Part 1: Cells reproduce by duplicating their chromosomes and other components and then distributing them into a pair of genetically identical daughter cells. This series of events is called the cell cycle. In the first part of this lecture, I provide a general overview of the cell-cycle control system, a complex regulatory network that guides the cell through the steps of cell division. I briefly describe the major components of this regulatory system and how they fit together to form a series of biochemical switches that trigger cell-cycle events at the correct time and in the correct order.
Part 2: Cyclin-dependent kinases (Cdks) are the central components of the control system that initiates the events of the cell cycle. In the second part of this lecture, I discuss my laboratory’s efforts to address the problem of how the Cdks trigger cell-cycle events. I describe our methods for identifying the protein substrates of the Cdks, and I discuss how these studies have led to important clues about how Cdks find their correct targets in the cell and how phosphorylation of those targets governs their function.
All Course Materials for this Session (Educators only) – Created by David Morgan
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.
Discuss strategies used to identify CDK substrates, and why some of these substrates (e.g. DNA replication proteins) bind CDK better during S-phase than during M-phase.
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 itâ€™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 itâ€™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 Itâ€™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 itâ€™s a little bit complicated.
00:27:56.16 So in this case, once again, itâ€™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.
David Morgan is a Professor in the Departments of Physiology and Biochemistry & Biophysics at the University of California, San Francisco (UCSF). He received an undergraduate degree in animal physiology from the University of Calgary in 1980, followed in 1986 by a PhD from UCSF, with Richard Roth. Following postdoctoral studies with William Rutter and… Continue Reading