CSF BIOL 3040 Exam 3 Learning Objectives

Principles of Cell Signaling: Extracellular signals, intracellular signaling pathways, effector proteins (Fig, 15-1)

• Extracellular signals - distance- paracrine (autocrine, contact-dependent, synaptic and endocrine (Fig. 15-2)

• Extracellular signals bind to specific receptors that are transmembrane on the target cells or inside the target cell. (Fig.15-3)

• Each cell responds to specific combinations of signals. (Fig. 15-4)

of signals. (15-10

• The same signal molecule can have different effects on different types of target cells (example - acetylcholine). (Fig. 15-5)

• Three classes of cell-surface receptors: Ion-channel coupled receptor, G-protein coupled receptors, Enzyme-coupled receptors. (Fig. 15-6)

• Cell-surface receptors relay signals via intracellular signaling molecules (second messengers- cAMP, calcium, DAG, IP3), protein molecular switches (switch on or off) regulated by kinases-serine-threonine kinases or tyrosine kinases, phosphatases, molecular switch- GTP-binding proteins, (Fig. 15-7) , GDP to GTP exchange -GEFs or GTP hydrolysis to GDP - GAPs, Monomeric GTPases (Fig. 15-8), sequence of two negative signals produces a positive signal (Fig. 15-9).

• Intracellular signals must be precise in a noisy cytoplasm, specificity- high affinity between signal proteins and binding proteins, only respond to high concentrations of signals, back-up activated signaling pathways that maintain robustness

Intracellular signaling complexes form at activated receptors- scaffold proteins, signaling complex assembles transiently on the receptor only after the binding of extracellular signal, activation of receptors leads to increased phosphorylation of specific phospholipids in the plasma membrane nearby (Fig. 15-10)

• Molecular interaction domains mediate interactions between intracellular signaling proteins -SH2, PTB -SH3, PH, adaptors (Fig. 15-11)

• Formation of large receptor clusters by multivalent interactions between proteins (Fig. 15-12)

• Relationship between signal and response varies in signaling pathways based on (7 points) : response timing, sensitivity, dynamic range of signaling-adaptation, persistence- positive feedback, signal processing, integration

of signals (Fig. 15-13), coordination of multiple responses.

• Speed of a response depends on the type of intracellular signaling change: phosphorylation, membrane potential, transcription, synthesis, degradation (Fig. 15-14), changes in intracellular signal protein concentrations depend on rate of synthesis of protein and the rate at which the protein degrades (Fig. 15-15)

• Types of responses to a gradually increasing signal - sigmoidal response, all-or-none response, hyperbolic response (Fig. 15-16)

• Positive feedback can generate All-or-None response (Fig. 15-17, Fig. 15-18)

• Negative feedback - short delay, long-delay (oscillatory response). (Fig. 15-19)

• Importance of examining individual cells to detect all or none responses. (Fig. 15-20)

• Cells can adjust their sensitivity to a signal - adaptation or de-sensitization, Fig. 15-21

• Signaling through G-protein coupled receptors (GPCRs) - (Fig. 15-22), Trimeric G-proteins relay signals from GPCRs - G-alpha, G-beta-gamma, GAPs, GEFs, (Fig. 15-22, Fig. 15-23), cAMP, Adenylyl cyclase, cAMP-

phosphodiesterase stimulatory G protein, inhibitory G protein, cholera toxin example (slide) (Fig, 15-24).

• Increase in cAMP in response to signal and ynthesis and degradation of cAMP (Fig. 15-25, 15-26).

• C-AMP-dependent activation of PKA (Fig. 15-27), cAMP binds and releases regulatory subunits from catalytic subunits of PKA that activates catalytic subunit, CRE response, (Fig. 15-28)

• Some G-proteins signal via phospholipids - PLC-beta - phosphatidylinositol 4,5-bisphosphate, IP3, DAG, PKC, calcium releases (Fig. 15-29), (Fig. 15-30)

• Nitric Oxide gas can mediate signaling between cells. (15-41)

• GPCR desensitization depends on receptor phosphorylation- GPCR kinase (GRK), Arrestin (Fig. 15-43)

Signaling through enzyme-coupled receptors - Receptor Tyrosine kinases (RTKs), Some subfamilies of RTKs (Fig.15-44), Activation of RTKs by dimerization, phosphorylated tyrosines on RTKs serve as docking sites for intracellular signaling proteins (Fig. 15-45), Activation of the EGF receptor kinase (Fig. 15-46), proteins with SH2 domains , PTB bind to phosphorylated tyrosines - phospholipase C-gamma (Fig. 15-47

