Cell Cycle Regulation PDF

PHA3110 – Molecular Biology, Q3 - 2025 Ellie Nguyen, PhD CELL CYCLE REGULATION  Learning objectives I. Describe the role of specific cyclin dependent kinases in the cell cycle II. Describe the activation and inactivation of Cdks III. Describe how...

PHA3110 – Molecular Biology, Q3 - 2025 Ellie Nguyen, PhD CELL CYCLE REGULATION  Learning objectives I. Describe the role of specific cyclin dependent kinases in the cell cycle II. Describe the activation and inactivation of Cdks III. Describe how DNA damage impacts the cell cycle IV. Describe the roles of tumor suppressor genes Rb and p53 on cell cycle V. Identify drugs targeting Cdk4/6  Cell Cycle Control Organ and body size are determined by three fundamental processes: cell growth, cell division, and cell survival, which are regulated by cell cycle control mechanism Each process above is tightly regulated — both by interactions between intracellular programs and by extracellular signal molecules. The extracellular signal molecules can be divided operationally into three major classes: mitogens – stimulate the rate of cell division; growth factors and survival factors. The intracellular programs typically includes checkpoints, Cyclins & Cdk, APC, Cdk inhibitors and tumor suppressor proteins.  are activated or inactivated in response to extracellular signal molecules  Cell Cycle checkpoints Checkpoints: points during the cell cycle at which the cell examines internal and external environmental conditions and "decides" whether or not to proceed with the cell cycle.  Once a cell passes through, the cell is committed to the next cell cycle event in a complete and irreversible manner.  Cell cycle arrest occurs at specific checkpoints (regulatory transitions) if cell does not pass. There are 3 major checkpoints  G1 check point: end of G1 phase  G2 check point: end of G1 phase  Metaphase (M) check point: middle of M phase The DNA damage checkpoint is primarily active in G1 and G2 phases  Repair DNA damage in G1 and G2 phase  Halt progression through G1 to S, and G2 to M phases Bruce Alberts et al., Molecular Biology of The Cell, 6e,  Cell cycle regulatory proteins Regulatory proteins for cell cycle are highly conserved proteins and similar in all eukaryotes  Correct sequential order of events at a specific time  One of each event per cell cycle, with complete, irreversible events The central intracellular components to regulate cell cycle is the enzyme Cyclin dependent kinases (Cdks) with changing cyclinical activity Bruce Alberts et al., Molecular  Cdks alone are inactive and require binding to cyclins for activation. Biology of The Cell, 6e, © 2014  Each Cdk-cyclin complex triggers specific cell cycle events. Cdks cause cyclical changes by phosphorylation of targeted proteins  Changes in targeted protein activity  Initiation and termination of cell cycle events  Cell Cycle Regulatory Proteins Cyclical activity of Cdks regulated by: 1. Activated by cyclins Cyclins bind to Cdks, activating them at specific phases of the cell cycle. Cyclin levels fluctuate, while Cdk levels remain relatively constant. 2. Activated by Cdk activating kinase (CAK) Phosphorylates cyclin bound Cdk to activate it 3. Inactivated by Cdk inhibitor proteins (CKI) Molecular Biology of the Cell (© Garland Science 2008) Interferes with the binding of cyclins to Cdk More important for regulating G1/S-Cdk and S-  Cdks and Cyclins Cyclins have no kinase activity and Cdks are inactive without tightly bound cyclin Cyclin – levels change throughout the cycle Each Cdk-cyclin complex triggers specific cell cycle events Once a cyclin completes its role, it is degraded by the ubiquitin-proteasome system Molecular Biology of the Cell (© Garland Science 2008)  APC for cyclin degradation Anaphase promoting complex (APC) is a proteolytic complex  