BIO 302 Cell Biology Lectures 2019 PDF

BIO 302 Cell Biology Cell Communication and Signal Transduction Pathway Text Chapter 15 edelbrock Cell Communication - Objectives ◼ Explain how cells communicate with each other and the environment ◼ Explain how a signal is sent through the cell to re...

BIO 302 Cell Biology Cell Communication and Signal Transduction Pathway Text Chapter 15 edelbrock Cell Communication - Objectives ◼ Explain how cells communicate with each other and the environment ◼ Explain how a signal is sent through the cell to regulate function ◼ Define quorum sensing in bacteria. ◼ Explain the general steps in signal transduction ◼ Explain the proteins involved in different types of cellular signaling Signaling Types ◼ External signal from environment ◼ Response to stimuli, temp, light, pressure ◼ Response to foreign cells, bacteria, virus ◼ Response to chemical or antigen ◼ External signal from the organism ◼ Hormones ◼ Cell-cell contact ◼ Internal signal from the cell ◼ Autocrine ◼ Signal cascades through cell  Bacteria – 2 Component System ◼ All bacteria have 2-component signaling systems ◼ Sensor – Histidine protein kinase ◼ Effector – Response regulator Aspartic acid protein kinase ◼ Kinases – protein which can transfer (add) a phosphate anion to another protein ◼ Addition of phosphate anion to a protein changes its conformation and makes it active or inactive (phosphorylation) ◼ Phosphatases – proteins which remove phosphate from another protein  Bacteria – 2 Component System Steps ◼ Receive Input stimulus (osmolarity, pH, temp., antibiotics, etc.). ◼ Autophosphorylation of Sensor (histidine kinase). ◼ Transfer of phosphate to Response Regulator (aspartic acid). ◼ Response – Gene regulation (turn on or off). ◼ Dephosphorylation of effector (turn off signal). ◼ Phosphorylated aspartate is unstable ADP ATP ATP ADP 1 P P His Asp 2 P P His Asp 3 P P 4 His Asp Asp His  Prokaryotic Communication ◼ In order for bacteria to effectively invade the host tissue it may need to communicate with other bacteria. ◼ Example: in order for bacteria to gain a foothold in the host it may need to produce an enzyme that will breakdown host defense proteins. One bacteria can not produce enough enzyme to do the job. So the bacteria must know how many bacteria are present to ensure the enzyme will be effective against the host.  Bacteria Quorum Sensing ◼ Cell density dependent response: ◼ Signal to make sure enough bacteria is present ◼ Bacteria sends out an autoinducer (AI) ◼ The AI migrates back into the bacteria ◼ AI concentration is diluted if not many bacteria around so chances of activating are low ◼ As bacteria grows and colony density increases each bacteria is sending out AI ◼ Concentration of AI in the area of infection increases until a trigger point is reached ◼ At the trigger point all of the bacteria will turn on a gene that produces a response and attacks the host. (Example LasA in opportunistic bacteria in a cystic fibrosis patient)  Quorum Sensing AI AI A Bacterial Colony Gene A Gene B The bacteria transcribes Gene A which makes protein A. Protein A creates and releases AI into the host environment. Other bacteria are doing the same thing. As the bacterial colony density increases the concentration of AI increases and diffuses back into the bacteria.  Quorum Sensing Eventually the concentration of AI is high and migrates back into all of the bacteria. AI binds to regulatory sequences and turns on Gene A to produce more AI (positive feedback) and turns on B to do damage. Gene B only works effectively if released in large quantities by all the bacteria at once. AI AI AI AI AI A AI AI AI AI Gene A Gene B Enzyme at high concentration Enzyme attacks host tissue Prokaryotic Cell Communication ◼ Bacteria use 2 component signaling and Quorum Sensing in an integrated fashion. ◼ Component systems often have several relay points ◼ There are many signals coming into the bacteria at the same time (pH changes, temperature, host immune response) ◼ All of these signals must be integrated into the appropriate response for the bacteria to survive.  