GTPase Ras signaling mediates signaling by most RTKs (Fig. 15-48), GEFs, GAPs, adaptor, Ras mutations- cancer, transient activation of Ras (Fig. 15-49), MAPKKK, MAPKK, MAPK (Fig. 15-50), MAPK modules by scaffold proteins

in yeast-example (Fig. 15-51

• PI-3 kinase signaling -PI(3,4,5)P3 - PH domains (Fig. 15-53), PI3-Kinase-AKT signaling (Fig. 15-54), mTOR signaling (Fig. 15-55)

• RTKs and GPCRs activate overlapping signaling pathways (Fig. 15-56)

• Alternative signaling routes in gene regulation - Notch receptor- contact-dependent, Delta, gamma-secretase (Alzheimer's example), receptor cleavage (Fig. 15-59, 15-60)

• Wnt signaling pathway (Fig. 15-61)

• Nuclear receptors are ligand modulated transcription regulators, hydrophobic signal molecules (Fig. 15-65), activation of nuclear receptors (Fig. 15-66)

• Four phases of cell cycle- G1, S, G2 and M (G0-resting phase and late G1- Start/Restriction point) (Fig. 17-1, 17-2, 17-3, 17-4).

• Tools for studying cell cycle progression - Microscopy- Cell Morphology observations, BrdU- S phase (fluorescent antibodies that recognize BrdU), Flow Cytometry for cell cycle profile based on analysis of DNA content. (Fig. 17-5, 17-6, and 17-7).

• Cell cycle control system- checkpoints in different phases (Fig. 17-8), Cell cycle control by Cyclin-Cdks (Fig. 17-9, 17-10), Four classes of Cyclins- G1, G1/S, S and M-phase cyclins, Cyclins levels change throughout cell cycle (Fig. 17-10). Four Cdks in vertebrates and they associate with different cyclins (Table 17-1). Structural basis of Cdk activation- T-loops, Cyclins and CAK (Fig. 17-11).

• Regulation of Cdk activity by phosphorylation- inhibitory Wee1 kinase phosphorylations are removed by Cdc25 phosphatase to activate Cdks. (Fig. 17-12). Binding of CKIs inactivate Cdk Cyclin complexes- example-p27- Fig. 17-13.

• Protein Phosphatases reverse the effect of CDKs- PP2A subunits (scaffold, catalytic and regulatory-B55 or B56). Regulation of PP2A-B55 by M-Cdk in mitosis through Greatwall and Ensa (17-14 and 17-15)

• Cell-cycle control system function as biochemical switches - positive feedback and mitotic regulatory circuit (Fig. 17-16, and 17-17)

Protein degradation by APC/C complex-ubiquitin ligase- plays an important role in the final stages of mitosis at metaphase to anaphase transition. APC/C destroys S and M phase cyclins. APC/C activated in mid-mitosis and turned off in late G1. APC/C activity regulated by binding to Cdc20 in mid-mitosis and by binding to Cdh1 in late mitosis through early G1. SCF-ubiquitin ligase have the F-box factors that bind to substrates such as CKIs (Fig, 17-18, 17-19 and Table 17- 2).

• Sequential activation of Cdks during cell cycle (17-20).

• S phase- DNA synthesis phase. S-Cdk initiates DNA replication once per cell cycle. Control of chromosome duplication. Pre-replicative (preRC) complexes are loaded with inactive helicases in early G1. (Fig. 17-21, 17-22).

• Replication initiation- ORC (origin recognition complex) stays bound to Ori elements and with the help of Cdc6 recruits the MCM -DNA helicase. Cdt1 also loads helicase. S-Cdk activates helicase and inactivates ORC, Cdt1 and Cdc6 by different mechanisms to prevent re-firing of origins till next S-phase. Chromatin structure is also duplicated and marked on newly synthesized DNA. However, the exact mechanism is unknown. (Fig. 17-21, 17-22).

• Cohesins (SMC and SCC subunits) forms a ring complex surrounding sister chromatids that hold them together till metaphase. DNA catenation also keeps newly synthesized DNA strands together but removed by early M. (Fig. 17-23).

