ArticleLiterature Review

Signaling Through Scaffold, Anchoring, and Adaptor Proteins

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Abstract

The process by which extracellular signals are relayed from the plasma membrane to specific intracellular sites is an essential facet of cellular regulation. Many signaling pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine residues of target proteins. Recently, it has become apparent that regulatory mechanisms exist to influence where and when protein kinases and phosphatases are activated in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks or by placing enzymes close to their substrates is discussed.

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... Secondly, it is becoming apparent that kinase anchoring and other adaptor molecules play a critical role in mediating substrate phosphorylation by restricting enzyme subcellular distribution (Faux and Scott., 1996a;Faux and Scott., 1996b;Pawson and Scott. 1997). In the case of glutamate receptors, A-kinase binding proteins (AKAPs) are important in mediating PKA regulation of this receptor. The molecule AKAP-79 has been implicated in the control of AMPA receptors (Rosenmund et al, 1994), and the molecule 'yotiao', also an AKAP, in that of the NMDA-type receptors (Westphal et al., 1999a;Westpha ...
... Previous studies have shown that the PKC-pl isoform is targeted to substrates by anchoring proteins, such as RACK-1, a homologue of Gprotein p subunits (Ron et al., 1994;Pawson and Scott, 1997; Mochly- Rosen and Gordon, 1998). This molecule binds a number of PKC isoforms, but has a significant preference for pi, and is thought to bind PKC when in an active form, and to potentiate this activity (Ron et al., 1994). ...
... The direct binding of kinases to ion-channel subunits has been shown in a number of cases, for example the binding of the protein tyrosine kinases src and fyn to the acetylcholine receptor (Fuhrer and Hall, 1996), the tyrosine kinase lyn to the AMP A -type glutamate channel, and the simultaneous binding of the tyrosine kinase src and the catalytic subunit of PKA to a Ca2+-dependent potassium channel . Secondly, kinases (and phosphatases) have been shown to be located near to, or at their substrate, an ion-channel in this case, by an intermediate anchoring molecule (Faux and Scott, 1996a;Faux and Scott, 1996b;Pawson et al., 1997). The A-Kinase Anchoring Proteins (AKAPS) are the best characterised of these molecules (Colledge and Scott, 1999). ...
Thesis
GABAA receptors are the main sites of inhibitory neurotransmission in the brain. They are also the sites of action of a number of clinically important drugs such as the benzodiazepines and barbiturates. Phosphorylation has been identified as an important mechanism by which neurons control the function of these receptors. GABAA receptors are hetero-pentamers which are assembled from the subunit classes α, β, γ, σ and ϵ. Subunits have a distinctive topology which includes 4 transmembrane domains, extracellular amino-, and carboxy termini, and a large intracellular domain between transmembrane regions 3 and 4. In vivo most benzodiazepine sensitive receptors are believed to be composed of α, β, and γ subunits. Intracellular domains of receptor subunits contain consensus phosphorylation sites for a range of protein kinases, including the cAMP-dependent kinase (PKA), the Ca2+-phospholipid dependent kinase (PKC) and the non-receptor tyrosine kinase src. Studies with subunit cDNAs, transiently expressed in heterologous cells, has identified principally the β and γ2 subunits as substrates in vivo for these kinases. To date there have been few studies on the phosphorylation of the native receptor. Using polyclonal antibodies, which were raised for this study, the tyrosine phosphorylation of the γ2 subunit has been able to be studied for the first time in neurones. Using an antibody which recognises the first 29 amino-terminal amino-acids of the γ2 subunit, this protein was shown to be tyrosine phosphorylated in adult rat brain lysate. It has been previously shown that the tyrosine kinase src phosphorylates the residues Y365 and Y367 of the γ2 subunit in HEK293 cells, causing upregulation of receptor function. The second antibody successfully raised in this study recognises the γ2 subunit when it is specifically phosphorylated on these two tyrosine residues. This allowed a more direct analysis of γ2 subunit tyrosine phosphorylation in adult rat brain lysate, and cultured cortical neurons. Results indicated that in cortical neurons phosphorylation of the γ2 subunit, on Y365 and Y367, is under very tight regulation. At present, there is little known as to how kinases or any other signalling molecules interact with GABAA receptors in neurones. Previous studies have identified serine and tyrosine kinase activities co-purifying with the receptor from brain preparations. To identify how protein kinases and related signalling molecules are targetted to the GABAA receptor, the intracellular domains of receptor subunits were used as baits to probe rat brain extracts. This approach has identified a very intimate association between PKC, the 'Receptor for Activated C-kinase', RACK-1, and the GABAA receptor β1 and β3 subunits. These interactions were initially identified by analysis of proteins bound to receptor fusion proteins in in vitro 'pull-down' assays. Both molecules were identified as binding directly and independently to the receptor by 'gel-overlay' assays. Confirmation of the in vivo existence of this complex was performed by co-precipitation of RACK-1 and PKC with the GABAA receptor from transfected HEK293 cells and from cultured cortical neurons. The functional significance of the RACK-1 interaction was investigated electrophysiologically in HEK293 cells, by disrupting the binding of RACK-1 with a polypeptide corresponding to its binding site on the GABAA β1 subunit. Finally, in cortical neurons, the β3 subunit was shown to be highly basally phosphorylated, due to the activity of closely associated PKC.
... Considering the large volume of atomic events for PPI, the molecularly docking was done using Cluspro 2.0, a CAPRI-based docking assessment [44], and PatchDock is a surface geometry-based ranking system [45] web server application. The generated results from the respective web servers are verified for the ranking patterns and analyzed for the effective PPI process. ...
... The results are quite impressive as we expected for the Cluspro 2.0 webserver data was matched with the data from the PatchDock for the SlpA interaction with Erb2 showed the ΔG (free energies) -34. 45 Kcal/mol and SlpA interaction with αIIB-β3 showed the ΔG -51. 19 Kcal/mol, the results are compared in Table 2. ...
Article
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Host microbial interactions had significant factors in maintains homeostasis and immune-related activity. One such interaction made by Lactobacillus sp. with Surface layer proteins (Slps) had been studied through a computational approach. Erb3 and αIIB-β3, which are epithelial surface layer receptors, are subjected to interact with the Slp homology model. Both cell surface receptors were subjected to interact through computational docking, followed by molecular dynamics simulations through the coarse-grain method to explore the conformational stability. Through the implementation of the molecular docking for the surface layer protein A, we have shown the surface layer protein A, protein-protein interactions are higher in cellular receptors with epidermal growth factor receptor at an-34.45 ΔG and-51.19 ΔG through molecular docking with Erb3 and αIIB-β3. This study shows the unique interaction of Slp with the epithelial surface receptors like Erb3 and αIIB-β3, which are multipurpose applications in microbial-based drug therapeutics.
... In the past two decades, SUMOylation has emerged as an important regulatory mechanism in various cellular processes involved in human cancer [28,29]. Qu et al. provided evidence that SUMOylation of Grb2 (Growth factor receptor-bound protein 2), a key adaptor protein involved in several TRK dependent signaling pathways [30], enhances the formation of Grb2-Sos1 complex and up-regulates the ERK activities, which is required for promoting migration and tumorigenesis [30]. SUMOylation also regulates nuclear accumulation and signaling activity of the ErbB4, a member of the EGFR of RTK [31]. ...
... In the past two decades, SUMOylation has emerged as an important regulatory mechanism in various cellular processes involved in human cancer [28,29]. Qu et al. provided evidence that SUMOylation of Grb2 (Growth factor receptor-bound protein 2), a key adaptor protein involved in several TRK dependent signaling pathways [30], enhances the formation of Grb2-Sos1 complex and up-regulates the ERK activities, which is required for promoting migration and tumorigenesis [30]. SUMOylation also regulates nuclear accumulation and signaling activity of the ErbB4, a member of the EGFR of RTK [31]. ...
Article
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Posttranslational modifications (PTMs) of proteins, the major mechanism of protein function regulation, play important roles in regulating a variety of cellular physiological and pathological processes. Although the classical PTMs, such as phosphorylation, acetylation, ubiquitination and methylation, have been well studied, the emergence of many new modifications, such as succinylation, hydroxybutyrylation, and lactylation, introduces a new layer to protein regulation, leaving much more to be explored and wide application prospects. In this review, we will provide a broad overview of the significant roles of PTMs in regulating human cancer hallmarks through selecting a diverse set of examples, and update the current advances in the therapeutic implications of these PTMs in human cancer.
... Similar to protein interactions, peptide binding is mediated by "hot spot" hydrophobic residues that have a high frequency of leucine residues [68,69]. Peptide domains within protein structures have been shown to exist throughout entire protein structures [4,104] and could be released intact after the proteolytic degradation of proteins by proteasome (Ferro, E.S. and Klitzke, C.F., unpublished data). Although the participation of proteasome-generated peptides in PPI could seem obvious [18], most previous studies have not considered the fact that free natural InPeps could affect protein interactions within cells. ...
... These data suggesting that binding to multiple protein regions/proteins could be an intrinsic property of peptides. Once peptides bind to protein motifs/domains of related functions, from the same or distinctive proteins, they could act synergically, producing functional consequences of biological/pharmacological relevance [4,104]. Thus, the flexible and complex structure of peptides, compared to that of small molecules of rigid and more specific binding sites, could became an advantage for therapeutic application of peptides as inhibitors of intracellular PPI. ...