Host-Pathogen Relationship ◼ Hosts and pathogens are in a constant evolutionary state. As one evolves to survive it puts selective pressure on the other to evolve and survive. ◼ A bacteria may evolve to enter the host cell better. - The host will respond and evolve to prevent entrance. ◼ The host may evolve to recognize the bacteria faster. - The bacteria may evolve to disguise itself and avoid recognition. ◼ The cycle continues – constant evolutionary pressure ◼ This involves constant communication between the host’s cells and the pathogen and the environment.  Host-Pathogen Communication Bacterial Quorum sensing may communicate with Host system through hormone recognition. Conversely, host may sense bacteria through hormone receptors. Good or Bad News?  Eukaryotic Signal Transduction ◼ General Steps ◼ Synthesis of signaling molecule ◼ Release of signaling molecule by the signaling cell ◼ Transport of signal to target cell ◼ Interaction of signal with target cell ◼ Membrane level ◼ Cytosol ◼ Nucleus ◼ Change in cell behavior ◼ Up or down gene regulation ◼ Removal of signal from target cell  Eukaryotic Signal Transduction Properties ◼ Fast response – Organism must respond rapidly to changes in internal or external environment e.g. fight or flight. General Statements: ◼ Mediated through peptide hormones and catecholamines ◼ Fully active stored in vesicles - have a 1 day or more supply ◼ Short half-life (seconds - minutes) ◼ Released by exocytosis (rapid vs diffusion) ◼ Release to interstitial fluid or blood stream is immediate ◼ Signal travels short distance to target cell ◼ Membrane receptor at target cell ◼ Mechanism at target cell is fast also  Eukaryotic Signal Transduction Properties ◼ Slow response – Organism does not need a rapid response e.g. growth process ◼ Mediated through steroid and growth hormones ◼ Synthesis is slow ◼ Stored in an inactive precurser form ◼ Released primarily by diffusion ◼ Signal travels longer distance to target cell ◼ Travel is mediated by carrier proteins ◼ Slow degradation halflife (hours – days) ◼ Nuclear or cytosolic receptor ◼ Mechanism through gene action slow  Eukaryotic Cell to Cell Signal Transduction ◼ Three Primary Mechanisms ◼ Contact – The signaling cell directly contacts the target cell. ◼ Major mechanism of immune system ◼ Gap Junctions – The signaling molecule is transferred through specialized cell-cell channels (connexons) ◼ Secretion – release of signals into interstitial fluid or bloodstream  Eukaryotic Signal Transduction ◼ Types of Secretion ◼ Endocrine – The signaling molecule is from a cell that is remote in location from the target cell. ◼ Hormones, insulin, growth factors ◼ Paracrine – The signaling molecule is produced by a cell that is adjacent to the target cell. ◼ Neurotransmitters, immune response ◼ Autocrine – The target cell produces its own signaling molecule. ◼ Growth factors, immune response ◼ Community effect – similar to Quorum Sensing in prokaryotes Eukaryotic Signaling  Exocytosis ◼ Process by which vesicles fuse with plasma membrane and release proteins or signal molecules ◼ Vesicles formed in the trans-Golgi ◼ Two secretory pathways: ◼ Constitutive – continuously producing proteins, lipids, etc for normal function and maintenance ◼ Regulated – specialized process of secretory cells that selected proteins and molecules are secreted through clathrin coated vesicles Exocytosis  Signal Transduction ◼ Various types of signal transduction pathways are present in higher organisms. ◼ G-Protein Coupled Receptors ◼ Phosphotidylinositol (lipid) ◼ Receptor Tyrosine-Linked Kinases ◼ Receptors with intrinsic enzymatic activity ◼ Tyrosine kinase receptors ◼ Receptor tyrosine phosphatases ◼ Receptor serine/threonine kinases ◼ Receptor guanylate cyclases ◼ Nuclear Receptors ◼ Apoptosis  Signal Transduction and Secondary Messengers ◼ Signaling cascades involve a relay of protein activation usually by phosphorylation ◼ MAP (mitogen-activating