinase, like Cdk1,
00:02:14.28 you simply provide that kinase with a version of ATP in which the gamma phosphate
00:02:20.00 is labeled with a radioactive tag.
00:02:22.28 And then when that protein kinase uses that ATP it will then transfer
00:02:27.24 that P-32 onto its substrates.
00:02:31.11 Now, unfortunately this cannot be used to identify unknown substrates
00:02:34.24 of Cdks because if you take a pure Cdk and some gamma labeled P-32-ATP
00:02:40.16 and put that into a crude cell lysate, you will get not only the labeling of the Cdk's targets
00:02:46.12 but the labeling of all the other protein kinase targets in that lysate because
00:02:49.15 that ATP can be used by any kinase.
00:02:52.22 And so Kevan Shokat's idea was to avoid this problem by using
00:02:56.15 so called analog-sensitive protein kinases.
00:02:58.29 And the strategy is based on the fact that protein kinases
00:03:02.07 tend to contain a large hydrophobic residue in the wall
00:03:05.19 of the adenine binding pocket of their active site.
00:03:08.21 And so the basic strategy is to mutate that large hydrophobic side chain there
00:03:12.28 to a glycine residue resulting in the formation of an extra pocket in
00:03:16.18 the side of that ATP binding site. And as a result
00:03:20.00 this mutant kinase can now use a bulky ATP analog in which extra moieties
00:03:24.25 have been added to the adenine base of the ATP.
00:03:27.28 And so for example, N6-benzyl-ATP can be used by the analog sensitive Cdk1 kinase
00:03:32.28 but cannot be used by a wild type kinase because that bulky ATP analog
00:03:37.04 can't fit into the wild type active site.
00:03:41.14 And so, of course, if you put a radio-label on the gamma phosphate
00:03:44.05 of this bulky ATP analog and then add this kinase to a crude cell lysate
00:03:48.19 what you hope to get is the specific labeling of just the direct targets of
00:03:52.23 that protein kinase and no other kinases in the cell lysate
00:03:56.00 because those other kinases can't use this bulky ATP analog.
00:04:00.12 So this method...we developed this method in collaboration with Kevan Shokat
00:04:04.04 a number of years ago and applied it to the yeast Cdk1 kinase
00:04:08.28 and the results with that are shown in the next slide.
00:04:12.09 So this slide shows an autoradiograph of a protein gel
00:04:15.14 in which we've separated the reaction products
00:04:16.28 from three different reactions, two of which are control reactions
00:04:20.03 and the third of which is the experimental reaction.
00:04:23.09 In the first lane what you see is what happens when you add this radiolabeled N6-benzyl-ATP
00:04:28.16 this bulky ATP to a crude cell extract made from yeast.
00:04:33.05 And the result is that you get very little labeling of anything in that cell extract
00:04:36.14 because that ATP analog cannot be used by the protein kinases in that crude cell extract.
00:04:42.14 The next lane is another control in which we're mixing the purified protein kinase Cdk1-as1
00:04:48.23 together with a cyclin partner and then adding that to some N6-benzyl-ATP
00:04:54.01 that's radio-labeled on its gamma position and the result then is that you
00:04:57.10 see auto-phosphorylation of the cyclin subunit of the Cdk-cyclin complex.
00:05:01.21 And so that results in a background band in the experimental lane over here.
00:05:07.03 But the third lane is really the crucial lane in which all three components
00:05:09.22 have been added. And so we're adding a purified kinase...analog sensitive kinase
00:05:13.14 with the bulky ATP analog and the cell extract and the result
00:05:17.21 is that you see a whole raft of different proteins being radio-labeled
00:05:22.06 in the cell extract and those proteins are presumably the direct targets of
00:05:27.00 Cdk1-cyclin complexes in that lysate.
00:05:31.12 So we obtained this result a number of years ago and then
00:05:33.29 dedicated quite a lot of effort to identifying
00:05:36.03 these various radiolabeled bands in this cell lysate.