• Mitosis- Stages of mitosis-prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis. Activation of M-Cdk by M-cyclin binding, CAK and removal of Wee1 kinase inhibitory phosphorylation by Cdc25 phosphatase.

Negative and positive feedback by M-Cdk. (panel 17-1, Fig. 17-16)

• Condensin - five subunit complex helps in chromosome condensation and sister chromatid resolution (Fig. 17-24,

17-25).

• Mitotic spindle- astral microtubules, non-kinetochore or interpolar microtubules, kinetochore microtubules (Fig. 17- 26). Centrosome duplication and organization- spindle poles (Fig. 17-27), motor proteins govern spindle assembly and function- Kinesin-5, kinesin-14, kinesin-4/10 and dynein (Fig. 17-28). Centrosome duplication (S phase)- semiconservative-centriole replication and separation, centrosome maturation- increase in gamma-tubulin ring complexes (Fig. 17-29)

• M-Cdk initiates spindle assembly- activates centrosome separation and kinesin-5 activation. Other kinases - Aurora kinase and plk also important for mitosis. M-Cdk phosphorylates lamins and breaks down nuclear lamina/membrane to help spindle attachment. Mitotic chromosomes promote bipolar spindle assembly with help of motor proteins, Ran-GTPase (Fig. 17-30) - acentrosomal spindle organization by motor proteins (Fig. 17-31, 17-32)

• Kinetochore attach sister chromatids to spindle- lateral attachment to microtubules by Ndc80 complex (Fig. 17-33, 17-34). After trials, microtubules plus ends capture the kinetochores in correct end-on orientation (Fig. 17-35). Biorientation is achieved by trial and error- tension stabilizes the correct bipolar attachment and this requires Aurora-B kinase (Fig. 17-36)

• Multiple forces act on chromosome in the spindle and maintain them in metaphase plate- two poleward forces (microtubule flux) and polar ejection force or polar wind -kinesin-4 and kinesin-10 motor proteins (Fig. 17-37).

• APC/C triggers sister chromatid separation- separase activation by degradation of securin and by inhibition of Cdk. Cohesin cleaved by separase. APC/C activity stimulated by M-Cdk phosphorylation and increase in Cdc20 levels in mitosis. (Fig. 17-38, 17-39)

• Improperly attached chromosomes block sister chromatid separation- activation of spindle assembly checkpoint- Mad2) prevents metaphase to anaphase transition (Fig. 17-40)

• Chromosome segregation in Anaphase A and Anaphase B -stages and differences (Fig. 17-41)

• Segregated chromosomes packaged in nuclei-telophase.

Cytokinesis- contractile ring formation- actin-myosin filaments, myosin filaments introduction (Chapter 16) cleavage furrow formation (Fig. 17-42, 17-43), midbody (Fig. 17-44).

• Microtubules of mitotic spindle determine plane of animal cell division: Rho GTPase signaling, Ect2, Centralspindilin (Fig. 17-45, 17-46, 17-47).

• Phragmoplast guides cytokinesis in higher plants (Fig. 17-48, 17-49).

• Membrane bound organelles must be distributed to daughter cells. Some cells reposition their spindle to divide asymmetrically (Fig. 17-50). Mitosis can occur without cytokinesis (Fig. 17-51).

Control of cell division and growth- mitogens-e.g. PDGF, growth factors and survival factors., Mitogens stimulate G1-Cdk and G1/S-Cdk activities, Ras-Myc-G1-Cdk-Rb (retinoblastoma) inhibition- E2F activation- G1/S cyclin synthesis-S-Cdk activation and positive feedback mechanisms (Fig. 17-59).

• DNA damage arrests cell cycle in G1, S and G2 phases (Fig. 17-60 and lecture slides on Chk1/Cdc25), p53-p21, p21 (CKI), Mdm2-p53, Chk1-Cdc25 pathways (Fig. 17-60).

• Cell growth regulation- PI3K-mTOR- activation (Fig. 17-61).

• Potential mechanisms for coordinating cell growth and division (Fig. 17-62).

• Cancer-definition, history of cancer (PPT. slides), traits of cancer- neoplasms, benign, malignant, metastasis (Fig. 20-1), cancer types, cancer incidence and mortality in US (Fig. 20-2), benign versus malignant tumors (Fig. 20-3), growth of a typical human tumor (Fig. 20-4), most cancers derive from single abnormal cell, Philadelphia chromosome (Fig. 20-5).

