BMS100_PHL1.13-F22_Cell signalling II and transcription_STUDENT.pdf

Cell signalling II Intracellular signalling and the central dogma Dr. Rhea Hurnik BMS 100 Outline: Cell signalling II 1) Model 1: second messenger activates a protein kinase cAMP àPKA Ca2+ à CaM Kinase Ras ààà Map Kinase DAG + Ca2+ à PKC PI 3 Kinase-AKT pathway 2) Model 2: Activated cell membrane re...

Cell signalling II Intracellular signalling and the central dogma Dr. Rhea Hurnik BMS 100 Outline: Cell signalling II 1) Model 1: second messenger activates a protein kinase cAMP àPKA Ca2+ à CaM Kinase Ras ààà Map Kinase DAG + Ca2+ à PKC PI 3 Kinase-AKT pathway 2) Model 2: Activated cell membrane receptor directly activates a transcription factor JAK àSTAT Smad 3) Model 3: Pathways involved in regulated proteolysis Wnt-Beta catenin pathway NF𝜅B 4) Model 4: Intracellular receptors Steroid hormones Thyroid hormones 5) Model 5: non-coding RNA affecting transcription miRNA lncRNA Signaling affecting gene transcription The signalling pathways you discussed yesterday were relatively fast, today’s pathways are slower since they involved gene transcription and altered protein synthesis Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-13. Page 826 Pathways affecting transcription regulators Intracellular signalling cascades can affect transcription in a variety of ways: § They can affect transcription factors § They can affect co-activators or co-repressors § They can affect histones remodelling Transcription factor Binds a DNA sequence directly to affect transcription Co-activator/ Co-repressor Binds a transcription factor to affect transcription * Doesn’t binds DNA directly Model 1 Second messenger in a signalling cascade activates a protein kinase involved in transcription regulation + Enzyme 2nd messenger + Protein kinase Protein kinase TF Modifies gene transcription Nucleus Model 1 – shutting it off Second messenger in a signalling cascade activates a protein kinase involved in transcription regulation + Enzyme 2nd messenger + Protein kinase Inhibitory enzyme that will shut off the signal + Protein kinase TF Modifies gene transcription Nucleus 1) i) cAMP An increase in cytosolic cAMP concentration is stimulated by which cell membrane receptor? cAMP activates cAMP-dependent protein kinase (aka PKA) § Where have we seen this before? 1) i) cAMP activates PKA PKA is a multimeric protein § Binding of cAMP causes dissociation of catalytic and regulatory subunits Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-26. Page 835 1) i) cAMP activates PKA PKA is a multimeric protein § Released catalytic subunits are free to phosphorylate specific target proteins Target proteins can include: § Transcription factors => effects occurs over hours § Adjacent phosphodiesterase Rapidly lowers cAMP levels to shut off the signal § Review from last day, also Other enzymes => effect occurs within seconds Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-26. Page 835 1) i) cAMP activate PKA to affect transcription To affect transcription regulators: § 1. Activated PKA enters the nucleus and activates CREB § CREB = CRE-binding protein § How do think PKA activates CREB? CREB is a transcription factor for genes with a CRE § CRE = cAMP response element § CREB stimulates transcription of genes with a CRE Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-27. Page 836 1) i) cAMP activate PKA to affect transcription To affect transcription regulators: § 2. Activated CREB also recruits a co-activator called CREB-binding protein (CBP) Further promotes transcription of a gene with a CRE Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-27. Page 836 1) i) Putting it all together Reminder of the full pathway Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-27. Page 836 1) ii) CaM Kinase Ca2+/ calmodulin-dependent kinases (CaMKinase) can also phosphorylate transcription regulators to increase or decrease transcription § How is CaM-Kinase activated? § CaM-Kinase can also phosphorylate CREB to increase transcription of genes with a CRE CaM-kinase Adapted from Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-27. Page 836 1) ii) Putting it all together Reminder of the full pathway CaM-kinase CaM-kinase Adapted from Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-27. Page 836 and Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-29. Page 838 1) iii) PKC When activated, protein Kinase C (PKC) functions similarly to PKA How was PKC activated? § It phosphorylates target proteins, which can: Activate/inhibit proteins in the cell Activate/inhibit transcription factors OR co-activator/ coinhibitors to alter gene transcription 1) iii) Putting it all together Reminder of the full pathway Activate or inhibit transcription factors Activate or inhibit co-regulators Adapted from Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-29. Page 838 1) iv) Ras Ras is a protein activated by receptor tyrosine kinases § Active Ras then triggers a series of activation-viaphosphorylation reactions ending with the activation of MAP kinase Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-49. Pg 