scholarly journals iTRVZ: liquid nano-platform for signal integration on the plasma membrane

2021 ◽  
Author(s):  
Taka Aki Tsunoyama ◽  
Christian Hoffmann ◽  
Bo Tang ◽  
Koichiro M Hirosawa ◽  
Yuri L Nemoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Specific Protein ◽  
Signalling Pathways ◽  
Signal Integration ◽  
Signalling Molecules ◽  
Raft Domains

Signalling is one of the most important functions of the cellular plasma membrane (PM). A variety of extracellular signalling molecules bind to their specific receptors in the PM, and the engaged receptors in turn trigger various cytoplasmic signalling cascades. These signalling pathways are intertwined and affect each other, in a process called crosstalk, which enables the cells to fine tune the overall signal. The crosstalk of different receptor signalling pathways has been examined quite extensively, but the platform responsible for signal integration has never been discovered. Here, using single-molecule imaging, we found a nanometer-scale (50-80 nm) liquid-like protein assembly on the PM cytoplasmic surface (at a density of ~2-μm apart from each other on average, with a lifetime of ~10 s), working as the signal transduction and integration platform for receptors, including GPI-anchored receptors (GPI-ARs), receptor-type tyrosine kinases (RTKs), and GPCRs. The platform consists of integrin, talin, RIAM, VASP, and zyxin, and is thus termed iTRVZ. These molecules are known as focal-adhesion constituents, but iTRVZ is distinct from focal adhesions, because iTRVZ exists on both the apical and basal PMs and lack vinculin. The iTRVZ formation is driven by specific protein-protein interactions, liquid-liquid phase separation, and interactions with actin filaments and raft domains via PI(4,5)P2. iTRVZ integrates and amplifies the GPI-AR and RTK signals in a strongly non-linear fashion, and thus works as an AND gate and noise filter. These findings greatly advance our understanding of the mechanism for crosstalk between signalling pathways.

246 EVIDENCE OF FOCAL ADHESION ASSEMBLY IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 221
Author(s):  
B. R. Sessions ◽  
H. Rutigliano ◽  
C. J. Davies ◽  
K. L. White
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Signalling Pathways ◽  
Bovine Oocyte ◽  
Signalling Molecules ◽  
Sperm Binding ◽  
Bovine Oocytes ◽  
Oocyte Membrane

Integrins are located on the plasma membrane of bovine oocytes and have been implicated in bovine fertilization and activation (Campbell et al. 2000; Sessions et al. 2006). In response to signals from the extracellular matrix, activated integrins cluster at focal adhesions and interact with the cytoskeleton via signalling molecules, including focal adhesion kinase (FAK), α-actinin, phosphotyrosine, and talin. Immunohistochemistry was used to illustrate the formation of focal adhesions at the site of sperm binding in bovine oocytes during fertilization. To confirm antibody specificity, proteins were recovered from bovine oocyte lysate, separated by electrophoreses, and exposed to Western blotting. Specificity was confirmed based on antibody binding to the correctly sized bovine protein in the blot. Bovine oocytes were recovered, matured in vitro, and exposed to spermatozoa as described previously. Antibodies were then used to directly identify co-localization of FAK, α-actinin, phophotyrosine, and talin with each other in addition to localization with β-integrin isotypes within the bovine oocyte plasma membrane. Appropriate controls were performed in which all steps were duplicated with the elimination of the primary antibodies, to confirm that immunohistochemistry was specific for the proteins of interest. The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin β subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm–oocyte binding in the bovine.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

Proteins with SH2 and SH3 domains couple receptor tyrosine kinases to intracellular signalling pathways

1993 ◽  
Vol 340 (1293) ◽  
pp. 279-285 ◽  
Keyword(s):  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Receptor Protein ◽  
Signalling Pathways ◽  
Protein Protein Interactions ◽  
Sh3 Domains ◽  
Ras Pathway ◽  
Sh2 Domains ◽  
Intracellular Signalling Pathways ◽  
Src Homology

The targets of receptor protein-tyrosine kinases are characterized by Src homology 2 (SH2) domains, that mediate specific interactions with receptor autophosphorylation sites. SH 2-mediated interactions are important for the activation of biochemical signalling pathways in cells stimulated with growth factors. A distinct protein module, the SH3 domain, is frequently found in polypeptides that contain SH2 domains, and is also implicated in controlling protein-protein interactions in signal transduction. Evidence suggesting that SH2 and SH3 domains act synergistically in stimulation of the Ras pathway is discussed.

scholarly journals Controlling protein function by fine-tuning conformational flexibility

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sonja Schmid ◽  
Thorsten Hugel
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Protein Function ◽  
Specific Protein ◽  
Cellular Protein ◽  
Steady States ◽  
Fine Tuning ◽  
Cell Protein ◽  
Global Changes ◽  
Protein Regulation

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90’s ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins.

scholarly journals S-Acylation of plant immune receptors mediates immune signaling in plasma membrane nanodomains