00:05:38.05 And to make very long story short, we ended up using proteomic libraries
00:05:42.24 to individually identify substrates and in the end
00:05:45.27 we came up with a list of about 181 proteins in cell extracts that
00:05:50.04 are rapidly modified by Cdk1-cyclin complexes.
00:05:53.27 And so this list of proteins provided us with our initial list of Cdk substrates.
00:05:59.23 These substrates are involved in a wide range of different cellular processes;
00:06:02.25 many of which are known to be connected to the cell cycle in some way
00:06:05.20 and are likely to represent important targets of Cdk1 throughout the cell cycle.
00:06:11.01 But for various reasons we decided that this list of substrates was
00:06:14.02 incomplete and also, because it was done in vitro we wanted to get
00:06:18.03 another approach that would allow us to identify, comprehensively, a larger number
00:06:23.09 of Cdk substrates that were modified in vivo by Cdk1.
00:06:26.23 And so the second method we've been using more recently has been to use
00:06:30.17 quantitative mass spectrometry approaches to identify all the phosphorylation
00:06:34.10 sites in the cell that are dependent on Cdk1.
00:06:37.18 In other words, phosphorylation sites whose levels decrease abruptly
00:06:40.18 when you inhibit the protein kinase activity of Cdk1.
00:06:45.12 And that method begins again with the analog sensitive Cdk1 mutant.
00:06:49.17 Now, another advantage of these analog sensitive mutants is that not only do they bind
00:06:53.24 bulky ATP analogs, but they also bind bulky inhibitors that can only fit into the active site
00:06:59.24 of the analog sensitive kinase but not the active site of a wild type kinase.
00:07:05.02 And so, for example, this inhibitor here 1-NM-PP1 binds with extremely high affinity to
00:07:10.05 analog sensitive Cdk1 but has essentially no affinity for the wild type kinase
00:07:14.06 or for any other kinase in the yeast cell.
00:07:16.23 And so we could use this analog sensitive Cdk1 to make a yeast strain in which
00:07:22.02 we can inhibit Cdk1 in vivo rapidly and specifically.
00:07:26.22 And so we did that a number of years ago. We created a yeast strain in which
00:07:29.12 the endogenous Cdk1 protein is replaced with the analog sensitive protein
00:07:33.23 and in that yeast strain it is now possible to almost completely and specifically inhibit
00:07:38.05 Cdk1 activity within minutes by the addition of 1-NM-PP1 to the culture medium.
00:07:44.19 So we used that strain in
00:07:46.08 this quantitative mass spectrometry approach that I want to tell you about
00:07:49.16 which was done in a collaboration with Judith Villen and Steve Gygi of Harvard University.
00:07:55.11 And the basic approach that we used is illustrated in this slide and in the next two slides as well.
00:08:01.11 It begins, as I said, with the analog sensitive yeast strain cdk1-as cells
00:08:05.06 in which Cdk1 can be inhibited specifically with the 1NM-PP1 inhibitor.
00:08:10.21 And what we do is we grow two parallel cultures of this yeast strain.
00:08:14.14 One culture, the so called light culture, is grown in regular lysine and arginine
00:08:19.21 whereas the so called heavy culture is grown in a different form of lysine and arginine
00:08:23.11 in which carbon-13 and nitrogen-15 have replaced the usual carbon-12 and nitrogen-14.
00:08:29.21 And so, as a result, after growth in this medium for some time, all the proteins in these cells
00:08:34.03 have been labeled with slightly heavier than average
00:08:37.04 lysine and arginine residues which means that all
00:08:39.10 the peptides derived from this culture will have a slightly higher mass
00:08:43.01 in the eventual mass spectrometry analysis
00:08:45.21 and that will allow us to identify the peptides coming from these two lysates.
00:08:50.22 So we treat the heavy culture with the inhibitor 1-NM-PP1 for a brief period, 15 minutes.
00:08:56.03 And then we harvest these cells after the inhibitor treatment.
00:09:00.08 Harvest the cells, mix them together, lyse them, break them open,
00:09:04.03 and then treat all the resulting proteins in those cell lysates
00:09:08.18 with trypsin to break them all down into tryptic peptides.