Ubiquitin ligase – polyubiquitinate proteins for degradation  Destruction of cyclins especially M- and S- cyclins Activation of the anaphase promoting complex (APC) which catalyzes:  Destruction of cohesion in sister chromatids in anaphase of mitosis  Inactivation of mitotic cyclin dependent kinase (M-Cdk) Molecular Biology of the Cell (© Garland Science 2008)  Activation of Cdks by Cyclins and CAKs Cyclins are activated by Cdk activating kinase(CAK) CAK phosphorylation is required for optimal activity of Cdk. CAK can only phosphorylate cyclin bound Cdk The active site of an unbound Cdk is obscured by a protein loop. Binding of a cyclin exposes the active site by moving the protein loop, partially activating the protein. Phosphorylation of the active site by CAK results in Bruce Alberts et al., Molecular Biology of The Cell, 6e, full activation of Cdk © 2014  Inactivation of Cdks by CKIs Cyclins are inactivated by Cdk inhibitor proteins (CKI) to prevent unchecked cell division The CKI such as p27 that bind to both the cyclin and the Cdk and distorts the Cdk active site rendering it inactive Loss or mutation of CKI contributes to cancer and cell cycle disorders. Molecular Biology of the Cell (© Garland Science 2008)  Types of Cyclins G1-cyclin (D) – binds Cdk to create the complex G1 – Cdk or Cdk4/6, which helps regulate the activities of G1/S cyclins. Levels fall after metaphase. S-cyclin (A)- binds Cdk after restriction point to create the complex S–Cdk, which is required for initiation of DNA replication. Levels fall in late G2-phase. M-cyclin (B) – binds Cdk to create the complex M–Cdk, which promotes mitosis, specifically prophase to metaphase. Levels fall after metaphase G1/S cyclin (E)- binds Cdk in G1 phase to create the complex G1/S–Cdk, which drives cell through restriction point to commit to S-phase. Levels fall in S-phase.  +G1/S-cyclin (E) +S-cyclin (A) +M-cyclin (B) Molecular Biology of the Cell (© Garland Science 2008)  Practice Questions 1. Which regulatory protein inhibits activity of Cdks? 2. What is the biochemical effect of anaphase promoting complex (APC) on cyclins? 3. What type of cyclin binds to M-Cdk?  S-Cdk Initiates DNA replication  Activates the protein assembly needed for DNA replication  Stimulates histones biosynthesis Simultaneously prevent re-replication  Once replication has started, it causes breakdown of the replication assembly  Also causes export of components from the nucleus +S-cyclin (A)  Ensures one DNA replication per cell cycle Molecular Biology of the Cell (© Garland Science 2008)  Levels remain high during G2 and early mitosis  M-Cdk M-Cdk is activated at the end of the G2 phase  Increase in M-cyclin expression during G2 phase  Accumulates during G2 phase  Triggers events from Prophase to Metaphase DNA replication checkpoint (G2/M- transition)  Check and repair unreplicated DNA/incomplete replication/DNA damage  M-Cdk drives mitotic entry, but the DNA checkpoint keeps it inactive until DNA replication and DNA repair are complete +M-cyclin (B) Bruce Alberts et al., Molecular Biology of The Cell, 6e,  M-Cdk Spindle-attachment/assembly checkpoint  A critical cell cycle checkpoint that ensures accurate chromosome alignment and segregation during mitosis (M) phase. Damaged spindles or improper chromosome attachment to spindle will:  Blocks activation of APC to prevent inactivation of M-Cdk  Sister chromatids are not separated  Metaphase arrest +M-cyclin (B) Molecular Biology of the Cell (© Garland Science 2008)  M-Cdk Stimulates APC activity  Stimulates Cdc20, an activating subunit of APC that is stimulated by M-Cdk Activation of the APC  Activated APC inactivates M-Cdk, S-Cdk and other proteins at the end of metaphase  Causes dephosphorylation of M-Cdk and S-Cdk targets  Causes metaphase to anaphase transition  Leads to separation of the sister chromatids (triggers +M-cyclin (B) anaphase)  Loss of most Cdk activity in anaphase Molecular Biology of the Cell (© Garland Science 2008)  Cdk At the end of mitosis, Cdk activity is almost zero because of inhibition of cyclin gene transcription  APC levels remain high to degrade M-cyclin (B) and M-Cdk subsequently  Increased expression of CKIs, such as p27  Cdk inactivation triggers chromosome decondensation and nuclear envelope reformation.  