protein) kinase cascade ◼ Many signaling cascades involve secondary messengers ◼ cAMP – cyclic AMP ◼ Lipids - DAG diacylgylerol ◼ Calcium NOTE ◼ In the following slides there will be discussion on ligands, proteins and the receptor. Keep the terminology clear between the Receptors and the Ligand or protein that interacts with the receptor. ◼ Keep the G-Protein terms separate from the Receptor terms G-Protein Coupled Receptors ◼ G-Protein coupled receptors are membrane bound receptors that: ◼ Link G-Proteins to outside signals ◼ Have ligand binding domains on the ectoplasmic face ◼ Have cytosolic domains that activate G- proteins ◼ Examples of downstream effectors include: ◼ Beta-Adrenergic Receptor ◼ Rhodopsin  G-Protein Coupled Receptors Seven membrane-spanning regions N-terminal segment on the exoplasmic face C-terminal segment on the cytosolic face of the plasma membrane G-protein coupled receptor family includes: light-activated receptors (rhodopsins) odorant receptors in the mammalian nose receptors for various hormones neurotransmitters (GABA) G-Proteins ◼ G-Proteins are proteins that ◼ Bind to GTP and GDP ◼ Respond rapidly ◼ Act as on/off switches ◼ Have an active (GTP bound) conformation ◼ Have an inactive (GDP) conformation ◼ Activate downstream proteins ◼ Interact with membrane receptors G-Protein Coupled Receptor Membrane anchored G-protein bound to GDP in an inactive form. Ligand binds to receptor and allows G-protein to interact with the receptor GTP exchanges for GDP on alpha subunit Active (GTP form) Alpha subunit dissociates from Beta-Gamma subunit and finds effector and binds to it G-Protein Coupled Receptor Effector action occurs in this case formation of cAMP as a secondary messenger Note: Arrestin may bind to inhibit further signal tranduction. GTP is hydrolyzed to GDP and the Alpha complex dissociates from the effector protein. Inactive heterotrimeric G-protein reforms and is ready for next signal http://www.youtube.com/watch?v=V_0EcUr_txk&feature=related  G-Protein Signaling 1. Ligand binds to G-Protein Coupled Receptor (GPCR) (Example: Beta Adenergic Receptor) 2. Receptor changes conformation and attracts G-protein to cytosolic domain 3. Upon binding of the G-protein to the GPCR, GDP is exchanged for GTP which activates the G-Protein 4. The Ga subunit dissociates from the Gβγ subunits and engages the downstream effector protein (Example: Adenylyl Cyclase) 5. The downstream effector signals the response through secondary messengers (Example: cAMP)  G-Protein Signaling 6. The GTP hydrolyzes to GDP and the Ga leaves the effector 7. The G-protein reforms in its inactive conformation with GDP and the alpha, beta and gamma subunits. 8. Additional secondary systems may be present 1. Kinase of GPCR and binding of arrest protein 2. Signal transduction of G-Protein Gβγ subunits through independent pathway 3. Secondary messengers GPCR Control Many Cellular Responses  Phophatidyinositol Signaling 1 1 2 4 PKC 3 1. Phosphtidylinositol is phosphorylated 2x to produce PI(4,5)P2 2. PI specific phospholipase cleaves the 3 phosphorylated inositol from the lipid producing 2 secondary messengers: Cytosolic Inositol Triphosphate Smooth ER Membrane bound Diacyl glycerol 3. IP3 is a ligand for a Calcium channel on the smooth ER, causing Ca2+ release 4. DAG can activate Protein kinase C which is a serine- threonine kinase Enzymatic Activity ◼ Receptor Tyrosine Kinases ◼ Receptors which can auto-phosphorylate and pass signal to cell effector ◼ Receptor Tyrosine-Linked Kinases ◼ Receptors which lack kinase activity. These receptors rely on another molecule to phosphorylate and pass signal to cell effector. Hence these receptors are Linked to a Tyrosine Kinase. ◼ We will not discuss RTLK see text for more detail if interested Intrinsic Enzymatic Activity ◼ Receptor Tyrosine Kinases (RTK) ◼ Transmembrane protein receptor ◼ Ligand binding domain – ectoplasmic face ◼ Activating domain – cytosolic face ◼ Auto-phosphorylation of receptor ◼ Transfer of phosphate to