Chap. 19 The Eukaryotic Cell Cycle Topics Overview of the Cell Cycle and Its Control Molecular Mechanisms for Regulating M & S Phase Events Mitogen-stimulated Entry of Cells into the Cell Cycle Surveillance Mechanisms in Cell-cycle Regulation Goals Learn the roles of 1) cyclins and cyclin- dependent protein kinases (CDKs), & 2) ubiquitin-protein ligases in regulation of the cell cycle. Learn the molecular mechanisms for regulation of mitosis and S-phase events. Learn how mitogens propel quiescent cells into the cell cycle. Learn how checkpoint mechanisms ensure quality control in cell cycle events. Cell division during C. elegans early embryogenesis

Two fundamental processes occur with each cell cycle-- chromosomes replicate, and then they segregate equally to two daughter cells. The mechanisms by which these processes occur are similar in all eukaryotic cells. Processes occurring during the cell cycle are highly regulated and coordinated. The cell cycle is regulated primarily at the DNA replication and mitosis steps. The master controllers of the cell cycle are 1) heterodimeric protein kinases composed of a regulatory subunit (a cyclin) and a catalytic subunit (a cyclin-dependent kinase, CDK), 2) two ubiquitin-protein ligases, and 3) regulatory phosphatases. Cyclin-CDKs phosphorylate and thereby regulate the activities of numerous cell proteins that participate in replication and division. The bound cyclins regulate the activities of the CDKs. Ubiquitin-protein ligases participate in the timed destruction of cyclins and other key proteins and thereby ensure passage through the cell cycle is irreversible. In the absence of regulation, cells replicate and divide uncontrollably, leading to diseases such as cancer. Overview of the Cell Cycle and Its Control

Regulating Protein Function by Degradation The proteolytic degradation (turnover) of proteins is important for regulatory processes, cell renewal, and disposal of denatured and damaged proteins. Lysosomes carry out degradation of endocytosed proteins and retired organelles. Cytoplasmic protein degradation is performed largely by the molecular machine called the proteasome. Proteasomes recognize and degrade ubiquinated proteins (Fig. 3.29). Ubiquitin is a 76-amino- acid protein that after conjugation to the protein, targets it to the proteasome. In ATP-dependent steps, the C-terminus of ubiquitin is covalently attached to a lysine residue in the protein. Polyubiquitination then takes place. The proteasome degrades the protein to peptides, and released ubiquitin molecules are recycling.

Major Events in the Cell Cycle The cell cycle proceeds via four phases in cycling (replicating) somatic cells. These phases are designated the G 1, S, G 2, and M phases (Fig. 19.1). In G 1 phase, cells synthesize many of the proteins that will be used for DNA synthesis and chromosome replication during S phase. G 2 follows S and is a transitional period preceding M phase. M phase is a multistage period wherein chromosomes separate and the cell divides. In a dividing mammalian cell, the four phases of the cell cycle typically require 9 h, 10 h, 4.5 h, and 30 min respectively. Many cells in adult multicellular organisms do not proliferate and never, or at least rarely, divide. These cells exit the cell cycle in G 1 phase and enter a quiescent phase called G 0.

Review of M Phase Processes (I) From an ultrastructural standpoint, M phase processes are the most complex. In comparison, few changes are visibly apparent in most cells during interphase, which consists of the combined G 1, S, and G 2 phases. M phase is subdivided into 4 main periods-- prophase, metaphase, anaphase, and telophase (Fig ). In prophase, replicated chromosomes condense and become visible. In prometaphase, the nuclear membrane retracts and the mitotic apparatus known as the spindle forms. Kinetochores assemble at centromeres and attach the chromosomes to the mitotic spindle fibers. In metaphase, chromosomes line up on the metaphase plate in the center of the spindle.

Review of M Phase Processes (II) In anaphase, sister chromatids of each duplicated chromosome separate and are drawn toward the two spindle poles. Then in telophase, the mitotic spindle disassembles, chromosomes decondense, the nuclear envelope reforms surrounding the chromosomes, and the cell undergoes cytokinesis--the physical division of the cytoplasm.

