week 1 lec 3 Cell Cycle Regulation: Cyclin Degradation, APC, Phosphorylation, and CDK Inhibitors

Cyclone Degradation, APC, Phosphorylation, and CDK Inhibitors

This lecture focuses on the regulation of the cell cycle, elaborating on mechanisms controlling CDK activity beyond just cyclin binding. It covers cyclin degradation, the Anaphase Promoting Complex (APC), phosphorylation events, and CDK inhibitors.

Cyclins and Cell Cycle Progression

Cyclins are expressed at different phases of the cell cycle, such as G1 cyclins in gap one and S cyclins in the synthesis phase. These cyclins promote specific phase activities and prepare the cell for the next phase. The lecture emphasizes the importance of regulating CDK activity, as CDK proteins are master controllers of the cell cycle. Multiple mechanisms ensure CDKs are active only at appropriate times.

Protein Turnover and Ubiquitin

Protein Turnover Rate

Protein turnover rate refers to the lifespan of a protein within a cell, ranging from minutes to days, depending on the protein and cellular conditions.

Irreversible Protein Degradation

Irreversible protein degradation means a specific instance of a protein is destroyed. However, the cell can create new instances of the protein. Cyclin degradation is crucial for unidirectional cell cycle progression.

Ubiquitination

Proteins destined for destruction are tagged with ubiquitin molecules through ubiquitination. Ubiquitin, a highly conserved 76 amino acid protein found in all eukaryotes, is essential for life and represents a ubiquitous protein degradation mechanism.

Ubiquitin Proteosome Pathway

  • Cyclins are tagged with ubiquitin and destroyed via the ubiquitin proteosome pathway.

  • The pathway involves enzymes E1, E2, and E3.

    • E1 (ubiquitin activating enzyme) activates ubiquitin using ATP.

    • E2 (ubiquitin conjugating enzymes) receives ubiquitin from E1.

    • E3 (ubiquitin ligase) binds to the target protein.

  • The activated ubiquitin is transferred to the target protein, often requiring polyubiquitination for destruction.

  • The target protein is directed to the proteosome, a barrel-like structure filled with proteases that break down the protein into amino acids.

  • Ubiquitin molecules are recycled and can be reactivated, while amino acids form new proteins.

  • Human cells have one E1 enzyme but multiple E2 and E3 enzymes, determining the specificity of target protein binding.

Cell Cycle Regulation

Cyclin degradation, regulated through the ubiquitination proteosome pathway, plays a fundamental role in cell cycle regulation.

Destruction Box

Cyclins are targeted for destruction due to a specific amino acid sequence called the destruction box, recognized by the E3 ubiquitin ligase. Destruction boxes are present in other proteins undergoing programmed proteolysis.

Half-life of a Protein

The amino acid at the N-terminal of the destruction box determines the half-life of a protein. Stabilizing amino acids result in long half-lives (over 20 hours), while destabilizing amino acids lead to short half-lives (3 minutes to 30 minutes).

M Cyclin Degradation

For instance, M cyclin destruction box sequences commonly have arginine as the N-terminal amino acid, leading to a half-life of approximately 3 minutes. The APCE3 ligase recognizes the destruction box in mitotic cyclins.

Anaphase Promoting Complex (APC)

APC Activation

The Anaphase Promoting Complex (APC) is an E3 ubiquitin ligase activated by MPF (M cyclin-CDK complex), promoting mitosis. APC targets M cyclins for destruction, leading to the progression of the cell cycle from mitosis into the G1 phase.

Targets of APC

The targets of APC are determined by protein binding partners.

Role in Metaphase-Anaphase Transition

  • When bound to CDC20, APC promotes the transition from metaphase to anaphase.

  • The APCCDC20 complex directs APC to the securin-separase complex.

  • Separase is an enzyme responsible for separating sister chromatids, but it's inactive when bound to securin.

  • APCCDC20 ubiquitinate securin, leading to its degradation by the proteosome, which activates separase.

  • Separase cleaves cohesin (the protein that binds sister chromatids), facilitating sister chromatid separation and movement to opposite poles during anaphase.

Role in Mitosis-G1 Transition

  • When bound to CDH1, APC targets M cyclins for destruction, aiding the progression from mitosis to the G1 phase.

  • Similar mechanism also prevents M cyclin accumulation before it's needed.

Regulation of APC Activity

The activity of APCCDH1 is controlled by phosphorylation. When CDH1 is phosphorylated, it cannot bind to APC, inactivating the complex. G1 cyclin-CDK complex phosphorylates CDH1 in favorable conditions, allowing M cyclin to accumulate. During late anaphase, CDC14 removes the phosphate group from CDH1, enabling it to bind to APC, targeting M cyclin for destruction and allowing the transition from mitosis to G1.