00:09:12.06 And then Judit Villen in the Gygi lab has developed a wide range of
00:09:15.27 powerful methods for purifying the phospho-peptides out of that tryptic peptide mixture.
00:09:20.20 And then we then subject those phospho-peptides
00:09:22.25 to mass spectrometry as shown in the next slide.
00:09:26.23 There are two basic forms of mass spectrometry that are applied to these phospho-peptide mixtures.
00:09:31.07 The first, on top, is to use conventional tandem mass spectrometry methods
00:09:35.15 to actually fragment these peptides and use those fragments
00:09:38.07 to determine their sequence.
00:09:40.02 And so, by this approach we can determine the sequence of all the phospho-peptides
00:09:44.04 coming out of these yeast lysates and just as importantly, we can identify
00:09:47.10 the precise site of the phosphorylation on those peptides.
00:09:50.29 And so by doing this, Judit was able to produce a list
00:09:54.12 of about 10,000 phosphorylation sites on 2,000 different proteins in the yeast lysate.
00:10:00.25 And then, in addition to determining sequence, we also quantify all the peptides
00:10:06.12 and determine the relative amount of the so called light and heavy peptides.
00:10:10.19 What this means is that every peptide coming out of these phospho-peptide mixtures
00:10:15.15 comes in both a light form which originally came from the light medium culture
00:10:19.13 and a heavy form that originally came from the inhibitor-treated heavy culture.
00:10:23.17 And they can be distinguished based on this slight
00:10:25.08 mass difference of their lysines and arginines.
00:10:28.12 And what we're looking for, of course, are peptides that look like this:
00:10:30.29 where the heavy peptide is much less abundant than the light peptide.
00:10:34.16 And that means that that peptide's abundance was inhibited
00:10:37.23 or decreased as a result of Cdk1 inhibition and therefore
00:10:41.02 that phosphorylation site on that peptide represents a
00:10:43.23 Cdk1-dependent phosphorylation site in vivo.
00:10:47.23 And so by applying this approach to the many phosphorylation sites identified here
00:10:52.18 we came up with a list of about 547 phosphorylation sites on about 308 proteins
00:10:58.10 that were clearly Cdk1-dependent and represent likely candidates for Cdk1 targets in vivo.
00:11:04.13 This list of targets included many of the same proteins
00:11:07.02 we had identified in our previous screen in vitro
00:11:09.12 and so for those proteins at least we have very good evidence that these
00:11:11.29 proteins are kinase substrates both in vitro and in vivo.
00:11:17.11 Now the list of substrates includes a wide range of proteins involved in a wide range of processes.
00:11:23.20 I'm not expecting you to see or read any of the gene names on these lists here.
00:11:26.27 This slide is simply meant to illustrate that we have lists of proteins involved in
00:11:30.14 a wide range of interesting processes. Some of these processes are totally expected.
00:11:35.00 For example, DNA replication, spindle behavior, kinetochores and cytokinesis are all
00:11:40.08 processes in which we expect Cdks to be involved in regulating some aspect of those processes.
00:11:45.10 There's also a few surprises here as well.
00:11:47.27 Protein translation, chromatin structure, and nuclear transport and the secretory pathway
00:11:53.09 all have a number of Cdk substrates involved
00:11:56.08 in those processes and so one might imagine
00:11:58.15 that this will lead to some new understanding of how Cdks might control those processes
00:12:02.17 as well as the more conventional cell cycle regulated processes.
00:12:07.14 But for the rest of this lecture today, I'm not going to talk in detail about
00:12:10.21 any specific substrates or processes, but instead I'm
00:12:13.14 going to tell you how we used our lists of substrates
00:12:16.02 to address some interesting general questions on how cell cycle progression is
00:12:20.10 controlled by Cdks in general.
00:12:23.13 And so we're going to address two questions in the remainder of this lecture.
00:12:26.14 The first one of which is shown on this next slide.
00:12:30.15 And that question is this one: How do different cyclins trigger different cell cycle events?