Mechanism of Cell Cycle Regulation (I) The molecular mechanisms by which the cell cycle is controlled in a typical eukaryotic cell is presented in Fig below. The initiation of the cell cycle occurs with the receipt of a signal (e.g., a growth factor ligand) by a cell in G 0 or G 1. The signal induces synthesis of G 1 and G 1 /S phase cyclin-CDKs, which then activate transcription of genes encoding DNA synthesis enzymes and S phase cyclin-CDKs. S phase cyclin- CDKs initially are held in check by inhibitors until G 1 /S phase cyclin-CDKs phosphorylate the inhibitors. This triggers their polyubiquitination by SCF ubiquitin ligase and degradation by proteasomes. The released S phase cyclin-CDKs then phosphorylate regulatory proteins bound to chromosomal replication origins, promoting initiation of DNA synthesis. The synthesis of mitotic cyclin-CDKs increases in S and G 2 phases. The activities of these complexes initially are blocked by phosphorylation of CDK subunits, and then are activated later by dephosphorylation. Once activated, mitotic cyclin-CDKs phosphorylate a large number of proteins that control chromosome condensation, retraction of the nuclear envelop, formation of the mitotic spindle, and alignment of chromosomes at the metaphase plate.

Subsequently, the anaphase promoting complex (APC/C), another ubiquitin ligase, polyubiquitinates a protein called securin which helps hold the sister chromatids of metaphase chromosomes together. The degradation of securin by proteasomes initiates anaphase and sister chromatids separate. Later in anaphase, APC/C polyubiquitinates mitotic cyclins leading to their degradation. Due to the loss of mitotic cyclin-CDK kinase activity proteins responsible for chromosomal condensation, etc. are dephosphorylated. Chromosomes then decondense, and nuclear membranes are re-synthesized. Cells next move forward into telophase where cytokinesis occurs, completing the cell cycle. In the ensuing G 1 phase, replication origin regulators are synthesized and pre-replication complexes assemble at origins. This prepares cells for another round of DNA synthesis in the next S phase. Due to degradation of regulatory proteins at the G 1 /S, metaphase/anaphase, and anaphase/telophase boundaries, the passage of cells through the cell cycle is irreversible. The G 1 /S transition (“START”) is a major checkpoint after which passage through the cycle becomes independent of mitogens (e.g., growth factors). Mechanism of Cell Cycle Regulation (II)

Mechanism of Cell Cycle Regulation (III)

APC/C Regulation of Sister Chromatid Separation Metaphase chromosomes are held together at centromeres via ring-like proteins called cohesins (Fig b, left). Once spindle-assembly checkpoint processes have been satisfied (see below), a protein called Cdc20 triggers sister chromatid separation (Fig right). Cdc20 activates the APC/C ubiquitin ligase which polyubiquitinates a protein called securin which is an inhibitor of the enzyme called separase. Once securin is degraded by proteasomes, separase cleaves the Scc1 component of cohesins resulting in their disassembly and separation of sister chromatids to the spindle poles.

Regulation of Initiation of DNA Replication by S phase Cyclin-CDKs (I) In eukaryotic cells, DNA synthesis occurs simultaneously at multiple replication origins which initiate DNA synthesis only once per cell cycle. This ensures that the number of chromosomes per cell is correctly maintained. At the end of M phase when all M phase cyclins are degraded, the dephosphorylated forms of MCM helicases and two initiation factors assemble along with the ORC (origin recognition complex) at replication origins (Step 1, Fig ). Then when S phase cyclin-CDKs are activated at the end of G 1, S phase cyclin-CDKs and the DDK kinase phosphorylate MCM helicases and the two initiation factors (Step 2). Phosphorylation causes ORC and the two factors to disassemble.