Article
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Intracellular peptides (InPeps) generated by proteasomes were previously suggested as putative natural regulators of protein–protein interactions (PPI). Here, the main aim was to investigate the intracellular effects of intracellular peptide VFDVELL (VFD7) and related peptides on PPI. The internalization of the peptides was achieved using a C-terminus covalently bound cell-penetrating peptide (cpp; YGRKKRRQRRR). The possible inhibition of PPI was investigated using a NanoBiT® luciferase structural complementation reporter system, with a pair of plasmids vectors each encoding, simultaneously, either FK506-binding protein (FKBP) or FKBP-binding domain (FRB) of mechanistic target of rapamycin complex 1 (mTORC1). The interaction of FKBP–FRB within cells occurs under rapamycin induction. Results shown that rapamycin-induced interaction between FKBP–FRB within human embryonic kidney 293 (HEK293) cells was inhibited by VFD7-cpp (10–500 nM) and FDVELLYGRKKRRQRRR (VFD6-cpp; 1–500 nM); additional VFD7-cpp derivatives were either less or not effective in inhibiting FKBP–FRB interaction induced by rapamycin. Molecular dynamics simulations suggested that selected peptides, such as VFD7-cpp, VFD6-cpp, VFAVELLYGRKKKRRQRRR (VFA7-cpp), and VFEVELLYGRKKKRRQRRR (VFA7-cpp), bind to FKBP and to FRB protein surfaces. However, only VFD7-cpp and VFD6-cpp induced changes on FKBP structure, which could help with understanding their mechanism of PPI inhibition. InPeps extracted from HEK293 cells were found mainly associated with macromolecular components (i.e., proteins and/or nucleic acids), contributing to understanding InPeps’ intracellular proteolytic stability and mechanism of action-inhibiting PPI within cells. In a model of cell death induced by hypoxia-reoxygenation, VFD6-cpp (1 µM) increased the viability of mouse embryonic fibroblasts cells (MEF) expressing mTORC1-regulated autophagy-related gene 5 (Atg5), but not in autophagy-deficient MEF cells lacking the expression of Atg5. These data suggest that VFD6-cpp could have therapeutic applications reducing undesired side effects of rapamycin long-term treatments. In summary, the present report provides further evidence that InPeps have biological significance and could be valuable tools for the rational design of therapeutic molecules targeting intracellular PPI.
... This suggests that leptin acts on a subset of cellular PKA that is localized near the plasma membrane. It is widely documented that a high level of regulation and specificity of PKA signaling is maintained by a family of scaffolding proteins known as A-kinase anchoring proteins (AKAPs), which target PKA and its signaling partners to distinct subcellular regions, thereby creating PKA signaling microdomains and nanodomains (19,31,32). To test if AKAPs are involved in targeting PKA to the cell membrane for leptin AKAP79/150 and K ATP channel trafficking in β-cells signaling, we used the PKA-AKAP interaction disruptor peptide st-Ht31 (50 μM) (33). ...
... PKA is typically activated by cAMP, which binds to PKA regulatory subunits and relieves autoinhibition to unleash the catalytic subunits. Alternatively, inhibiting the opposing activity of PP2B could also enhance net PKA signaling (11,31,(47)(48)(49). Our results above implicating that AKAP150 KD/ AKAP79ΔPIX cells have increased basal PKA signaling are consistent with an inhibitory role of PP2B on steady-state PKA signaling. ...
Article
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The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic β-cell, which secretes insulin upon glucose stimulation. Leptin is known to suppress glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the β-cell surface, which increases K+ conductance and causes β-cell hyperpolarization. We have previously shown that leptin-induced KATP channel trafficking requires protein kinase A (PKA)-dependent actin remodeling. However, whether PKA is a downstream effector of leptin signaling or PKA plays a permissive role is unknown. Using FRET-based reporters of PKA activity, we show that leptin increases PKA activity at the cell membrane and that this effect is dependent on NMDA receptors, CaMKKβ, and AMPK, which are known to be involved in the leptin signaling pathway. Genetic knockdown and rescue experiments reveal that the increased PKA activity upon leptin stimulation requires the membrane-targeted PKA-anchoring protein AKAP79/150, indicating PKA activated by leptin is anchored to AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, leads to increased surface KATP channels even in the absence of leptin stimulation. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate KATP channel trafficking in β-cells, hence insulin secretion. The study further advances our knowledge in the downstream signaling events that may be targeted to restore insulin secretion regulation in β-cells defective in leptin signaling, such as those from obese individuals with type 2 diabetes.
... The spatial organisation of enzymatic activity can be achieved via scaffold, anchoring and adapter proteins 50 . Many talin ligands are enzymes, or known enzyme substrates 27,44,45 , and there are many enzymes linked to IACs 46 and located in dendritic spines 47 . ...
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The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cell's computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 µm in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information.
... Cells accommodate to changes occurring in their microenvironment due to the information transmitted by adhesion receptors on neighboring cells, which regulate receptor-mediated cellular interactions through participation in the signal transduction pathways [1,4,5]. These receptors use their cytoplasmic domains that often contain specific motifs (including proline-rich motifs, phosphorylation sites, FERM and PDZ domain-binding motifs) to interact with appropriate specific interaction motifs in cytoplasmic proteins (SH3, Src-homology-3; SH2, Src-homology-2; FERM or PDZ domains, respectively) [6][7][8][9]. Cell-cell adhesion and contact between adjacent cells take place through multiprotein complexes located in intercellular junctions. In vertebrates, these cellular structures mostly comprise adherens junctions (AJs), gap junctions (GJs), desmosomes, and tight junctions (TJs) [10][11][12][13][14][15]. ...
Article
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The F11 Receptor (F11R), also called Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is a transmembrane glycoprotein of the immunoglobulin superfamily, which is mainly located in epithelial and endothelial cell tight junctions and also expressed on circulating platelets and leukocytes. It participates in the regulation of various biological processes, as diverse as paracellular permeability, tight junction formation and maintenance, leukocyte transendothelial migration, epithelial-to-mesenchymal transition, angiogenesis, reovirus binding, and platelet activation. Dysregulation of F11R/JAM-A may result in pathological consequences and disorders in normal cell function. A growing body of evidence points to its role in carcinogenesis and invasiveness, but its tissue-specific pro- or anti-tumorigenic role remains a debated issue. The following review focuses on the F11R/JAM-A tissue-dependent manner in tumorigenesis and metastasis and also discusses the correlation between poor patient clinical outcomes and its aberrant expression. In the future, it will be required to clarify the signaling pathways that are activated or suppressed via the F11R/JAM-A protein in various cancer types to understand its multiple roles in cancer progression and further use it as a novel direct target for cancer treatment.
... Perhaps ELMO1 provides an accessory protein or scaffold for these processes specific for cell wall proteins, possibly specific for those involved in adhesion. Scaffold proteins, which recruit and link proteins in a complex, facilitate the activity of enzymatic and signal transduction pathways (Lim et al., 2019;Pawson and Scott, 1997). There are examples of enzyme complexes in the biosynthetic pathways of carbohydrates in wheat (Tetlow et al., 2008), the likely formation of complexes in flavonoid synthesis, (Burbulis and Winkel-Shirley, 1999) and nonenzymatic proteins involved in starch metabolism in the chloroplast of A. thaliana (Lohmeier-Vogel et al., 2008). ...
Article
Plant growth, morphogenesis and development involve cellular adhesion, a process dependent on the composition and structure of the extracellular matrix or cell wall. Pectin in the cell wall is thought to play an essential role in adhesion, and its modification and cleavage are suggested to be highly regulated so as to change adhesive properties. To increase our understanding of plant cell adhesion, a population of ethyl methanesulfonate-mutagenized Arabidopsis were screened for hypocotyl adhesion defects using the pectin binding dye Ruthenium Red that penetrates defective but not wild-type (WT) hypocotyl cell walls. Genomic sequencing was used to identify a mutant allele of ELMO1 which encodes a 20 kDa Golgi membrane protein that has no predicted enzymatic domains. ELMO1 colocalizes with several Golgi markers and elmo1-/- plants can be rescued by an ELMO1-GFP fusion. elmo1-/- exhibits reduced mannose content relative to WT but no other cell wall changes and can be rescued to WT phenotype by mutants in ESMERALDA1, which also suppresses other adhesion mutants. elmo1 describes a previously unidentified role for the ELMO1 protein in plant cell adhesion.
... The Nck SH2/SH3 domains couple sites of tyrosine phosphorylation to the activation of downstream signaling partners that regulate cytoskeletal remodeling, gene transcription, protein translation, and cell survival (Li et al., 1992;Pawson and Scott, 1997;McCarty, 1998;Pawson and Nash, 2003;Pesti et al., 2012;Oeste and Alarcon, 2015;Porter and Day, 2016). However, depending on the extracellular stimulus and cell type, Nck1 and Nck2 may be capable of selectively regulating different responses within the cell (McCarty, 1998). ...
Article
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The Nck family of modular adaptor proteins, including Nck1 and Nck2, link phosphotyrosine signaling to changes in cytoskeletal dynamics and gene expression that critically modulate cellular phenotype. The Nck SH2 domain interacts with phosphotyrosine at dynamic signaling hubs, such as activated growth factor receptors and sites of cell adhesion. The Nck SH3 domains interact with signaling effectors containing proline-rich regions that mediate their activation by upstream kinases. In vascular biology, Nck1 and Nck2 play redundant roles in vascular development and postnatal angiogenesis. However, recent studies suggest that Nck1 and Nck2 differentially regulate cell phenotype in the adult vasculature. Domain-specific interactions likely mediate these isoform-selective effects, and these isolated domains may serve as therapeutic targets to limit specific protein-protein interactions. In this review, we highlight the function of the Nck adaptor proteins, the known differences in domain-selective interactions, and discuss the role of individual Nck isoforms in vascular remodeling and function.