substrate (kinase activity) ◼ Cascade effect of kinases ◼ Results in interaction of downstream proteins ◼ Example: Growth Factor Receptor  Receptor Tyrosine Kinases ◼ RTK pathways are involved in gene regulation ◼ Cell proliferation ◼ Cell differentiation ◼ Promotion of cell survival ◼ Modulation of cellular metabolism ◼ Example Downstream Effector: RTKs transmit a growth signal to Ras, a GTPase switch protein that passes on the signal on to downstream components  Receptor Tyrosine Kinases L L R R R R ATP P P P P P Ligand binds to RTK, Dimer RTK, ADP receptor dimerizes Multiple Tyr PO4 sites autophosphorylates P P P P P P P P BP BP PO Binding protein activate cascade Activated Substrate protein HO carries signal to downstream Substrate protein inactive target  Receptor Tyrosine Kinases ◼ Signal Transduction ◼ Ligand binding and dimerization of receptor tyrosine kinase ◼ Different dimerization combinations possible ◼ Autophosphorylation of receptor ◼ Phosphorylation of substrate with or without binding protein ◼ Multiple binding proteins can build a protein scaffold allowing a specific signal cascade ◼ Activation of signal cascade  Effector: Activation of Ras ◼ Ras is a protooncogene that mediates mitogenic stimulus of growth hormones ◼ Ras is a membrane anchored protein ◼ Inactive Ras is bound to GDP ◼ Activated Ras is bound to GTP ◼ Active Ras activates Raf ◼ Raf activates the MAP kinase pathway leading to cell proliferation ◼ Ras is mediated by Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs)  Activation of Ras 1. Guanine Exchange Factor (GEF) replaces GDP with GTP to activate Ras – On Switch * Exchange can be inhibited by a Guanine dissociation inhibitors (GDI) 2. Activated Ras signals mitogenic response (MAP kinase active) 3. GTPase Activating Protein (GAP) enables Ras to hydrolyze GTP to GDP – Ras Inactive Over 30% of human cancer involves 4. Ras-GDP (Off Switch) a mutation in Ras proteins which Inactive Ras-GDP is ready for activate the MAP kinase pathway. the next signaling event Raf→MAP Cell Growth MAP Kinase Pathway Mitogen activated protein (MAP) kinase cascade involves a signal cascade through several Protein Tyrosine Kinase. Often the signal is iniatated by Ras. Ras then activates Ras-Activated Factor (Raf) and a multi-step phosphorylation cascade occurs between the downstream protein kinases. Eventually, a transcription factor is activated and induces gene expression. A MAP kinase phosphatase can be produced to down regulate the growth response  Nuclear Receptors ◼ Type 1 – Nuclear Receptor ◼ Bound in an inactive form in the cytoplasm or nucleus with Heat Shock Proteins (HSP) ◼ Ligand dependent ◼ Steroids, estrogen, progesterone, glucocorticoid receptors, etc. ◼ Type 2 - Nuclear Receptors – ◼ Bound to the gene and is repressed ◼ Ligand dependent ◼ Thyroid hormones, Retinoids, Vitamin D ◼ Orphan receptors – no identified ligand Receptor Domain ◼ DNA binding Domain – highly conserved ◼ Ligand Binding Domain –gives specificity to the ligand ◼ Allows homo or hetero dimerization ◼ Homodimerization ER with ER (steroids) ◼ Heterodimerization VDR with RAR ◼ N-Terminal Domain – variable, contains phosphorylation sites ◼ Activating Function – activates co- activators or co-repressors  Type I - Nuclear Receptors ◼ Binding of steroid with receptor ◼ Conformational changes and release from carrier ◼ Translocation to nucleus ◼ Dimerization of receptor/ligand complex ◼ DNA Interaction ◼ Gene Transcription Note: Some steroids translocate to the nucleus and then bind with receptors, Difference between the 2 models is where the binding takes place (cytoplasm or nucleus)  Nuclear Receptors – Steroid #1 HSP HSP Nucleus Steroid Dissociation Translocation HSP S HSP Dimerization R R S DNA binding Receptor Complex Binding Coactivator Recruitment CoAct Gene Transcription Response Element Cytoplasm  Nuclear Receptors – Steroid #2 HSP HSP Nucleus Steroid Dissociation S HSP Dimerization HSP R S R Translocation DNA binding Receptor Complex Binding CoAct Gene Transcription