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Principles of cell signaling Lecture 2 Johan Lennartsson Molecular Cell Biology (1BG320), 2014 [email protected] 1 A cell senses signals from its surroundings via receptor proteins G protein-coupled receptors Enzyme-linked receptors TNF family receptors Ion channel receptors Nuclear receptors 2 Phosphorylation is a post-translational modification commonly used in signal transduction Phosphate groups in proteins: added by enzymes called kinases carry two negative charges can engage in hydrogen bond networks stable against spontaneous hydrolysis removed by enzymes called phosphatases Phosphorylation is a chemically stable but reversible modification and is therefore often used by the cell to regulate protein function Phosphorylation Phosphorylation occurs on hydroxyl groups (Ser, Thr &Tyr) and is catalyzed by enzymes denoted kinases Tyrosine (Tyr, Y) Serine (Ser, S) Threonine (Thr, T) Kinases are group into: tyrosine kinases serine/threonine kinase 4 Phosphorylation Mg2+ + + The phosphate group is donated from ATP (g-phosphate) The negatively charged ATP is stabilized by interaction with Lysines from the kinase protein and Mg2+ ions Electrons on the hydroxyl group oxygen makes a nucleophilic attack on the P atom, and ADP functions as a leaving group 5 Dephosphorylation Electrons on the Cys makes nucleophilic attack on P atom in the phosphoprotein and the substrate protein functions as a leaving group Target protein dephosphorylation The Cys thiol group is regenerated by a nucleophilic attack by a water molecule that is activated by glutamine residue in the phosphatase Phosphatase regeneration 6 Phosphorylation regulates enzymatic activities Inactive state Active state Electrostatic effects can cause structural changes in proteins 7 Phosphorylation regulates binding activities The SH2 domain is a compactly folded domain consisting if about 100 a.a. In humans there are 120 different SH2 domains within 115 proteins Variations in the amino acids forming the second socket in different SH2 domains allow them to bind to phosphotyrosine adjacent to different sequences. 8 A cell senses signals from its surroundings via receptor proteins G protein-coupled receptors Enzyme-linked receptors TNF family receptors Ion channel receptors Receptor tyrosine kinases Cytokine receptors Nuclear receptors 9 Receptor tyrosine kinases Ligands for receptor tyrosine kinases are called growth factors Growth factors act in endocrine, paracrine, juxtacrine and autocrine fashion Growth factors are important for proper development and function of multicellular organisms since they participate in cell – cell communication regarding cell proliferation, position and identity (differentiation) Many growth factors and components in the signal transduction pathways they activate are proto-oncogenes or tumor suppressor genes 10 Families of receptor tyrosine kinases 58 Receptor tyrosine kinases (RTKs) in 20 subfamilies 11 Receptor tyrosine kinases are activated by ligand-induced dimerization Transphosphorylation (=autophosphorylation) increased catalytic activity docking sites for intracellular signaling proteins 12 Receptor tyrosine kinase activation Activation of RTKs: dimerization 14 EGF receptor dimerization 15 RTK dimerization places two kinase domains in optimal position for autophosphorylation 16 Example: dimerization of c-Kit c-Kit is a receptor tyrosine kinase important for hematopoietic stem cells, as well as melanocytes and germ cells Deregulated c-Kit activity has been found in several types of cancer, e.g. leukemia and GIST The process of ligand-induced dimerization and kinase activation has been well studied 17 Schematic structure of c-Kit Extracellular domain: 5 Ig-like domains (D1-D5) out of which D1-D3 binds ligand, SCF Single transmembrane helix Intracellular tyrosine kinase domain that contains a kinase insert region important for signal transduction 18 Early experiments indicating that c-Kit may undergo dimerization upon SCF binding Truncated receptors lacking the cytoplasmic domain inhibit signaling Antibodies against the cytoplasmic domain activate the kinase domain 19 Western blotting (immunoblotting, WB, Ib) 20 Chemical cross-linking experiments suggested ligand-induced c-Kit dimerization Protein 2 Lys .. Protein 1 Lys Protein2-NH .. HN-Protein 1 receptor dimer receptor monomer 21 EMBO J. 1991 10(13):4121-8 The c-Kit ligand (SCF) is itself dimeric In many cases growth factors are bivalent, i.e. they have two identical binding surfaces hydrophobic interactions salt bridges at the periphery of binding surface hydrogen bonds (H20-mediated) Covalent disulfide bonds (Cys-S-S-Cys) found in SCF 22 Proc. Natl. Acad. Sci. U. S. A. (2000) 97,:7732–7737 EMBO J (2000) 19, 3192–3203 c-Kit dimerization is driven by the bivalent nature of its ligand (SCF) EMBO J (2007) 