... p66Shc belongs to the ShcA family of adaptor proteins, which also contains p46 and p52 isoforms [111]. p66Shc comprises of five domains, namely 100 amino acids long SH2 domain at the C-terminal [112,113], PTB domain, proline, and glycine-rich collagen homology (CH1) domain [91,96,114], the cytochrome c binding domain (CB), and a unique 110amino acid long CH2 domain present only in p66Shc isoform at N-terminal. CH2 domain is proline and glycine-rich and contains unique serine phosphorylation sites at serine 36 and serine 54 [95]. ...
Article
This review examines the concerted role of Epidermal Growth Factor Receptor (EGFR) and integrins in regulating Reactive oxygen species (ROS) production through different signaling pathways. ROS as such are not always deleterious to the cells but they also act as signaling molecules, that regulates numerous indespensible physiological fuctions of life. Many adaptor proteins, particularly Shc and Grb2, are involved in mediating the downstream signaling pathways stimulated by EGFR and integrins. Integrin-induced activation of EGFR and subsequent tyrosine phosphorylation of a class of acceptor sites on EGFR leads to alignment and tyrosine phosphorylation of Shc, PLCγ, the p85 subunit of PI-3 K, and Cbl, followed by activation of the downstream targets Erk and Akt/PKB. Functional interactions between these receptors result in the activation of Rac1 via these adaptor proteins, thereby leading to Reactive Oxygen Species. Both GF and integrin activation can produce oxidants independently, however synergistically there is increased ROS generation, suggesting a mutual cooperation between integrins and GFRs for redox signalling. The ROS produced further promotes feed-forward stimulation of redox signaling events such as MAPK activation and gene expression. This relationship has not been reviewed previously. The literature presented here can have multiple implications, ranging from looking at synergistic effects of integrin and EGFR mediated signaling mechanisms of different proteins to possible therapeutic interventions operated by these two receptors. Furthermore, such mutual redox regulation of crosstalk between EGFR and integrins not only add to the established models of pathological oxidative stress, but also can impart new avenues and opportunities for targeted antioxidant based therapeutics.
... The scaffold proteins bind protein to protein that usually function in sequence. The adaptor proteins recruit proteins that usually contain protein-protein interaction domains, and also protein kinases and their substrates to complex [163]. ...
Article
Insects possess an immune system that protects them from attacks by various pathogenic microorganisms that would otherwise threaten their survival. Immune mechanisms may deal directly with the pathogens by eliminating them from the host organism or disarm them by suppressing the synthesis of toxins and virulence factors that promote the invasion and destructive action of the intruder within the host. Insects have been established as outstanding models for studying immune system regulation because innate immunity can be explored as an integrated system at the level of the whole organism. Innate immunity in insects consists of basal immunity that controls the constitutive synthesis of effector molecules such as antimicrobial peptides, and inducible immunity that is activated after detection of a microbe or its product(s). Activation and coordination of innate immune defenses in insects involve evolutionary conserved immune factors. Previous research in insects has led to the identification and characterization of distinct immune signalling pathways that modulate the response to microbial infections. This work has not only advanced the field of insect immunology, but it has also rekindled interest in the innate immune system of mammals. Here we review the current knowledge on key molecular components of insect immunity and discuss the opportunities they present for confronting infectious diseases in humans.
... Some of the best-studied adaptor protein modules include phosphotyrosine-binding (PTB) and Src homology 2 (SH2) domains, both binding to phosphorylated tyrosine residues; the Src homology 3 (SH3) domain, binding to proline-rich sequences with the consensus PXXP; and the pleckstrin homology (PH) domain, interacting with phospholipids. 3,5 In addition, many cytoplasmic adaptor proteins contain a sterile α motif (SAM), an interaction domain with various functions including homo-and heterotypic association as well as RNA-binding. 6,7 For an in-depth discussion of the relevant adaptor protein modules, the reader is referred to previously published reviews. ...
Article
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Intracellular adaptor proteins are indispensable for the transduction of receptor‐derived signals, as they recruit and connect essential downstream effectors. The SLy/SASH1‐adaptor family comprises three highly homologous proteins, all of them sharing conserved structural motifs. The initial characterization of the first member SLy1/SASH3 (SH3 protein expressed in lymphocytes 1) in 2001 was rapidly followed by identification of SLy2/HACS1 (hematopoietic adaptor containing SH3 and SAM domains 1) and SASH1/SLy3 (SAM and SH3 domain containing 1). Based on their pronounced sequence similarity, they were subsequently classified as one family of intracellular scaffold proteins. Despite their obvious homology, the three SLy/SASH1‐members fundamentally differ with regard to their expression and function in intracellular signaling. On the contrary, growing evidence clearly demonstrates an important role of all three proteins in human health and disease. In this review, we systematically summarize what is known about the SLy/SASH1‐adaptors in the field of molecular cell biology and immunology. To this end, we recapitulate current research about SLy1/SASH3, SLy2/HACS1, and SASH1/SLy3, with an emphasis on their similarities and differences.
... Specific binding m odules h ave b e en identified (P aw son an d Scott, 1997). For receptor tyrosine k in ases, cytosolic co m p o n en ts bind either SH 2 or PTB dom ains (specifically recognising p h o spho-tyrosine re sid u es (P aw so n a n d Gish, 1992). ...
Thesis
Protein phosphorylation plays an essential role in a diverse array of signalling cascades and regulates many cellular processes. Protein kinase Cs (PKCs) constitute one of the families of kinases involved in phosphorylating substrates on serine or threonine residues. These kinases were initially identified as being receptors for tumour promoters (phorbol esters) and the conditions required to activate different isoforms determine the subgroup classification of the 10 isoforms. Classical PKCs (α,β1, βII and γ) depend upon Ca2+ and lipids (DAG, PS- phospholipids.) Novel PKCs (δ, ϵ, η, μ and θ) are insensitive to Ca2+ but are activated by lipids, DAG and phospholipids. The atypical PKCs (ζ, i and λ) differ greatly. These proteins are insensitive to Ca2+ and phorbol ester binding. The lack of knowledge on the control of the atypical PKCs has made the role of the atypical PKCs more elusive. Nevertheless, PKC ζ has been implicated in cell growth and differentiation. Moreover, PKC ζ is thought to be involved in a plethora of signal transduction pathways, including the Ras and MEK/MAPK pathways. The related atypical PKC i may be involved in UV induced apoptosis and insulin signalling. The aims of this thesis are to define the control and biological role of the atypical PKCs - primarily focusing on PKC ζ. As one approach, the project attempted to create a knockout mouse. This would help define a biological end point and therefore permit elucidation of the inputs. This study led to the identification of a pseudogene and its origin is described. As a second approach to investigate PKC ζ control, various direct paths were followed - ranging from searching for potential binding proteins and cellular localisation, to analysis of activation by lipids and phosphorylation. These studies have provided evidence for the dynamic control of PKC ζ (and PKC i) through a kinase cascade involving the lipid kinase PI3-kinase, the lipid responsive PDK1 and finally phosphorylation of PKC ζ at a site defined as threonine 410. The operation of this pathway and its influence on PKC ζ autophosphorylation (in vivo) and activity (in vitro) are presented.
... Many different conserved protein domains exist which bind a variety of specific target sequences. Multiple domains are often present in single molecules which enables the formation of large, multiprotein complexes which produce a regulated response to extracellular signals (reviewed in Pawson, 1995;Bork et al., 1997;Pawson and Scott, 1997). Some of these conserved domains are discussed below. ...
Thesis
Rac is a member of the Rho family of low molecular weight GTPases (p21s) which is involved in diverse processes including regulation of the actin cytoskeleton and transcriptional activation. Chimaerin, a multidomain GTPase activating protein (GAP) downregulates Rac by increasing its intrinsic rate of GTP hydrolysis. Two splice variants of the chimaerin gene differ in tissue and developmental expression patterns and α2-chimaerin contains an N terminal SH2 domain which is absent from αl-chimaerin. The distribution and morphological effects of the chimaerins, α2-chimaerin SH2 domain mutants and potential α2-chimaerin targets in NIE 115 neuroblastoma cells were investigated. The distribution of α1-chimaerin was predominantly cytoskeletal and α2-chimaerin cytosolic. In transiently transfected NIE 115 cells, α1-chimaerin was concentrated in the perinuclear region and its expression induced cell rounding, whilst α2-chimaerin was expressed throughout flattened, neurite bearing cells. A point mutation in the SH2 domain of α2-chimaerin induced an α1-chimaerin-like protein distribution and morphology. The effects of long term chimaerin overexpression on cell morphology and potential protein interactions were also investigated. Overexpression of α2-chimaerin induced an enlarged, flattened morphology and neurite outgrowth in the presence of serum, whilst overexpression of α1-chimaerin induced a rounded morphology with multiple peripheral actin microspikes and inhibited neurite outgrowth. p35, the neuronal cdk5 regulator and also an 130 kDa tyrosine phosphorylated protein were immunoprecipitated with chimaerin from these cell lines. Similarly an 180 kDa tyrosine phosphorylated protein was identified as a potential target of the α2-chimaerin SH2 domain. Investigation into the effects of chimaerin on activation of the transcription factor NFKB demonstrated cell type specific differences in NFKB signalling pathways between HeLa and NIE 115 cells. These results suggest that functional differences in the chimaerin isoforms are specified by the divergent N terminal sequences.
... It is largely unclear why and how nature chooses OGT to fulfill its duties in such a ubiquitous manner. To achieve that, one possibility is that some OGT interactors serve as scaffold, anchoring, or adaptor proteins that contribute to recruiting active OGT molecules into cellular complexes or by placing OGT close to their substrates, as they do for other post-translational modifications (e.g., phosphorylation) [63,64]. Indeed, among the OGT interactors, quite a few are well-known scaffold proteins (e.g., several 14-3-3 family proteins including YWHAE, YWHAG, YWHAH, and YWHAZ), anchoring proteins (e.g., AKAP2, AKPA12), and adaptor proteins (e.g., importin α). ...
