BIOL 200 7.2 CDK-Cyclin Regulation

Cyclins & CDKs

-these work together to control the cell cycle

Frog Egg Experiment

-extracted cytosol from frog eggs that were at a known stage of division

  • injected it into a cell that was stalled at the end of interphase

  • compared with cells at the same stage that had been injected with cytosol from cells also in interphase

  • active agent that promotes next stage of cell cycle is in cytosol of cell

  • control of DNA synthesis and mitosis is positive → active agent turns on the process in the recipient cell (capable to respond but hasn’t initiated process itself)

  • cells can be advanced into next stage by appropriate factors

-after this discovery → replicated using cytosol from mitotic cells from different species and taking cytosol from one species and using it to induce mitosis in another species

-’factor’ in cytosol was universal and helped to promote the maturation of cell → maturation promoting factor (MPF)

-MPF = Activated Cyclin-CDK Complex

  • active agent is a protein (Cyclin-Dependant Kinase — CDK)

    • regulates activity of a large number of other proteins required for cell cycle progression by phosphorylating them

  • second protein (cyclin)

    • interacts with CDK

    • acts as a regulatory unit

  • CDK can only perform its function as a kinase when cyclin is bound

  • if cyclin removed from CDK → CDK inactivated

  • these 2 proteins combine to produce a single enzyme → cyclin-CDK complex

Cyclin-CDK Complex

-CDK concentrations in cell are more or less constant throughout the cell cycle

  • enzymatic activity becomes activated and deactivated throughout

-cyclin shows a cyclical pattern of increasing concentrations

  • coincides with increasing enzymatic activity of CDK

  • concentrations decrease to almost nothing

  • change in cyclin concentration coincides with end of CDK enzymatic activity

  • cycling of cyclin concentration and CDK activity that allow the cell to progress through checkpoints and the cell cycle

Four Major Classes of Cyclins

-increasing cyclin concentrations always precedes the passage through a checkpoint

  • cyclin-CDK activity must hit threshold before checkpoint is passed

  • M-cyclin: activity of CDK decreases rapidly not long after checkpoint has ben passed

  • S-cyclin: activity of CDK is activated at one checkpoint and stays high throughout rest of cycle

Phosphorylation Controls Cyclin-CDK Enzymatic Activity

-while the binding of cyclin to CDK is necessary for CDK activity, not sufficient on its own

-cyclin-CDK complex must be phosphorylated by other kinases

-activity of CDKs are tightly controlled by cell

  • cell moving into next stage of cell cycle before it is ready can be disastrous

-cyclin-CDKs are at the heart of a complex signalling pathway

  • involving hundreds of enzymes fighting each other to activate or deactivate cyclin-CDK complex

-balance between activators and deactivators occurs when conditions are just right

  • correct phosphates are in place on the cyclin-CDK complex

  • kinase is able to function

ex

  • addition of phosphate group to M-cyclin-CDK by kinase Wee1 results in inhibition of complex

  • Wee1 thought to respond to intracellular cues to ensure cell doesn’t progress through G2/M checkpoint unless they are big enough to divide

  • once cell has reached appropriate size → Wee1 protein is degraded (can’t phosphorylate M-CDK) → another protein Cdc25 removes phosphates from M-cyclin-CDK complex → activating complex

-in yeast → Wee1 and Cdc25 are key regulators of M-CDK

  • antagonistic relation discovered using genetic experiments where the dosage of genes was experimentally manipulated

  • increasing gene dosage (gof) → increases enzyme concentration

  • decreasing gene dosage (lof) → decreases enzyme concentration

-relationship between amount of protein and difference in when they go through mitosis is evidence of the complexity of the control that cyclin-CDK complexes are under

Specific Cyclins & their Role in the Cell Cycle

M-CDK Controls the G2/M Checkpoint and Re-entry into G1

-transition from G2 phase into M phase is complex

-requires complete rearrangement of cytoplasm

  • shutting down transcription & translation

  • preparing all organelles for separation → shut down

  • building a second MTOC for mitotic spindle

  • completely rearranging cytoskeleton

-M-CDK-cyclin enzymatically active at the end of G2 phase → primary control for this translation

