Regulators of Cell Cycle Progression

Transcription

1 Regulators of Cell Cycle Progression Studies of Cdk s and cyclins in genetically modified mice reveal a high level of plasticity, allowing different cyclins and Cdk s to compensate for the loss of one another. Cdk1 is capable of substituting for the all the other Cdk s.

2 Regulators of Cell Cycle Progression The activity of Cdk s is regulated by four mechanisms: 1. Association of Cdk s and cyclin partners formation of specific Cdk/cyclin complexes is controlled by cyclin synthesis and degradation.

3 Regulators of Cell Cycle Progression 2. Activation of Cdk/cyclin complexes requires phosphorylation of threonine around position 160. This is catalyzed by an enzyme called CAK (for Cdk-activating kinase), which is composed of Cdk7/cyclin H. 3. Inhibitory phosphorylation of tyrosine near the Cdk amino terminus, catalyzed by the Wee1 protein kinase. The Cdk s are then activated by dephosphorylation by Cdc25 protein phosphatases.

4 Regulators of Cell Cycle Progression 4. Binding of inhibitory proteins Cdk inhibitors (CKIs). In mammalian cells, there are two families of Cdk inhibitors: Ink4 and Cip/Kip

5 Regulators of Cell Cycle Progression The combined effects of these multiple modes of Cdk regulation are responsible for controlling cell cycle progression in response to checkpoint controls and to extracellular stimuli. Proliferation of animal cells is regulated largely by extracellular growth factors that control progression through the restriction point in late G 1. This implies that intracellular signaling pathways ultimately act to regulate components of the cell cycle machinery.

6 Regulators of Cell Cycle Progression The D-type cyclins provide one link between growth factor signaling and cell cycle progression. Growth factors stimulate cyclin D1 synthesis through the Ras/Raf/MEK/ERK pathway, and are synthesized as long as growth factors are present.

7 Regulators of Cell Cycle Progression Cyclin D1 is also rapidly degraded, so the intracellular concentration rapidly falls if growth factors are removed. As long as growth factors are present through G 1, Cdk4,6/cyclin D1 complexes drive cells through the restriction point. Defects in cyclin D1 regulation could contribute to the loss of growth regulation characteristic of cancer cells. Many human cancers arise as a result of defects in cell cycle regulation.

8 Regulators of Cell Cycle Progression Rb is a key substrate protein of Cdk4, 6/cyclin D complexes, and is frequently mutated in many human tumors. It was first identified in retinoblastoma, a rare inherited childhood eye tumor. Rb is the prototype of a tumor suppressor gene a gene whose inactivation leads to tumor development. Rb plays a key role in coupling cell cycle machinery to the expression of genes required for cell cycle progression.

9 Regulators of Cell Cycle Progression In G 0 or early G 1, Rb binds to E2F transcription factors, which suppresses expression of genes involved in cell cycle progression. Rb is phosphorylated by Cdk4,6/cyclin D complexes as cells pass through the restriction point, and dissociates from E2F, allowing transcription to proceed.

10 Regulators of Cell Cycle Progression Progression through the restriction point and entry into S phase is mediated by activation of Cdk2/cyclin E complexes. In G 0 and early G 1, Cdk2/cyclin E is inhibited by p27 (Cip/Kip family). Then, cyclin E synthesis is stimulated by E2F following phosphorylation of Rb, and transcription of p27 is inhibited by growth factor signaling. The resulting activation of Cdk2/cyclin E leads to activation of MCM helicase and initiation of DNA replication.

11 Figure Cdk2/cyclin E and entry into S phase

12 Regulators of Cell Cycle Progression Cell cycle arrest at DNA damage checkpoints is mediated by related protein kinases, ATM and ATR, that are activated in response to DNA damage. They then activate a signaling pathway that leads to cell cycle arrest, DNA repair, and sometimes, programmed cell death. ATM is activated by double-strand breaks, ATR is activated by single-stranded or unreplicated DNA. They phosphorylate and activate the checkpoint kinases Chk2 and Chk1.

13 Figure Cell cycle arrest at the DNA damage checkpoints

14 Regulators of Cell Cycle Progression Chk1 and Chk2 phosphorylate and inhibit Cdc25 phosphatases, which are required to activate Cdk1 and Cdk2. Inhibition of Cdk2 results in cell cycle arrest in G 1 and S. Inhibition of Cdk1 results in arrest in G 2.