Article
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Interactions between proteins are essential to any cellular process and constitute the basis for molecular networks that determine the functional state of a cell. With the technical advances in recent years, an astonishingly high number of protein–protein interactions has been revealed. However, the interactome of O-linked N-acetylglucosamine transferase (OGT), the sole enzyme adding the O-linked β-N-acetylglucosamine (O-GlcNAc) onto its target proteins, has been largely undefined. To that end, we collated OGT interaction proteins experimentally identified in the past several decades. Rigorous curation of datasets from public repositories and O-GlcNAc-focused publications led to the identification of up to 929 high-stringency OGT interactors from multiple species studied (including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, and others). Among them, 784 human proteins were found to be interactors of human OGT. Moreover, these proteins spanned a very diverse range of functional classes (e.g., DNA repair, RNA metabolism, translational regulation, and cell cycle), with significant enrichment in regulating transcription and (co)translation. Our dataset demonstrates that OGT is likely a hub protein in cells. A webserver OGT-Protein Interaction Network (OGT-PIN) has also been created, which is freely accessible.
... SH3 in turn interacts with a phosphorylated proline residue on other targeted proteins. Through this they lead to stimulation of other downstream molecules [44]. Ras near the cell membrane is a guanine nucleotide binding protein that shuttles between GTP/GDP bound form. ...
Chapter
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Skin aging is an inescapable phenomenon that leads to a functional decline of the skin along with emergence of characteristics features such as coarse skin, wrinkles, loss of elasticity and an overall aged appearance. While chronological aging is inevitable occurring with time, photoaging is contributed by Ultraviolet radiation and reactive oxygen species principally which can boost the skin aging process. These processes can however be ameliorated with the help of treatment strategies, one of them being supplementation with antioxidants. This chapter summarizes diverse mechanisms underlying skin aging with regards to Ultraviolet radiation and reactive oxygen species along with role of antioxidants in impeding these processes. Further, it provides a glimpse towards possible future explorations and challenges dominating the field of skin aging.
... Structurally, SLK displays an N-terminal Ste20 catalytic kinase domain (amino acids 1-338), a predicted central unstructured region (amino acids 339-788) and a C-terminal coiled-coil domain (amino acids 789-1202) (Fig. 1). In addition to the catalytic core, the central domain of the kinase contains a consensus SH3-binding site (P-P-E-P-E) located at position 735, suggesting that SLK may interact with SH3-domaincontaining proteins (Pawson and Scott, 1997;Sabourin et al., 2000). ...
Article
Over the past 20 years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target.
... Instead, they are known to be authentic protein domains for PDE4. Their action is equivalent to other protein domains, such as SH2 and SH3 domains, while having the ability to undergo self-folding(Pawson and Scott, 1997).Figure 1.8. The schematic illustration of PDE4 isoenzymes.Long forms contain UCR1 and UCR2, whilst short forms only haveUCR2. ...
Thesis
The alteration of intracellular cyclic adenosine monophosphate (cAMP) levels plays important regulatory roles in both physiological and pathological conditions, such as cancer. As the most aggressive form of brain tumour, glioblastoma is currently incurable due to limited treatment modalities. Low level of intracellular cAMP levels has been reported to be a feature of brain tumours. Thus, it is hypothesised that increasing cAMP concentrations by targeting regulatory proteins involved in the cAMP signalling pathways may offer advantages in preventing or treating glioblastoma. The overarching goal of this study, therefore, is to determine the dynamic effects of cAMP modulation on glioblastoma cell proliferation. The efficacies of compounds targeting various proteins involved in cAMP pathways were first investigated. The mechanisms explored were elevation of cAMP level through phosphodiesterases (PDEs) inhibition, adenylyl cyclase (AC) activation, as well as modulation via β-adrenoceptor (β-AR) and G proteins. A series of compounds were evaluated applying various assaying techniques and pharmacological tools using cAMP accumulation assays, cell proliferation, caspase-3/7 activation, and flow cytometry to determine cell cycle. It was demonstrated that increasing cAMP levels by multiple PDE inhibition or AC stimulation resulted in cell growth suppression on both rat and human glioblastoma models. The study was also extended to identify the role of possible crosstalk between calcium through SOCE (store-operated calcium entry) and cAMP pathways, which both were found to contribute to cell growth modulation. The effect of the elevation of intracellular cAMP on cell proliferation was further explored through the direct activation of adenosine A2A receptor (A2AR) and inhibition of cAMP degradation via PDE10A. Previous computational studies revealed that the triazoloquinazoline-based compounds (compound 1-6), initially known as PDE10A inhibitors, are bound at the orthosteric site of A2AR. To validate the computational results, these compounds were characterised using NanoBRET-based ligand binding studies with HEK293T expressing Nluc-A2AR and functional assays in lung cancer cell lines and glioma/glioblastoma cell models, which both cell models expressed endogenous levels of PDE10A and A2AR. The study highlighted that compounds 1 and 5 were dual-target ligands to A2AR and PDE10A, whereas compound 3 appeared to be a pan-agonist of adenosine receptors (ARs), and compound 4 was more potent when A2BR was expressed. Compound 2 seems to possess toxic effects that may be independent of action to A2AR or PDE10A. Lastly, preliminary studies were conducted to investigate the possibility of biased signalling by RAMPs on protease-activated receptor 4 (PAR4) and calcitonin-like receptor (CLR). Using PAR4 transiently transfected HEK293T cells, both cognate ligand and agonist peptide were used to profile PAR4 signalling including RAMPs-trafficking to the plasma membrane, promoting intracellular calcium release and recruiting β-arrestins. The effects of RAMPs were also investigated in HUVECs and cardiomyocytes focusing on the effect of endogenous ligands on cell growth. Whilst RAMPs altered PAR4 initial signalling events in promoting β-arrestin recruitment, the study on heterodimer complex of RAMPs and CLR on cell growth further corroborated that signalling bias can be translated into physiological responses in HUVECs and cardiomyocytes. To conclude, these studies provided evidence on how the alterations of intracellular cAMP levels affected cell proliferation in numerous cancer models, and that the cAMPmediated anti-proliferative effect was cell-line dependent. Targeting multiple PDEs suppressed cell growth in cancer-derived cells, therefore providing a viable target to reduce tumour progression. Given the critical role of PAR4 in platelet aggregation and pro-proliferative of calcitonin peptide family, this research may have important implications for the role of RAMPs in cardiovascular pathologies.
... Additionally, the protein domains are linked to each other for creating multi-domain protein structures to gain a wide range of functional property 79,80 . This can be exemplified through flexible architectures of signaling pathways like mitogen-activated protein kinase (MAPK) cascades 81,82 . This pathway is associated with controlling for various biological processes such as metabolism, cellular morphology, cell cycle progress, and gene expression in the influence of any extracellular signals or stimuli 83 and cellular signaling and pathogenesis-related structure development 64,84 . ...
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Blast disease incited by Magnaporthe oryzae is a major threat to sustain rice production in all rice growing nations. The pathogen is widely distributed in all rice paddies and displays rapid aerial transmissions, and seed-borne latent infection. In order to understand the genetic variability, host specificity, and molecular basis of the pathogenicity-associated traits, the whole genome of rice infecting Magnaporthe oryzae (Strain RMg_Dl) was sequenced using the Illumina and PacBio (RSII compatible) platforms. The high-throughput hybrid assembly of short and long reads resulted in a total of 375 scaffolds with a genome size of 42.43 Mb. Furthermore, comparative genome analysis revealed 99% average nucleotide identity (ANI) with other oryzae genomes and 83% against M. grisea, and 73% against M. poe genomes. The gene calling identified 10,553 genes with 10,539 protein-coding sequences. Among the detected transposable elements, the LTR/Gypsy and Type LINE showed high occurrence. The InterProScan of predicted protein sequences revealed that 97% protein family (PFAM), 98% superfamily, and 95% CDD were shared among RMg_Dl and reference 70-15 genome, respectively. Additionally, 550 CAZymes with high GH family content/distribution and cell wall degrading enzymes (CWDE) such endoglucanase, beta-glucosidase, and pectate lyase were also deciphered in RMg_Dl. The prevalence of virulence factors determination revealed that 51 different VFs were found in the genome. The biochemical pathway such as starch and sucrose metabolism, mTOR signaling, cAMP signaling, MAPK signaling pathways related genes were identified in the genome. The 49,065 SNPs, 3267 insertions and 3611 deletions were detected, and majority of these varinats were located on downstream and upstream region. Taken together, the generated information will be useful to develop a specific marker for diagnosis, pathogen surveillance and tracking, molecular taxonomy, and species delineation which ultimately leads to device improved management strategies for blast disease. Since the traditional times, both cereal and cereal products act as pre-eminent and substantial carbohydrate food resources for much of the human population, especially in Asian countries 1,2. Among the pre-harvest production constraints, the global cultivation of many cereals including rice and pearl millet is mostly affected by a blast disease-causing filamentous ascomycete fungus, Magnaporthe (Hebert) Barr (anamorph: Pyricularia) 3. This devastating hemibiotrophic pathogen belongs to the family Magnaporthaceae and is of principal concern due to the wide distribution, rapid aerial transmissions, seed-borne latent infection, and associated yield losses 2,4,5. Morphologically, it causes white, bluish, or greyish water-soaked lesions in all foliar parts such as grain, leaf, OPEN
... Additionally, the protein domains are linked to each other for creating multi-domain protein structures to gain a wide range of functional property 79,80 . This can be exemplified through flexible architectures of signaling pathways like mitogen-activated protein kinase (MAPK) cascades 81,82 . This pathway is associated with controlling for various biological processes such as metabolism, cellular morphology, cell cycle progress, and gene expression in the influence of any extracellular signals or stimuli 83 and cellular signaling and pathogenesis-related structure development 64,84 . ...
