Cell Signaling & The Cell Cycle Lecture Notes PDF

https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling Ext...

https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling https://www.youtube.com/watch?v=XOaiWl-nW1k Overview of cell structure Chapter 17 Cell Signaling Extracellular Signal Molecules Bind to Specific Receptors Introduction Loading… In general, cells can only respond to a signal if they possess ____________ receptor Types of Receptors Receptor types include: – ________ Steroid hormone receptors – G-protein _______ coupled receptors (GPCRs) – Loading… Receptor protein-______ tyrosine kinases (RTKs) – Non-Receptor protein-tyrosine kinases – Ligand gated channels – Specific receptors such as B-and T-cell receptors  Besides kinases and phosphatases, GTP-binding proteins are common to signaling pathways A hormone binding to a GPCR as below stimulates thyroid cell proliferation. How would you expect inhibitors of cAMP Phosphodiesterase (PDE) to impact the proliferation? Figure 16.15 Association of downstream signaling molecules with receptor tyrosine kinases 1. What type of receptor is this? 2. ___________ domains bind to specific phosphotyrosine-containing peptide sequences of the activated receptors. Figure 16.16 Activation of ______________ tyrosine kinases Confusing name: these are receptors similar to RTK’s, but kinase activity is not intrinsic to the receptor, but in an associated protein kinase. Ligand binding induces dimerization of receptor with its associated (nonreceptor) tyrosine kinases Dimerization leads to the activation of the associated nonreceptor tyrosine kinases via cross-phosphorylation. The activated kinases then phosphorylate tyrosine residues of the receptor, creating phosphotyrosine-binding sites for downstream signaling molecules. Figure 16.18 Integrin signaling via FAK – a non-receptor tyrosine kinase Integrins link cells to ECM. Also serve as receptors to activate signaling pathways Can control movement and other aspects of cell behaviour in response to cell- matrix interactions. Binding of integrins to the extracellular matrix leads to integrin clustering and activation of the nonreceptor tyrosine kinase FAK by autophosphorylation. Src binds to the FAK autophosphorylation site and phosphorylates FAK on additional tyrosine residues Those serve as binding sites for additional downstream signaling molecules. Figure 16.19 Ras proteins and Ras-MAP kinase cascades Ras – Ras is a G protein embedded in the membrane by a lipid anchor. – Ras is active when bound to GTP and inactive when bound to GDP. Loading… – Ras-GTP binds and activates downstream signaling proteins – Approximately 30% of human cancers contain mutant versions of Ras that cannot hydrolyze GTP to GDP. – Oncogenic Ras leads to unrestrained cell proliferation, a hallmark of cancer What is an oncogene? The steps of a generalized Ras-MAP kinase cascade 1. Binding of growth factor 2. Autophosphorylation of receptor 3. Recruitment of Grb2-Sos (Sos is GEF for Ras) GEF 4. Activation of Ras 5. Recruitment of RAF to membrane by RAS. Phosphorylation and activation of RAF. 5-7. 3 step phosphorylation scheme resulting in an activated transcription factor 8. MAPK translocates into nucleus and phosphorylates and activates TFs 9. Increase in transcription of specific genes involved in cell proliferation. 3 kinase scheme is characteristic of all MAP kinase cascades Figure 16.20 Activation of Ras, Raf, and ERK downstream of receptor tyrosine kinases Growth factor binding leads to autophosphorylation and formation of binding sites for the SH2 domain of a guanine nucleotide exchange factor (GEF). GEF recruited to the plasma membrane, where it stimulates Ras GDP/GTP exchange. The activated Ras–GTP complex activates the Raf protein kinase. Raf phosphorylates and activates MEK, MEK activates and phosphorylates ERK ERK phosphorylates a variety of nuclear and cytoplasmic target proteins. Figure 17.21 Induction of immediate-early genes expression by ERK- an example Activated ERK translocates to the nucleus, phosphorylates the transcription factor Elk-1. Elk-1 binds to the serum response element (SRE) in a complex with serum response factor (SRF). Phosphorylation stimulates the activity of Elk-1 as a transcriptional activator, leading to immediate-early gene induction. Figure 17.22 Pathways of MAP kinase activation in mammalian cells Cells have multiple MAP- kinase cascades that control distinct cellular processes Do not memorize Figure 17.23 A scaffold protein for the ERK MAP kinase cascade Scaffold proteins organize signaling cascades Why? https://www.youtube.com/watch?v=oDjDUUhGVsI Mapk signaling  Reminder: Second messengers Substance released into interior of cell because of binding of the first messenger (ligand) to receptor on outer surface of cell First messengers bind