-M-cyclin concentrations peak in metaphase → crashes and deactivates M-CDK

Kinase

-manages transition by phosphorylating several other proteins

-phosphate group changes conformation of protein → affect its function

-some targets of activated M-cyclin-CDK complex

  • histone H1

    • changes in chromatin configuration and in conjunction with other proteins leads to chromatin condensation

  • condensins

    • class of DNA binding proteins that bind to chromatin and help with chromosome condensation

  • nuclear lamins

    • phosphorylated lamins have a lower affinity for each other → nuclear lamins fall apart

    • disassembly of nuclear lamina results in break up of nuclear envelope into ER

  • structural proteins of the nucleolus

    • dispersion of nucleolar proteins

    • disintegration of nucleolus

    • vital for condensation of chromosomes

  • variety of protein kinases that regulate the cytoskeleton

    • arrangement of cytoskeleton as the cell disassembles the interphase microtubule network and forms the mitotic spindle

  • cdc25

    • M-CDK activating phosphate

    • creates a positive-feedback loop → further activation of M-CDK-cyclin → M-CDK activity rises increasingly rapidly as more M-CDK becomes active

  • anaphase promoting complex (APC)

    • activated

    • at the beginning of anaphase → protein complex degrades cohesin proteins that bind sister chromatids together → releases 2 separate chromosomes

      • APC also activates enzymes that tag M-cyclin for degradation

      • ensures that M-CDK will be properly deactivated at the end of its usefulness

      • cell can’t complete mitosis and return to cytoplasm to its interphase state unless MCDK is inactive

Control of G1/S Checkpoint and S-Phase Progression

-requires 2 cyclin classes

-transition from G1 to S-phase controlled by G1/S cyclin-CDK complexes

  • checkpoint passed → G1/S cyclin degraded and S-cyclin takes over

  • in some cases → 2 cyclins can bind to same CDKs → re-activated when new cyclin binds (and enzyme is properly phosphorylated)

-in order for cell to pass into S-phase (and replicate DNA) → must pass through G1/S checkpoint

  • there must be no DNA damage

  • protein that checks for damage is a transcription regulator → p53

    • protein that is constantly translated and degraded as long as it remains inactive

  • when DNA damage detected → p53 becomes phosphorylated → stops degradation → binds to promoter sequence for a CDK inhibitor (p21) → activating transcription & subsequent translation

  • p21 blocks the activity of cyclin-CDK complex

-once checkpoint has passed → G1/S cyclin helps to initiate S-phase via phosphorylation of replication machinery to help it assembly

-G1/S cyclin degraded and S-cyclin takes over

-S-cyclin-CDK complex activates DNA helicase and promotes assembly of the rest of the replication machinery required

-as it is activating replication → prevents replication from being able to happen more than once a cell cycle

  • does this by phosphorylating key enzymes in replication (cdc6)

    • phosphorylation of cdc6 result in it being tagged for degradation

    • without cdc6 → replication can’t be initiated

-before replication, the chromosome consists of a single (double-stranded) DNA molecule (chromatid)

-after replication, the chromosome consists of 2 DNA molecules (sister chromatids)

  • connected at centromere

  • complete with full complement of histones and any other proteins required for chromosome packing

-2 sister chromatids are held together along their length via a protein (cohesin)

  • must be degraded just prior to chromatid separation during anaphase

-S-CDK remains active right until start of mitosis despite S-phase being long over before that

  • proves that different cyclins are influenced by each other

Deactivation of Cyclin-CDK Complex

-once cyclin-CDK complex has completed its job → deactivated

  • via shutting down transcription of cyclin genes and degrading the cyclin proteins that already exist

-negative feedback loops are built in so that shutting down CDKs is possible

-one of phosphorylation targets of M-CDK is anaphase promoting complex (APC)

  • tags proteins for ubiquitination → marks them for degradation by proteasome

  • concentration of cyclin then drops rapidly

    • cyclin degraded → CDK deactivated → next step of cell cycle can begin

    • APC is deactivated in G1

      • one of the phosphorylation targets of activated G1/S-cyclin CDK complex