Посещений:
Контроль Клеточных Циклов

Принципы

Overview: The Cell-Cycle Control System
David O Morgan
from The Cell Cycle: Principles of Control
Chapter 3: The Cell-Cycle Control System
© 1999-2004 New Science Press Ltd




Figure 1-3.1  |  A simplified view of the cell-cycle control system


Figure 1-3.2  |  Cyclin-dependent kinase activation


Figure 1-3.3  |  Overview of cell-cycle control

References
Hartwell LH, Weinert TA: Checkpoints: controls that ensure the order of cell cycle events. Science 1989, 246:629-634.
Morgan DO: Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 1997, 13:261-291.
Murray AW, Kirschner MW: Dominoes and clocks: the union of two views of the cell cycle. Science 1989, 246:614-621.


Figure 3-1.1  |  Table of cyclin-dependent kinases that control the cell cycle


Figure 3-1.2  |  Cyclin-dependent kinase structure

References

De Bondt HL, et al.: Crystal structure of cyclin-dependent kinase 2. Nature 1993, 363:595-602.
Morgan DO: Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 1997, 13:261-291.
Ubersax JA, et al.: Targets of the cyclin-dependent kinase Cdk1. Nature 2003, 425:859-864.


Figure 3-4.1  |  The structural basis of Cdk activation

References

Brown NR, et al.: The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat Cell Biol 1999, 1:438-443.
De Bondt HL, et al.: Crystal structure of cyclin-dependent kinase 2. Nature 1993, 363:595-602.
Honda R, et al.: The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles. EMBO J 2005, 24:452-463.
Jeffrey PD, et al.: Mechanism of CDK activation revealed by the structure of a cyclin A-CDK2 complex. Nature 1995, 376:313-320.
Pavletich NP: Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and CIP and Ink4 inhibitors. J Mol Biol 1999, 287:821-828.
Russo AA, et al.: Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat Struct Biol 1996, 3:696-700.
The cell-cycle control system is a complex assembly of oscillating protein kinase activities
Система контроля клеточного цикла является регуляторной сетью, которая контролирует порядок и время событий клеточного цикла. Серия биохимических переключений запускает действие посредством трех главных регуляторных КПП (checkpoints) клеточного цикла: Start, который предопределяет вступление в цикл на поздней G1; G2/M checkpoint, когда контролируется вступление в митоз; и переход от метафазы к анафазе, когда инициируются финальные события митоза.
Центральными компонентами системы контроля клеточного цикла являются cyclin-dependent kinases (Cdks). Когда клетки проходят через клеточный цикл, то резкие изменения ферментативной активности этих киназ ведут к изменениям в состоянии фосфорилирования и тем самым в состоянии активации, белков, которые контролируют прохождение клеточного цикла. Концентрации Cdk белков постоянны в течение всего клеточного цикла; осцилляции в их активности зависят в первую очередь от соотв. осцилляций в уровнях регуляторных субъединиц, известных как cyclins, которые тесно связаны с Cdks и стимулируют их каталитическую активность. Разные типы циклинов продуцируются на разных стадиях клеточного цикла, давая в результате образование серии cyclin-Cdk комплексов. Эти комплексы управляют самостоятельными событиями клеточного цикла и поэтому мы будем их называть G1-, G1/S-, S- и M-Cdks. Основное внимание будет посвящено последним трем комплексам, которые контролируют прохождение через три главных КПП (checkpoints) (Рис.1).

