CDK Inhibitors: p19, p21, and p27

• p19 binds to the ATP binding site of cdk6, inhibiting catalytic activity and preventing cyclin binding through a conformational change.
• p21 is a universal cdk inhibitor that binds to all cdk2,4,6 complexes, likely blocking progression through all G1/S stages.
• In primary cultured cells, cdk-cyclin dimers are found as quaternary complexes with PCNA (DNA polymerase 8 component) and the inhibitor p21.
• At a 1:1 stoichiometry, p21 is not inhibitory; an increase in p21 subunits associated with the cdk-cyclin dimer inhibits kinase function.
• In transformed cells, cdk-cyclin complexes lack p21 and PCNA, suggesting p21's involvement in G1/S control.
• p27 has a sequence related to p21, binds promiscuously to cdk-cyclin complexes, and its overexpression blocks S phase progression
• p27 levels increase when cells enter a quiescent state with TGFB treatment.
• p21 and p27 block the catalytic subunit of cdk-cyclin dimers from being a substrate for CAK phosphorylation and prevent its catalytic activity.
• p21 and p27 are partially redundant
• the mechanism by which they inhibit the cell cycle does not depend on controlling RB because they can inhibit proliferation of cells that lack RB.
• p27 connects extracellular mitogens and the cell cycle, with an inverse correlation between p27 activity and proliferation ability.
• Tumor suppressors, including cki proteins, cyclins D1,2, and RB, emphasize the importance of the pathway from cki proteins to RB.
• cki proteins are needed to suppress unrestrained cell growth.
• SCF is an E3 ligase that targets the inhibitor Sic1.
• In S. cerevisiae, Sic1 binds to CDC28-CLB during G1, keeping the kinase inactive.
• Entry into S phase requires Sic1 degradation to release the kinase.
• The Sic1 target is recognized by the SCF complex, which functions as an E3 ligase and includes Cdc53, Skp1, and Cdc4.
• Cdc4 is the targeting component that binds to Sic1 with Skp1; Skp1 connects to Cdc53, which interacts with the E2 ligase (Cdc34).
• Cdc4 is an F-box protein, using the F-box motif to bind to Skp1
• Other SCF complexes exist with different F-box proteins as targeting subunits, while Cdc53 and Skp1 remain the same.
• SCFGrr1, with the F-box protein Grr1, degrades G1 cyclins.
• SCF substrates must be phosphorylated to be recognized, performed by cdk-cyclin complexes that are active at the appropriate cell cycle stage.
• SCF complexes are controlled by degrading the F-box subunits; SCFCdc4 targets Cdc4, creating an autoregulatory limitation on its activity, with feedbacks to maintain a supply of Cdc53-Skp1 cores.

Protein Degradation in Mitosis

• Mitosis, initiated by M phase kinase activation, requires cyclin and other protein degradation.
• Chromosome separation at anaphase requires the proteasomal system for protein degradation.
• The degradation system has at least three targets: cyclin A at metaphase, then two targets at anaphase (Pds1p and B cyclins).
• Pds1p degradation triggers sister chromatid separation, and B cyclin degradation inactivates M phase kinase.
• At the end of mitosis, substrate protein phosphorylations performed by M phase kinase needs to be reversed.
• The anaphase-promoting complex (APC), a large complex of 8 subunits, selects substrates degraded in anaphase and is found in both yeasts and vertebrates.
• APC functions as an E3 ubiquitin ligase for substrate protein binding, becoming active specifically during mitosis.
• CDC20 and Cdh1 are regulatory factors that bind to the APC and activate its ubiquitination activity
• CDC20 is necessary for the APC to degrade Pds1p, while Cdh1 is necessary for the APC to degrade Clb2 (yeast cyclin B), with differences in the timing of activation and persistence.
• Prior to anaphase, Pds1p binds to Esp1p, keeping it inactive.
• When the APC degrades Pds1p, Esp1p is released, causing the protein Scclp release from the sister chromatids
• Scc1p is a component of the cohesin complex that holds the chromatids together.
• The core of the cohesin complex is a heterodimer of SMC proteins,
• Cohesin complex also contains Smc1p and Smc3p, which are ATPases with coiled-coil domains; when Scclp is released, the chromatids are now free to segregate.
• Scclp is degraded later in anaphase by the APC.
• Cohesin complex formation occurs during S phase, and mutants in the locus ctf7 have sister chromatids that never associate because the gene acts during DNA replication.
• A cohesin complex, related to that of mitosis, may also form at meiosis.
• Cohesion is a target for a checkpoint and Progression through mitosis requires all kinetochores to be paired with their homologs.
• An unattached kinetochore triggers the Mad pathway, inhibiting CDC20 activity and the checkpoint is a surveillance system triggered by the presence of one.
• Mad proteins bind to CDC20 and prevent it from activating the APC
• All chromosomes attached signals Mad proteins release which activates APC, allowing anaphase.
• Bub proteins play a similar role in controlling Cdh1's ability to activate the APC.

Reorganization of the Cell at Mitosis

• Cell division culminates in chromosomes segregating into two diploid sets and cell component partitioning between daughter cells.
• Reorganization includes chromatin condensation, nuclear lamina dissolution, microtubule dissociation and reformation, and actin filament reorganization.
• Changes are reversible; post-separation, actin filaments resume normal form, microtubular spindle dissolves, nuclear envelope reforms, and chromosomes regain dispersed structure.
• Mitotic changes and their reversal depend on M phase kinase activation and inactivation.
• Histone H1 is a well-characterized substrate for M phase kinase which helps regulate chromosome condensation.
• Nuclear integrity is lost when lamina dissociates into lamins
• Lamins are phosphorylated causing lamina disassociation
• Phosphorylation on serine residues induces a structural change in the lamin subunits, performed directly by the M phase kinase.
• Phosphorylation and dephosphorylation of lamins accounts for nuclear envelope dissolution and reformation.
• Microtubules, composed of a-tubulin and B-tubulin dimers, extend from organizing centers in the centrosomes and at the kinetochores
• Within the centrosome, y-tubulin is part of a potential nucleating complex

Centrioles

• In animal cells, a centrosome contains a pair of centrioles surrounded by a dense amorphous region.
• The centriole is a small hollow cylinder with a wall consisting of a series of triplet fused microtubules.
• The centriole may be concerned with orienting the spindle or plays a role in establishing directionality for cell movement.
• Centrioles duplicate during interphase.
• Procentriole is elaborated perpendicular to the parental centriole, extended to full length later during interphase.
• The parental centriole at the mitotic pole is responsible for establishing the direction of the spindle.

Apoptosis

• Programmed cell death or apoptosis is a crucial control over total cell number.
• It involves the activation of a pathway that leads to suicide of the cell by a characteristic process in which the cell becomes more compact, blebbing occurs at the membranes, chromatin becomes condensed, and DNA is fragmented.
• The pathway is an active process that depends on RNA and protein synthesis by