Phosphorylation

Phosphorylation and CDK Activity

Phosphorylation can either activate or inhibit CDK activity. The phosphate groups bind to amino acid residues near the substrate-binding site, influencing its ability to bind target proteins.

Experimental Data in Fission Yeast

Experiments in fission yeast, where the point of no return is in G2, provide insights into cell cycle regulation.

Cell Size and G2 Phase

The size of fission yeast cells after division indicates the time spent in G2 phase.

Mutated Yeast Cells

  • Mutations leading to small cells indicate premature division due to shortened G2 phase and were named we1.

  • Mutations in a gene called CDC 25 lead to elongated cells that never divide, indicating a failure to transition from G2 into mitosis.

wee1 and CDC 25 Proteins

Both we1 and CDC 25 regulate MPF activity. CDC 25 promotes MPF activation, while we1 inhibits MPF activity.

Regulation of MPF Activity by V1 and CDC 25

  1. Inactive MPF complex exists before activation by phosphorylation.

  2. We1, an inhibitory kinase, adds a phosphate group at position Y15 on CDK, blocking the substrate-binding surface.

  3. CAC (CDK activating kinase) phosphorylates CDK at position T161, activating the MPF complex. However, the complex remains inactive due to the inhibitory phosphate group at Y15.

  4. CDC 25, a phosphatase, removes the inhibitory phosphate group, resulting in an active MPF complex with the phosphate group at T161.

Effects of Mutations

  • Without functional we1, inhibitory phosphate groups are not added to MPF, accelerating MPF activation and leading to premature entry into mitosis, resulting in small cells.

  • Without functional CDC 25, the inhibitory phosphate group is never removed from MPF, leading to cells stuck in G2 that never divide.

Expression Patterns of Cycling Proteins

The cell employs a complicated process of activating MPF due to the expression patterns of cycling proteins. M cyclins are expressed for an extended period before their expression reaches a peak, yet the associated activity isn't correspondingly high for that whole period. Inhibitory phosphorylation ensures that MPF does not become prematurely activated. Active and inactive MPF complexes accumulate; with one phosphorylation event, the cell transitions from G2 into mitosis.

CDK Inhibitors

CDK Inhibitors Defined

CDK inhibitors are proteins that bind to CDK and block its activity, preventing premature activation of CDK-cyclin complexes.

Kinds of CDK Inhibitors

There are different classes of CDK inhibitors with specific roles in different cell cycle phases and their role is to prevent premature activation of CDK cycling complexes.

INk-4

  • INk4 (inhibitor of CDK4) is a family of proteins that bind directly to CDK, preventing cyclin binding.

  • They specifically inactivate G1 cyclin-CDK complexes, integrating extracellular signals with the transition from G1 to S phase.

Cyclin Kinase Inhibitors

  • Cyclin kinase inhibitors (CKIs or CIPs) are another family of proteins targeting different cell cycle steps.

  • Instead of blocking cyclin binding, they bind to the complete CDK-cyclin complex, physically blocking the active site of CDK.

Protein Naming Convention

Proteins designated with "P" followed by a number are named based on their molecular weight in kilodaltons.

Inhibition of CDK Function by P16

  1. Cell cycle progression occurs when P16 is inactive or not expressed.

  2. E2F transcription factors are bound and inactivated by RB protein.

  3. G1 cyclin-CDK complex phosphorylates RB, causing it to release E2F.

  4. E2F then promotes the transcription of genes essential for S phase.

  5. When P16 is active, it prevents the formation of the G1 cyclin-CDK complex.

  6. E2F remains bound to RB, inhibiting the transcription of S phase genes.

  7. Cell cycle progression is arrested, and the cell remains in phase G1.

Function of P27

  • P27 of cyclin kinase inhibitors binds to cyclin-CDK complexes.

  • P27 blocks the active site of the enzyme, preventing catalytic activity.

  • Mice lacking functioning P27 leads to larger body sizes and excessive cell numbers within organs because it regulates cell cycle and a regulatory mechanism is lost, resulting more cells, larger body size, and hyperplasia.

  • The transition inhibited by P27 is G1 to S phase.

Review of Cell Cycle Regulation

  • Cell cycle checkpoints monitor events like cell size, DNA damage, and chromosome attachment to mitotic spindles.

  • If errors exist, the cell cycle pauses for correction.

  • Regulation involves cyclin degradation and inhibitory phosphorylation.

  • These mechanisms ensure high fidelity, resulting in viable daughter cells.

  • Regulatory mechanisms include formation of cyclin CDK complexes, cyclin degradation via the proteosomal pathway, activation events such as phosphorylation, and checkpoints (DNA damage, spindle attachment).