Regulation of Initiation of DNA Replication by S phase Cyclin-CDKs (II) S-phase cyclin-CDKs also phosphorylate MCM helicase activators (red) (Step 2). Subsequently, origins are unwound by active MCM helicases, DNA polymerases load onto the origins, and bidirectional DNA synthesis ensues (Step 3). The phosphorylated forms of initiation factors cannot rebind DNA at origins, and they are degraded by the SCF proteasome. Only after S phase and mitotic cyclin-CDKs are degraded at the end of mitosis can the initiation factors be synthesized and accumulate in their dephosphorylated states, and then assemble again at replication origins. This ensures that DNA replication occurs only once per cell cycle.

Mitogen-stimulated Gene Expression in G 0 -arrested Mammalian Cells Cells in G 0 do not synthesize cyclins or CDKs. The transition of quiescent cells from G 0 to G 1 and resumption of the cell cycle is triggered by growth factors in serum (mitogens). Shortly after binding to receptors, growth factors turn on the transcription of early response genes using TFs that preexist in the cell. Among the early response genes are c- fos, c-jun, and c-myc These genes turn on the transcription of delayed-response genes. Included within the latter are the G 1 cyclin-CDKs and a TF called E2F, which is controlled by the Rb gene (next slide). The synthesis of G 1 cyclin-CDKs propels the cell into G 1. Prior to the START point, the withdrawal of growth factors leads to rapid degradation of G 1 cyclin-CDKs and return to G 0. At the restriction point, G 1 cyclin-CDKs reach irreversibly high levels and cells are committed to enter S phase. After the restriction point, growth factors are no longer needed for completion of the cycle. One role of TGFß is inhibition of G 1 cyclin-CDKs.

Rb and the START Point Rb is the prototype tumor suppressor gene. Inactivation of Rb leads to tumors of the retina in children. Rb also is inactivated in many other tumors. In non-proliferating cells, Rb protein binds to E2F, and the complex activates histone deacetylases leading to gene silencing (Fig b). When the expression of the G 1 cyclin-CDKs (cyclin D-CDK4/6) are turned on by a mitogen, Rb is phosphorylated and active E2F is released. E2F activates transcription of genes needed for passage into S phase, namely genes encoding DNA synthesis enzymes, Cyclins E & A (G 1 /S phase cyclins), CDK2, and itself. Cyclins E/A-CDK2 (G 1 /S cyclin-CDKs) also phosphorylate Rb. This occurs even if the mitogen is withdrawn and is the key control allowing the cell to pass through the restriction point. In S, G 2, and mitosis, S-phase and mitotic cyclin- CDKs continue to phosphorylate Rb. Only after degradation of mitotic cyclins at the end of mitosis is Rb dephosphorylated. Rb then can inhibit E2F in early G 1 and in G 0 - arrested cells.

Checkpoints in Cell-cycle Regulation To minimize mistakes in cell cycle events and transmission of damaged DNA or otherwise abnormal chromosomes to daughter cells, numerous quality control checkpoints regulate passage of cells through the cell cycle. For example, DNA-damage checkpoints occur at several steps (Fig ). If damage is detected, the cell cycle is arrested and the damage repaired, if possible. Severe DNA damage may trigger apoptosis (Chap. 21).

The Spindle-assembly Checkpoint In nondisjunction, chromosomes segregate in anaphase prior to attachment of the kinetochores of all sister chromatids to mitotic spindle fibers. This results in unequal segregation of chromosomes to daughter cells (below left). In trisomy 21, nondisjunction occurs 95% of the time in meiosis I during gametogenesis in the mother. To prevent nondisjunction, a regulatory mechanism involving the Mad2 protein which is known as the spindle-assembly checkpoint operates just prior to anaphase (Fig ). Mad2 binds to kinetochores that have not bound to microtubules of the mitotic spindle. Kinetochore binding activates Mad2, and it in turn inhibits the activity of Cdc20 which controls the APC/C ubiquitin ligase (Fig ). This delays degradation of securin and anaphase. Only after all kinetochores have bound to the spindle is Mad2 inactivated, releasing Cdc20 to trigger securin degradation.