856 1) iv) Ras triggers activation of MAPK MAP Kinase (Erk) can enter the nucleus to phosphorylate transcription factors § This activates transcription factors needed for immediate early genes Some of these newly translated proteins then turn on other genes § Eg. FYI for now –some of these other genes include G1 cyclins needed for the regulation of the cell cycle 1) v) PI 3 Kinase AKT pathway A common pathway for growth factors is the PI 3 Kinase-AKT pathway § Let’s review it! § AKT activates a wide variety of targets, including Various transcription factors § Eg. AKT can activate CREB mTOR complex 1 § Activates a variety of targets, including transcription factors involved in ribosome production for increased protein synthesis 1) v) PI 3 Kinase AKT pathway Can activate transcription factors (eg. CREB) AKT *Note: consecutive inhibition (ie. Inhibition of an inhibitory molecule) results in activation Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 17-64. Pg 1017 Model 2 Activated receptor activates a transcription factor that travels to the nucleus to modify gene transcription + TF TF Modifies gene transcription Nucleus Model 2 – shutting it off Activated receptor activates a transcription factor that travels to the nucleus to modify gene transcription + TF TF Transcription of inhibitory proteins to turn signal off Modifies gene transcription Nucleus 2) i) JAK Janus Kinase (JAK) is a cytosolic tyrosine kinase § JAK is activated by a cytokine ligand binding to its cell membrane receptor JAK phosphorylates and activates transcription factors called STATS § STAT = signal transducers and activators of transcription Once activated, STATS travel to the nucleus and regulate gene transcription 2) i) JAK activates STAT Note the much more direct route to gene transcription than some of the other pathways we’ve looked at. Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-56. Pg 864 2) ii) Smad Initiated by activation of receptor serine/threonine kinases § Activated by TGF-beta and BMP ligands TGF- β = Transforming growth factor Beta BMP = Bone morphogenetic protein § Once activated, the receptor will bind and phosphorylate a transcription factor, Smad The specific Smad activated depends on the ligand Specific Smad proteins are FYI Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-57. Pg 866 2) ii) Smad forms complex Specific Smad proteins are FYI Once Smad is activated, it dissociates from the receptor and forms a complex with a coSmad (co-Smad) § This complex travels to the nucleus and associates with other translation factors and co-regulators to control transcription Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-57. Pg 866 3) Model 3 Activation of a receptor triggers destruction of an inhibitory protein of a transcription factor. The transcription factor can the travel to the nucleus to modify gene transcription + T.F. T.F. Modifies gene transcription Nucleus 3) Model 3 Activation of a receptor triggers destruction of an inhibitory protein of a transcription factor. The transcription factor can the travel to the nucleus to modify gene transcription + Protein Protein Transcription of inhibitory proteins to turn signal off Modifies gene transcription Nucleus 3) i) Without Wnt Wnt regulates the proteolysis of a multi-functional protein β-catenin § Without Wnt signalling, β-catenin is phosphorylated and targeted via ubiquitylation for destruction by a βcatenin degradation complex One protein in this complex is APC Ubiquitylation targets β-catenin for destruction by proteosome § Wnt-responsive genes are kept silent by an inhibitory complex of transcription regulatory proteins Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-60. Pg 870 Eg. FYI (for now) Myc **All molecules except Beta-catenin & APC are FYI 3) i) Wnt binding frees Beta-catenin Wnt regulates the proteolysis of a multi-functional protein βcatenin § With Wnt signalling, the β-catenin degradation complex is disrupted § Unphosphorylated β-catenin travels to the nucleus § Binding of β-catenin displaces the co-repressor (FYI “Groucho”) and functions as a co-activator th Molecular Biology of the Cell (Alberts et al) 6 ed. Figure 15-60. Pg 870 Eg. FYI (for now) Myc 3) ii) NF𝜅B In response to acute inflammatory ligands (IL-1, TNF⍺), a cell surface receptor is activated § Activated receptor triggers the ubiquitylation and phosphorylation to release and destroy an inhibitory protein complex (FYI – I𝜅B) bound to NF𝜅B Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-62. Pg 874 **All molecules except NFkB are FYI 3) ii) NFkB In response to acute inflammatory ligands (IL-1, TNFalpha), a cell surface receptor is activated § NF𝜅B travels to the nucleus and initiates transcription of NF𝜅B-responsive genes Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-62. Pg 874 **All molecules except NFkB are FYI 4) Intracellular receptors Small, hydrophobic ligands don’t need cell surface receptors since they can easily diffuse across the plasma membrane § Ligands: Steroid hormones, thyroid hormones, retinoids, vitamin D § Their receptors are located inside the