00:12:35.10 So I told you at the beginning of the lecture that
00:12:37.02 S-phase cyclin Cdk complexes initiate S-phase and mitotic Cdk-cyclin complexes
00:12:42.00 initiate M-Phase and there's good evidence from yeast genetics and elsewhere
00:12:46.00 that S-phase Cdk-cyclin complexes have a better intrinsic ability to
00:12:49.21 initiate S-phase than a mitotic cyclin-Cdk complex.
00:12:54.01 So there's something different about cyclin-Cdk complexes that are activated at S-phase
00:12:58.28 that allows them to more effectively activate the onset of S-phase.
00:13:02.05 And so, what is that difference?
00:13:04.12 Well, one obvious possibility is that the cyclin that associates with the Cdk
00:13:07.21 helps determine the substrate specificity of that Cdk.
00:13:11.20 So in budding yeast, for example, where there's only a single Cdk
00:13:14.20 associating with all these different cyclins,
00:13:16.08 one can imagine that associating with an S-phase cyclin
00:13:19.20 at the beginning of S-phase might target that Cdk for specific substrates involved in S-phase.
00:13:26.01 And so we decided we could address this question on a more global level
00:13:29.15 by actually analyzing the relative phosphorylation rate of
00:13:32.09 a wide range of Cdk substrates using purified S-phase Cdks and M-phase Cdks.
00:13:38.15 And specifically we carried out these studies using the S-phase cyclin Clb5 from budding yeast
00:13:44.08 and the M-phase cyclin Clb2 from budding yeast.
00:13:47.15 And Mart Loog, a post-doc in the lab, basically purified these two kinases
00:13:51.14 and then tested their activity towards about 150 different Cdk substrates
00:13:55.17 to look for substrates that were highly specific for one or the other.
00:13:59.21 Some of his early results are shown in this next slide
00:14:02.07 which gives you an illustration of the sort of thing we found.
00:14:06.03 Here we're looking at autoradiographs of protein gels in which three different proteins
00:14:11.00 listed across the top--Mcm3, Orc2, and Orc6
00:14:14.00 have been treated with either the mitotic cyclin-Cdk complex on the left
00:14:18.14 or the S-phase complex on the right.
00:14:20.22 And you can see, quite clearly, that these three proteins are all phosphorylated
00:14:24.08 much more rapidly by the S-phase Cdk-cyclin complex Clb5.
00:14:29.21 So Mart went ahead and did this exact same reaction with about 150 proteins as I said
00:14:35.06 and the results from those experiments are shown on this slide.
00:14:37.24 So this slide summarizes everything that he found.
00:14:40.15 What we're looking at here is a plot of about 150 proteins
00:14:44.06 each one of which is represented by these little circles on this plot.
00:14:47.10 And these circles are plotted according to the rate of their phosphorylation
00:14:52.02 by Clb2 on that axis and Clb5 on this axis.
00:14:55.22 And so most of the proteins are falling along the diagonal of this plot
00:14:59.21 indicating that they are equally well phosphorylated by both kinases.
00:15:02.24 In other words, they're not cyclin specific targets.
00:15:05.09 However, we found a quite large number of proteins over here on the right
00:15:09.17 especially these red circles here that represent proteins that
00:15:12.25 are far more rapidly phosphorylated by Clb5-Cdk1 than they are by Clb2-Cdk1.
00:15:19.18 So these proteins, and note by the way that this is a log phase scale here
00:15:23.03 so some of these proteins are 10 or over 100 or even 1000 fold more rapidly phosphorylated by
00:15:28.02 Clb5-Cdk1 than by Clb2-Cdk1. So these clearly represent proteins that are highly Clb5 specific.
00:15:35.05 That the cyclin is somehow determining or increasing
00:15:37.29 the rate of phosphorylation of these proteins.
00:15:40.14 So what are these proteins? Well, we were satisfied to see that at least five of them
00:15:46.07 are proteins known to be involved in DNA replication, especially Sld2 here.
