Signaling Pathways Controlling Gene Activity PDF

Signaling Pathways that Control Gene Expression  Overview: Major Classes of Cell-surface Receptors / cAMP/ PKA CREB – cAMP-response element binding protein  Signal Transduction Pathways are Complex Several classes...

Signaling Pathways that Control Gene Expression  Overview: Major Classes of Cell-surface Receptors / cAMP/ PKA CREB – cAMP-response element binding protein  Signal Transduction Pathways are Complex Several classes of receptors can transduce signals by more than one pathway Many genes are regulated by multiple Trasnscription factors – each TS factor can be activated by one or more extracellular signal Large amount of cross talk between signaling pathways – especially seen during early development Components of Major Signaling Pathways  Transforming Growth Factor β Signaling Transforming Growth Factor β (TGFβ) superfamily is a large class of signals involved in a wide range of developmental processes, including: – induction of transformed phenotype in cultured cells – induction of bone formation & growth – formation of mesoderm – expression of cell-adhesion & extracellular matrix molecules – signals for synthesis and secretion of growth factors – note: TGFβ has anti-proliferation effects on many mammalian cells where TGFβ signaling generally inhibits cell proliferation Complex cellular effects, but fairly simple signaling pathway: – TGFβ (signal) binds TGFβ-receptor, direct phosphorylation and TS factor activation  Human TGFβ Signals Human TGFβ isoforms (TGFβ1-3) are: – encoded by unique genes – tissue specific – developmentally regulated TGFβ is synthesized as a part of a larger precursor that is cleaved after secretion from the cell: – Pro-domain is cleaved, but remains non-covalently associated With the Mature domain via cysteine residues that form disulfide bonds Latent TGFβ complex in Extra Cellular Matrix: Pro-domain & mature domain stored in complex with latent TGFβ -binding protein (LTBP) Mature TGFβ is released as active homo- or hetero-dimer after proteolysis or LTBP conformational change – integrin or matrix proteins binding to LTBP can cause this  TGFβ Receptors 3 types of receptors: TGFβ RI, RII, & RIII – TGFβ-RIII: most abundant TGFβ receptor that binds and concentrates TGFβ at the cell surface, but does not have kinase activity – TGFβ-RII: dimeric transmembrane proteins that can bind TGFβ on the cell surface and has cytosolic Serine/Threonine kinase activity for TGFβ-RI – TGFβ-RI: dimeric transmembrane proteins that does not bind TGFβ outside the cell, but has cytosolic Serine /Threonine kinase activity for R-Smads TGFβ binding induces complex formation with 2 copies of TGFβ-RII & TGFβ-RI – TGFβ-RII phosphorylates & activates TGFβ-RI kinase activity – activated TGFβ-RI directly phosphorylate & activate TS factors called Smads  Smads – transcription factors Three types of Smads: – R-Smads: Receptor-regulated Smads (Smad2, Smad3) – Co-Smads (Smad4) – I-Smads: Inhibitory or antagonistic Smads (Smad7) Structure of R-Smads: R-Smad inactive when C unphosphorylated MH2 Linker DBD NLS – MH1 & MH2 domains associated MH1 – NLS is masked N – Can’t bind Co-Smad or DNA TGFβ-RI TGFβ-RI phosphorylates 3 kinase Serine residues on R-Smad DBD NLS PPP near C-term which MH1 MH2 N C separates the MH1 & MH2 Linker domains TGFβ Signaling Pathway TGFβ binds to TGFβ-RIII or TGFβ-RII – if binding is to RIII, TGFβ presented to RII – ligand-bound RII recruits and phosphorylates RI, causing its activation Activated TGFβ -RI phosphorylates Smad3 (R-Smad), exposing NLS Two phosphorylated Smad3 interact with Smad4 (Co-Smad) & importin in cytosol, forming large complex Entire complex translocates to nucleus – Ran-GTP causes dissociation of importin Smad complex associates w/ other TS factors forming activation complex TS begins (often growth-inhibitory genes) Dephosphorylation of Smads in nucleus results in their translocation to cytoplasm  I-Smads Regulate Smad Signaling I-Smads (Smad7) block the ability of TGFβ-RI to phosphorylate R-Smads – Induction of Smad7 inhibits intracellular signaling  Oncoproteins Regulate Smad Signaling Smad signaling is also regulated by other proteins, including SnoN & Ski (Sloan-Kettering Cancer Institute) – Identified as oncoproteins: cause abnormal cell proliferation SnoN & Ski bind to Smad2/Smad4 or Smad3/Smad4 complexes after TGFβ stimulation – Smad complexes can still bind to DNA control regions – block TS activation by DNA bound Smad complexes – Blocks TS of growth- inhibitory genes normally made by TGFβ pathway – How does that happen in this example?  