cell Receptors are all structurally similar and are part of a nuclear receptor superfamily **Structures are FYI Molecular Biology of the Cell (Alberts et al) 6th ed. Figure 15-642. Pg 876 4) Intracellular receptors continued Ligand diffuses into the cell and binds to its receptor to alter the ability of the receptor to control transcription of specific genes. § The receptor is BOTH the intracellular receptor AND a transcription factor § A co-regulator is often recruited as well. Model 4 Hormone ligand binds to an intracellular receptor that, when activated directly modified transcription *Receptor may be in the cytosol or already in the nucleus Modifies gene transcription Nucleus 4) i) Steroid hormones Steroid hormone diffuses into the cytoplasm and binds to the receptor in the cytosol § This displaces an inhibitory protein (FYI – hsp) bound to the inactive receptor § Receptor will dimerize and travel to the cell nucleus Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 , via Wikimedia Commons. 4) i) Steroid hormones Inside the nucleus the receptor will bind to a DNA sequence specific to the steroid receptor § Hormone response element (HRE) Coactivator will also bind and transcription will be initiated § (Not shown) Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 , via Wikimedia Commons. 4) ii) Thyroid hormone Thyroid hormone receptor is located in the nucleus, already bound to DNA § Binding of thyroid hormone can increase or decrease transcription of genes depending on the gene itself § Thyroid hormone receptors commonly form heterodimers with other nuclear receptors (FYI – RXR = retinoid X receptor) Thyroid Hormone *Note this image is showing the thyroid hormone entering the nucleus from the cytosol! 4) ii) Thyroid hormone Thyroid hormone receptor is located in the nucleus, already bound to DNA § Binding of thyroid hormone can increase or decrease transcription of genes depending on the gene itself Positively regulated genes will have increased transcription when thyroid hormone binds to its receptor Negatively regulated genes will have decreased transcription when thyroid hormone binds to its receptor 5) Modification by non-coding RNA Gene expression can also be regulated by noncoding RNAs: § miRNA Mature miRNA is ~21-30 nucleotides in length (FYI) Modulate translation of target messenger RNAs (mRNAs) Post-transcriptional silencing of gene expression by miRNA is a fundamental mechanism of gene regulation present in all eukaryotes Each miRNA can modulate activity of multiple protein-coding genes § lncRNA >200 nucleotides in length (FYI) Can bind chromatin to interfere or promote transcription Also involved in X chromosome inactivation 4)i) miRNA miRNA process: § Transcription of miRNA forms a primary transcript (pri-miRNA) § Further processing by a number of enzymes (eg. Dicer) produces smaller and smaller miRNA segments Active product is called miRNA 4) i) miRNA cont. miRNA process: § miRNA associates with proteins to form a RNA-induced silencing complex (RISC) § Base-pairing of the miRNA (within RISC) can either: Induce mRNA cleavage/destruction Repress translation The net effect is that miRNAs within a RISC complex act to silence mRNA post-transcription 4) ii) lcnRNA lncRNA can function is many ways to modify transcription by: § A) promote gene transcription § B) supress gene transcription § C) Promote chromatin modification directly § D) Stabilize protein complexes that modify chromatin structure Team activity time Colorectal cancer is the 4th most commonly diagnosed cancer in Canada. § The tumor itself (colorectal adenocarcinoma) tends to grow slowly and can get quite large before symptoms will present. § Initial presenting symptoms can include anemia with fatigue and weakness, bleeding with defecation, or bowel obstruction. 70-80% of nonfamilial colorectal cancers involve defects in genes for APC within the Wnt/β-catenin pathway, resulting in APC loss of function. APC loss of function has been implication is the pathogenesis of colonic tumours. Myc is a Wnt-responsive gene. § Myc is a gene that codes for the transcription factor MYC, which has been shown to drive cell proliferation. Team activity time - Questions 1. Draw out the basic schematic of the Wnt-β catenin pathway § Which model did it belong to? 2. In 2-3 bullet points clearly explain what would happen to gene transcription for Wnt-responsive genes. 3. In 2-3 bullet points connect the loss of APC to development of colon cancer. In your answer, please connect the role of Myc. References Boron, W. and Boulpaep, E. Medical Physiology (3rd ed). Elsevier Alberts et al. Molecular Biology of the Cell. Garland Science. Robbins and Cotran. Pathologic Basis of Disease. Elsevier Images: § Image adapted from: Boghog2, Public domain, via Wikimedia Commons. Retrieved from: https://commons.wikimedia.org/wiki/File:Type_ii_nuclear_receptor_action.png § Image adapted from: Ali Zifan 03:07, 10 July 2016 (UTC), CC BY-SA 4.0 , via Wikimedia Commons. Retrived from: https://commons.wikimedia.org/wiki/File:Regulation_of_gene_expression_by_steroid_hormone _receptor.svg