00:15:50.07 Sld2 is a protein whose phosphorylation is known
00:15:52.25 to be crucial for the initiation of DNA replication.
00:15:55.16 And so these proteins make perfect sense as Clb5 specific targets because those are
00:16:00.04 the proteins that we need to phosphorylate early in S-phase
00:16:02.22 to help drive progression through chromosome duplication.
00:16:07.27 So what this list of cyclin-specific substrates in hand, we next addressed
00:16:12.10 the mechanism underlying this cyclin specificity.
00:16:14.25 Why is it that Clb5-Cdk1 phosphorylates these proteins
00:16:18.06 so much more rapidly than Clb2-Cdk1?
00:16:21.13 Through kinetic studies we discovered that the reason for this higher rate of phosphorylation was
00:16:26.15 that these substrates have a much higher affinity for the Cdk-cyclin complex
00:16:30.05 when Clb5 is associated, suggesting that they might associate with that cyclin subunit.
00:16:36.16 In fact, there's previous suggestions of what
00:16:38.04 the mechanism for this association might be.
00:16:40.25 And those are based on the known crystal structures of Cdk-cyclin complexes from human cells.
00:16:46.06 So this shows the crystal structure of a Cdk-cyclin complex from humans
00:16:50.20 that illustrates very nicely the basic parts of the Cdk-cyclin complex
00:16:55.08 and where cyclin substrates typically associate with this complex.
00:16:59.01 Over on the left is the Cdk catalytic subunit and between these two lobes here
00:17:02.29 is an active site cleft in which you can see this ATP molecule binding right here.
00:17:08.19 Typically a protein substrate would bind along the surface of this protein kinase right here
00:17:12.29 in a way that the serine or threonine hydroxyl would be positioned in such a way
00:17:17.25 to allow the transfer of phosphate from that ATP onto the hydroxyl residue.
00:17:23.10 So, the primary site of substrate association with the Cdk-cyclin complex
00:17:27.00 is of course the active site, the place where that serine or threonine
00:17:30.21 associates with its sequence contacts to be phosphorylated.
00:17:35.16 However, this is probably not the only site of substrate association in a Cdk-cyclin complex.
00:17:39.28 There is considerable evidence from mammals and from yeast as well
00:17:43.13 that there is a docking site on this cyclin itself
00:17:46.20 that can also associate to some extent with parts of the substrate.
00:17:50.10 And this docking site is mostly composed of this large alpha-helix here
00:17:54.18 that contains a number of hydrophobic residues
00:17:57.06 that are together called the hydrophobic patch.
00:17:59.16 It is involved in associating with certain substrates
00:18:01.23 and enhancing activity towards those substrates.
00:18:07.02 So, we obviously hypothesized that perhaps this docking site on Clb5
00:18:10.16 exists on Clb5 and that this docking site is required
00:18:13.26 for the cyclin-specific phosphorylation that we saw in our experiments.
00:18:18.03 And so to test that the obvious approach was to mutagenize this docking site
00:18:21.09 through a number of single point mutations and then test whether that
00:18:25.08 has any impact on cyclin specificity and that is shown in this slide.
00:18:28.20 And the answer was a definite yes, that mutation of that docking site
00:18:32.27 completely abolishes the Clb5 specificity that we had seen.
00:18:35.23 So here again we're looking at autoradiographs of protein phosphorylation
00:18:39.27 by purified Clb2 on the left two lanes and Clb5 on the right.
00:18:43.20 And we're looking at the phosphorylation of 5 highly Clb5 specific proteins.
00:18:47.20 And you can see that the wild type Clb2, the wt here, phosphorylates these proteins
00:18:52.13 rather poorly whereas wild type Clb5 phosphorylates them extremely well.
00:18:57.18 Once again, indicating how specific these proteins are for Clb5.
00:19:01.03 However, if you mutate the hydrophobic patch or the docking site
00:19:04.15 on Clb5 you find that that specific phosphorylation is almost completely lost
00:19:09.12 indicating that that site is really required for the increased affinity
00:19:12.25 that Clb5-Cdk1 has for these substrates.