• Cancer cells contain somatic mutations, germline mutations, epigenetic changes, several successive mutations and age-dependency (Fig, 20-6), gradual development of cancers (E.g. lung cancer-Fig. 20-7).

• Stages of tumor progression (Fig. 20-8), clonal evolution (Fig. 20-9)-tumor heterogeneity, cancer as a microevolutionary process- natural selection (PPT. slide), genetic instability of tumors (Fig. 20-10), chromosome segregation defects-chromothripsis and aneuploidy (Fig. 20-11).

• Small population of stem cells in some cancers: lifetime of cancer cells is coordinated with division rates (Fig. 20-12), Cancer stem cells (Fig. 20-13).

• Summary of properties of cancer, Increased division and decreased death contribute to tumor growth (Fig. 20-14), cancer cells display altered proliferation, growth, lack of contact inhibition (Fig. 20-15), direction of

cell extrusion decides fate (Fig. 20-16), replicative senescence-bypass (telomerase) (PPT. slide), cell death - necrosis and anoxia in large tumor mass(20-17). Altered sugar metabolism- increased glycolysis- Warburg

effect (Fig. 20-18)

• Tumor stroma -microenvironment (Fig. 20-19) and its influence on tumor growth. Invasiveness and metastasis, CTCs, (Fig. 20-20)- EMT-epithelial to mesenchymal transition and MET- mesenchymal to epithelial transition (PPT. slide).

Cancer-critical genes- drivers versus passengers, gain of function versus loss of function mutations- oncogenes vs. tumor suppressors, (Fig. 20-21), tumor viruses-retroviruses-Rous Sarcoma Virus (PPT. slides), proto-oncogene to oncogene activation (Fig. 20-22), Ras oncogenic activation (PPT. slide), EGFR mutation (Fig. 20-23), Myc overproduction (Burkitt's lymphoma) (PPT. slide).

Tumor suppressor genes- Rb-hereditary and non-hereditary (Fig, 20-24), 6 ways of losing the remaining good copy of tumor suppressor gene (Fig. 20-25).

Sequencing of cancer genomes -distinct sequence changes in oncogenes versus tumor suppressors(Fig. 20-26), prevalence of aneuploidy (Fig. 20-27). Epigenetic changes- methylation and heterochromatin-silencing of tumor suppressor (PPT. slide Fig. and Fig.20-28). About 1% of genes in human genome are cancer-critical. Passenger versus driver mutations. Disruptions of few key pathways are common to many cancers (Fig, 20-29).

• Mutations in the PI3K/AKT/mTOR pathway -cancer cell growth and PTEN phosphatase (tumor suppressor) (Fig. 20- 30 and PPT. slide Fig). p53 pathway mutations (Fig. 20-31 and PPT. slide Fig). Different mechanisms such as mutations in EGF-receptor can activate the common Ras, PI3K pathways. Mouse models help to study cancer - luciferase imaging-e.g. prostrate cancer (Fig. 20-32). Oncogene collaboration in transgenic mice (Fig. 20-33). Heterogeneity in cancers as they progress (PPT. Slide).

• Development of colorectal cancers slowly by succession of mutations (Table 20-1), hereditary and non-hereditary colon cancers- APC mutations, FAP, HNPCC, mismatch repair mutations (Fig. 20-35).

• Colon tumor progression usually involves a series of mutations as the tumor develops (Apc loss, activation of Ras,

Smad4 loss, p53 loss) (Fig. 20-36).

• Barriers to metastasis and metastatic changes not completely understood (EMT-epithelial to mesenchymal

transition).

• Cancer prevention and treatment- some cancer incidence is related to avoidable environment factors (Fig. 20-37), some to random replication errors, Carcinogens- Ames test (Fig. PPT slide), carcinogen activation - Aflatoxin. Age- adjusted cancer death rates (Fig. 20-39). Viruses cause some cancers (DNA tumor viruses and RNA viruses) (Table 20- 2), papillomaviruses (Fig. 20-40-HPV), traditional therapies and cell cycle checkpoint responses.

• Drugs targeting oncogene addiction by targeting oncogene- E.g. Philadelphia chromosome- Bcr-Abl fusion oncogene

and Gleevac inhibitor, (Fig. 20-42, 20-43), Inhibitors of Ras-Mapk pathway (Fig. 20-44).

• Cancer drug resistance and combinatorial therapy and personalized cancer treatment (PPT. slides).