scholarly journals Mapping paths: new approaches to dissect eukaryotic signaling circuitry

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1853
Author(s):  
Nebibe Mutlu ◽  
Anuj Kumar
Keyword(s):  
Cell Signaling ◽  
Protein Interactions ◽  
Protein Localization ◽  
Specific Protein ◽  
Cellular Growth ◽  
Intracellular Signals ◽  
Allele Specific ◽  
Coordinate Changes ◽  
And Function ◽  
Computational Analyses

Eukaryotic cells are precisely “wired” to coordinate changes in external and intracellular signals with corresponding adjustments in the output of complex and often interconnected signaling pathways. These pathways are critical in understanding cellular growth and function, and several experimental trends are emerging with applicability toward more fully describing the composition and topology of eukaryotic signaling networks. In particular, recent studies have implemented CRISPR/Cas-based screens in mouse and human cell lines for genes involved in various cell growth and disease phenotypes. Proteomic methods using mass spectrometry have enabled quantitative and dynamic profiling of protein interactions, revealing previously undiscovered complexes and allele-specific protein interactions. Methods for the single-cell study of protein localization and gene expression have been integrated with computational analyses to provide insight into cell signaling in yeast and metazoans. In this review, we present an overview of exemplary studies using the above approaches, relevant for the analysis of cell signaling and indeed, more broadly, for many modern biological applications.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Pairing your Sox: Identification of Sox11 partner proteins and interaction domains in the developing neural plate

2020 ◽  
Author(s):  
Kaela S. Singleton ◽  
Pablo Silva-Rodriguez ◽  
Elena M. Silva
Keyword(s):  
Protein Interactions ◽  
Neural Development ◽  
Specific Protein ◽  
Neural Plate ◽  
Neural Fate ◽  
Mouse Cortex ◽  
Interaction Domains ◽  
Species Specific ◽  
And Function ◽  
Partner Proteins

AbstractSox11, a member of the SoxC family of transcription factors, has distinct functions at different times in neural development. Studies in mouse, frog, chick and zebrafish show that Sox11 promotes neural fate, neural differentiation, and neuron maturation in the central nervous system. These diverse roles are controlled in part by spatial and temporal-specific protein interactions. However, the partner proteins and Sox11-interaction domains underlying these diverse functions are not well defined. Here, we identify partner proteins and the domains of Xenopus Sox11(xSox11) required for protein interaction and function during neurogenesis. Our data show that Sox11 co-localizes and interacts with Pou3f2 and Ngn2 in the anterior neural plate and in early neurons, respectively. We also demonstrate that xSox11 does not interact with Ngn1, a high affinity partner of Sox11 in the mouse cortex, suggesting that Sox11 has species-specific partner proteins. Additionally, we determined that the N-terminus including the HMG domain of xSox11 is necessary for interaction with Pou3f2 and Ngn2, and established a novel role for the N-terminal 46 amino acids in the establishment of placodal progenitors. This is the first identification of partner proteins for Xenopus Sox11 and of domains required for partner protein interactions and distinct roles in neurogenesis.

scholarly journals Direct interaction of PIWI and DEPS-1 is essential for piRNA function and condensate ultrastructure inCaenorhabditis elegans

2019 ◽  
Author(s):  
KM Suen ◽  
F Braukmann ◽  
R Butler ◽  
D Bensaddek ◽  
A Akay ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Direct Interaction ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Piwi Domain ◽  
Small Ncrnas ◽  
Complex Forms ◽  
Rna Pathways ◽  
And Function

SummaryMembraneless organelles are platforms for many aspects of RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. To address this question, we investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule inCaenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 revealed an interaction with the constitutive P granule protein DEPS-1. Furthermore we identified a novel motif on DEPS-1, PBS, which interacts directly with the Piwi domain of PRG-1. This protein complex forms intertwining ultrastructures to build elongated condensatesin vivo. These sub-organelle ultrastructures depend on the Piwi-interacting motif of DEPS-1 and mediate piRNA function. Additionally, we identify a novel interactor of DEPS-1, EDG-1, which is required for DEPS-1 condensates to form correctly. We show that DEPS-1 is not required for piRNA biogenesis but piRNA function:deps-1mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. Our study reveals how specific protein-protein interactions drive the spatial organisation and function of small RNA pathways within membraneless organelles.

