Buněčný cyklus - principy regulace buněčného růstu a buněčného dělění Mitóza Dr. B. Duronio, The University of North Carolina at Chapel Hill Buněčný cyklus Kinázy závislé na cyklinech kontrolují buněčný cyklus Kontrola vstupu do mitózy - výsledek využití různých buněčných modelů Experimental Systems Important for Cell Cycle Studies Arbacia punctulata Xenopus laevisSchizosaccharomyces pombe Saccharomyces cerevisiae Dr. B. Duronio, The University of North Carolina at Chapel Hill Cyclin was Discovered in Sea Urchin Embryos Protein Level Time cyclin A cyclin B M M M can stimulate to lay lots of eggs Dr. B. Duronio, The University of North Carolina at Chapel Hill Frog life cycle 1 mm sperm tadpole feeds, grows and becomes an adult frog OOCYTE GROWS WITHOUT DIVIDING (MONTHS) FERTILIZATION FERTILIZED EGG DIVIDES WITHOUT GROWING (HOURS) The Maturation of Frog Eggs An Assay for Maturation Promoting Factor (MPF) Yoshio Masui, 1971 MPF Activity Peaks Before Each Cell Division Moreover, MPF has kinase activity Dr. B. Duronio, The University of North Carolina at Chapel Hill Purification of MPF: The Birth of Cyclin Dependent Kinases This is cdc2+!! (Cdc28 in S. cerevisiae) This is cyclin!! Which = cdc13+ in S. pombe Dr. B. Duronio, The University of North Carolina at Chapel Hill Fission yeast: Schizosaccharomyces pombe Dr. B. Duronio, The University of North Carolina at Chapel Hill Budding Yeast Saccharomyces cerevisiae Dr. B. Duronio, The University of North Carolina at Chapel Hill Permissive (low) temperature Restrictive (high) temperature Cdc Mutants Arrest at the Same Cell Cycle Phase Dr. B. Duronio, The University of North Carolina at Chapel Hill Cdc Genes Encode Proteins Needed for the G2-M Transition: Studies in S. pombe cdc2+ encodes a kinase Moreover = cdc28 in S. cerevisiae! Dr. B. Duronio, The University of North Carolina at Chapel Hill Phosphorylation of CDK Targets Changes Their Activity Now performs a cell cycle function Dr. B. Duronio, The University of North Carolina at Chapel Hill Jak jsou CDK regulovány? 1. prostřednictvím syntézy a odbourávání cyklinů 2. fosforylací 3. pomocí CDK inhibitory proteins (CKIs) Cyclin Destruction is Controlled by Ubiquitination Dr. B. Duronio, The University of North Carolina at Chapel Hill Představují cykliny jediný způsob regulace CDK? Exprese cyklinů v jednotlivých fázích BC Dr. B. Duronio, The University of North Carolina at Chapel Hill CDK jsou regulovány fosforylací Conformational Changes Associated with CDK Phosphorylation The T-loop blocks substrate access Free CDK CDK + Cyclin T161 phosphorylation Binding of cyclin moves the T-loop Phosporylation moves the T-loop more Cyclin Dependent Kinase Inhibitors (CKIs) The p21 Family of CDK inhibitors (p21CIP1/WAF1 , p27KIP1 , p57KIP2 ) CDK Cyclin active p21+ inactive CDK Cyclin p21 p27Ki p1 Cyclin A CDK 2 Russo et al. (1996) Nature 382:325 Cyclin ACDK 2 Jeffrey et al. (1995) Nature 376:313 Dr. B. Duronio, The University of North Carolina at Chapel Hill The INK4 Family of CDK inhibitors (p16INK4a , p15INK4b , p18INK4c , p19INK4d ) INK4+ CDK4/6 Cyclin D active CDK4/6 Cyclin D INK4 + inactive Russo et al. (1998) Nature 395:237 Brotherton et al. (1998) Nature 395:244 Dr. B. Duronio, The University of North Carolina at Chapel Hill CKIs Regulate the G1-S Transition (p16) (p21, p27) Dr. B. Duronio, The University of North Carolina at Chapel Hill Nature Reviews Cancer 2001, 1:222 Dr. B. Duronio, The University of North Carolina at Chapel Hill pRB Binds to the E2F Transcription Factor Dr. B. Duronio, The University of North Carolina at Chapel Hill G1 Cyclin CDKs Phosphorylate pRB Dr. B. Duronio, The University of North Carolina at Chapel Hill Proliferation Signals p16 Regulates pRB Phosphorylation Dr. B. Duronio, The University of North Carolina at Chapel Hill Nature Reviews Cancer 2001, 1:222 Kontrola buněčného cyklu úzce souvisí s: kontrolou buněčného růstu; přítomností růstových faktorů a dalších růstových stimulů a živin; působením ostatních buněk populace a mezibuněčné hmoty. The Difference Between Growth and Cell Division Cell Division No Growth Cell Division + Growth = Proliferation! Growth with No Cell Division Growth with No Cell Division: A Differentiated Neuron Dr. B. Duronio, The University of North Carolina at Chapel Hill Cell Division with No Growth: Early Development 1 mm sperm Dr. B. Duronio, The University of North Carolina at Chapel Hill Nature Reviews Cancer 2001, 1:222 Growth Factors act at the Restriction Point Growth Factors Induce Cell Cycle Progression Dr. B. Duronio, The University of North Carolina at Chapel Hill Nature Reviews Cancer 2001, 1:222 Zetterberg and Larsson, PNAS 82:5365 (1985) pm = post mitotic ps = pre-synthetic Dr. B. Duronio, The University of North Carolina at Chapel Hill Growth Factors Induce Gene Expression Dr. B. Duronio, The University of North Carolina at Chapel Hill Growth Factors Induce Cyclin D1 Expression Sherr and McCormick, Cancer Cell, Vol 2, 103-112 (2002) Mitogen Induced Cell Cycle Progression in Cell Culture Dr. B. Duronio, The University of North Carolina at Chapel Hill Cell Cycle Checkpoints Weinert and Hartwell, Science 246:629 (1989) Cell Cycle Checkpoints Improve Cell Viability How do Cell Cycle Checkpoints Work? Zhou and Elledge Nature 408, 433 - 439 (2000) Zhou and Elledge Nature 408, 433 - 439 (2000) Signal Sensor Transducer Effector Dr. B. Duronio, The University of North Carolina at Chapel Hill Dr. B. Duronio, The University of North Carolina at Chapel Hill Metaphase in a mammalian cell The Metaphase to Anaphase Transition The Spindle Assembly Checkpoint poškození DNA poruchy buň. cyklu poruchy buň. dělení nedostatek živin a růstových faktorů kontaktní inhibice vliv ECM zástava buň.cyklu (restriction point, checkpoints) setrvání v G0 fázi apoptóza dostatek živin a růstových faktorů mitogeny proliferace Deregulace buněčného cyklu a růst nádorových buněčných populací