Multiple regulatory mechanisms govern Cdk activity during the cell cycle


Каждый cyclin-Cdk комплекс способствует активации следующего этапа, гарантируя тем самым, что ход клеточного цикла будет происходить упорядоченно. Точное время изменений в активности Cdk управляется множественными механизмами. Концентрации циклинов особенно важны и мы увидим, как они регулируются с помощью комбинации изменений в экспрессии генов циклинов и скоростей деградации cyclin. Активность комплексов cyclin-Cdk далее модулируется путем добавления или удаления ингибирующего фосфорилирования и путём изменений в уровнях Cdk-ингибирующих белков.
G1/S-, S- и M-Cdks не активны в G1, гарантируя тем самым, что события клеточного цикла не будут запускаться несвоевременно до того как клетки подготовят себя к следующему циклу. Три ингибирующих механизма супрессируют активность этих Cdks во время G1. Два из них затрагивают cyclins: экспрессия основных генов cyclin супрессируется с помощью ингибирующего ген-регуляторных белков, и скорости деградации cyclin существенно возрастают благодаря активации важного белкового комплекса, наз. anaphase-promoting complex или APC, который специфически нацелен на S и M cyclins (но не на G1/S cyclins) для деградции (Рис.1). Третьим является присутствие высоких концентраций ингибиторов Cdk в G1.
Вступление в новый цикл начинается, когда сигналы из вне клетки (митогены, напр.) и изнутри (системы мониторинга клеточного роста, напр.) запускают комбинацию событий, которые дают волю экспрессии генов G1/S- и S-cyclin и активации G1/S-Cdks. Активность G1/S-Cdk возникает немедленно, т.к. G1/S cyclins не поставляются с помощью APC и т.к. G1 Cdk-inhibitory белки или не действуют на G1/S-Cdks (у дрожжей и мух) или удаляются от G1/S-Cdks с помощью др. механизмов (у млекопитающих). G1/S-Cdks непосредственно инициируют некоторые ранние события клеточного цикла, но их основная функция активировать S-Cdks прежде всего путем запуска деструкции Cdk-ингибирующих белков и инактивации APC, оба из которых противодействуют активности S-Cdk в G1. S-Cdks затем фосфорилируют белки, которые инициируют удвоение хромосом, тем самым запускают S фазу. Как только запускается S фаза, G1/S-Cdks способствуют своей собственной инактивации посредством стимулирования деструкции G1/S cyclins, и экспрессия гена G1/S-cyclin снижается.
К концу S фазы, включается экспрессия гена M-cyclin и концентрация M-cyclin возрастает, это ведет к накоплению M-Cdk комплексов во время G2. В большинстве типов клеток эти комплексы первоначально находятся в неактивном состоянии за счет ингибирующего фосфорилирования субъединиц Cdk. В начале митозов, однако, резкое удаление этого фосфорилирования ведет к активации всех M-Cdks. Они затем запускают в действие G2/M checkpoint. Сборка веретена и дрю ранних митотических событий ведут к выстраиванию удвоенных сестринских хроматид на митотическом веретене в метафазе.
Помимо управления клетками в метафазе, M-Cdks в конечном итоге стимулируют активацию APC, который запускает переход от метафазы к анафазе. Центральной функцией APC на этой стадии является стимуляция деструкции белков, которые удерживают сестринские хроматиды вместе. APC кроме того обусловливает деструкцию S и M cyclins, приводя в результате к инактивации всех основных активностей Cdk в позднем митозе. Снижение экспрессии генов S- и M-cyclin и повышение продукции Cdk-ингибирующих белков также происходит в позднем митозе. Возникающая в результате инактивация Cdks делает возможным дефосфорилирование их митотических мишеней, которые необходимы для разборки веретена и завершения M фазы. Низкие уровни Cdk неактивности поддерживаются вплоть до позднего периода в последующей G1, когда увеличение G1/S-Cdks снова настраивает клетки на новый цикл. Действие разных cyclin-Cdk комплексов и APC в ходе клеточного цикла суммированы на Рис.1.

The cell-cycle control system generates robust, switch-like and adaptable changes in Cdk activity


Комплексы cyclin-Cdk и др. регуляторы, которые управляют клеточным циклом собраны в сильно взаимосвязанную регуляторную систему, чья эффективность усиливается с помощью ряда важных свойств. Во-первых, система контроля клеточного цикла включает петли обратной связи и др. регуляторные взаимодействия, которые ведут к необратимой, включению-подобной активации и инактивации большинства cyclin-Cdk комплексов. Т.о., события клеточного цикла в целом запускаются по принципу всё-или-ничего, что позволяет клетке избежать повреждений, которые м.б. возникнуть, если бы события были инициированы только частично. Во-вторых, регуляторные взаимодействия между разными cyclin-Cdk переключениями гарантируют, что они происходят в правильном порядке и скоординированы др. с др. В-третьих, система контроля клеточного цикла чрезвычайно сильна: активация и инактивация любого cyclin-Cdk переключения управляется множественными механизмами, что позволяет системе оперировать хорошо при разных условиях и даже, если некоторые компоненты отсутствуют. Наконец, система способна адаптироваться, делая возможной подгонку времени основных регуляторных переключений к соотв. регуляторным импульсам от различных внутриклеточных и внеклеточных факторов.



