Please answer all of 5 After the completion of S phase F2F f

After the completion of S phase, F.2F function is no longer needed. pRb must be dephosphorylated so that it can once again bind to E2F. Name the type of enzyme responsible for removing phosphate groups from proteins. Knowing what you know about the cell cycle, explain why the RB gene would be considered a tumor suppressor gene. Below are listed four different mechanisms of regulating of cyclin/CDK activity. For each mechanism explain how that mechanism regulates cyclin/CDK activity. Phosphorylation by WeeI Dephosphorylation by Cdc 25 Binding by a cyclin dependent kinase inhibitor Degradation of cyclins by the proteasome

Solution

5. A. Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe (S. pombe). It has a molecular mass of 96 kDa and it is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1. It has homologues in many other organisms, including mammals.

Wee1 inhibits Cdk1 by phosphorylating it on two different sites, Tyr15 and Thr14. Cdk1 is crucial for the cyclin-dependent passage of the various cell cycle checkpoints. At least three checkpoints exist for which the inhibition of Cdk1 by Wee1 is important:

B. Cdc25 is a dual-specificity phosphatase first isolated from the yeast Schizosaccharomyces pombe as a cell cycle defective mutant. As with other cell cycle proteins such as Cdc2 and Cdc4, the \"cdc\" in its name refers to \"cell division cycle\". Dual-specificity phosphatases are considered a sub-class of protein tyrosine phosphatases. By removing inhibitory phosphate residues from target Cyclin-Dependent Kinases (Cdks), Cdc25 proteins control entry into and progression through various phases of the cell cycle, including mitosis and S (\"Synthesis\") phase.

Cdc25 activates cyclin dependent kinases by removing phosphate from residues in the Cdk active site. Also, the phosphorylation of M-Cdk (a complex of Cdk1 and cyclin B) activates Cdc25. Together with Wee1, M-Cdk activation is switch-like. The switch-like behavior forces entry into mitosis to be quick and irreversible. Cdk activity can be reactivated after dephosphorylation by Cdc25. The Cdc25 enzymes Cdc25A-C are known to control the transitions from G1 to S phase and G2 to M phase.

C. A cyclin-dependent kinase inhibitor (CKI) is a protein that interacts with a cyclin-CDK complex to block kinase activity, usually during G1 or in response to signals from the environment or from damaged DNA. In animal cells, there are two major CKI families: the INK4 family and the CIP/KIP family. The INK4 family proteins are strictly inhibitory and bind CDK monomers. Crystal structures of CDK6-INK4 complexes show that INK4 binding twists the CDK to distort cyclin binding and kinase activity. The CIP/KIP family proteins bind both the cyclin and the CDK of a complex and can be inhibitory or activating. CIP/KIP family proteins activate cyclin D and CDK4 or CDK6 complexes by enhancing complex formation.

In yeast and Drosophila, CKIs are strong inhibitors of S- and M-CDK, but do not inhibit G1/S-CDKs. During G1, high levels of CKIs prevent cell cycle events from occurring out of order, but do not prevent transition through the Start checkpoint, which is initiated through G1/S-CDKs. Once the cell cycle is initiated, phosphorylation by early G1/S-CDKs leads to destruction of CKIs, relieving inhibition on later cell cycle transitions. In mammalian cells, the CKI regulation works differently. Mammalian protein p27 (Dacapo in Drosophila) inhibits G1/S- and S-CDKs, but does not inhibit S- and M-CDKs.

D. Proteasomes are protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, proteasomes are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into shorter amino acid sequences and used in synthesizing new proteins. Proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein.

In structure, the proteasome is a cylindrical complex containing a \"core\" of four stacked rings forming a central pore. Each ring is composed of seven individual proteins. The inner two rings are made of seven subunits that contain three to seven protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. The outer two rings each contain seven subunits whose function is to maintain a \"gate\" through which proteins enter the barrel. These subunits are controlled by binding to \"cap\" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system.

The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress. The importance of proteolytic degradation inside cells and the role of ubiquitin in proteolytic pathways was acknowledged in the award of the 2004 Nobel Prize in Chemistry.

Cell cycle progression is controlled by ordered action of cyclin-dependent kinases (CDKs), activated by specific cyclins that demarcate phases of the cell cycle. Mitotic cyclins, which persist in the cell for only a few minutes, have one of the shortest life spans of all intracellular proteins. After a CDK-cyclin complex has performed its function, the associated cyclin is polyubiquitinated and destroyed by the proteasome, which provides directionality for the cell cycle. In particular, exit from mitosis requires the proteasome-dependent dissociation of the regulatory component cyclin B from the mitosis promoting factor complex. In vertebrate cells, \"slippage\" through the mitotic checkpoint leading to premature M phase exit can occur despite the delay of this exit by the spindle checkpoint.