to a single receptor species, second messengers may affect a variety of cellular activities Second messengers include phospholipid- derived molecules, nitric oxide, cAMP, … Phosphatidylinositol-Derived Second Messengers PI PIP PIP2 PIP3 Figure 16.24 The PI 3-kinase/Akt pathway A major pathway of signaling downstream of RTKs is based on a second messenger derived from the conversion membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to PIP3. PI 3-kinase recruited to RTK. Phosphorylates PIP2 to PIP3. Akt recruited to PM by its pleckstrin homology (PH) domain binding to PIP3. Akt activated by two other protein kinases (PDK1 and mTORC2) that also bind PIP3. Akt phosphorylates a number of target proteins That regulate proliferation and survival. Don’t memorize gene names, know generalities Figure 17.25 Regulation of FOXO - a target of Akt In the absence of growth factor, the FOXO transcription factor translocates to the nucleus and induces target gene expression. Growth factor stimulation leads to activation of Akt, which phosphorylates FOXO. This creates binding sites for the cytosolic chaperone 14-3- 3, which sequesters FOXO in an inactive form in cytoplasm. Figure 16.28 NF-κB signaling (a transcription factor) from the TNF receptor (unique receptor family) From previous lectures: ubiquitination, proteosome, NF-kB localization Activation of the tumor necrosis factor (TNF) receptor leads to the recruitment of adaptor proteins that activate IκB kinase. Phosphorylation (by IκB kinase) marks IκB for ubiquitylation and degradation by the proteasome NF-κB can translocate to the nucleus and activate transcription of its target genes. Proteosome inhibitor? Figure 16.31 Feedback inhibition of NF-κB Feedback loops and signaling dynamics One of the genes activated by NF-κB encodes IκB, generating a feedback loop that inhibits NF- κB activity. Figure 16.32 Crosstalk between the ERK and PI 3-kinase signaling pathways Crosstalk- the interaction of one signaling cascade The Ras/Raf/MEK/ERK with another and PI 3- kinase/Akt/mTORC1 pathways are connected by both positive and negative crosstalk https://www.youtube.com/watch?v=89W6uACEb7M Cell signaling Chapter 17 The Eukaryotic Cell Cycle Introduction Cells reproduce by the process of cell division. Cell division does not stop with the formation of the mature organism but continues in certain tissues throughout life. M_____ isos leads to cells that are genetically identical to their parent and serves as the basis for producing new cells. M____ eiosis leads to production of cells with half of the genetic content of the parent and is basis for producing new sexually reproducing organisms.  The Cell Cycle In vivo Cell cycles in vivo – Three cell types can be distinguished based on their capacity to grow and divide. Cells, such as nerve cells, muscle cells, or red blood cells, that are highly specialized and lack the ability to divide. Once these cells have differentiated, they remain in that state until they die. Cells that normally do not divide but can be induced to begin DNA synthesis and divide when given an appropriate stimulus. Examples include liver cells and lymphocytes. Cells that normally possess a relatively high level of mitotic activity. These include hematopoietic stem cells that produce blood cells and somatic stem cells that can divide and differentiate into epithelial cells. Cell cycles can range in length from 30 min (frog embryo) to several https://learn.genetics.utah.edu/content/cells/scale/ months in slowly growing tissues (e.g. mammalian liver). Figure 17.1 Phases of the cell cycle Figure 17.2 Embryonic cell cycles During early embryonic cell cycles, the egg cytoplasm is rapidly divided into smaller cells. These cells lack G1 and G2 phases and do not grow. Cell cycle consists of short S phases alternating with M phases. Figure 17.3 Determination of cellular DNA content Population of cells labeled with a fluorescent dye that binds DNA. Cells are passed through a flow cytometer, which measures the fluorescence intensity of individual cells. Data are plotted as cell number versus fluorescence intensity, which is proportional to DNA content. Two peaks corresponding to cells with DNA contents of 2n and 4n; these cells are in the G1 and G2/M phases of the cycle, respectively. Loading… Cells in S phase have DNA contents between 2n and 4n. Figure 17.4 Regulation of the cell cycle of budding yeast- yeast has been a primary model for cell cycle studies The cell cycle is regulated by growth and extracellular signals The cell cycle of Saccharomyces cerevisiae is regulated primarily at a point in G1 called START. Passage through START is controlled by the availability of nutrients, mating factors, and cell size. Yeast divide by budding. Figure 17.5 Regulation of animal cell cycles by growth factors Restriction