D103 Final Review
• Signaling enables a cell to receive information from its intra-cellular or extra-cellular environment and to respond to the signal.
• Most signals cannot cross the plasma membrane, thus require a plasma membrane localized receptor to transduce the signal.
• Metazoans use similar types of signaling that evolved from early primitive multi-cellular organisms. So, different model systems can be used to study the signaling.
• Abnormal signaling can have dramatic effect on both development and tissue homeostasis.
• The principles of signaling can be understood through familiarity with the mechanisms and function of conserved components used in signaling pathways.
• Individual components of a signaling pathway may be shared with other pathways.
• Many signaling molecules can be produced or removed quickly either by de novo synthesis and degradation, or by release and sequestration.
• Different cell types have different combinations of signaling machinery components that defines a cell response to a stimulus.
• Signal transduction routinely involves reversible changes in the activity and conformation of a protein structure due to non-covalent or covalent association with small molecules – e.g., phosphate, Ca 2+ , lipids.
• Modular domains composed of a series of conserved amino acids mediate interactions between intracellular signaling proteins and associated molecules.
• Nitric oxide (NO) gas acts as a signaling molecule in numerous mammalian tissues, including blood vessels.
• Cellular response to steroid hormones can be rapid (independent of transcription) or slow (transcription dependent).
• Lipid soluble hormones can diffuse through the plasma membrane and bind to nuclear hormone receptors that cause changes in gene expression.
• Nuclear hormone receptors usually have three important functional domains – a ligand binding domain; a DNA binding domain; and a transcriptional activation domain.
• Some hormones bind to receptors in the cytosol, while others bind to receptors that are already bound to DNA in the nucleus.
• Steroid hormone receptors are bound by chaperone proteins in the cytosol, which maintains them in an inactive state prior to binding ligand.
• In the absence of ligand, retinoid, vitamin D and thyroid hormone receptors are found in the nucleus where they usually prevent transcription. Upon binding of ligand, the receptors are converted to transcriptional activators.
• The steroid hormone intracellular receptor proteins function as both signal transducers and transcription factors to modulate expression of target genes.
• Dysfunction in nuclear hormone receptors or abuse of anabolic steroids can have dramatic effects on human development and homeostasis.
• GPCR are a large class of receptor proteins that each contains seven transmembrane a-helices.
• These receptors couple to heterotrimeric G-proteins, composed of an a, b and g subunits.
• GPCRs activate heterotrimeric G-proteins which in turn modulate the activity of plasma membrane-bound effector enzymes or ion channels.
• Stimulation of G-proteins by an activated receptor causes the G-protein a and bg subunits to undergo a conformational change, which results in the a-subunit exchanging GDP for GTP. Upon GTP bound, the a- subunit can transmit a signal.
• Usually, the a subunit regulates target effector proteins in the plasma membrane, although less frequently the bg subunits can also regulate a target effector protein.
• Target effectors of G-proteins include adenylyl cyclase, PLCb, cGMP phosphodiesterase and ion channels.
• GPCR signaling can be desensitized by a family of receptor specific kinases (GRKs).
• GPCR-associated signaling events play essential roles in development and tissue homeostasis.
• GPCR are dysregulated in many diseases (e.g., affected by common bacterial toxins).
• GPCRs are important drug targets.
• Second messengers comprise a wide range of small molecules.
• Messages are encoded by the change of their concentration, which conveys a signal from a source to a target.
• The concentration of second messenger is determined by a combination of its rate of synthesis and degradation via specific enzymes.
• Different second messengers function in discrete sub-cellular compartments (e.g., Ca2+ in cytosol, DAG in membrane).
• cAMP and cGMP are formed and degraded by similar enzymes and share similar effector proteins (i.e., protein kinases), suggesting their common evolutionary origin.
• Phospholipases, lipid kinases and lipid phosphatases regulate lipid second messengers.
• TGF-b receptor, cytokine receptor and receptor tyrosine kinases are important cell surface receptor family members that transduce signals into the interior of the cell
• Each class of receptor has multiple members and multiple ligands. They have broad functions in development and tissue homeostasis of multi-cellular species.
