SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.
TL;DR: A dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.
Abstract: Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.
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TL;DR: It is found that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation.
Abstract: The accumulation of unfolded proteins under endoplasmic reticulum (ER) stress leads to the activation of the multidomain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. We find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Further insight into ho...
77 citations
TL;DR: Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module, which has had important implications for understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.
Abstract: Tyrosine kinases were first discovered as the protein products of viral oncogenes We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals Several families of cytoplasmic tyrosine kinases have in common a core architecture, the "Src module," composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain Each of these families is defined by additional elaborations on this core architecture Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies
56 citations
Cites background from "SH2-catalytic domain linker heterog..."
...For example, sequence heterogeneity across SH2-kinase linkers in Src-family kinases has been shown to correlate with the degree of allosteric coupling of the SH3 and SH2 domains to the ATP binding site (Register et al. 2014)....
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TL;DR: Overall, it is concluded that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in function or regulatory properties.
Abstract: The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in function or regulatory properties
37 citations
TL;DR: The pharmaceutical industry has been developing compounds that bind to the ATP-binding site of protein kinases and potently disrupt protein-protein interactions between target protein kinase and their regulatory interacting partners, so learning to modulate allosteric processes will facilitate the development of protein- protein interaction modulators.
31 citations
TL;DR: It is found that Ang II induces podocyte injury via a Csk-dependent pathway and Knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn.
Abstract: Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway.
23 citations
References
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TL;DR: This chapter reviews the evidence implicating Src family kinases in specific receptor pathways and describes the mechanisms leading to their activation, the targets that interact with these kinases, and the biological events that they regulate.
Abstract: Src family protein tyrosine kinases are activated following engagement of many different classes of cellular receptors and participate in signaling pathways that control a diverse spectrum of receptor-induced biological activities. While several of these kinases have evolved to play distinct roles in specific receptor pathways, there is considerable redundancy in the functions of these kinases, both with respect to the receptor pathways that activate these kinases and the downstream effectors that mediate their biological activities. This chapter reviews the evidence implicating Src family kinases in specific receptor pathways and describes the mechanisms leading to their activation, the targets that interact with these kinases, and the biological events that they regulate.
2,455 citations
TL;DR: Crystal structures of inactive kinases have revealed a remarkable plasticity in the kinase domain that allows the adoption of distinct conformations in response to interactions with specific regulatory domains or proteins.
1,669 citations
TL;DR: This review highlights the analysis of those protein‐protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.
Abstract: Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.
1,240 citations
TL;DR: The crystal structure of the haematopoietic cell kinase Hck has been determined at 2.6/2.9 Å resolution and the conformation of the active site has similarities with that of inactive cyclin-dependent protein kinases.
Abstract: The crystal structure of the haematopoietic cell kinase Hck has been determined at 2.6/2.9 A resolution. Inhibition of enzymatic activity is a consequence of intramolecular interactions of the enzyme's Src-homology domains SH2 and SH3, with concomitant displacement of elements of the catalytic domain. The conformation of the active site has similarities with that of inactive cyclin-dependent protein kinases.
1,179 citations
TL;DR: Four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation are reported, in which the ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation.
851 citations