Article
Blast disease incited by Magnaporthe oryzae is a major threat to sustain rice production in all rice growing nations. The pathogen is widely distributed in all rice paddies and displays rapid aerial transmissions, and seed-borne latent infection. In order to understand the genetic variability, host specificity, and molecular basis of the pathogenicity-associated traits, the whole genome of rice infecting Magnaporthe oryzae (Strain RMg_Dl) was sequenced using the Illumina and PacBio (RSII compatible) platforms. The high-throughput hybrid assembly of short and long reads resulted in a total of 375 scaffolds with a genome size of 42.43 Mb. Furthermore, comparative genome analysis revealed 99% average nucleotide identity (ANI) with other oryzae genomes and 83% against M. grisea, and 73% against M. poe genomes. The gene calling identified 10,553 genes with 10,539 protein-coding sequences. Among the detected transposable elements, the LTR/Gypsy and Type LINE showed high occurrence. The InterProScan of predicted protein sequences revealed that 97% protein family (PFAM), 98% superfamily, and 95% CDD were shared among RMg_Dl and reference 70-15 genome, respectively. Additionally, 550 CAZymes with high GH family content/distribution and cell wall degrading enzymes (CWDE) such endoglucanase, beta-glucosidase, and pectate lyase were also deciphered in RMg_Dl. The prevalence of virulence factors determination revealed that 51 different VFs were found in the genome. The biochemical pathway such as starch and sucrose metabolism, mTOR signaling, cAMP signaling, MAPK signaling pathways related genes were identified in the genome. The 49,065 SNPs, 3267 insertions and 3611 deletions were detected, and majority of these varinats were located on downstream and upstream region. Taken together, the generated information will be useful to develop a specific marker for diagnosis, pathogen surveillance and tracking, molecular taxonomy, and species delineation which ultimately leads to device improved management strategies for blast disease.
... Src Homology 2 (SH2) domains and phosphotyrosine binding (PTB) domains can both interact with phosphotyrosine-containing peptide target sequences that are generated upon activation of a receptor tyrosine kinase; the specificity of the interactions are determined by the amino acid context of the phosphorylated amino acid residues (reviewed by Pawson, 1995 andPawson &Scott, 1997). These protein modules will be discussed in greater depth later in this chapter. ...
Thesis
Rac is a member of the Rho subfamily of low molecular weight GTPases (p21s) and is involved in diverse cellular processes. GTPase-activating proteins (GAPs) regulate p21 activity by increasing intrinsic GTPase activity. The chimaerins are a family of p21-Rac GAPS with distinct patterns of tissue and developmental distribution, α2 chimaerin contains an amino-terminal SH2 domain and is selectively expressed within the nervous system. SH2 domains bind specific phosphotyrosine-containing sequences and the presence of this domain may place α2 chimaerin within tyrosine kinase signalling pathways. Comparisons between SH2 domains suggest that the mechanism of target interaction of the chimaerin SH2 domain may be distinct from that of others. Affinity chromatography was used to detect potential α2 chimaerin SH2 domain target proteins in rat brain extracts; some of these proteins were tyrosine-phosphorylated. Tubulin and actin were isolated as targets and peptide sequence information was obtained for three other potential target proteins, two of which appeared to be novel sequences. Several different kinase activities bound α2 chimaerin SH2 domain affinity columns; one of these phosphorylated full length α2 chimaerin. Full length α2 chimaerin and its isolated SH2 domain bound a phosphotyrosine column. Amino acid residue substitutions were made in the α2 chimaerin SH2 domain at sites essential for function in other SH2 domains; certain point mutations affected phosphotyrosine-binding. α2 Chimaerin probes bound two previously identified putative α2 chimaerin target proteins of molecular mass 13kDa and 64kDa; these interactions were phosphotyrosine-independent. The interactions of the 13kDa and 64kDa proteins with α2 chimaerin differed in their sensitivity to point mutation of the chimaerin SH2 domain. Specific antibodies have been raised to these proteins to facilitate further studies. Results suggest that substrates of the α2 chimaerin SH2 domain may include both tyrosine-phosphorylated and non-tyrosine phosphorylated proteins.
... Néanmoins le phénotype observé au niveau du lignage EPr est moindre que celui observé dans les embryons Fgfr1-/- (Kang et al., 2017;Molotkov et al., 2017). Ces embryons Fgfr2-/-présentent une forte réduction du nombre de cellules NANOG positives comparés aux embryons simples mutants pour Fgfr1-/- (Kang et al., 2017 (Pawson and Scott, 1997). Une fois GRB2 fixé sur ces protéines co-effectrices, du GDP est relargué ce qui favorise la fixation de GTP sur la protéine RAS permettant ainsi son activation par hydrolyse de ce GTP. ...
Thesis
A E3.5 jours de développement (E3.5), l’embryon murin se compose d’une monocouche de cellules externes correspondant au Trophectoderme (TE) et d’une masse cellulaire interne (MCI), hétérogène, constituée de deux sous-populations de cellules précurseurs : les cellules épiblastiques (Epi) et les cellules d’endoderme primitif (EPr). NANOG, marqueur épiblastique et GATA6, marqueur de l’EPr, sont co-exprimés à E3.5 dans la MCI puis adoptent une expression exclusive au sein de leur lignage respectif. La différenciation du lignage EPr nécessite l’expression de GATA6 et l’activation de la voie Récepteur Tyrosine Kinase (RTK) activée par le FGF (RTK-FGF) pour l’induction de gènes cibles de GATA6 tels que Sox17 et Gata4.Au cours de ma thèse, j’ai, dans un premier temps, étudié la relation GATA6/voie RTK-FGF lors de l’induction de l’expression des gènes de différenciation de l’EPr. J’ai utilisé des cellules souches embryonnaires murines ES sauvages ou mutantes pour Gata6 (ES Gata6-/-), dans lesquelles j’ai surexprimé différentes formes mutantes de Gata6 inactivées sur les différents résidus identifiés comme potentiellement phosphorylables par la voie RTK-FGF. Ainsi, j’ai analysé l’expression protéique des gènes Sox17 et Gata4 ainsi que des expressions ARN de ces cibles et d’autres gènes caractéristiques exprimés dans l’EPr dans les différentes conditions de surexpression des formes de Gata6 en absence ou présence d’inhibiteurs de la voie RTK-FGF. Ainsi, j’ai pu mettre en évidence que la transmission du signal s’effectue au travers de récepteur au FGF et qu’il existe une compensation entre les branches RTK-MEK-ERK et RTK-PI3K ciblant le résidu Sérine 37 de GATA6. Enfin, les résidus S34 et T509 sont nécessaires et les résidus S34, S37 et T509 semblent coopérer, au travers d’un mécanisme pour le moment non détaillé, pour l’induction des gènes cibles exprimés au sein de l’EPr.Dans un second temps, j’ai débuté la caractérisation phénotypique du rôle des facteurs Dickkopf1 (DKK1), un inhibiteur de la voie WNT/β-caténine, et NOGGIN, un inhibiteur de la voie des Bone Morphogenic Protein (BMP) lors de la différentiation de l’EPr en endoderme pariétal (EP) et viscéral (EV). A l’aide de modèles de souris KO pour DKK1 et NOGGIN, croisées en fond C57Bl6 pur, j’ai pu observer que l’expression d’OCT4 était maintenue au sein des embryons homozygotes mutants pour Dkk1 et double homozygotes mutants pour Dkk1 et Noggin. Cependant, le mécanisme potentiel de compensation ou de coopération de ces deux marqueurs n’est pour le moment pas détaillé précisément et mérite l’analyse d’un plus grand nombre d’embryons mutants.
... 14-3-3 proteins are a family of highly conserved acidic proteins that are widely expressed, with seven isoforms in mammals . Known as scaffolding and chaperon proteins, 14-3-3 proteins exist as homo-and/or heterodimers and interact with a vast number of proteins in varied cells involved in a wide range of cell signaling pathways, which regulate diverse cell functions such as cell growth, cell development, apoptosis and cell motility Morrison, 1994;Pawson and Scott, 1997). Many kinases are regulated in a 14-3-3 proteindependent manner, including RAF kinases, apoptosis signal-regulation kinase 1, calciumdependent protein kinases, phosphatidylinositol 4-kinases, leucine-rich repeat protein kinase-2, ...
... Despite their lack of enzymatic activity, these adapter proteins play important roles in both normal and oncogenic cell signaling [10][11][12]. Upon their membrane translocation, GRB2 and SHC recognize the tyrosine phosphorylation of RTKs using a Src homology 2 (SH2) domain [13] and phosphotyrosine binding (PTB) domain [14], respectively. ...
Article
RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS. Fullsize Image
... The specificity of protein phosphorylation requires the spatialtemporal maintenance of cAMP and the molecules responsible (PKA, PDA, and ACs) for the phosphorylation of target proteins. The task is carried out by a kinase anchoring protein (AKAP) by confining the PKA holoenzyme and PDA in a specific cellular region [40,41]. Therefore, the presence of PKA at the anterior region in close proximity to PAC indicates the involvement of AKAP. ...
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Euglena gracilis is a photosynthetic flagellate. To acquire a suitable position in its surrounding aquatic environment, it exploits light and gravity primarily as environmental cues. Several physiological studies have indicated a fine-tuned relationship between gravity sensing (gravitaxis) and light sensing in E. gracilis. However, the underlying molecular mechanism is largely unknown. The photoreceptor photoactivated adenylyl cyclase (PAC) has been studied for over a decade. Nevertheless, no direct/indirect interaction partner (upstream/downstream) has been reported for PAC. It has been shown that a specific protein, kinase A (PKA), showed to be involved in phototaxis and gravitaxis. The current study reports the localization of the specific PKA and its relationship with PAC.
