Bioc 325 Lecture 2 2023 PDF

Roles of Structural Domains and Scaffold Proteins in Signal Transduction 1 1. Introduce the structure, function, and different types of signaling structural domains 2. Appreciate the roles of structural domains in signal propagati...

Roles of Structural Domains and Scaffold Proteins in Signal Transduction 1 1. Introduce the structure, function, and different types of signaling structural domains 2. Appreciate the roles of structural domains in signal propagation between signaling proteins in a signaling cascade 3. Introduce the kinds of scaffold proteins and their importance in signal propagation 2  Signaling proteins serve as building blocks and have special structural domains that help in their cross-recognition, binding, and signal propagation receptor ligand Signaling proteins Cellular response Structural domains Signal propagation Signal ligand initiation active receptor inactive receptor 3  Intracellular signaling is the series of events which translate a specific message into a biological response in target cells.  Initial step involves the interaction between ligand and its cognate receptor, which in turn, induces stoichiometric and conformational changes on the ligand-receptor complex.  All known signal transduction cascades within the cell involve one or, in most cases, two basic mechanisms: ◦ Protein-Protein interactions ◦ Protein Phosphorylation 4 Protein-Protein Interactions are essential:  Signal propagation through the intracellular space requires proteins to interact with each other and thereby to acquire the ability to transfer the message from one site to another.  Importance: Maintain signal specificity until the final target is achieved (ex. gene promoters).These non- covalent interactions do not occur randomly, but rather in a tightly controlled mode.  Protein interactions are restricted to authorized partners.  Protein modules (interaction motifs) are active players in these specific recognition processes. 5 Protein Phosphorylation  Protein phosphorylation is one of the most popular mechanisms that are important in transducing information along protein cascades  Protein phosphorylation occurs when a protein kinase interacts with tyrosine, threonine or serine residues  Chemical modification is viewed by cellular proteins as a signal that switches on or switches off downstream signaling events. 6 Structural Domains 7  Definition: ◦ Domains are regions of similar architecture within proteins with specific spatial organization and that serve as functional elements ◦ Are modular: they form modules (compact regions) that are repeated within the same polypeptide or recur in various protein structures ◦ Some domains have different structure but same function example: PTB (Phosphotyrosine Binding) and SH2 (they both bind p~Tyr) ◦ Some domains have same protein folding structure but may not function the same (ex: PTB domains and PH (Pleckstrin homology) domains) ◦ Most proteins contain at least two structural domains and many signaling molecules have more. 8  Structural domains play roles as key functional elements.  However, ◦ A particular structural domain in a protein may confer a specific function, but it does not guarantee it. ◦ This is due to: mutations and/or shuffling of exons within gene encoding the domain (insertion, deletion, duplication) 9  Structural Domains A B Protein Catalytic interaction domains domains 10  Structural Domains A B Protein Catalytic interaction domains domains 11  Have enzymatic activity  Examples: ◦ protein kinase domains (as in: PKA, PKC, RTKs) ◦ protein phosphatase domains (as in: PTEN) 12  R dimer Binding of four molecules of cAMP to the regulatory subunits of inactive PKA tetramer results in the dissociation of the tetramer and the release of active free catalytic monomers (open Inactive substrate-binding sites). tetramer Active C monomers 13  Catalytic Domain of RTKs: A tyrosine protein kinase domain Receptor tyrosine kinases (RTKs): have intrinsic Tyr kinase activity through a cytoplasmic tyrosine kinase domain that catalyzes phosphorylation of protein substrates including the receptors themselves 14 PTEN acts as a dual- specificity