Coactivator Recruitment Response Element Cytoplasm  Selective Estrogen Response Modulators SERMS – Selective Estrogen Receptor Modulators: Raloxifene Hydrochloride –Agonist in bone tissue for Osteoporosis Tamoxifen – Antagonist for breast cancer treatment Osborne, C. K.; Zhao, H.; Fuqua, S. A. Selective estrogen receptor modulators: structure, function, and clinical use. J Clin Oncol 2000, 18, 3172-3186. Type II Receptors (Thyroid) ◼ Translocation to Nucleus ◼ Binding to receptors ◼ Remove corepressor signal ◼ Activate gene Transcription  Nuclear Receptors – Type II Inactive Repressed CoRep Retinoic Acid e.g. RA Nucleus Translocation CoRep Receptor Complex Binding Release of Co-Repressor HAT Coactivator Recruitment CoAct Gene Transcription Response Element Cytoplasm Apoptosis ◼ Signaling pathway that leads to cell death ◼ Controlled cell-death pathway ◼ Response to normal development - removal of webbing between fingers ◼ Response to bacterial or viral infection mediated through immune system ◼ Response to DNA damage and mutation  Apoptosis ◼ Apoptosis involves ◼ Initiation ◼ External through “Death Domain” receptors ◼ Internal through stress detection ◼ Targeting by an immune cell ◼ Activation of caspases (cystine/aspartic acid cleavage enzymes - proteases) ◼ Cell shrinkage ◼ Nuclear break-up ◼ Surface Bleb formation ◼ Engulfment by phagocytes The Mitochondria and Apoptosis 1. Internal cellular damage causes stress 2. Stress is sensed by the RER (affects protein synthesis) or by DNA damage sensing proteins 3. Signal sent to activate “BCL-2 proteins” Bcl-2 anti-apoptotic – survival signal Bax, Bad pro-apoptotic 4. Activation of Bax or Bad cause permeability of the mitochondrial membrane 5. Cytochrome c is released and serves as a messenger for Apoptotic Protease Activating Factor-1 to convert Procaspase 9 to act caspase 9 6. Caspase 9 in turn activates the major executioner caspase (caspase-3) that targets the nucleus Receptor-mediated Apoptosis Pathway 1. Tumor necrosis Factor bind to its receptor 2. Death domains are activated and recruit adaptor proteins (FADD and TRADD) 3. Adaptor proteins provide activation of Procaspase 8 into active caspase 8 enzyme 4. Caspase 8 cleaves Procaspase 3 to active caspase 3 5. Caspase 3 in the major executioner caspase that targets the nucleus 6. Caspase 8 can target the mitochondria Good Video: http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/rece ptors.htm 1465-9921-7-53-2 FADD and TRADD – death domain adaptor proteins Procaspases – inactive form of a caspase CAD –Caspase activated DNase ICAD – Inhibitor of CAD Bid – BCL2 interacting domain tBid – truncated BID Apaf – apoptotic protease activating factor http://www.youtube.com/watch?v=9KTDz-ZisZ0  Integration of Signals ◼ Just as in prokaryotes, eukaryotes must integrate various signals occurring at the same time. ◼ Convergence – multiple signals come to the same effector, cell must base response on combination, timing, and strength of these signals ◼ Divergence – The same ligand may signal through different pathways based upon tissue type, receptor makeup, other signals ◼ Crosstalk – different signaling cascades and pathways communicate with one another so multiple responses may occur from one signaling event causing multiple downstream events involving other pathways. BIO 302 Cell Biology Cellular Division and Cell Cycle Control Chapter 13, 14 Text Edelbrock  Cell Division and Cell Cycle Objectives ◼ Describe the cell cycle ◼ Identify the proteins involved in cellular division ◼ Describe interaction of proteins during cellular division ◼ Identify the cellular checkpoints during division ◼ Understand the cellular control of the division process ◼ Understand the fundamentals of cell cycle regulation ◼ Understand the experimental evidence for cell cycle control Cell Cycle Overview Cell Cycle Overview ◼ Interphase - Non Mitotic Events ◼ G1 – Normal growth and metabolism, ◼ S – Synthesis phase for DNA, chromosome duplication ◼ G2 – Cell growth prior to Mitosis, Organelles