1-3 The Cell-Cycle Control System
David O Morgan
from The Cell Cycle: Principles of Control Chapter 1: The Cell-Cycle Control System © 1999-2005 New Science Press Ltd

Cell-cycle events are initiated at three regulatory checkpoints

The cell-cycle control system drives progression through the cell cycle at regulatory transitions called checkpoints (Figure 1-3.3). The first is called Start or the G1/S checkpoint. When conditions are ideal for cell proliferation, G1/S- and S-phase cyclin–Cdk complexes are activated, resulting in the phosphorylation of proteins that initiate DNA replication, duplication of the centrosome and other early cell-cycle events. Eventually, G1/S- and S-phase Cdks also promote the activation of M-phase cyclin–Cdk complexes, which drive progression through the second major checkpoint at the entry into mitosis (G2/M checkpoint). M-phase cyclin–Cdks phosphorylate proteins that promote spindle assembly, bringing the cell to metaphase.
The third major checkpoint is the metaphase-to-anaphase transition, which leads to sister-chromatid segregation, completion of mitosis and cytokinesis. Progression through this checkpoint occurs when M-phase cyclin–Cdk complexes stimulate an enzyme called the anaphase-promoting complex, which causes the proteolytic destruction of cyclins and of proteins that hold the sister chromatids together. Activation of this enzyme therefore triggers sister-chromatid separation and segregation. Destruction of cyclins leads to inactivation of all Cdks in the cell, which allows phosphatases to dephosphorylate Cdk substrates. Dephosphorylation of these substrates is required for spindle disassembly and the completion of mitosis, and for cytokinesis.

Cell-cycle progression in most cells can be blocked at checkpoints

In the cells of the early animal embryo, the Cdk activities of the cell-cycle control system are linked together to form a rigidly programmed oscillator that is essentially autonomous – that is, it can generate appropriately timed waves of Cdk activity without external input. This system is ideal for cells that must divide as rapidly as possible and are not affected by external influences. The control system of most cell types, however, includes additional levels of regulation that allow cell-cycle progression to be adjusted by various intra and extracellular signals. Most cells, for example, initiate a new cell cycle only when stimulated by external signals, thereby ensuring that new cells are made only when needed. Similarly, initiation of cell-cycle events in most cells is responsive to surveillance mechanisms that monitor the progress of previous events. If the cell fails to complete DNA replication, for example, a negative signal blocks the initiation of mitosis. Later events are thus dependent on the completion of earlier events.
To allow regulation of cell-cycle progression, the cell-cycle control system of most cells is supplemented by molecular braking mechanisms that can be used, if necessary, to inhibit the Cdks and other regulators that drive progression through the three major checkpoints. If environmental conditions are not appropriate for cell proliferation, inhibitory signals prevent activation of G1/S- and S-phase Cdks – thereby blocking progression through Start. Similarly, the failure to complete DNA replication blocks entry into mitosis by inhibiting M-phase cyclin–Cdk activation. Delays in spindle assembly inhibit the proteolytic machinery that drives the metaphase-to-anaphase transition, thereby preventing sister-chromatid segregation until the spindle is ready. By these and numerous other mechanisms, the cell is able to arrest cell-cycle progression at an appropriate point when conditions are not ideal and continue it when they are.
The cell-cycle control system can thus be viewed as a linked series of tightly regulated molecular switches, each of which triggers the initiation of cell-cycle events at a specific regulatory checkpoint. We discuss the molecular components and design of this system in Chapter 3. First, in Chapter 2, we review the wide range of experimental organisms in which cell-cycle control is studied.

