Alberts • Johnson • Lewis • Morgan • Raff • Roberts • Walter

Molecular Biology of the Cell Sixth Edition

Chapter 17 The Cell Cycle Regulation

Copyright © Garland Science 2015

Five Phases of the Cell Cycle

• G1 - primary growth phase • S – synthesis; DNA replicated • G2 - secondary growth phase – collectively these 3 stages are called interphase • M – phase – Mitosis – cytokinesis

G0 PHASE

Resting cells Terminally differentiated cells 4

Spindle assembly Checkpoints G1 checkpoints: • Cell size • Nutrients • growth factors • DNA damage

G2 Checkpoints: • Cell size • DNA damage

• Cell cycle controlled by Cdk-cyclin activity and levels • phosphorylation & dephosphorylation • Degradation of cyclins by proteasome CDK: cyclin dependent kinase

Structural basis for CDK activation

• Active site blocked by T-loop • Binding of cyclin causes the T-loop to move out of the active site • Phosphorylation of Cdk2 at Thr in T-loop further activates the enzyme by changing the shape of the T-loop improving the ability of the enzyme to bind its protein substrates

Regulation of Cdk activity by phosphorylation

added by CAK

• The active cyclin–Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site • Removal of these phosphates by the phosphatase Cdc25 activates the cyclin–Cdk complex

Inhibition of a cyclin–Cdk complex by a CKI

Cdk inhibitor protein

• The p27 (the CKI Cdk inhibitor protein) binds to both the cyclin and Cdk in the complex, distorting the active site of the Cdk. • It also inserts into the ATP-binding site, further inhibiting the enzyme activity

Regulation of cyclin levels • The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control • This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. • Destruction box is recognized by corresponding ubiquitin ligase

Polyubiquitination of Mitotic Cyclins • Regulated degradation of mitotic cyclins occurs in late anaphase via the APC-ubiquitin pathway which makes use of a “destruction box” sequence common to the N-terminus of mitotic cyclins • This ubiquitin pathway involves E1 (ubiquitin activating enzyme) , E2 (ubiquitin conjugating enzyme) & E3 (ubiquitin ligase or APC) • Cyclins are finally degraded by proteasome

Regulated degradation of mitotic cyclins

• • • •

Anaphase-Promoting Complex (APC/C) is an E3 ubiquitin ligase APC/C is activated in mitosis by association with Cdc20, which recognizes specific amino acid sequences on M-cyclin and other target proteins. With the help of E1 and E2 proteins, the APC/C assembles polyubiquitin chains on the target protein. The polyubiquitylated target is degraded by the proteasome

Examples: • • • •

The Regulatory Aspects of DNA Replication M-Cdk drives entry into mitosis Mitogen stimulation of cell-cycle entry How DNA damage arrests the cell cycle in G1

The Regulatory Aspects of DNA Replication • Genetic studies with S. cerevisiae have dissected the regulatory aspects of DNA replication • Control of S-phase by regulated proteolysis of sic 1 – Sic 1 is a CKI specific for the S-phase cdk-cyclin complex. – proteolysis is initiated by the G1 phase cdk-cyclin which phosphorylates sic1 making it a target for cdc34 ubiquitin conjugating enzyme and the ubiquitin ligase SCF.

The regulatory aspects of DNA replication • Regulation of pre-replication complexes: • cdk’s simultaneously activate initiation of replication and prevent re-initiation at origin by phosphorylation and disassociation of Mcm helicase from the ORCs (origin recognition complex) which are permanently associated with replication origins. • For the Mcm’s to reassemble at the origins they need to be dephosphorylated which does not occur until mitosis.

P P

The regulatory aspects of DNA replication

The replication origin is bound by the ORC throughout the cell cycle. In early G1, Cdc6 associates with the ORC, and these proteins bind the DNA helicase (Mcm). The helicase also associates with a protein called Cdt1. Using energy provided by ATP hydrolysis, the ORC and Cdc6 proteins load two copies of the DNA helicase, in an inactive form, around the DNA next to the origin, forming the prereplicative complex (preRC). At the onset of S phase, S-Cdk stimulates the assembly of several initiator proteins on each DNA helicase, while another protein kinase, DDK, phosphorylates subunits of the DNA helicase. As a result, the DNA helicases are activated and unwind the DNA. DNA polymerase and other replication proteins are recruited to the origin, and DNA replication begins. The ORC is displaced by the replication machinery and then rebinds. S-Cdk and other mechanisms also inactivate the preRC components ORC, Cdc6, and Cdt1, preventing formation of new preRCs at the origins until the end of mitosis

M-Cdk drives entry into mitosis 1. Cdk1 associates with M-cyclin as the levels of M-cyclin gradually rise. The resulting M-Cdk complex is phosphorylated on an activating site by the Cdkactivating kinase (CAK) and on a pair of inhibitory sites by the Wee1 kinase.

2. The resulting inactive M-Cdk complex is then activated at the end of G2 by the phosphatase Cdc25. Cdc25 is further stimulated by active M-Cdk, resulting in positive feedback. This feedback is enhanced by the ability of M-Cdk to inhibit Wee1.

Mitogen stimulation of cell-cycle entry 1. Mitogens bind to cell-surface receptors to initiate intracellular signaling pathways. Via activation of the small GTPase Ras, a MAP kinase cascade is activated. 2. Increased expression of numerous immediate early genes, including the transcription regulatory protein Myc. Myc increases the expression of many delayed-response genes, including some that lead to increased G1-Cdk activity (cyclin D-Cdk4), which triggers the phosphorylation of members of the Rb family of proteins. 3. This inactivates the Rb proteins, freeing the gene regulatory protein E2F to activate the transcription of G1/S genes (cyclin E, cyclin A). The resulting G1/SCdk and S-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop. E2F proteins also stimulate the transcription of their own genes, forming another positive feedback loop

How DNA damage arrests the cell cycle in G1 When DNA is damaged, various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell-cycle arrest. The first kinase at the damage site is either ATM or ATR, depending on the type of damage. Additional protein kinases, called Chk1 and Chk2, are then recruited and activated, resulting in the phosphorylation of the transcription regulatory protein p53. Mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. Phosphorylation of p53 blocks its binding to Mdm2; as a result, p53 accumulates to high levels and stimulates transcription of numerous genes, including the gene that encodes the CKI protein p21. The p21 binds and inactivates G1/S-Cdk and S-Cdk complexes, arresting the cell in G1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes a further increase in p53