... Protein−protein (or ligand) interactions (PPIs) play a central role in numerous biological processes, such as cellular signal transduction and proliferation. 1 The accurate estimation of binding affinity and kinetics is essential for understanding the molecular mechanism of PPIs and the development of PPI inhibitors. 2,3 Biolayer interferometry (BLI) is widely used for the study of diverse biomolecular interactions because the binding affinity and kinetics can be measured in one experiment between biosensor-immobilized ligands and analytes in a well. ...
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Quantitative analysis of protein-protein interactions (PPIs) using biolayer interferometry (BLI) requires effective suppression of nonspecific binding (NSB) between analytes and biosensors. In particular, the study of weak interactions (i.e., K D > 1 μM) requires high concentrations of analytes, which substantially increases NSB. However, there are only a few so-called NSB blockers compatible with biomolecules, which limits the use of BLI in the accurate analysis of weak interactions. The present study aims to identify a new NSB blocker for the quantitative analysis of weak PPIs using BLI. We find that saccharides, especially sucrose, are potent NSB blockers and demonstrate their compatibility with other blocking additives. We also demonstrate the effects of the new NSB blocker by characterizing the binding between nonstructural protein 1 of the influenza A virus and human phosphoinositide 3-kinase. We anticipate that the new NSB-blocking admixture will find broad applications in studying weak interactions using BLI.
... Phosphorylated tyrosine residues of RTKs function as docking sites for a wide range of proteins. These proteins include phospholipase C (PLC-g), phosphatidylinositol 3 0 kinase (PI3K), RAS guanosine triphosphatase (GTPase) activating protein (GAP), and SRC family tyrosine kinases (Pawson and Scott 1997). These molecules contain noncatalytic domains called SRC homology (SH) regions 2 and 3. SH2 domains bind preferentially to tyrosine-phosphorylated proteins while SH3 domains may promote binding to membranes or the cytoskeleton. ...
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Growth factors are relatively small and stable soluble polypeptides that mediate short-range cell-to-cell interactions. Growth factors and their cognate receptors, which often harbor enzymatic activity, function in the framework of modules regulating various cellular processes, such as proliferation, differentiation, and migration. These modules are evolutionarily conserved, and in many cases their primary developmental function is the determination of cell lineage through heterotypic cellular interactions. Many growth factors are versatile, promoting appropriate biological outcomes of several cell lineages, while others are more limited in scope. Growth factor expression is highly regulated, such that excessive growth factor activity is often associated with pathogenic hyper-proliferation (e.g.,cancer, fibrosis, and psoriasis).In addition to discussing the role of certain growth factors in different diseases and the implications for cancer, we also provide approved drugs against specific growth factors for patient treatment.
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A core network of ubiquitously expressed glutamate-synapse-associated proteins mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we sought to classify the diversity of activity-dependent remodeling across brain regions using quantitative protein interaction network (PIN) analysis. We first compared the response of cultured neurons to distinct stimuli, and defined PIN parameters that differentiate input types. We next compared the response of three different brain regions maintained alive in vitro to an identical stimulus, and identified three qualitatively different PIN responses. Finally, we measured the PIN response following associative learning tasks, delay and trace eyeblink conditioning, in three brain regions, and found that the two forms of associative learning are distinguished from each other using brain-region-specific network mechanisms. We conclude that although the PIN of the glutamatergic post-synapse is expressed ubiquitously, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity.
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The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro , and reverses EMT, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1, and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.
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Historically, the diffusion of chemical signals through the cell was thought to occur within a cytoplasmic soup bounded by the plasma membrane. This theory was predicated on the notion that all regulatory enzymes are soluble and moved with a Brownian motion. Although enzyme compartmentalization was initially rebuffed by biochemists as a ‘last refuge of a scoundrel', signal relay through macromolecular complexes is now accepted as a fundamental tenet of the burgeoning field of spatial biology. A-Kinase anchoring proteins (AKAPs) are prototypic enzyme-organizing elements that position clusters of regulatory proteins at defined subcellular locations. In parallel, the primary cilium has gained recognition as a subcellular mechanosensory organelle that amplifies second messenger signals pertaining to metazoan development. This article highlights advances in our understanding of AKAP signaling within the primary cilium and how defective ciliary function contributes to an increasing number of diseases known as ciliopathies.
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Molecules that induce interactions between proteins, often referred to as “molecular glues”, are increasingly recognized as important therapeutic modalities and as entry points for rewiring cellular signaling networks. Here, we report a new PACE-based method to rapidly select and evolve molecules that mediate interactions between otherwise non-interacting proteins: rapid evolution of Protein-Protein Interaction Glues (rePPI-G). Proof-of-concept evolutions demonstrated that rePPI-G reduces the “hook” effect of the engineered molecular glues, due at least in part to tuning the interaction affinities of each individual component of the bifunctional molecule. Altogether, this work validates rePPI-G as a continuous, phage-based evolutionary technology for optimizing molecular glues, providing a strategy for developing molecules that reprogram protein-protein interactions.
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1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (tetrahydrocurcumin, THC) is a major bioactive metabolite of curcumin, demonstrating the potential anti-inflammatory, antioxidant and neuroprotective properties, etc. In this study, it was found that Aβ induced decreased cell viability, cell cycle arrest and apoptosis in BV-2 cells, which were ameliorated by THC. In vivo, THC administration rescued learning and memory, and reduced Aβ burden in the hippocampus of APP/PS1 mice. By proteomic analysis of the hippocampus of mice, 157 differentially expressed proteins were identified in APP/PS1 mice treated with THC (comparing with APP/PS1 mice), which also suggested that the effects of THC on the cell cycle and apoptosis were mostly related to the “Ras signaling pathway”, etc. In APP/PS1 mice, the down-regulation of Gab2 and K-Ras, and the up-regulation of caspase-3, TGF-β1 and TNF-ɑ were observed; THC attenuated the abnormal expression of Gab2, K-Ras, caspase-3 and TNF-ɑ, and up-regulated TGF-β1 and Bag1 expression. In BV-2 cells, Aβ induced the down-regulation of Gab2, K-Ras and TGF-β1, and the overexpression of caspase-3, PARP1, cleaved-PARP1 and TNF-ɑ, which were restored by THC. Moreover, THC up-regulated Bag1 expression in Aβ-treated BV-2 cells. The decreased transcriptional expression of Ccnd2 and Cdkn1a were also observed in Aβ-treated BV-2 cells, and THC alleviated the down-regulation of Ccnd2. For the first time, we identified that the action of THC in preventing AD was associated with inhibition of cell cycle arrest and apoptosis of microglia via the Ras/ERK signaling pathway, shedding new light on the role of THC in alleviating the progression of AD.
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Nearly all proteins are synthesized in the cytosol. The majority of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. To better understand cellular biology, molecular and chemical technologies to properly characterize protein trafficking (and mistrafficking) have been developed and applied. Herein, we take a biochemical perspective to review technologies that enable spatial and temporal measurement of protein distribution, focusing on both the most widely adopted methodologies and exciting emerging approaches.
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Extensive cellular and behavioral studies have led to the postulation that memories are encoded by changes in synaptic strength between neurons, as demonstrated by the correlation between long-term changes in animal behavior and long-term changes in neuronal connections underlying a specific behavior in invertebrate animals, or even in vertebrate animals, where cellular models of synaptic plasticity using genetic approaches, such as Long-Term Potentiation (LTP) and Long-Term Depression (LTD), have been shown to depend on long-term changes in synaptic activity implicated in behavioral learning and memory. Long-term memory (LTM) is crucial for animal's survival and thus represents a mechanism that underlies fundamental neurobiological events in the nervous system of vertebrate and non-vertebrate species including the human. Long-term changes in synaptic connectivity as well as long-term behavioral changes (both activities that underlie several of the properties of LTM and are used as a parameter to explain the long-lasting enhancement of neuronal function after a stimulus) have been demonstrated to rely on signals that initially occur in the cell body. LTP is a form of synaptic plasticity widely accepted as a cellular model for stabilization of synapses in neurobiological phenomena such as development and learning and memory. Much of the experimental work concerning LTP in learning has been focused on the NMDA receptor dependent forms of LTP. But several questions have arisen regarding if LTP equals memory. If LTP has a real role in memory, a more appropriate hypothesis should be stated by postulating that activity-dependent synaptic plasticity and multiple forms of memory known to exist, share a common core; that is, the synaptic plasticity and memory hypothesis states that activity dependent synaptic plasticity is induced at appropriate synapses during memory formation. Synaptic plasticity is a physiological phenomenon that induces specific patterns of neural activity sustained by chemical and molecular mechanisms, that gives rise to changes in synaptic efficacy and neural excitability that long outlast the events that trigger them. Based on the various properties of synaptic plasticity discovered, LTP may be proposed as a suitable neuronal mechanism for the development of several memory systems, including initial encoding and storage of memory traces and initial phases of trace consolidation over time. Such memory processing made up by LTP or LTD most probably occur as a network specific process, making LTP a universal mechanism for encoding and storage of memory traces and, what gets encoded, is part of a network property rather than mechanisms working at individual synapses. For example, the type of information processed at the hippocampus is quite different from the information processed by the amygdala, and such information should remain if the mechanisms of plasticity operating in each brain area are conserved. Decades of research have demonstrated that LTP in the hippocampus is induced by synaptic activity and that cytoplasmic membrane-bound molecule(s) are required to transduce extracellular signals mediated by receptor-activation into activation of intracellular signaling processes. Most of these processes depend on intracellular calcium activity, and thereby on calcium-dependent mechanisms that are recruited for LTP induction and expression. For instance, NMDA receptors have been shown to be essential for initiation of LTP, but the expression of this phenomenon is brought about primarily by AMPA receptors. Induction of LTP in CA1 hippocampal region has been shown to depend on increases of intracellular calcium and activation of specific calcium-dependent molecules such as the calcium/calmodulin-dependent protein kinase (CaMKII), whose cell expression is confined predominantly at postsynaptic densities. Moreover, long-term expression of LTP requires protein synthesis, where transient signals will be linked to activation of specific genes that ultimately will determine growth and remodeling of potential active synapses. Different types of synapses may express and use a different set of molecules mediating activation of intracellular signaling pathways to initiate and maintain synaptic
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Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.