replicate ◼ Mitosis (M) – mitotic events ◼ Prophase, Prometaphase, metaphase, anaphase, telophase, cytokinesis  Cell Cycle Overview – Eukaryotic ◼ Different types of cells exhibit different propensities for cellular division: ◼ Inability to divide ◼ Nerve cells, Red Blood Cells ◼ Some ability under certain conditions ◼ liver after partial hepatectomy, cells referred to as being in Go (G zero) ◼ High ability to divide – ◼ Adult stem cells, epithelial cells, sperm cells ◼ Cancer cells (transformed cells), fibroblasts in culture Johnson & Rao, 1970 ◼ Hypothesis: There are cellular factors which are present during different phases of the cell cycle to control correct progression of events. ◼ Fusion of cells at different stages in the cell cycle may cause these factors to alter the other cell’s behavior ◼ Fuse Mitotic with Interphase Cells  Johnson & Rao G1 or G2 cell Mitotic Cell Fuse M Cell with a G1 or G2 cell. The nuclear membrane of G1 and G2 dissolves and the chromatin condenses.  Cell Fusion Results Cells Fused Results: Second Phase stops until other phase gets to that point. G1, S, or G2 Disappearance of nuclear +M membrane, chromosome condensation G1 G1 nuclei DNA begins to replicate +S G2 No DNA Synthesis in G2 nuclei, +S delayed entry into M  Johnson & Rao ◼ Conclusions: ◼ Cytoplasmic factors are involved in signaling and mediating cell cycle events ◼ Protein factors involved are diffusible Control of the Cell Cycle – 4 Protein Groups ◼ Cyclins – cytoplasmic proteins which regulate the cell cycle ◼ G1 Cyclins ◼ S-phase Cyclins ◼ M-phase Cylcins ◼ Cyclin Dependent Kinases (CDKs) ◼ G1 CDKs ◼ S-phase CDKs ◼ M-phase CDKs ◼ Proteins involved in Degrading cyclins ◼ Ubquitin ligases - Anaphase Promoting Complex ◼ Proteolytic enzymes (26S Proteasome complex) ◼ Inhibitory proteins  Models for Studying Cell Cycle ◼ Yeast – Used budding and mutant assays to identify Cell Cycle proteins. ◼ Oocytes - Starfish, sea urchins, and Xenopus laevis (African Frog) – Eggs are large enough to see and are synchronized after fertilization. ◼ Cultured Human Cells - Harder to work with but are growth factor specific. Oocytes – Sea Urcin Highly synchronized cells, many divisions before out of phase DNA Replicated Proteins expressed are those needed for replication and Cell Cycle Can extract proteins and identify Extracted proteins are in large quantity since cells are in synchrony same proteins are expressed  MPF – Maturation Promoting Factor progesterone fertilization Interphase Fertilized G2 Meiosis 1 Meiosis 2 Egg Prepare cytosol from M-2 and microinject into G2 egg – would G2 initiate Meiosis 1 Conclude that progesterone induces factors present in M-2 that cause cell division  Oocyte Experiment ◼ Meiosis used to study cell division process ◼ Progesterone stimulates M1 → M2 ◼ Cytoplasm from M2 injected into G2 eggs ◼ Cytoplasm induced G2 → M1 → M2 ◼ Repeat with 2nd and 3rd generation w/o progesterone stimulation to rule out that the progesterone was the cause ◼ Conclude that factors present in cytoplasm cause cell cycle progression ◼ They called this the Maturation Promoting Factor, but didn’t know what it was.  Cell Division Cycle Mutants - Yeast ◼ Separate Researchers were working on the same problem using yeast as a model: ◼ Create mutants from yeast cells, plate and grow colonies ◼ Look for temperature sensitive mutants ◼ Yeast will grow at 250C but not at 350C ◼ Grow colony of mutant and shift temperature ◼ If colony is all in the same growth arrest – assume that the mutation disrupted the cell cycle  Cell Division Cycle Mutants 35OC Replicate Plating Ts mutant 25OC Culture mutant colony – shift to 35OC Not a CDC mutant different CDC mutant all in the same stages of cell cycle stage  Putting it together ◼ cdc2 was the name given to the yeast mutant the protein was later cloned and purified ◼ cdc2 mutant was homologous to known protein kinases ◼ Antibody against Cdc2 recognized the MPF of Xenopus ◼ MPF = Cyclin B + Cdc2 (dimer with kinase activity)  Eukaryotes Model: Assembly and