15 Figure Role of p53 in G 1 arrest In mammalian cells, arrest at the G1 checkpoint is also mediated by protein p53, which is phosphorylated by both ATM and Chk2. p53 is a transcription factor, and its increased expression leads to induction of Cdk inhibitor p21. p21 inhibits Cdk2/cyclin E complexes, leading to cell cycle arrest in G1. The gene encoding p53 is frequently mutated in human cancers. Loss of p53 prevents G1 arrest in response to DNA damage, so the damaged DNA is replicated and passed on to daughter cells.

16 The Events of M Phase M phase involves a major reorganization of virtually all cell components: Chromosomes condense, the nuclear envelope breaks down, the cytoskeleton reorganizes to form the mitotic spindle, and the chromosomes move to opposite poles. Cell division (cytokinesis) usually follows.

17 The Events of M Phase Mitosis is divided into four stages: 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase

18 Figure Fluorescence micrographs of chromatin, keratin, and microtubules during mitosis of newt lung cells

19 The Events of M Phase Prophase appearance of condensed chromosomes (two sister chromatids). The chromatids are attached at the centromere, where proteins bind to form the kinetochore (site of eventual attachment of the spindle). The centrosomes (which duplicated during interphase) separate and move to opposite sides of the nucleus. There they serve as the two poles of the mitotic spindle, which begins to form during late prophase.

20 The Events of M Phase In higher eukaryotes the end of prophase corresponds to the breakdown of the nuclear envelope (open mitosis). In some unicellular eukaryotes (e.g., yeasts) the nuclear envelope remains intact (closed mitosis).

21 The Events of M Phase Prometaphase transition period between prophase and metaphase. Microtubules of the spindle attach to the kinetochores of condensed chromosomes. The chromosomes shuffle back and forth until they align on the metaphase plate. At this point, the cell has reached metaphase.

22 The Events of M Phase Most cells remain only briefly at metaphase before proceeding to anaphase. The link between sister chromatids breaks, and they separate and move to opposite poles of the spindle. Mitosis ends with telophase, during which nuclei re-form and chromosomes decondense. Cytokinesis usually begins during late anaphase and is almost complete by the end of telophase.

23 The Events of M Phase Cdk1/cyclin B protein kinase (MPF) acts as a master regulator of M phase transition. It activates other mitotic protein kinases and directly phosphorylates structural proteins involved in the cellular reorganization.

24 The Events of M Phase Condensation of chromatin is a key event in mitosis. Chromatin condenses nearly a thousandfold during the formation of metaphase chromosomes. Transcription ceases during chromatin condensation. Chromatin condensation is driven by condensins, members of a class of structural maintenance of chromatin (SMC) proteins. Condensins and cohesins (another family of SMC proteins) contribute to chromosome segregation.

25 The Events of M Phase Cohesins bind to DNA in S phase and maintain the linkage between sister chromatids. Condensins are activated by Cdk1/cyclin B phosphorylation, and replace most of the cohesins, so sister chromatids remain linked only at the centromere.

26 Figure The action of cohesins and condensins

27 The Events of M Phase Breakdown of the nuclear envelope involves changes in all of its components: Nuclear membranes fragment Nuclear pore complexes dissociate The nuclear lamina depolymerizes due to phosphorylation of lamins by Cdk1

28 Figure Breakdown of the nuclear envelope

29 The Events of M Phase The Golgi apparatus fragments into small vesicles at mitosis, which may be absorbed into the ER or distributed to daughter cells at cytokinesis. Golgi breakdown is mediated by phosphorylation of proteins by Cdk1 and other protein kinases. Centrosome maturation and spindle assembly involve protein kinases of the Aurora and Pololike kinase families, located at the centrosome. The rate of microtubule turnover increases five- to tenfold during mitosis, resulting in depolymerization and shrinkage of the interphase microtubules.

30 The Events of M Phase The number of microtubules emanating from the centrosomes also increases, so the interphase microtubules are replaced by large numbers of short microtubules radiating from the centrosomes. Breakdown of the nuclear envelope allows spindle microtubules to attach to chromosomes at the kinetochores. Chromosomes in prometaphase shuffle back and forth between the centrosomes and the center of the spindle due to activity of microtubule motors at the kinetochore and centrosomes.

31 Figure Electron micrograph of microtubules attached to the kinetochore of a chromosome

32 The Events of M Phase The balance of forces acting on the chromosomes leads to their alignment on the metaphase plate in the center of the spindle. The spindle consists of kinetochore and chromosomal microtubules attached to the chromosomes, and polar microtubules, which overlap with one another in the center of the cell.