00:19:18.20 So we conclude that an interaction, probably simultaneous between
00:19:22.01 this docking site and the active site, allows specific Clb5 substrates to interact
00:19:26.19 with the Clb5-Cdk complex in a high affinity fashion
00:19:30.10 that allows more rapid phosphorylation of those proteins.
00:19:34.20 And so that leads us to at least a partial answer for the question that I first posed
00:19:39.04 which is: How do different cyclins drive different cell cycle events?
00:19:43.07 Well, part of the answer appears to be that the associated cyclin that associates
00:19:47.01 with the Cdk helps target that Cdk to specific substrates.
00:19:51.01 And so S-phase Cdk-cyclin complexes when they're activated
00:19:54.14 at the end of G1 tend to phosphorylate more rapidly the proteins
00:19:57.25 that are most important for initiating S-phase.
00:20:02.06 OK, now I want to turn to an entirely different sort of general question
00:20:05.05 that we also used our substrate lists to address.
00:20:09.13 And in particular, we used our recent mass spectrometry analysis
00:20:12.11 and our 547 Cdk1-dependent phosphorylation sites to address this question.
00:20:17.20 And this is a much more general question that just...that goes beyond
00:20:21.28 issues of simple cell cycle control but reaches into areas involved in
00:20:25.26 the general issues of phospho-regulation. And the question is this one:
00:20:30.10 How is it that phosphorylation changes the function of a protein?
00:20:33.10 How is it that the addition of a phosphate group to a protein changes that protein's function
00:20:37.20 in a way that allows it to initiate cell cycle events or do other things?
00:20:41.27 And there are typically a couple of different approaches
00:20:44.09 or different mechanisms that are thought to be involved in changing protein function.
00:20:48.03 And the first and possibly most commonly imagined mechanism is this one here
00:20:51.25 the so-called allosteric switch. And the idea with this mechanism is that the placement
00:20:56.13 of a phosphate on a protein in a very specific location
00:20:59.06 causes a precise conformational change in that protein that then
00:21:02.24 initiates some change in its function, its enzymatic activity or its association with something.
00:21:08.21 Now, this mechanism, of course, requires that the position of that phosphorylation site
00:21:11.29 is extremely precise and conserved. In other words,
00:21:15.10 you can't put a phosphate just anywhere on a protein and
00:21:17.23 cause this very precise conformational change.
00:21:20.08 It has to be extremely well positioned
00:21:21.21 and because of that it’s very difficult to evolve that sort of phospho-regulation.
00:21:26.23 That kind of phosphorylation cannot appear randomly very easily
00:21:31.22 and achieve the kind of regulation that is required.
00:21:35.08 So the alternative mechanism is this one--which I call bulk electrostatics.
00:21:40.08 And this mechanism suggests that the position of the phosphorylation
00:21:43.14 does not require such precise position of the phosphorylation.
00:21:47.27 The basic idea here is that the placement of clusters of phosphorylation sites
00:21:51.22 on the surface of the protein, typically on a loop or a disordered region on the surface
00:21:56.01 can result in interesting regulation such as interference with association with another protein
00:22:01.16 or for that matter, promotion of association with phosphate binding proteins.
00:22:06.04 And so this very simple mechanism of phospho-regulation
00:22:08.18 can occur by the placement of clusters of phosphates in a general region
00:22:13.19 of a protein but the exact position of each of those phosphates is not absolutely important,
00:22:18.06 not critical and therefore the position of those phosphates can shift during evolution
00:22:22.05 in different proteins. And so for that reason this mechanism is much more easily evolved.
00:22:28.10 It’s very easy to imagine that random mutations
00:22:30.09 could result in the appearance of phosphorylation sites
00:22:32.19 on the surface of certain proteins where they interact with other proteins
00:22:35.17 and that can result in regulatory possibilities that could be selected for.
00:22:41.08 And so, both of these mechanisms are known to be important in different cases.
00:22:46.12 There are examples of proteins that are regulated in both of these ways.