• TGF-b and cytokine receptors transduce signals to the nucleus via gene regulatory proteins, including Smads (TGF-b) and STATs (cytokine)
• Receptor tyrosine kinases transduce signals to the nucleus via the signaling pathways like Ras-MAPK pathway
• These signal transduction pathways share similar principles
phosphorylation of amino acids as a method to regulate binding of other proteins to the cytoplasmic tails of the transmembrane receptors
conformational change of proteins as the cause or a consequence of their interaction
initially low kinase activity that can be enhanced by a phosphorylation-dependent conformational change in a kinase domain
masking of a protein targeting sequence (e.g., NLS) as a method to control sub-cellular localization
de-phosphorylation or ubiquitination-mediated degradation as a method of regulating activity
• Defects in these signaling pathways are associated with common human genetic disorders including developmental defects and cancer
• Signaling pathways have evolved specific behavior to suit the cellular function controlled by that system
• Signaling systems can respond continuously or abruptly to a gradually increasing signal
• The speed of a cellular response is dependent on the rate of turnover of signaling molecules
• Allosteric interaction and cooperative binding allow a cell to produce graded activation of signaling processes over a narrow concentration range – e.g., activation of (a) PKA by cAMP; (b) calmodulin by Ca 2+
• Positive feedback can turn a graded response to a signal into a binary response
• Negative feedback can limit the level and duration of a response
• Adaptation allows cells to adjust their sensitivity to an extra-cellular signal, enabling them to respond to the same percentage of change in a signal over a wide range of stimulus strengths
• Cells have evolved several different methods to achieve specificity in response to signals in a “noisy” environment
• Cells need scaffolding proteins and post-translational modification to mediate a specific binding between signaling molecules
• Scaffold proteins allow a cell to control specificity and efficiency of subcellular signaling
• Crosstalk between signaling pathways enhances sophistication of cellular response to individual signals
• The eukaryotic cell cycle consists of four phases - G1, S, G2 and M (shortest)
• Cyclin dependent kinases (CDKs) regulate the biochemical and structural reorganization of the cell as it moves through each phase of the cell cycle
• CDKs have kinase domain, but must interact with cyclins to become active
• Different cyclins help direct CDKs to their correct cellular targets
• During G1, growth factors stimulate cell mass by increasing synthesis of macromolecules while mitogens stimulate cell division by increasing activity of cyclin / CDK
• Phosphorylation of Rb by cyclin/CDK in G1 allows gene expression to drive the cell to enter S-phase
• S-phase cyclin/CDKs control initiation of DNA replication
• During cell cycle, the cellular machinery can identify defects in the integrity of the process at a series of "checkpoints”
• Cells e.g., with incomplete DNA replication or chromosome alignment on spindle cannot pass a checkpoint and must repair the problem or die
• CDK activity can be regulated by cyclins, phosphorylation and binding of CKIs
• Cyclins and other components of the cell cycle machinery are regulated by ubiquitination, which causes degradation of the components in the proteasome
• Degradation of proteins synthesized only once during different stages of the cell cycle is an effective method to ensure the process of cell division to move in only one direction
• Cells in G0 can be stimulated to re-enter the cell cycle at G1
• Cells must exit the cell cycle (to G0) to undergo terminal differentiation
• Biochemical approaches allow the identification of M-cyclin as a key component of maturation promoting factors (MPFs)
• Studies used model systems that provided large quantities of biological material (frogs, sea urchins, surf clams)
• 35 S-methionine labeling of synchronously developing frog and clam embryos plus biochemical fractionation was used to identify newly synthesized molecules that were candidates for MPFs
• Maturation of frog oocytes in vitro was an important assay for MPF activity
• Biochemical fractionation, assay and cloning identified cyclins and cyclin-dependent kinases as components of MPFs
• Cyclins were shown to be synthesized from maternal products during each cell cycle in early Xenopus embryo development
• MPF activity (i.e., M-cyclin/Cdk) is dependent on cyclin B
• Cyclin B must be degraded prior to exiting mitosis
• Cyclin B is degraded by polyubiquitination / proteosome, which is regulated by an Ub-E3 ligase activity called anaphase promoting complex (APC).