Ensuring proper mitotic exit and transition into G1 phase Molecular Biology of the Cell (© Garland Science 2008)  Retinoblastoma in G1 Rb (Retinoblastoma) serves as a brake in G1 cells Rb = tumor suppressor gene  Inhibits cell cycle progression  Inhibits transcription of S-phase genes and keeps cells in G1  Inactivated by G1-Cdk  Must be inactivated for cells to progress to S-phase  Rb mutation causes cancer Molecular Biology of the Cell (© Garland Science 2008)  G1-Cdk G1-Cdk  Inactivates Rb protein by phosphorylation  Leads to increase gene expression of proteins including G1/S cyclins and S-cyclins  Cell progression to S-phase Molecular Biology of the Cell (© Garland Science 2008) G1-Cdk is activated by favorable extracellular signals, oncogenes and growth factors which cause an increase in G1-cyclin expression and consequently increased G1-Cdk activity  p53 DNA damage in G1  Inhibits G1/S-Cdk and S-Cdk  Prevents cells from crossing the restriction point as a DNA damage checkpoint DNA damage activates the tumor suppressor gene p53: At least 50% of all  Stimulates gene transcription including CKIs such as p21 human cancers  p21 binds and inhibits G1/S-Cdk and S-Cdk have p53 mutations  Blocks entry to the S-phase Molecular Biology of the Cell (© Garland Science 2008)  G1-Cdk Under favorable conditions, G1- Cdk is activated. G1-Cdk - inactivates Rb, thereby relieving DNA damage in G1 the inhibitory effect of Rb- allowing progression to S-phase Retinoblastoma (Rb) + Tumor suppressor protein Suppresses the transcription of G1/S cyclin p53 and S-cyclin, thereby - - preventing progression to S- phase + S- G1/S-Cyclin P21 (Cdk Inhibitory Cyclin protein) + + Cdk Cdk - - G1/S-Cdk S-Cdk The G1/S Cdk and the S-Cdk are essential for transitioning from the G1- phase to the S-phase as well as ensuring proper DNA replication during the Progression to S- S-phase. Phase  CdK4/6 (G1-CdK) Inhibitors Cdk4/6 are overexpressed in some hormone dependent breast cancer Palbociclib (Ibrance®), ribociclib (Kisqali®), and abemaciclib (Verzenio®) are Cdk4/6 inhibitor drugs  FDA-approved for treatment of breast cancer  Disrupt the mitogenic and hormone signals driving cell proliferation  Induce cytostatic (G1-cell cycle arrest) to slow down or stop the growth of cancer cells  Prevents cells from transitioning from G1 to S phase https://www.sciencedirect.com/science/article/pii/S2468294220300125?via%3Dihub Summary Of Cell Cycle Control  Summary of Cell Cycle Regulation Retinoblastoma – tumor suppressor gene that keeps cell in G1 Cyclins – regulates the activity of cdks, undergo a cycle of expression and degradation M-Cdk – responsible for the events from prophase to metaphase S-Cdk – stimulates DNA replication G1/S-Cdk – controls the cell transition from the G1 to the S- phase, inactivated by retinoblastoma G1-Cdk – inactivates retinoblastoma and activated by growth factors APC – a ubiquitin ligase. It inactivates M-Cdk, breaks down cohesion, and triggers anaphase p53 – detect presence of DNA damage and stimulates expression of p21 p21 & p27 – a cyclin dependent kinase inhibitor (CKI) that inhibits S-Cdk and G1/S-Cdk Cdk4/6 inhibitors - are approved for treatment of breast cancer  Practice Questions 1. Which Cdk initiates DNA replication? 2. Which one serves as tumor suppressor gene? 3. What type of cancer treatment is approved by FDA to use Cdk4/6 inhibitors?