2019 ◽  
Author(s):  
Dongqin Chen ◽  
Nagib Ahsan ◽  
Jay J. Thelen ◽  
Gary Stacey
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Critical Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Immune Signaling ◽  
Immune Receptors ◽  
Similar Phenotype ◽  
External Stimuli

SUMMARYS-Acylation is a reversible protein post-translational modification mediated by Protein S-acyltransferases (PATs). Here, we demonstrate that three plant immune receptors P2K1 (DORN1), FLS2 and CERK1, representing three distinct families of receptor like-kinases, are S-acylated by Arabidopsis PAT5 and PAT9 on the plasma membrane (PM). Mutations in Atpat5 and Atpat9 resulted in an elevated plant immune response, increased phosphorylation and decreased degradation of FLS2, P2K1 and CERK1 during immune signaling. Mutation of key cysteine residues S-acylated in these receptors resulted in a similar phenotype as exhibited by Atpat5/Atpat9 mutant plants. The data indicate that S-acylation also controls localization of the receptors in distinct PM nanodomains and, thereby, controls the formation of specific protein-protein interactions. Our study reveals that S-acylation plays a critical role in mediating spatiotemporal dynamics of plant receptors within PM nanodomains, suggesting a key role for this modification in regulating the ability of plants in respond to external stimuli.

scholarly journals Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

1992 ◽  
Vol 12 (4) ◽  
pp. 1835-1845 ◽  
Author(s):  
C Seidel-Dugan ◽  
B E Meyer ◽  
S M Thomas ◽  
J S Brugge
Keyword(s):  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Fold Increase ◽  
Soft Agar ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Src Homology ◽  
In Cells

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.

scholarly journals Targeting TLR/IL-1R Signalling in Human Diseases

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Maria Loiarro ◽  
Vito Ruggiero ◽  
Claudio Sette
Keyword(s):  
Autoimmune Diseases ◽  
Protein Interactions ◽  
Signalling Pathways ◽  
Toll Like Receptor ◽  
Protein Protein Interactions ◽  
Signalling Molecules ◽  
Adaptive Immune ◽  
Therapeutic Drugs ◽  
Clinical Benefits ◽  
Functional Features

The members of Toll-like receptor/Interleukin (IL)-1 receptor (TLR/IL-1R) superfamily play a fundamental role in the immune response. These receptors detect microbial components and trigger complex signalling pathways that result in increased expression of multiple inflammatory genes. On the other hand, an aberrant activation of TLR/IL-1R signalling can promote the onset of inflammatory and autoimmune diseases, raising the interest in the development of therapeutic strategies for the control of their function. In this review, we illustrate the structural and functional features of TLR/IL-1R proteins and discuss some recent advances in the approaches undertaken to develop anti-inflammatory therapeutic drugs. In particular, we will focus on inhibitors, such as decoy peptides and synthetic mimetics, that interfere with protein-protein interactions between signalling molecules of the TLR/IL-1R superfamily. Given their central role in innate and adaptive immune responses, it is foreseen that pharmaceutical modulation of TLR/IL-1R signalling pathways by these drugs might yield clinical benefits in the treatment of inflammatory and autoimmune diseases.

scholarly journals The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

2020 ◽  
Author(s):  
Ricardo Capone ◽  
Ajit Tiwari ◽  
Arina Hadziselimovic ◽  
Yelena Peskova ◽  
James M. Hutchison ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Protein Interactions ◽  
Strong Association ◽  
Amyloid Β ◽  
Membrane Vesicles ◽  
Precursor Protein ◽  
Plasma Membrane Vesicles ◽  
Amyloidogenic Processing ◽  
Raft Domains

AbstractProcessing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer’s disease. The cleavage of APP by β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) peptides are essential steps in this pathway. Biochemical evidence suggests amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid ordered phase membrane raft domains. However, direct evidence that C99 preferentially associates with rafts has remained elusive. Here, we test this idea by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles. We find that C99 is essentially excluded from ordered domains in HeLa cells, SH-SY5Y cells and neurons, instead exhibiting a strong (roughly 90%) affinity for disordered domains. The strong association of C99 with disordered domains occurs independently of its cholesterol binding activity, homodimerization, or the familial Alzheimer disease Arctic mutation. Finally, we confirm previous studies suggesting that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying either that amyloidogenic processing of the protein occurs in disordered regions of the membrane, that processing involves a marginal sub-population of C99 found in rafts, or that as-yet-unidentified protein-protein interactions involving C99 in living cells drive it into rafts to promote its cleavage therein.

scholarly journals The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function

2019 ◽  
Vol 20 (22) ◽  
pp. 5718 ◽  
Author(s):  
Abdelkarim ◽  
Banerjee ◽  
Grudzien ◽  
Leschinsky ◽  
Abushaer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Hypervariable Region ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Gtpase Domain ◽  
Ras Gtpases ◽  
And Function

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein–protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein–protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src

1992 ◽  
Vol 12 (4) ◽  
pp. 1835-1845
Author(s):  
C Seidel-Dugan ◽  
B E Meyer ◽  
S M Thomas ◽  
J S Brugge
Keyword(s):  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Fold Increase ◽  
Soft Agar ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Src Homology ◽  
In Cells

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.

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