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Phosphorylation forms an important part of the signalling system that cells use for decision making and regulation of processes such as celll division and differentiation. To date, a large portion of identified phosphosites are not known to be targeted by any kinase. At the same time around 30% of kinases have no known target. This knowledge gap stresses the need to make large scale, data-driven computational predictions. In this paper, we have created a machine learning-based model to derive a probabilistic kinase-substrate network from omics datasets. We show that our methodology displays improved performance compared to other state of the art kinase-substrate predictions, and provides predictions for more kinases than most of them. Importantly, it better captures new experimentally-identified kinase-substrate relationships. It can therefore allow the improved prioritisation of kinase-substrate pairs for illuminating the dark human cell signalling space.
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Chapter
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Phosphorylation plays a vital role in signal transduction and cell cycle. Identifying and understanding phosphorylation through machine-learning methods has a long history. However, existing methods only learn representations of a protein sequence segment from a labeled dataset itself, which could result in biased or incomplete features, especially for kinase-specific phosphorylation site prediction in which training data are typically sparse. To learn a comprehensive contextual representation of a protein sequence segment for kinase-specific phosphorylation site prediction, we pretrained our model from over 24 million unlabeled sequence fragments using ELECTRA (Efficiently Learning an Encoder that Classifies Token Replacements Accurately). The pretrained model was applied to kinase-specific site prediction of kinases CDK, PKA, CK2, MAPK, and PKC. The pretrained ELECTRA model achieves 9.02% improvement over BERT and 11.10% improvement over MusiteDeep in the area under the precision-recall curve on the benchmark data.
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Thesis
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Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.
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NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K. T.-Y., Ho, A. M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P. G.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.
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Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1PH contains a pleckstrin homology (PH) domain, and the IH2PTB contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated that the IH2PTB in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1PH or the SAIN regions. In 32D cells the IH1PH was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70s6k phosphorylation. In contrast, the IH2PTB and the SAIN regions were not required for these insulin actions; however, the IH2PTB improved the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis. The sensitivity of these responses was partially reduced by deletion of either the IH1PH or the IH2PTB and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.
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Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.
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The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.
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Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC.
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Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.
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Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.
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Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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Shc phosphorylation in cells following growth factor, insulin, cytokine, and lymphocyte receptor activation leads to its association with Grb2 and activation of Ras. In addition to being a cytoplasmic substrate of tyrosine kinases, Shc contains an SH2 domain and a non-SH2 phosphotyrosine binding (PTB) domain. Here we show that the Shc PTB domain, but not the SH2 domain, binds with high affinity (ID50 approximately equal to 1 microM) to phosphopeptides corresponding to the sequence surrounding Tyr250 of the polyoma virus middle T (mT) antigen (LLSNPTpYSVMRSK). Truncation studies show that five residues amino-terminal to tyrosine are required for high affinity binding, whereas all residues carboxyl-terminal to tyrosine can be deleted without loss of affinity. Substitution studies show that tyrosine phosphorylation is required and residues at -5, -3, -2, and -1 positions relative to pTyr are important for this interaction. 1H NMR studies demonstrate that the phosphorylated mT antigen-derived sequence forms a stable beta turn in solution, and correlations between structure and function indicate that the beta turn is important for PTB domain recognition. These results show that PTB domains are functionally distinct from SH2 domains. Whereas SH2 domain binding specificity derives from peptide sequences carboxyl-terminal to phosphotyrosine, the Shc PTB domain gains specificity by interacting with beta turn-forming sequences amino-terminal to phosphotyrosine.
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Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein that can transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. Shc binds a variety of tyrosine-phosphorylated growth factor receptors presumably via its carboxyl-terminal SH2 domain. We cloned a fragment of Shc when screening a bacterial expression library with tyrosine-phosphorylated epidermal growth factor (EGF) receptor. Surprisingly, this fragment encodes the amino terminus of Shc, a region that has no significant similarity to an SH2 domain. When expressed as a glutathione S-transferase fusion protein, this amino-terminal domain binds to autophosphorylated EGF receptor, as well as HER2/neu and TrkA receptors. This fragment acts like an SH2 domain in that it does not bind non-phosphorylated EGF receptor or EGF receptor with all tyrosine phosphorylation sites mutated or deleted. Our data define a novel domain in Shc that has the potential to interact with growth factor receptors and other tyrosine-phosphorylated proteins.
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Phosphorylation of molecules involved in synaptic transmission by multifunctional protein kinases modulates both pre- and post-synaptic events in the central nervous system. The positioning of kinases near their substrates may be an important part of the regulatory mechanism. The A-kinase-anchoring proteins (AKAPs; ref. 3) are known to bind the regulatory subunit of cyclic AMP-dependent protein kinase A with nanomolar affinity. Here we show that anchoring of protein kinase A by AKAPs is required for the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate channels. Intracellular perfusion of cultured hippocampal neurons with peptides derived from the conserved kinase binding region of AKAPs prevented the protein kinase A-mediated regulation of AMPA/kainate currents as well as fast excitatory synaptic currents. This effect could be overcome by adding the purified catalytic subunit of protein kinase. A control peptide lacking kinase-binding activity had no effect. To our knowledge, these results provide the first evidence that anchoring of protein kinase A is crucial in the regulation of synaptic function.
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We have previously used an overlay assay technique to detect proteins that interact with protein kinase C (PKC) (Hyatt, S. L., Klauck, T., and Jaken, S. (1990) Mol. Carcinogenesis 3, 45-53). In some cases, binding proteins were also identified as substrates. Therefore, we used the overlay assay approach to screen a rat kidney lambda gt11 cDNA library to isolate and identify additional PKC substrates. Two clones have now been characterized. 35A is the rat homologue of the myristoylated alanine-rich C kinase substrate (MARCKS)-related F52 cDNA, whereas 35H is a partial cDNA with substantial homology to the 3' end of beta-adducin. Both cDNAs encode proteins that bind phosphatidyl-serine (PS) and are substrates for PKC. Phosphorylation decreased both PS and PKC binding activities. Both proteins contain high density positive charge domains similar to that found in the major PKC substrate MARCKS. These results demonstrate that PKC interactions with certain substrate proteins are of sufficiently high affinity to facilitate their isolation via interaction cloning.
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Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units. Similarly, in mammalian cells the cyclic adenosine 3',5'-monophosphate-dependent protein kinase (PKA) and phosphatase 2B [calcineurin (CaN)] are complexed by an A kinase anchoring protein, AKAP79. Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN. The subcellular distributions of PKC and AKAP79 were similar in neurons. Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes.
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Protein dephosphorylation by phosphatase PP1 plays a central role in mediating the effects of insulin on glucose and lipid metabolism. A PP1C-targeting protein expressed in 3T3-L1 adipocytes (called PTG, for protein targeting to glycogen) was cloned and characterized. PTG was expressed predominantly in insulin-sensitive tissues. In addition to binding and localizing PP1C to glycogen, PTG formed complexes with phosphorylase kinase, phosphorylase a, and glycogen synthase, the primary enzymes involved in the hormonal regulation of glycogen metabolism. Overexpression of PTG markedly increased basal and insulin-stimulated glycogen synthesis in Chinese hamster ovary cells overexpressing the insulin receptor, which do not express endogenous PTG. These results suggest that PTG is critical for glycogen metabolism, possibly functioning as a molecular scaffold.
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The oriented peptide library technique was used to investigate the peptide-binding specificities of nine PDZ domains. Each PDZ domain selected peptides with hydrophobic residues at the carboxyl terminus. Individual PDZ domains selected unique optimal motifs defined primarily by the carboxyl terminal three to seven residues of the peptides. One family of PDZ domains, including those of the Discs Large protein, selected peptides with the consensus motif Glu-(Ser/Thr)-Xxx-(Val/Ile) (where Xxx represents any amino acid) at the carboxyl terminus. In contrast, another family of PDZ domains, including those of LIN-2, p55, and Tiam-1, selected peptides with hydrophobic or aromatic side chains at the carboxyl terminal three residues. On the basis of crystal structures of the PSD-95-3 PDZ domain, the specificities observed with the peptide library can be rationalized.
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The mating response pathway of the yeast Saccharomyces cerevisiae includes a heterotrimeric guanine nucleotide-binding protein (G protein) that activates a mitogen-activated protein MAP kinase cascade by an unknown mechanism. An amino-terminal fragment of the MAP kinase scaffold protein Ste5p that interfered with pheromone-induced cell cycle arrest was identified. A haploid-specific interaction between the amino terminus of Ste5p and the G protein beta subunit Ste4p was also detected in a two-hybrid assay, and the product of a signaling-defective allele of STE4 was defective in this interaction. In cells with a constitutively activated pheromone response pathway, epitope-tagged Ste4p was coimmunoprecipitated with Ste5p. Thus, association of the G protein and the MAP kinase cassette via the scaffolding protein Ste5p may transmit the G protein signal.