Cancer & Loss of TGFβ Signals TGFβ signaling generally inhibits cell proliferation Loss of various components of the signaling pathways contributes to abnormal cell proliferation & malignancy. – Many cancers are caused by gene mutations in the TGFβ signaling pathway (either TGFβ receptors or Smad proteins) and are therefore resistant to growth inhibition by TGFβ. Example: In most human pancreatic cancers, a deletion to the Smad 4 gene occurs – Smad 4 protein is not produced or is non-functional – proteins that inhibit cell proliferation upon stimulation by TGFβ are not synthesized  OTHER RECEPTORS There are two broad categories of receptors that activate tyrosine kinases 1. those in which the tyrosine kinase enzyme is an intrinsic part of the receptor’s polypeptide chain called Receptor Tyrosine Kinases (RTK) 2. cytokine receptors – the receptor and kinase are separate polypeptides encoded by different genes EPO- Erythropoietin (RBC) TPO-thrombopoietin (platelets) G-CSF-granulocyte colony-stimulating factor (WBC)  Cytokine Signaling Cytokines are small secreted proteins that control growth and differentiation of different cell types – Cytokines can act in an autocrine, paracrine, and endocrine manner Cytokines bind to their cell surface cytokine receptor and turn on intracellular signaling cascade resulting in various responses: – increasing or decreasing expression of membrane proteins (including cytokine receptors) – cell proliferation (T-cells, B-cells) – secretion of effector molecules Examples of cytokines and responses: – Interleukin-2: T cell & NK cell proliferation – Interleukin-4: B cell proliferation – Erythropoietin: regulates RBC production – Thrombopoietin: platelet formation  Cytokine Signaling Cytokine receptor: two monomeric transmembrane proteins each associated with JAK (Janus Kinases), a separate cytosolic protein tyrosine kinase Cytokine binds to its receptor causes: – Receptor dimerization – tyrosine phosphorylation and activation of JAKs – tyrosine phosphorylation of receptor tails  Recruitment of Cytosolic Proteins to Receptor Tails Phosphorylated Tyrosine on the activated receptor tail recruit a number of proteins that contain SH2 (Src Homology domain 2) or PTB (Phosphotyrosine binding) domains The recruited proteins are then phosphorylated (by the JAK) which enhances their activity Those proteins go on to signal and activate transcription. Src= acronym for Sarcoma  Cytokines & the JAK/STAT Pathway Ligand binds to cytokine receptor JAK is phosphorylated & activated JAK phosphorylates tyrosine residues on the receptor tail Recruitment of SH2 domain containing STAT proteins – STAT: Signal Transducers and Activators of Transcription STATs are phosphorylated by JAK Phosphorylated STATs dissociate from the receptor and dimerize, exposing NLS STAT dimer translocates to the nucleus results in binding to enhancer sequences & activation of TS of target genes  Short Term Regulation of Cytokine Receptors Dephosphorylation of tyrosines on JAK by phosphatase SHP1 – activation involves phosphorylation, so inactivation involves dephosphorylation SHP1 binds to a phosphotyrosine on the receptor tail and removes phosphate from JAK prevents further activation occurs w/in a few minutes SHP1 – Src homology phosphatase 1  Long Term Regulation of Cytokine Receptors SOCS (suppressor of cytokine signaling) inhibit or terminate long term signaling – SOCS protein expression is induced by STAT proteins in Epo stimulated cells Mechanisms: – Signal blocking: SOCS binds to phosphotyrosine residues on EpoR or JAK2 – Protein degradation: SOCS target proteins for ubiquitin- proteasome degradation summary  Receptor Tyrosine Kinases (RTKs) RTKs are similar to cytokine receptors but the cytosolic domain has its own intrinsic tyrosine kinase activity Examples of RTK ligands include Growth factors and Insulin – NGF (nerve), PDGF (platelet), FGF (fibroblast), EGF (epidermal) Ligand binding causes receptor dimerization, kinase activation, phosphorylation of tyrosine (Y) on receptor tails  Ligand binding to RTK & Ras Activation Phosphorylated tyrosines on RTK tails recruit adapter proteins with SH2 & PTB domains These adaptor proteins couple activated RTKs to various components of signaling pathways, such as Ras activation Ras is a monomeric, GTP-binding switch protein – Inactive Ras bound to GDP; Active Ras bound to GTP Ras is not directly linked to cell-surface receptors Ras is anchored to plasma membrane by hydrophobic anchor Ras - Rat sarcoma virus  Ligand binding to RTK & Ras Activation Activated RTK recruits SH2 domain containing GRB2 GRB2 recruits SOS protein (which has GEF activity) SOS replaces GDP with GTP on RAS GRB2 -Growth Factor Receptor Bound Protein 2 Sos – (son of sevenless) it is a GEF (Guanine nucleotide exchange factor) Active Ras Turns on MAPKinase Pathway Ras/MAP kinase pathway: Ras binds to & activates Raf (S/T kinase) Raf activates MEK (kinase) MEK activates MAPK (kinase) MAPK translocates to the nucleus inducing TS – MAPK regulates activity of many TS factors MAPK- mitogen activated protein kinase Ras/MAPK Pathway Mutations to Components of MAPK Pathway Linked to Cancer  Role of Scaffold Proteins Upstream components of MAPK cascades assemble into large pathway-specific complexes stabilized by scaffold proteins Scaffold proteins associate different kinases of a signaling pathway to prevent accidental phosphorylation of other substrates – allowing kinases of one pathway to interact w/ each another, but not w/ kinases in other pathways  Insulin & Protein Kinase B (PKB) Insulin binds to insulin receptor (RTK) which can: 1. Activate Ras-MAPK pathway leading to changes in gene expression, or 2. Activate PKB/Akt, a S/T kinase How does Insulin lower blood glucose? – In fat & muscle cells: PKB/Akt causes movement of GLUT4 transporters from intracellular vesicles to the plasma membrane GLUT4: increases import of glucose by fat and muscle cells – In liver & muscle cells: PKB/Akt stimulates glycogen synthesis by causing glycogen synthase activation PKB/Akt phosphorylates and inactivates an inhibitor of GS Summary