26, 891–901 23 EMBO J (2000) 19:3192–3203 Proc. Natl. Acad. Sci. U. S. A. (2000);97:7732–7737 Interactions between D4 in the receptor monomers are essential for the formation of a functional dimer SCF 24 Proc. Natl. Acad. Sci. U. S. A. (2008) 105:7681-7686 The interactions between D4 (and D5) regions bring the transmembrane close to each other 25 Cell (2007), 213-215 Dimerization places the two internal kinase domains in optimal position for autophosphorylation Note: one turn of the a-helix corresponds to 3.6 amino acids Kinase activity +++ +++ + ++ +++ +/- - ++ 26 Mol. Biol. Cell. (2000) 11(10):3589-99 Alternative splicing affecting the rigid a-helical region may produce receptor variants with different kinase activities 27 J. Biol. Chem., (2003) 278 (11): 9159-9166 Ligand-induced dimerization of c-Kit The bivalent nature of SCF brings to c-Kit monomers into a dimeric state (but inactive) Interactions between the D4 (and D5) in the extracellular domains brings the transmembrane helices (and intracellular kinase domains) close to each other Dimerization puts the kinase domains in an orientation relative each other that is functional 28 Consequences of RTK activation Autophosphorylation creates binding sites for signal transduction molecules (SH2 & PTB domains) Bound signal transduction molecules are activated (localization & phosphorylation) 29 Src homology 2 (SH2) domain The pocket of the SH2 domain is highly specific for phospho-Tyr because of the bulky size of the tyrosine. The other side of the pocket is more variable and allows specific recognition of the residues at the C-terminal of the phosphotyrosine One side of the pocket is lined with conserved basic amino acids and binds the phosphotyrosine 30 Src domains interact with phosphorylated tyrosine residues (pTyr) 31 Attraction of SH2-domain containing proteins depends on the amino acid sequences outside the kinase domain 32 Src homology 3 (SH3) domain SH3 domains are important for constitutive protein-protein interactions and they have an elongated binding cleft, where hydrophobic pockets contact the proline-rich peptide helix Proline Poly-Pro helix Recognize proline rich motifs with minimal sequence PxxP, where x is any amino acid Specificity is maintained by amino acids either amino (or carboxyl) terminal to the prolines Side view top view Signal transduction proteins often contain multiple domains About 80 interaction domains have been identified and signaling proteins often contains several What are the consequences for a signaling protein to bind to the receptor? 34 Activation of signal transduction molecules Conformational change: catalytic domain changes into a shape optimal for catalysis Activation of signal transduction molecules Translocation: substrate and activator are become co-localized at a particular region of the cell Activation of signal transduction molecules Phosphorylation: Addition of phosphate group causes a conformational change and/or promote complex formation 37 RTK inactivation In order for the cell to respond in an appropriate manner to a growth factor the signaling has to be terminated This occurs both at the level of intracellular pathways but also at level of the receptor 38 RTKs are often internalized through clathrincoated pits after their activation Adaptors and structural proteins forming the clathrin cage Dynamin is a GTPase needed for budding of the vesicle Actin is a component of cytoskeleton 39 Endocytosis through clathrin-coated pits A coated pit and a coated vesicle formed during receptormediated endocytosis (TEMs) 40 Clathrin trimer interact and form a lattice that surrounds the vesicle 41 There are many endocytic pathways besides clathrin-coated pits Independent of uptake mechanism, most vesicles fuse early endosomes endosome with larger vesicles called early Endocytic vesicles fuse with early endosome Rab proteins (G-proteins) on the endocytosed vesicle interact with proteins (Rab effector protein) on the endosome As the membranes come closer to each other there is a energetic barrier against membrane fusion. The energy barrier is caused by the hydrophilic membrane surfaces are solvated by water and these molecules have to be removed before fusion can take place SNARE proteins on the vesicle and target compartment provide energy to overcome this energy barrier 43 Endocytic vesicles fuse with early endosome SNAREs on the vesicle selectively binds to SNAREs on the acceptor membrane Starting from the N-terminal, the SNAREs interact and form a zipper-like structure This forces the membranes to make contact, leading to their fusion The endosome is an important sorting station Endosomal sorting Recycle back to plasma membrane multivesicular bodies (e.g. RTK) 45 Activated RTKs become ubiquitinated and this is important for intracellular sorting c-Cbl RTK c-Cbl RTK endocytosis, endocytosis, lysosomal