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THE proteins Gab1 and the related DOS (for 'daughter of seven-less') each bind to substrates of tyrosine kinases like Grb2 or Corkscrew, and act in signalling pathways downstream of tyrosine kinase receptors1–3. Here we show that Gab1 interacts directly with the c-met-encoded receptor tyrosine kinase but not with a number of other tyrosine kinases from different subfamilies. A newly identified proline-rich domain of Gab1 is responsible for the binding of this protein to the tyrosine-phosphorylated bidentate docking site4,5 in c-Met. Expression of Gab1 in epithelial cells is sufficient to induce the c-Met-specific activities6–9, including branching morphogenesis. Thus we have discovered a new phosphotyrosine interaction domain in Gab1 and shown that Gab1 is the substrate of the c-Met receptor tyrosine kinase that mediates epithelial morphogenesis.
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The nuclear magnetic resonance structure of the phosphotyrosine binding (PTB) domain of Shc complexed to a phosphopeptide reveals an alternative means of recognizing tyrosine-phosphorylated proteins. Unlike in SH2 domains, the phosphopeptide forms an antiparallel beta-strand with a beta-sheet of the protein, interacts with a hydrophobic pocket through the (pY-5) residue, and adopts a beta-turn. The PTB domain is structurally similar to pleckstrin homology domains (a beta-sandwich capped by an alpha-helix) and binds to acidic phospholipids, suggesting a possible role in membrane localization.
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The amount of protein phosphatase 1 (PP1) activity in rabbit skeletal muscle associated with membranes (predominantly sarcoplasmic reticulum) is similar to that bound to glycogen-protein particles. Membrane-vesicle-associated (sarcovesicular) PP1 can be solubilised with 0.5% Triton X-100 (but not 0.5M NaCl) and is complexed to a protein that is structurally and functionally very similar or identical to the G subunit which targets PP1 to glycogen-protein particles. This conclusion is based on immunoblotting and immunotitration experiments using two different preparations of G-subunit-specific antibodies, binding of Triton-solubilised sarcovesicular enzyme to glycogen, stimulation of phosphorylase phosphatase activity by glycogen, phosphorylation of the same tryptic peptides by cyclic-AMP-dependent protein kinase (A-kinase) and release of catalytic subunit following phosphorylation by A-kinase. Membrane-association is not mediated via glycogen because sarcovesicular PP1 is (1) not released by digestion with alpha-amylase or at dilutions which fully dissociate the glycogen-bound enzyme, and (2) is solubilised by Triton X-100 (whereas glycogen-associated PP1 is not). These findings demonstrate that sarcovesicular PP1 is highly homologous to, or the same as, glycogen-associated PP1G and raises the possibility that a common targetting subunit may direct PP1 to different subcellular locations.
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ANCHORING of ion channels at specific subcellular sites is critical for neuronal signalling, but the mechanisms underlying channel localization and clustering are largely unknown (reviewed in ref. 1). Voltage-gated K+ channels are concentrated in various neuronal domains, including presynaptic terminals, nodes of Ranvier and dendrites, where they regulate local membrane excitability. Here we present functional and biochemical evidence that cell-surface clustering of Shaker-subfamily K+ channels is mediated by the PSD-95 family of membrane-associated putative guanylate kinases, as a result of direct binding of the carboxy-terminal cytoplasmic tails to the K+ channel subunits to two PDZ (also known as GLGF or DHR) domains in the PSD-95 protein. The ability of PDZ domains to function as independent modules for protein-protein interaction, and their presence in other junction-associated molecules (such as ZO-1 (ref. 3) and syntrophin), suggest that PDZ-domain-containing polypeptides may be widely involved in the organization of proteins at sites of membrane specialization.
Article
IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.
Article
Solution structures of two Src homology 3 (SH3) domain-ligand complexes have been determined by nuclear magnetic resonance. Each complex consists of the SH3 domain and a nine-residue proline-rich peptide selected from a large library of ligands prepared by combinatorial synthesis. The bound ligands adopt a left-handed polyproline type II (PPII) helix, although the amino to carboxyl directionalities of their helices are opposite. The peptide orientation is determined by a salt bridge formed by the terminal arginine residues of the ligands and the conserved aspartate-99 of the SH3 domain. Residues at positions 3, 4, 6, and 7 of both peptides also intercalate into the ligand-binding site; however, the respective proline and nonproline residues show exchanged binding positions in the two complexes. These structural results led to a model for the interactions of SH3 domains with proline-rich peptides that can be used to predict critical residues in complexes of unknown structure. The model was used to identify correctly both the binding orientation and the contact and noncontact residues of a peptide derived from the nucleotide exchange factor Sos in association with the amino-terminal SH3 domain of the adaptor protein Grb2.
Article
Src homology 2 (SH2) domains bind specifically to tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes. A protein domain was identified that bound specifically to the tyrosine-phosphorylated form of its target protein but differs from known SH2 sequences. Phosphotyrosine-binding (PTB) domains were found in two proteins: SHC, a protein implicated in signaling through Ras; and SCK, encoded by a previously uncharacterized gene. The PTB domain of SHC specifically bound to a tyrosine-phosphorylated 145-kilodalton protein. PTB domains are an alternative to SH2 domains for specifically recruiting tyrosine-phosphorylated proteins into signaling complexes and are likely to take part in signaling by many growth factors.
Article
Specificity of protein kinases and phosphatases may be achieved through compartmentalization with preferred substrates. In neurons, adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase (PKA) is localized at postsynaptic densities by association of its regulatory subunit with an A kinase anchor protein, AKAP79. Interaction cloning experiments demonstrated that AKAP79 also binds protein phosphatase 2B, or calcineurin (CaN). A ternary complex of PKA, AKAP, and CaN was isolated from bovine brain, and colocalization of the kinase and the phosphatase was established in neurites of cultured hippocampal neurons. The putative CaN-binding domain of AKAP79 is similar to that of the immunophilin FKBP-12, and AKAP79 inhibited CaN phosphatase activity. These results suggest that both PKA and CaN are targeted to subcellular sites by association with a common anchor protein and thereby regulate the phosphorylation state of key neuronal substrates.
Article
Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.
Article
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Article
Src homology 2 (SH2) domains mediate assembly of signaling complexes by binding specifically to tyrosine-phosphorylated proteins. A phosphotyrosine binding (PTB) domain has been identified which also binds specifically to tyrosine-phosphorylated targets, but is structurally different from SH2 domains. Expression cloning was used to identify targets of PTB domains. PTB domains bound to phosphotyrosine within a sequence motif, asparagine-X1-X2-phosphotyrosine (where X represents any amino acid), that is found in many signaling proteins and is not recognized by SH2 domains. Mutational studies indicated that high affinity binding of PTB domains may require a specific conformation of the motif.
Article
Background: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. Results: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. Conclusion: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.
Article
The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses.
Article
In a yeast two-hybrid screen we identified a member of the 14-3-3 family of proteins that can bind to Bcr. 14-3-3 beta binds to the serine/threonine rich region B in the kinase domain encoded by the first exon. In this paper we show by co-immunoprecipitation that Bcr binds to Raf in vivo and we argue that this interaction is mediated by 14-3-3 dimers, based on the following findings. First, 14-3-3 isoforms bind to both Raf and Bcr. Second, Bcr does not bind to Raf directly in the two-hybrid system, but co-expression of 14-3-3 beta allows complex formation. Third, Bcr, 14-3-3 proteins and Raf co-elute in gel filtration and in sequential ion exchange chromatography and the three proteins can be co-immunoprecipitated from the the separate fractions, indicating that they are present in a ternary complex. Moreover, approximately 10 times more Raf is bound to Bcr, and vice versa, in the membrane fraction (where Raf is activated) than in the cytosolic fraction. We suggest a new function for 14-3-3 proteins as a novel type of new function for 14-3-3 proteins as a novel type of adaptor which acts by dimerization and binding to different proteins.
Article
The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref. 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes. Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution. Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove. The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins. The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family.
Article
A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation. We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution. Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains. The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep. Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface. At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C.
Article
The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction. The peptide binds in an extended conformation and makes primary interactions with the SH2 domain at six central residues: PQ(pY)EEI. The phosphotyrosine and the isoleucine are tightly bound by two well-defined pockets on the protein surface, resulting in a complex that resembles a two-pronged plug engaging a two-holed socket. The glutamate residues are in solvent-exposed environments in the vicinity of basic side chains of the SH2 domain, and the two N-terminal residues cap the phosphotyrosine-binding site. The crystal structure of Src SH2 in the absence of peptide has been determined at 2.5 A resolution, and comparison with the structure of the high affinity complex reveals only localized and relatively small changes.
Article
A fundamental question in signal transduction is how stimulation of a specific protein kinase leads to phosphorylation of particular protein substrates throughout the cell. Recent studies indicate that specific anchoring proteins located at various sites in the cell compartmentalize the kinases to their sites of action. Inhibitors of the interactions between kinases and their anchoring proteins inhibit the functions mediated by the kinases. These data indicate that the location of these anchoring proteins provides some of the specificity of the responses mediated by each kinase and suggest that inhibitors of the interaction between the kinases and their anchoring proteins may be useful as therapeutic agents.
Article
The transduction of a signal is a change in form of the signal as it is passed from one carrier to another. The root "duce" means "to lead" in Latin; thus, a signal is led through a cell by steps of transduction (the same root is in the words seduce and duct as well as II Duce). The earliest transduction steps that were elucidated involved massive release of small molecule "second messengers", originally cAMP, that flooded a cell with information. With the understanding that such proteins as tyrosine kinases and Ras relatives are signal transducers, came the realization that many signaling pathways are more precise, sending controlled and probably weakly amplified signals to specific targets. These intracellular signals are often maintained in macromolecular form rather than being passed to small molecules.
Article
A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.