Exam 4 The Cell Cycle Cell Cycle ordered series of events that leads to cell duplication and division o M phase mitosis meiosis small fraction of the cycle o Interphase the rest of the cell cycle G1 Phase Gap 1 period between the completion of previous mitosis and the initiation of DNA synthesis for the next mitosis Some cells exit the cell cycle into a post mitotic non proliferating state G0 G zero S phase period of DNA synthesis for chromosome duplication G2 phase gap 2 period between the completion of DNA replication and the initiation of mitosis o o o o o o The cell cycle is tightly regulated Progression relies on cell growth and successful passage through several checkpoints Ensures the accuracy and fidelity of chromosome replication Chromosome replication and cell division must occur in the proper order and proper time in every cycle In many cells growth and division rates are coupled The two processes can be regulated independently as well Cell size is determined by the rate of growth and duration of the cell cycle Depend on extracellular signals Signaling relies on post translational modifications Phosphorylation of tyrosines serines and threonines REVERSIBLE o o Ubiquitination on lysines Triggers protein degradation IRREVERSIBLE Cell growth is driven by PI 3 kinase pathways Receptor tyrosine kinases RTKs in response to growth factors recruit and activate PI 3 kinases PI3K at plasma membrane PI3K is a lipid kinase that binds to phosphor tyrosine residues in the cytoplasmic domains of activated RTKs brings PI3K in proximity to phospholipids on the cystolic face of the membrane PI3K phosphorylates the 3 position of inositols to generate PI 3 4 5 P3 and other phosphatidylinositides o Accumulation of PI 3 4 5 in the membrane leads to activation of a serine threonine kinase called Akt Once activated phosphorylates many proteins in the cytoplasm and nucleus mTOR one target a kinase that promotes cell growth Activation of complexes containing mTOR results in phosphorylation of targets involved in protein synthesis and metabolism S6 kinase a substrate of mTOR that phosphorylates ribosomal protein S6 Results in increased translation of mRNAs 4E BP inhibits translation initiation factor elF4E o o Phosphorylation releases elF4E from 4E BP inhibition and allows it to initiate translation Growth can be balanced by protein degradation in the proteasome o Two major protein degradation pathways Lysosome pathways Proteasome pathways Proteasome very large macromolecular machine consisting of 50 protein subunits that act collectively to degrade many cellular proteins o Hydrolyzes ATP to provide the energy needed for degradation Protein lifespan is controlled by regulated protein degradation Important functions of protein degradation Removes proteins that are misfolded damaged or potentially toxic to the cell Controlled degradation of normal proteins provides a powerful mechanism to maintain appropriate levels of proteins and their activities Regulated degradation also permits rapid responses to changing conditions Ubiquitin marks proteins for degradation by the proteasome o Multiple ubiquitin molecules can be attached covalently to proteins via a multi step process called poly ubiquitin recognized by the proteasome Process involves several enzymes Specificity of poly ubiquitination is achieved through E3 Ub ligases Each recognizes a specific signal in a given protein for poly ubiquitination Cell cycle progression is controlled by protein expression phosphorylation and degradation Passage through the cycle is controlled by heterodimeric protein complexes consisting of Regulatory subunit cyclin Catalytic subunit cyclin dependent kinase CDK Depend on cyclins for activation of their kinase activity and for access to substrates Early G1 cyclin D CDK4 6 Late G1 S cyclin E CDK2 S cyclin A CDK2 o o o o M cyclin A A B CDK1 o Together cyclin CDK complexes protein phosphatases and ubiquitin ligases regulate cell cycle transitions The synthesis and degradation of cyclins control progression through the cell cycle o The concentrations of cyclin proteins oscillate during the cell cycle while the concentrations of the CDKs remain steady o o o o o o o o o o o o o o CDK activity is tightly regulated o Activation by cyclin binding and T loop phosphorylation Binding of a cyclin to a CDK alters the conformation of an area called the T loop Leads to partial activation of the CDK Phosphorylation by a CDK activating kinase CAK of a specific T loop residue induces an additional conformational change that allows substrate access and full CDK activation Inhibitory phosphorylation of the active site by Wee1 kinases Wee1 phosphorylates and reduces the activity of CDKs o De phosphorylation of inhibitory sites by Cdc25 phosphatases Thereby increasing the activity of the CDK Physical inhibition by CDK inhibitors CKIs The inhibitory effects of Wee1 can be antagonized by Cdc25 mediated removal of phosphates from a CDK Binding of these molecules causes a large rearrangement in the structure of the CDK active sites rendering it inactive Cyclin CDKs and Ub ligases regulate transitions o Mid G1 G1 cyclin CDKs activate the transcription of genes required for DNA replication and assemble pre replication complexes at origins SCF ubiquitin ligase initiated passage through the restriction point by polyubiquitinating inhibitoes of S phase cyclin CDKs Mitotic cyclin CDKs cyclin B CDK1 Marking them for proteasome mediated degradation o After this restriction point is passed S phase cyclin CDKs activate DNA replication origins Accurate DNA replication must occur once and only once during each cell cycle Late G2 mitotic cyclin CDKs trigger entry into mitosis o o APC polyubiquitinates securing to promote anaphase With the help of Cdc14 phosphatase polyubiquitinates mitotic cyclins to promote telophase Phosphorylate a number of cellular proteins and sets the stage for the dramatic changes in mitosis Condensins chromosome condensation Nuclear lamins nuclear envelope breakdown Microtubule associated proteins chnages in MT dynamics ER or Golgi associated proteins reorganization of the ER and the Golgi o When chromosomes have segregated sufficiently at anaphase Cdc14 phosphatases is activated Dephosphorylates and activates a protein that directs the APC to mitotic cyclins Dephosphorylates many proteins that have been phosphorylated by cyclin B CDK1 Mitotic cyclins are polyubiquitinated by the APC and degraded by the proteasome Resulting