lysosomal degradation degradation RTK RTK endocytosis, endocytosis, proteasomal proteasomal degradation degradation endocytosis, signaling signaling ? ? Ubiquitinated receptors are sorted into multivesicular bodies by ESCRT complexes on the endosomal surface Cytoplasm Ligand binding region Endosomal lumen 47 Exp Cell Res. 2009 315(9):1619-26 Multivesicular bodies will form/fuse with lysosomes Lysosomes are organells that have a low internal pH (<5) and contain acid hydrolase enzymes (including proteases) that degrade proteins and other cell debris 48 A cell senses signals from its surroundings via receptor proteins G protein-coupled receptors Enzyme-linked receptors TNF family receptors Ion channel receptors Receptor tyrosine kinases Cytokine receptors Nuclear receptors 49 Cytokine receptors have no intrinsic kinase activity but associate with Jak family of tyrosine kinases Jak family of tyrosine kinases associated with cytokine receptors (in non-stimulated cells in an inactive form) Cytokine receptor dimerization/oligomerization leads to Jak apposition leading transphosphorylation and activation Active Jak:s tyrosine phosphorylate intracellular part of the receptor on multiple sites Four members in the Jak family: Jak1, Jak2, Jak3 and Tyk2 50 Cytokine receptors frequently contain protein subunits shared with other receptor complexes 51 Since protein subunits are shared there is frequent redundancy in cytokines signaling 52 A cell senses signals from its surroundings via receptor proteins G protein-coupled receptors Enzyme-linked receptors TNF family receptors Ion channel receptors Nuclear receptors 53 G-protein coupled receptors (GPCR:s) GPCR constitute a large family of cell surface receptors (there are about 800 GPCRs in humans) GPCR can function as monomer and dimers Diversity through dimerization 54 G-protein coupled receptors (GPCR:s) Based on available structural data on GPCR these conclusions can be drawn regarding their achitecture: Highly similiar transmembrane regions consisting of 7 helices (good basis for homology modeling of other GPCRs for this region) Ligandbinding 55 G-protein coupled receptors (GPCR:s) The extracellular ligand-binding domain display larger differences (consistent with the different types of ligands they bind) G-protein coupled receptors (GPCR:s) In the cytoplasmic domain there are variations in the length and secondary structure of the loops connecting the transmembrane helices (this can explain G-protein selectivity) 57 G-protein coupled receptors (GPCR:s) Ligand activated GPCRs utilize intracellular heterotrimeric G-proteins to initiate signal transduction 58 G-protein coupled receptor activation Ligand binding to GPCR result in conformational change in the transmembrane helices that creats a G-protein binding surface on the intracellular side Ga GTP GDP GTP GTP Binding of Ga subunit to the GPCR stimulate release of its bound GDP Since the concentration GTP is higher that GDP in the cytoplasm this will most likely result in an exchange to GTP. Ga-GTP dissociates from the receptor 59 G-protein coupled receptor activation Active conformation Inactive conformation 60 G-protein coupled receptor activation 61 Ga and Gbg subunits engage downstream effectors 62 The heterotrimeric G-protein cycle The enzymatic activity of the Ga subunit is a GTPase activity meaningthat it hydrolyses GTP into GDP, thus turning itself off. The level of GDP vs GTP bound can be regulated by interactions with other proteins The heterotrimeric G-protein cycle The GDP and GTP bound states of the Ga protein has different conformations Effector protein The switch region(s) have different conformations depending on whether GDP or GTP is bound to Ga 64 There are several types of Ga with different, sometimes opposite, abilities to activate downstream targets 65 Termination of GPCR activity Similar to that of RTK:s ! 66 Downregulation of GPCR activity: b-arrestin GRK bg subunit Signal tranduction a subunit GRK-mediated GPCR phosphorylation promotes the binding of β-arrestin which result in: targeting of many GPCRs for internalization in clathrin-coated vesicles uncoupling of GPCR from heterotrimeric G proteins binding and activation 67 Downregulation of GPCR activity GPCRs are removed from cell surface and degraded The affinity of the GPCR for its ligand is in some cases decreased when Ga subunit binds to GTP (= is active). This will limit the number of Ga that can be activated. Binding of Ga to adenylate cyclase will enhance the intrinsic GTPase actvity of a-subunit leading to its inactivation. This will limit the time adenylate cyclase is active. The PKA-induced substrate phosphorylation is transient and in the absence of PKA activity will be dephosphorylated. This will limit the signal produced cAMP phosphodiesterases hydrolyze cAMP into AMP and this will terminate the signal 68