scholarly journals Sinner or Saint?: Nck Adaptor Proteins in Vascular Biology

2021 ◽  
Vol 9 ◽  
Author(s):  
Mabruka Alfaidi ◽  
Matthew L. Scott ◽  
Anthony Wayne Orr
Keyword(s):  
Protein Interactions ◽  
Vascular Biology ◽  
Adaptor Proteins ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Cell Phenotype ◽  
Protein Protein Interactions ◽  
Cytoskeletal Dynamics ◽  
Phosphotyrosine Signaling ◽  
And Function

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.

scholarly journals Fractionation of the Epithelial Apical Junctional Complex: Reassessment of Protein Distributions in Different Substructures

2005 ◽  
Vol 16 (2) ◽  
pp. 701-716 ◽  
Author(s):  
Roger Vogelmann ◽  
W. James Nelson
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Structure ◽  
Specific Protein ◽  
Junctional Complex ◽  
Protein Protein Interactions ◽  
Peripheral Membrane Proteins ◽  
Apical Junctional Complex ◽  
Important Regulator ◽  
And Function

The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures.

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.

scholarly journals Membrane composition influences the conformation and function of the dopamine transporter in vivo

2019 ◽  
Author(s):  
Wendy M. Fong ◽  
Kevin Erreger ◽  
Se Joon Choi ◽  
India Reddy ◽  
Christopher W. Johnson ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Transmembrane Protein ◽  
Specific Protein ◽  
Biochemical Properties ◽  
Membrane Composition ◽  
Loss Of Function ◽  
And Function

SummaryThe biophysical and biochemical properties of membrane lipids can alter the conformation and function of membrane-spanning proteins, yet the specific, physiological consequence in vivo of changing the membrane milieu for a specific protein has been rarely investigated. Using various genetic approaches to eliminate expression of the membrane-associated protein Flotillin-1, we have found that the lipid environment of the dopamine transporter (DAT) is necessary for mice to respond to amphetamine but not cocaine, because the localization of DAT to cholesterol-rich membranes is required for a DAT conformation that is essential for reverse transport of dopamine. Furthermore, a conditional rather than constitutive loss-of-function approach was necessary to reveal this phenotype, indicating a broader role for membrane-protein interactions that are modulated by Flotillin-1. Taken together, these findings demonstrate how interaction of a transmembrane protein with its membrane environment can regulate distinct events in the vertebrate brain that give rise to specific behavioral outcomes.

scholarly journals Altered chromatin localization of hybrid lethality proteins in Drosophila

2018 ◽  
Author(s):  
J.C. Cooper ◽  
A. Lukacs ◽  
S. Reich ◽  
T. Schauer ◽  
A. Imhof ◽  
...  
Keyword(s):  
Protein Localization ◽  
Evolutionary Genetics ◽  
Hybrid Male ◽  
Hybrid Incompatibility ◽  
Telomeric Sequences ◽  
Sequence Composition ◽  
Over Expression ◽  
Allele Specific ◽  
Single Complex ◽  
And Function

AbstractUnderstanding hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, the interaction of at least three genes is necessary for hybrid male lethality: Hmr mel, Lhr sim, and gfzf sim. All three hybrid incompatibility genes are chromatin associated factors. While HMR and LHR physically bind each other and function together in a single complex, the connection between either of these proteins and gfzf remains mysterious. Here, we investigate the allele specific chromatin binding patterns of gfzf. First, our cytological analyses show that there is little difference in protein localization of GFZF between the two species except at telomeric sequences. In particular, GFZF binds the telomeric retrotransposon repeat arrays, and the differential binding of GFZF at telomeres reflects the rapid changes in sequence composition at telomeres between D. melanogaster and D. simulans. Second, we investigate the patterns of GFZF and HMR co-localization and find that the two proteins do not normally co-localize in D. melanogaster. However, in inter-species hybrids, HMR shows extensive mis-localization to GFZF sites, and this altered localization requires the presence of gfzf sim. Third, we find by ChIP-Seq that over-expression of HMR and LHR within species is sufficient to cause HMR to mis-localize to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans as well as a molecular interaction between gfzf and Hmr in the form of altered protein localization.

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