00:22:49.17 But we thought that perhaps our giant list of Cdk substrates would allow us to
00:22:53.11 address the relative importance of these two mechanisms more generally.
00:22:58.06 And so what we did is we took those 547 Cdk1-dependent phosphorylation sites
00:23:03.06 and aligned them with homologous sequences from other species
00:23:07.17 to see how well these phosphorylation sites are actually conserved.
00:23:10.27 And the basic idea here was that if we found that
00:23:13.11 sites are generally, extremely well preserved that might argue for this sort of mechanism,
00:23:17.24 but sites that drift during evolution might argue for this sort of mechanism.
00:23:23.23 So the next slide gives you an illustration of the sort of thing that we found.
00:23:27.14 Now, here we are aligning a bunch of different protein sequences
00:23:29.29 and I don't expect you to actually read these sequences.
00:23:32.17 The important thing is that there are these little yellow boxes that represent
00:23:35.15 a SP or TP di-peptide motifs that are the consensus sequences for Cdk phosphorylation.
00:23:42.09 And along the very top here is the sequence of a part of a protein
00:23:45.27 called Shp1 that we identified two phosphorylation sites in our mass spectrometry experiments.
00:23:51.24 Those sites are site A and site B.
00:23:53.26 One site is over here in a region of the protein that is known
00:23:57.05 to form into a globular domain and anther site is here in a region
00:24:00.21 that's predicted to form a disordered domain.
00:24:03.16 And these other sequences that lie below this top sequence from budding yeast
00:24:06.26 are the sequences of orthologous proteins from various yeast species
00:24:11.25 whose genomes have been sequenced
00:24:13.20 starting with the most closely related yeast here and
00:24:15.25 moving all the way down to the most distantly related yeast at the bottom.
00:24:20.03 And so these protein alignments tell us some very simple things.
00:24:23.19 First of all, site A here is very well conserved in evolution
00:24:27.10 and you can see that almost all of the orthologs of this protein in all these other yeast species
00:24:31.14 contain a likely Cdk consensus site at the exact same position in this highly conserved region.
00:24:38.25 So site A appears to represent an example of the kind of site I mentioned
00:24:42.09 in the left side of the previous slide, a site that is highly conserved in evolution.
00:24:46.27 But site B is not. Site B is very poorly conserved and disappears essentially
00:24:51.15 after a few species and is no longer found at that position in other orthologs.
00:24:57.24 However, if you look in this region of these other species' proteins, you find that
00:25:01.21 SP and TP di-peptide motifs appear scattered throughout this region
00:25:05.08 in a larger number of these yeast homologous proteins,
00:25:08.20 suggesting that even though the initial position here in budding yeast has not been preserved,
00:25:14.09 the Cdk phosphorylation of this region has been conserved in evolution
00:25:18.21 but the exact position of the phosphorylation sites
00:25:20.23 has been shifting dramatically over evolution.
00:25:23.27 So this is obviously consistent with the second idea,
00:25:26.13 that precise phosphorylation site positioning is not
00:25:29.16 required here because these sites might be involved in some more simple, general
00:25:34.03 regulatory mechanism involving association with phosphate binding proteins
00:25:38.03 or interference with protein binding.
00:25:41.11 So we did this exact same alignment for all 547 of our phosphorylation sites
00:25:46.09 and then in the next slide what I'm going to show you
00:25:47.28 is a somewhat complex graphic that illustrates the results that we found from that.
00:25:53.14 And this was done in collaboration with Brian Tuch, a graduate student
00:25:56.22 working in the laboratory of Sandy Johnson at UCSF.
00:26:00.09 And this top plot here represents a hierarchically clustered
00:26:02.27 clustergram as we call it that illustrates the conservation of the precise position of
00:26:08.26 phosphorylation sites in orthologs of the proteins we identified.
00:26:13.29 And so what we're looking at here is a graphic in which there are 547 columns
00:26:18.04 in this graphic, each one of which represents a single Cdk1 dependent phosphorylation site
00:26:23.14 that we identified by mass spectrometry.