Point in animal cells is similar to START in yeast If growth factors are not available during G1, the cells enter a quiescent stage of the cycle called G0. Figure 17.7 Cell cycle checkpoints monitor progress through the cell cycle Checkpoints ensure that damaged or incompletely replicated DNA is not passed on to daughter cells. DNA damage checkpoints in G1, S, and G2 lead to cell cycle arrest in response to damaged or unreplicated DNA. The spindle assembly checkpoint arrests mitosis if the chromosomes are not properly aligned on the mitotic spindle. Checkpoints are surveillance mechanisms that halt the progress of the cell cycle if 1) chromosomal DNA is damaged or 2) other critical processes have not been completed, such as replication during S phase or chromosome alignment during M phase – If a checkpoint sensor detects a defect, it triggers a response that temporarily arrests the cell cycle – If DNA damage to severe, can transmit a signal that leads to death of cell or conversion to permanent cell cycle arrest (senescence) Regulators of cell cycle progression Eukaryotic cell cycles are controlled by a conserved set of kinases (CDKs) and their regulatory subunits (Cyclins) that trigger the major cell cycle transitions. Studies in: frog oocytes Yeast Sea urchin embryos were key to this discovery. Regulators of cell cycle progression: Identification of MPF Frog oocytes arrested in G2, progesterone results in entry into M phase. Exp: G2-arrested oocytes (recipient oocytes) microinjected with cytoplasm extracted from oocytes that had undergone the transition from G2 to M (donor oocytes). Induced the G2 to M transition in the absence of hormone (progesterone) Interpretation: a cytoplasmic factor, called maturation promoting factor (MPF), can induce entry into the M phase of meiosis. Key Experiment, Ch. 17 Can you read this graph as it relates to the previous slide? Figure 17.9 Properties of S. cerevisiae cdc28 mutants Temperature sensitive mutants defective in cell cycle progression were important tools in cell cycle research. Why is this a Ts mutant? Yeast Cdc28 is a conserved protein kinase known as Cdk1 (a cyclin-dependent kinase) Figure 17.10 Accumulation and degradation of cyclins in sea urchin embryos The cyclins accumulate throughout interphase and are rapidly degraded toward the end of mitosis. The totality of studies tell us…. Cell Cycle Control Cyclin-dependent kinases (Cdks) Protein kinases are essential in regulation of the cell cycle. Entry into the M phase in mammals is triggered by activation of a protein kinase, maturation promoting factor (MPF), a type of Cdk. Cdks have two subunits: a kinase and a regulatory subunit, cyclin. Fluctuations of cyclin and MPF levels Increased concentration of cyclin during the cell cycle. activates the kinase. The cyclin levels fluctuate predictably during the cell cycle. Figure 17.11 Structure of MPF MPF is a dimer consisting of cyclin B and the Cdk1 protein kinase Figure 17.12 MPF regulation by phosphorylation and cyclin association Cdk1 complexes with cyclin B during G2. Cdk1 is phosphorylated on Thr161, which is required for Cdk1 activity, and on Tyr15 and Thr14—which inhibits Cdk1 activity. Dephosphorylation of Tyr15 and Thr14 activates MPF at G2 to M transition. MPF activity is terminated toward end of M by proteolytic degradation of cyclin B, mediated by ubiquitin ligase called anaphase promoting complex (APC) Followed by dephosphorylation of Cdk1. Figure 17.13 Complexes of cyclins and cyclin-dependent kinases Multiple cyclins and Cdks drive progression through the phases of the cell cycle. In animal cells, progression through: -the G1 restriction point -the G1 to S transition -through S phase -G2 to M transition  Cell Cycle Control Cyclin Binding – Cyclin binds to the catalytic subunit of Cdk. – Different cyclins are transcribed at different points in the cell cycle. – Cyclin-Cdk complexes phosphorylate and thereby regulate other proteins involved in cell divsion. Table 17.1 Cdk Inhibitors Cdks can also be regulated by inhibitors What else regulates Cdk activity? Figure 17.14 Induction of D-type cyclins D-type cyclins are a critical link between growth factor signaling and cell cycle progression through the restriction point Growth factors induce synthesis of D-type cyclins via the Ras/Raf/MEK/ERK signaling pathway Upregulation of Cyclin D linked to many cancers Remember…. Figure 17.15 Cell cycle regulation: Rb is a target of cdk4,6/cyclin D In its underphosphorylated form, Rb binds to members of the E2F family, repressing transcription of E2F-regulated genes. Phosphorylation of Rb by Cdk4, 6/cyclin D results in its dissociation from E2F. E2F stimulates expression of target genes encoding proteins required for cell cycle progression. Rb is associated with many cancers. Prototype of tumor suppressor genes