• In cells with a G1 phase, Cdh1 is the co-factor for APC required to complete mitosis. Cdh1 is degraded at the G1/S phase of the next cell cycle
• Genetic experimental approaches using yeast identified many important genes that regulate eukaryotic cell division
• The small genome, short life-cycle, haploid phase of life cycle, and ability to grow in large quantity were important factors make yeast as an ideal model for cell cycle study
• Conditional temperature sensitive (ts) mutants enable mutants to be propagated in a haploid state
• Functional complementation of mutant yeast by the transformation with plasmid libraries containing individual yeast genes is a powerful approach to identify the mutated genes
• The phenotype of cdc mutants reveals the stage in the cell cycle when the protein is required
• Cdc2 and Cdc13 constitute MPF in S. pombe
• cdc2 encodes M-Cdk, while cdc13 encodes M-cyclin
• Cdk is regulated by two kinases (Wee1 and CAK), which prepare the MPF for activation during mitosis, and Cdc25, a phosphatase, which "releases the brakes" on MPF at mitosis
• Cyclins are degraded by polyubiquitination / proteosome, which is regulated by an Ub-E3 ligase activity called anaphase promoting complex (APC/C)
• Chromosomes replicate and centrosomes duplicate prior to entry into M-phase
• In prophase, centrosomes separate, and each nucleates one pole of the mitotic spindle
• Chromosomes condense during prophase and the lifespan of MTs shortens due to reduced activity of MT-stabilizing MAPs, with persistent catastrophe activity
• During prophase, intracellular membranes become disassembled e.g., Golgi, mitochondria
• Pro-metaphase begins with the breakdown of the nuclear envelope
• During pro-metaphase, chromosomes begin to become captured by MTs emanating from the developing mitotic spindles
• Once both kinetochores on a chromosome are captured, the chromosome can align on the metaphase plate
• The spindle checkpoint involves signaling from unoccupied kinetochores that prevent anaphase from commencing until all kinetochores are properly attached to the spindle poles
• Separation of sister chromatids is mediated by a protease called separase, which is activated via removal of its inhibitory partner protein securin, by APC/C-Cdc20 E3 ligase complex
• Microtubule motors separate chromatids and elongate the mitotic spindle
• Cytokinesis is the final step of M phase
• Cells will divide as two at the step of cytokinesis
• Actin filaments and myosin II form contract ring to proceed with cytokinesis
• Centraspindlin recruits GEF to midbody and activates RhoA to form contract ring
• Vesicle transport contribute to cytokinesis
• Defect in cytokinesis will form bi-nuclear cells
• Apoptosis regulates cell number in multi-cellular animals by controlling cell death
• Apoptosis and necrosis have distinct morphological features
• Apoptosis removes cells that are unwanted or are potentially dangerous (e.g., pre-cancerous)
• An imbalance in apoptosis is associated with many diseases
• Apoptosis is mediated by a family of proteases called caspases
• Caspases exist as pro-caspases that are activated via either extracellular or intracellular signaling pathways
• The activation involves pro-caspases that have minimal proteolytic activity, being brought into proximity by adaptor molecules
• The BCL2 and Fas family of proteins are involved in control of intrinsic and extrinsic cell death pathways, respectively.
• BCL2 anti- and pro-apoptotic members antagonize each other's function. Survival signals favor the anti-apoptotic members; absence of survival factors enhances pro-apoptotic BCL2 activity
• A key event in intrinsic apoptosis involves release of cytochrome c from mitochondria
• Survival molecules increase cell numbers by preventing apoptosis
• Extracellular survival molecules are often rate-limiting in promoting cell survival
• Extracellular signals that block apoptosis include growth factors, cytokines, cell adhesion molecules
• Cancer is composed of many discrete diseases.
• Development of cancer is a cellular evolutionary process.
• 80 % of human cancer is carcinoma - derived from epithelia where cell division is more frequent and where tissues are exposed to physical and chemical damage.
• Multiple mutagenic events are required for a tumor to progress to full-blown cancer.
• These mutations facilitate rounds of natural selection for cells that are best suited to proliferate and undergo metastasis.
• Cancer cells are genetically unstable and can evade normal controls on cell proliferation, differentiation, senescence and death.
• Cancer cell metabolism is shifted to utilize large amount of glucose despite the presence of oxygen (Warburg effect).
• Cancer-causing genes can be categorized as recessive (e.g., tumor suppressor genes) or dominant (oncogenes).
• Familial (inherited) cancers are often associated with inheritance of a defective copy of a tumor suppressor gene - e.g., Rb in retinoblastoma, BRCA1 in breast cancer, p53 in many forms of cancer.
• A small proportion of individuals infected with certain viruses have increased susceptibility to develop cancer due to presence of viral gene products that interfere with normal cellular signaling system.
• Many different cellular pathways can be affected in cancer cells