Activation of the cyclin and cyclin dependent kinase (CDK) complex - T-loop Cyclin YT YT CDK CDK Cdc Cyclin Cyclin YP T YP TP CDK CDK Thr kinase Still Inactive  Assembly and Activation of MPF Cyclin Cyclin P phosphatase Y T YP T P CDK CDK Active Cyclin/CDK complex: Cyclin and CDK together as dimer Threonine is Phosphorylated Proteasome Tyrosine is not phosphorylated Degrades the cyclin T-Loop open No inhibitors present P Y T CDK Phosphatase YT Inactive CDK CDK  What to Know ◼ Activation of Cyclin/Cyclin Dependent Kinase ◼ Dimerization between cyclin and cyclin dependent kinase (CDK) protein (Cdc2 in yeast) ◼ Enzyme cleft blocked by T-Loop ◼ Requires systematic phosphorylation and dephosphorolation events ◼ Requires active kinases and phosphatases ◼ Cleft is opened allowing Cyclin/CDK to target proteins involved in that phase ◼ Proteasome degrades Cyclin Cell Cycle Control ◼ Mammalian System similar to Yeast ◼ Cyclins – Factors which dimerize with Cyclin Dependent Kinases (CDKs) ◼ Cyclin Dependent Kinases – proteins which dimerize with Cyclins and have kinase activity (yeasts cdcs) ◼ Example targets of an active complex ◼ Transcription Factors ◼ Nuclear envelope ◼ Chromatin remodeling ◼ Nucleolin protein ◼ Myosin – prevents contraction  Mammalian Cell Cycle Cyclin B Cdk 1 M Cyclin D Cyclin A G2 Cdk 4 & 6 Cdk 2 G1 S Cyclin E Cdk 2  Cell Cycle Regulation Cyclin D/CDK 4,6 – Controls M → G1 phase transition. Highest concentration in G1. Cyclin D/CDK 4,6 G1 Cyclin E/CDK 2 – Controls G1 → S phase transition Highest concentration in S. Cyclin A/CDK 2 – Controls S → G2 phase transition. M Cyclin B/CDK 1 S Cyclin E/CDK 2 Highest concentration in G2. Cyclin B/CDK 1 – Controls G2 → M phase transition. G2 Highest concentration in M. Cyclin A/CDK 2  Proteasome ◼ Barrel shaped proteolytic complex that degrades ubiquitin-targeted sequences. ◼ Rings not identical identify different AA peptide bonds ◼ Cap structure binds ubiquitin and unfolds protein  Ubiquitin Mediated Proteasome Degradation Ubiquitin Polyubiquitin Cyclin isopeptidase Cyclin Degradation CDK ATP 1. Ubiquitin ligase adds a polyubiquitin chain to cyclin Ubiquitin 2. Cyclin and CDK separate and Cyclin taken to 26S Proteasome Ligase 3. Polyubiqutin chain removed and ADP Cyclin recycled by polyubiquitin isopeptidase. Cyclin 4. Cyclin unfolded and threaded into the proteasome. 5. Proteasome cleaves the Cyclin into amino acids. CDK ATP ADP Tyr Ser Ala Leu Iso Val Gly Asp Cyclins have a ubiquitin recognition sequence A specific targeting sequence is recognized by proteins which bind ubiquitin to the protein (Cyclin) which needs to be destroyed  Cyclin/Cdk Inhibitors G1 phase proteins S phase proteins CyclinD Cdk 2 Cdk 4 CyclinE 2ATP CyclinE 2ADP P P RB RB E2F E2F  Cyclin/Cdk Inhibitors G1 phase proteins p21 CyclinD Cdk 4 CyclinD 2ATP p21 Cdk 4 2ADP RB RB E2F E2F  S-Phase Gene Transcription and p21 ◼ Cyclin D and Cdk4 complex builds up during G1 phase ◼ Normally this complex adds a phosphate group to RB (retinoblastoma) ◼ RB normally inhibits gene transcription by binding to the transcription factor E2F ◼ When RB is phosphorylated it releases E2F which transcribes Cyclin E and Cdk 2 (S- phase proteins) ◼ P21 inhibits the ability of Cyclin D/Cdk4 from releasing E2F and therefore is a cell cycle inhibitor  p53 Normally Inactive Cell signal good growth conditions p53 MDM2 Ub p53 Ub Ub No DNA damage When, cell conditions are good the p53 protein is expressed continually but gets degraded by ubiquitin proteasome pathway. This is done by interaction with MDM2 protein, which is an ubiquitin ligase.  p53 Normally Inactive Cell signal - Poor growth conditions UV radiation p53 DNA gets damaged, uv as an example MDM2 p21 When, cell conditions are bad the MDM2 protein is blocked and no longer can target p53 for Cyclin destruction. p53 is a transcription p21 factor for p21. Then p21 stops CDK the cell from going through the cell cycle. The cell tries to repair p21 blocks the cyclin/cyclin the DNA damage. dependent kinase from being active