Definitions

checkpoint: a regulated transition point in the cell cycle, where progression to the next phase can be blocked by various negative signals. This term is sometimes defined to include the signaling mechanisms that monitor cell-cycle events and send the information to the control system; in this book the term is used to define the transition point in the cell cycle where these mechanisms act.
cyclin: positive regulatory subunit that binds and activates cyclin-dependent kinases, and whose levels oscillate in the cell cycle.
cyclin-dependent kinase (Cdk): protein kinase whose catalytic activity depends on an associated cyclin subunit; key component of the cell-cycle control system.
G2/M checkpoint: important regulatory transition where entry into M phase can be controlled by various factors such as DNA damage or the completion of DNA replication.
metaphase-to-anaphase transition: cell-cycle transition where the initiation of sister-chromatid separation can be blocked if the spindle is not fully assembled. Also called the M/G1 checkpoint, but this is not an ideal term because it does not coincide with the boundary between M phase and G1.
Start: major regulatory transition at the entry into the cell cycle in mid to late G1, also called the G1/S checkpoint or the restriction point (in animal cells). Progression past this point is prevented if cell growth is insufficient, DNA is damaged or other preparations for cell-cycle entry are not complete. Unlike cells arrested at the G2/M checkpoint or metaphase-to-anaphase transition, cells prevented from passing Start do not arrest at this point but typically exit the cell cycle into a prolonged nondividing state from which a return to the cycle is a lengthy process.


3-1 Cyclin-Dependent Kinases
David O Morgan
from The Cell Cycle: Principles of Control Chapter 3: The Cell-Cycle Control System © 1999-2005 New Science Press Ltd

The cyclin-dependent kinases are a small family of enzymes that require cyclin subunits for activity

The cyclin-dependent kinases (Cdks) are a family of serine/threonine protein kinases whose members are small proteins (~34–40 kD) composed of little more than the catalytic core shared by all protein kinases. By definition, all Cdks share the feature that their enzymatic activation requires the binding of a regulatory cyclin subunit. In most cases, full activation also requires phosphorylation of a threonine residue near the kinase active site.
Although originally identified as enzymes that control cell-cycle events, members of the Cdk family are involved in other cellular processes as well. Animal cells, for example, contain at least nine Cdks, only four of which (Cdk1, 2, 4 and 6) are involved directly in cell-cycle control (Figure 3-1.1). Another family member (Cdk7) contributes indirectly by acting as a Cdk-activating kinase (CAK) that phosphorylates other Cdks, as we discuss in section 3-3. Cdks are also components of the machinery that controls basal gene transcription by RNA polymerase II (Cdk7, 8 and 9) and are involved in controlling the differentiation of nerve cells (Cdk5).
We will focus on the small number of Cdks for which there is clear evidence of a direct role in cell-cycle control (Figure 3-1.1). In the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae (see section 2-1), all cell-cycle events are controlled by a single essential Cdk called Cdk1. Cell-cycle events in multicellular eukaryotes are controlled by two Cdks, known as Cdk1 and Cdk2, which operate primarily in M phase and S phase, respectively. Animal cells also contain two Cdks (Cdk4 and Cdk6) that are important in regulating entry into the cell cycle in response to extracellular factors.
Cdk function has been remarkably well conserved during evolution. It is possible, for example, for yeast cells to proliferate normally when their CDK1 gene is replaced with the human CDK1 gene. This and other evidence clearly illustrates that Cdk function, and thus the function of the cell-cycle control system, has remained fundamentally unchanged over hundreds of millions of years of eukaryotic evolution.
Cdks exert their effects on cell-cycle events by phosphorylating a large number of proteins in the cell. During mitosis in particular, when many aspects of cellular architecture and metabolism are altered, Cdks phosphorylate hundreds of distinct proteins. These Cdk substrates are phosphorylated at serine or threonine residues in a specific sequence context that is recognized by the active site of the Cdk protein. In most cases, the the target serine (S) or threonine (T) residue is followed by a proline (P); it is also highly favorable for the target residue to have a basic amino acid two positions after the target residue. The typical phosphorylation sequence for Cdks is [S/T*]PX[K/R], where S/T* indicates the phosphorylated serine or threonine, X represents any amino acid and K/R represents the basic amino acids lysine (K) or arginine (R).

