BCHM3972 Human Molecular Cell Biology (Advanced) 2013 Course University of Sydney

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1 BCHM3972 Human Molecular Cell Biology (Advanced) 2013 Course University of Sydney Page 2: Immune Mechanisms & Molecular Biology of Host Defence (Prof Campbell) Page 45: Infection and Implications for Cell Function (Dr Newsome) Page 67: Cellular Signaling Mechanisms Metabolism, Growth & Malignancy (Prof Conigrave) Page 82: Cell Proliferation & Apoptosis (Prof Christopherson) Page 97: Systems Biology (Prof James) 1

2 Cell Proliferation & Apoptosis (Prof Christopherson) Lecture 1 Control of the Cell Cycle Mitogens stimulate growth TGF-β may inhibit growth Differentiated cells do not proliferate Many growth factors (>20) lead to a binary decision to grow or arrest o Cell cycle clock = network of interactions Oncogenes and tumour suppressor genes disrupt cell cycle clock Mammalian Cell Cycle G1 = expression of proteins and growth S phase = synthetic phase (~6.4 x 10^9 base pairs per diploid genome) G2 phase = delays transition from S phase to M phase. Cell prepares for mitosis Mitosis (M phase) = Prophase, metaphase, anaphase, telophase, cytokinesis Checkpoints in Cell Cycle 1. DNA damage checkpoint in late G1 blocks S phase entry if DNA damaged 2. DNA damage checkpoint in S phase DNA replication halted if genome is damaged. In human cells, there are >40 proteins involved in DNA repair 3. G2/M phase checkpoint entrance into M phase blocked if DNA replication is incomplete 4. Metaphase checkpoint cannot progress into anaphase if chromatids are not assembled properly on mitotic spindle ATR Kinase Activated by single strand breaks or double strand breaks in DNA ATR phosphorylates proteins including p53, which arrests the cell cycle Mutant: lacking ATR kinase o Chromosome breaks are common due to loss of DNA repair Many cancers inactivate checkpoint controls à accumulate mutations Response to Extracellular Signals During Cell Cycle Period of G1 phase up to R point = when cells are responsive to mitogenic GFs and TGF-β (inhibitory) o After this point, the cell is committed to proliferation o Once committed, the cycle continues even without GFs o However, metabolic, genetic or physical disasters can halt the cycle Embryonic cells grow without GF stimulation o prb regulation is not present in embryonic cells Growth of most somatic cells must be controlled (10 14 cells) Most cancers de-regulate the R point 82

3 Cyclins and CDKs CDK2-cyclin A o PSTAIRE helix binds cyclins specifically o Activation loop phosphorylated on a Thr for CDK activity o Cyclin A determines specificity of CDK2 for S phase progression CDK2-cyclin E o During late G1, CDK2 phosphorylates proteins for entry into S phase Control of Cell Cycle Progression Protein kinases are involved in promoting progression o Phosphorylation of other proteins prior to S activates sites of DNA replication o Phosphorylation of histones in preparation for S and M phases o Phosphorylation of lamin and nucleoporins dissolves nuclear membrane in early M phase CDKs depend on cyclins o CDKs are serine threonine kinases o Control hundreds of responder molecules o CDKs have ~40% sequence similarity with each other o Cyclin A binds CDK2 to increase CDK2 activity 400,000x Cyclin-CDK partners and control of cell cycle o During G1, CDK4 and CDK6 associate with cyclins D1, D2, D3 o After R point, cyclins E1 and E2 associate with CDK2 to ph late substrates for S entry o In S phase, cyclins A1 and A2 replace E cyclins with CDK2 for S phase progression o Later in S phase, CDK2 is replaced with CDC2/CDK1 o In G2, cyclins B1 and B2 replace cyclins A1 and A2 with CDC2/CDK1 o At M phase, the CDC2/Cyclin B1 and B2 complexes trigger PMAT 83

4 CDK activity is regulated by changing levels of cyclins o Afterwards, cyclins are ubiquitinylated and rapidly degraded by the proteasome D cyclins Cyclins D1, D2 and D3 do not change dramatically, but are controlled by mitogenic GFs and inhibitory TGF-β D type cyclins inform Cell Cycle Clock of environmental conditions D cyclins are controlled by many signals. Examples: o HER2/Neu receptor induces D1 (breast cancer target) o Bcr/Abl induces D2 Cyclin D1 can associate with Estrogen Receptor. Interaction of D1 with ER mimics effect of estrogen by inducing gene expression and promoting proliferation. Cyclin D1 is overexpressed in most breast cancers Cyclin D1 also can associate with Transcription Factor C/EBP-beta, and may block cell differentiation Coordination of subsequent cyclin-cdk complexes occurs depending on existing cyclin-cdk complexes. 84

5 Lecture 2 CDK Inhibitors Cyclin D-CDK4/6 complexes are inhibited by p16, p15, p18, p19 Other cyclin-cdk complexes are inhibited by p57, p27, p21 Specific actions: o p27 block the ATP binding site of CDK2 o p16 inhibits CDK6 (and 4) by distorting the cyclin-binding site (reduces affinity for ATP and D cyclins) EXCEPTION: p21 and p27 stimulate cyclin D-CDK4/6 in early to mid-g o Inhibit most cyclin-cdk complexes in late G1, S, G2 and M phases o In late G1, increasing amount of cyclin D-CDK4/6 removes p21 and p27 from cyclin E-CDK2 à R point transition Cell Cycle Control by TGF-beta TGF-β modulates levels of CDKIs blocks cell cycle TGF-β strongly induces expression of p15 and weakly induces p21 Roles of p21: o DNA strand breaks induce p21, which blocks cyclin-cdk complexes past R point. When damage is repaired, p21 block is removed o p21 also binds to Proliferating Cell Nuclear Antigen (PCNA) and halts DNA synthesis until repair is completed Cell Cycle Control by Mitogens Mitogens oppose effect of CDKIs stimulate growth Mitogens stimulates receptor tyrosine kinase, which activates PI3K pathway Akt/PKB phosphorylates p21 and p27, confining them to the cytoplasm and reducing inhibition of cell cycle Control by TGF-β and mitogens 85