protein phosphatase: It dephosphorylates: tyrosine- serine- and threonine- phosphorylated proteins PIP2: phosphatidyl inositol (4,5) biphosphate 15 PIP3: phosphatidyl inositol (3,4,5) triphosphate  Structural Domains A B Protein Catalytic interaction domains domains 16  No enzymatic activity  Main role: provide sites of interaction with other proteins  Examples: ◦ SH2 and SH3 domains 17 The adaptor protein Grb2: growth factor receptor bound protein-2 Grb2 binds: phospho tyrosine of RTKs through SH2 domain Proline-rich residues on Sos through SH3 domains 18 1. Domains that bind oligopeptide motifs: SH2, SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 19 1. Domains that bind oligopeptide motifs: SH2, SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 20 Nomenclature: SH2=Src homology region 2  Protein interaction domains that bind to short motifs containing a phosphotyrosine  Examples of SH2-domain containing proteins:  Receptor tyrosine kinases (RTKs) and non-RTKs  Phospholipase Cγ (PLCγ)  the regulatory subunits of phosphoinositide-3 kinase (PI3K)  some protein tyrosine phosphatases (PTPs). 21 Nomenclature: SH3=Src homology region 3  Protein interaction domains that bind to short motifs containing proline-rich sequences  Proline-rich sequences: Generally consist of 8–10 residues of Pro and include at least two prolines in a PxxP motif  SH3 domains have a hydrophobic binding surface  Examples of SH3-domain containing proteins: Adaptor proteins such as Grb2 and Nck 22  Oncogene (2004) 23, 7918–7927. Src Family kinases include 9 non-receptor tyrosine kinases that are involved in the regulation of fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, besides regulating specialized cell signals. 23 Closed inactive Open active conformation conformation 24 Nomenclature: PhosphoTyrosine Binding domains Asp-Pro-X-pTyr  They bind phosphotyrosine residues (N-P-X-pY ), but recognize sequences that are quite distinct from those recognized by SH2 domains  Ability to bind to acidic phospholipid head groups, such as those of the phosphoinositides Importance !!!!!!  could translocate to membrane surfaces where they may then encounter their peptide ligands 25 The PTB domain of insulin receptor substrate-1(IRS-1). 26 1. Domains that bind oligopeptide motifs: SH2,SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 27 Nomenclature: PH domains: Pleckstrin Homology domains  Physiological ligands: phosphoinositides and βγ-subunits of Gi-proteins  There are seven main phosphoinositides: mainly found in membranes of organelles and plasma membrane  Role in protein–protein interactions (Ex: GRK2 [G-protein receptor kinase 2] interaction with activated β-adrenergic receptors)  Examples of PH-domain containing proteins: pleckstrin, GRK2, PLC, Akt.. 28  IP3: inositol PH domain triphosphate Site of IP3 binding G-protein receptor kinase 2 (GRK2) has a PH domain of a β-sandwich structure, which is formed by seven antiparallel strands. 29 Initial attachment of PLCγ1 to cell membrane through its PH domain (binds PIP3) 30 31 1. Domains that bind oligopeptide motifs: SH2,SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 32  1. EF-hand motifs: Nomenclature: E and F denote helical regions of the muscle protein parvalbumin, in which the motif was first identified E F  EF hand motif is a helix-loop-helix motif that recognizes and binds calcium  Usually occur as adjacent pairs  fold 33  1. EF-hand motifs of Calmodulin: EF-hand structure of calmodulin E helix E helix One of the EF-hands of loop calmodulin showing a bound Ca2+ ion (green F helix sphere). Seven oxygen atoms form the coordination shell around calcium (shown as red spheres). loop F helix 34  2. C2 Domains: Nomenclature: ‘second conserved’ regulatory domain of PKCβ  Recognize and bind calcium 35 2. C2 Domains: 36 1. Domains that bind oligopeptide motifs: SH2,SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 37 Nomenclature: Zinc fingers: Zn2+ -binding modules  C2H2: largest family of zinc fingers in which Zn2+ is coordinated by two cysteines and two histidines by forming a stable complex 38 C H 39 1. Domains that bind oligopeptide motifs: SH2,SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 40  Are catalytic domains (have intrinsic activity)  Example: catalytic subunits of PKA, PKC, RTKs… (discussed earlier) 41 1. Domains that bind oligopeptide motifs: SH2,SH3, and PTB domains 2. Phosphoinositide-binding motifs and domains: PH domains 3. Ca2+ binding domains: EF-hand motif and C2 domains 4. Zinc finger domains: C2H2 zinc finger domain 5. Protein kinase domains 6. PDZ domains 42 Nomenclature: PDZ is an acronym combining the first letters of three proteins ◦ post synaptic density protein (PSD95), ◦ Drosophila disc large tumor suppressor (Dlg1), ◦ zonula occludens-1 protein (zo-1)  PDZ domains bind to the C-terminal residues of some ion channels and receptors. They could also bind to phosphoinositides  Function: ◦ help anchor transmembrane proteins (receptors and ion channels) to the cytoskeleton ◦ hold together signaling complexes 43  InaD: scaffold that links PKC to PLCβ, in the microvillar compartment of Drosophila photoreceptors , which are attached to it through PDZ domains. 1. Light falls on eye, which light activates rhodopsin 4 (receptor) 1 2. Coupling of receptor to Gq 3. Activation of PLC that leads 2 to the generation of DAG 4. DAG releases Ca2+ from TRP InaD (transient receptor potential) 3 channels leading to membrane depolarization 5 A loss of InaD causes a retardation of the 5. Desensitization of TRP channels due to return to the resting state and hence a phosphorylation by eye-PKC loss of visual resolution. 44  A PDZ binding motif recognizes and binds a PDZ domain on other signaling molecules PDZ binding Motif of PTEN PDZ-based protein PDZ domain 45 Proline Rich Proline Rich 46 PDZ- and nonPDZ-based Scaffold Proteins 47 Definition: Are proteins that associate with two or more other proteins to enhance the efficiency and /or specificity of a cellular pathway Scaffold Proteins 1 2 nonPDZ- PDZ-based based 48  Scaffold Proteins 1 2 nonPDZ- PDZ-based based InaD PSD-95 NHERF MAGUK proteins 49 A: InaD B: PSD-95 C: NHERF postsynaptic Na H Exchanger density protein 95 regulatory factor 50 InaD coordinates the Drosophila melanogaster phototransduction cascade 51 Harris, B. Z. et al. J Cell Sci 2001;114:3219-3231 52 MAGUK Proteins: Nomenclature: MAGUK: Membrane-Associated Guanylate Kinases  Are characterized by having: ◦ PDZ domain ◦ SH3 domain ◦ GUK (guanylate kinase) domain (inactive kinase) 53 MAGUK Proteins: Examples of higher-order organization of PDZ domains found in signalling proteins 54 Harris, B. Z. et al. J Cell Sci 2001;114:3219-3231  Scaffold Proteins 1 2 nonPDZ- PDZ-based based AKAP JAK-STAT β-arrestin 55 1 2 3 AKAP: JAK-STAT β-arrestin A-kinase anchoring protein β-arrestin interacts with the AKAP is needed for receptor phosphorylated receptor, phosphorylation by PKA interfering with the coupling of G protein 56  JAK-STAT as a scaffold in the signaling pathway downstream of cytokine receptors:  Tyk2 and Jak1 are examples of Janus Kinases (JAKs)  STAT1 and STAT2 are examples of STATs (Signal Transducer and Activators of Transcription). 57  JAK-STAT as a scaffold in the signaling pathway downstream of cytokine receptors: (Figure Legend)  Unlike the RTKs, the cytokine receptors lack intrinsic catalytic domains. Instead, once the cytokine binds to its receptor, the Janus Kinases (JAKs), which are non-covalently associated with the receptor, phosphorylate the receptor at specific intracellular tyrosine residues.  The receptor, once phosphorylated at specific tyrosine residues, serves as a docking site, for transcription factors termed STATs (Signal Transducer and Activators of Transcription). STATs bind phosphorylated receptor via their SH2 domains).  This is followed by phosphorylation of STATs by the JAKs, dimerization of the STATs (through their SH2 domains), and their translocation to the nucleus to activate gene expression. 58  G-protein coupled receptors (GPCRs) could bind PDZ- and nonPDZ- based scaffold proteins: ◦ Third Intracellular Loop (IC3) = interaction with non-PDZ protein (ex: b- arrestin) ◦ COOH-end = binds PDZ based scaffold (ex:PSD - 95) ◦ Distal part of COOH end = binds non-PDZ protein 59 G-Protein Coupled receptors 60