The active site of cyclin-dependent kinases is blocked in the absence of cyclin

All protein kinases have a tertiary structure comprising a small amino-terminal lobe and a larger carboxy-terminal lobe. ATP fits snugly in the cleft between the lobes, in such a way that the phosphates are oriented outwards, toward the mouth of the cleft. The protein substrate binds at the entrance of the cleft, interacting mainly with the surface of the carboxy-terminal lobe. Nearby residues catalyze the transfer of the terminal ?-phosphate of ATP to a hydroxyl oxygen in the protein substrate.
Cdks have the same two-lobed structure as other protein kinases (Figure 3-1.2), but with two modifications that make them inactive in the absence of cyclin. These modifications have been revealed by detailed crystallographic studies of the structure of human Cdk2. First, a large, flexible loop – the T-loop or activation loop – rises from the carboxy-terminal lobe to block binding of protein substrate at the entrance of the active-site cleft. Second, in the inactive Cdk several important amino-acid side chains in the active site are incorrectly positioned, so that the phosphates of ATP are not ideally oriented for the kinase reaction. Cdk activation therefore requires extensive structural changes in the Cdk active site.
Two alpha helices make a particularly important contribution to the control of Cdk activity. The highly conserved PSTAIRE helix of the upper kinase lobe (also known as the ?1 helix) interacts directly with cyclin and moves inward upon cyclin binding, causing the reorientation of residues that interact with the phosphates of ATP. The small L12 helix, just before the T-loop in the primary sequence, changes structure to become a beta strand upon cyclin binding, also contributing to reconfiguration of the active site and T-loop. We discuss the structural basis of Cdk activation in more detail in section 3-4. First, we will describe the cyclins and other regulators that influence activation.

Definitions

L12 helix: a small alpha helix adjacent to the T-loop in the active site of Cdk2 (residues 147–151), which changes structure to a beta strand upon cyclin binding.
PSTAIRE helix: alpha helix in the amino-terminal lobe of Cdks (also known as the ?1 helix), which interacts with cyclin and is moved inward upon cyclin binding, resulting in reorientation of key active-site residues. The name of this helix comes from its amino-acid sequence, which is conserved among all major Cdks.
T-loop: a flexible loop adjacent to the active site of Cdks, named for the threonine whose phosphorylation is required for maximal activity. Sometimes called the activation loop.


3-4 The Structural Basis of Cdk Activation
David O Morgan
from The Cell Cycle: Principles of Control Chapter 3: The Cell-Cycle Control System © 1999-2005 New Science Press Ltd

The conformation of the Cdk active site is dramatically rearranged by cyclin binding and phosphorylation by CAK

Cdk activation is understood in structural detail from X-ray crystallographic studies of human Cdk2 in various states of activity (Figure 3-4.1). As described earlier (see section 3-1), the active site of Cdk2 is located in a cleft between the two lobes of the kinase (Figure 3-4.1a). ATP binds deep within the cleft, with its phosphates oriented outwards. The protein substrate would normally interact with the entrance of the active-site cleft, but this region is obscured in the inactive Cdk2 monomer by the T-loop. Key residues in the ATP-binding site are also misoriented in the Cdk2 monomer, further suppressing its activity.
Cyclin A binding has a major impact on the conformation of the Cdk2 active site (Figure 3-4.1b). Several helices in the cyclin box contact both lobes of Cdk2 in the region adjacent to the active-site cleft, resulting in extensive conformational changes in Cdk2. The most obvious change occurs in the T-loop, in which the L12 helix has been changed into a beta strand, and which no longer occludes the binding site for the protein substrate, but lies almost flat at the entrance of the cleft. Major changes also occur in the ATP-binding site, leading to the correct positioning of the ATP phosphates for the phosphotransfer reaction. Cyclin A structure is unaffected by Cdk2 binding but provides a rigid framework against which the pliable Cdk2 subunit is molded.
The T-loop of Cdk2 contains Thr 160, the threonine residue whose phosphorylation by the Cdk-activating kinase (CAK) further increases the activity of the cyclin A–Cdk2 complex (see section 3-3). Following phosphorylation, the phosphate on Thr 160 is inserted in a cationic pocket and acts as the central node for a network of hydrogen bonds spreading outward to stabilize neighboring interactions in both the Cdk and cyclin. The T-loop is flattened and moves closer to cyclin A (Figure 3-4.1c), and this region serves as a key part of the binding site for protein substrates containing the [S/T*]PX[K/R] phosphorylation site described earlier (Figure 3-4.1d).
Crystallographic studies of Cdk activation have so far focused entirely on human Cdk2 and its partner cyclin A. This complex probably serves as a good representative for the entire Cdk family, but the details of Cdk activation appear to be different in some complexes. There is evidence, for example, that the same Cdk, when bound by different cyclins, possesses different amounts of kinase activity toward the [S/T*]PX[K/R] sequence. It is therefore likely that different cyclins do not induce precisely the same conformational changes in the associated Cdk subunit.
Сайт создан в системе uCoz