00:26:26.17 And then each row in this graphic represents
00:26:29.22 how that phosphorylation site aligns with its orthologs in other species,
00:26:34.06 the same yeast species that I showed in the previous slide
00:26:36.23 starting with the most closely related yeast species
00:26:39.03 and then working to the most distantly related ones.
00:26:41.28 And in each column a yellow box indicates that that phosphorylation site
00:26:47.06 is precisely conserved in its position in that orthologous sequence.
00:26:51.03 In other words, over here on the left this yellow box at the top moves down
00:26:55.16 for a few species and then disappears indicating that this site is only precise...
00:26:59.24 these columns here, these phosphorylation sites
00:27:02.12 are conserved only in the closely related
00:27:04.21 yeasts species but then are lost in all distantly related species.
00:27:10.03 And so by looking at this graph you can see that there's only
00:27:12.10 a small group of phosphorylation sites, these ones in here especially
00:27:16.06 and note particularly these ones here that are conserved
00:27:19.04 throughout all the yeast homologs that we identified.
00:27:21.23 And so this small number of phosphorylation sites, perhaps 30 or 40 of them are
00:27:26.12 at most, are preserved in large numbers of yeast species, indicating that
00:27:31.29 the precise position of phosphorylation
00:27:33.17 has been conserved in a relatively small number of cases.
00:27:37.16 OK, so how do we then test the possibility that instead of precise positioning
00:27:44.01 during evolution that we're looking at drifting phosphorylation site positioning?
00:27:48.28 And that required the development of another graphic which is shown below here
00:27:52.17 in which I'll take you through slowly because it’s a little bit complicated.
00:27:56.16 So in this case, once again, it’s another hierarchically clustered graphic in which
00:28:00.22 there are 547 columns, each representing a different phosphorylation site
00:28:05.15 identified in our analyses. But in this case...
00:28:08.17 and once again, the rows represent alignments with orthologs, orthologous proteins
00:28:13.00 from other yeast species. But in this case the yellow box
00:28:16.02 doesn't indicate precise positioning of phosphorylation site, but instead
00:28:19.18 indicates that the ortholog of that particular protein in these other yeast species
00:28:24.03 has a statistically enriched frequency of Cdk consensus sites, SP and TP motifs.
00:28:30.28 In other words, these yellow boxes represent proteins
00:28:33.21 in which the frequency of SP and TP motifs is far greater than that expected by chance.
00:28:39.00 And so these large numbers of proteins here represent proteins in which
00:28:43.08 even though the precise site of phosphorylation is not conserved
00:28:46.19 as shown up here, these proteins do contain a high frequency of
00:28:50.12 Cdk consensus sites whose positions are clearly drifting during evolution.
00:28:56.04 And so these large number of proteins over here on the right side of this clustergram
00:28:59.24 may represent proteins in which the precise position of phosphorylation
00:29:03.21 does not matter but the regulation of those proteins by phosphorylation
00:29:07.17 is conserved despite that. And so clearly we'd like to think that that evidence
00:29:14.00 tends to suggest that this mechanism on the right here
00:29:16.03 these easily evolved bulk electrostatic mechanism
00:29:19.05 is a major mechanism by which phospho-regulation can easily be evolved
00:29:23.10 and that drifting phosphorylation sites especially clusters of phosphorylation sites
00:29:26.19 on disordered regions is really crucial for
00:29:30.10 the regulation of many different Cdk substrates.
00:29:34.28 So with that, I want to leave us with the question that we started out this whole lecture with
00:29:39.20 and that is: How do Cdk's drive cell cycle events?
00:29:42.04 Well, clearly our list of Cdk substrates, the ones I'm showing here and
00:29:45.21 the many that aren't shown here, probably contain the answer to this question.
00:29:50.06 Clearly through the detailed analysis of large numbers of these substrates
00:29:53.03 and that will lead us to a much better understanding of how Cdks
00:29:57.17 drive the events of cell cycle and how they alter all these different processes
00:30:02.04 in the cell to initiate cell cycle events.