A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Medicinal Chemistry Perspectives on Recent Advances in Src Kinase Inhibitors as a Potential Target for the Development of Anticancer Agents: Biological Profile, Selectivity, Structure-Activity Relationship.

The physiological Src proto-oncogene is a protein tyrosine kinase receptor that served as the essential signaling pathway in different types of cancer. Src kinase receptor is divided into different domains: a unique domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain, and a regulatory tail, which runs from the N-terminus to the C-terminus. Src kinase inhibitors bind in the kinase domain and are activated by phosphorylation. The etiology of cancer involved various signaling pathways and Src signaling pathways are also involved in those clusters. Although the dysregulation of Src kinase resulted in cancer being discovered in the late 19th century it is still considered a cult pathway because it is not much explored by different medicinal chemists and oncologists. The Src kinase regulated through different kinase pathways (MAPK, PI3K/Akt/mTOR, JAK/STAT3, Hippo kinase, PEAK1, and Rho/ROCK pathways) and proceeded downstream signaling to conduct cell proliferation, angiogenesis, migration, invasion, and metastasis of cancer cells. There are numerous FDA-approved drugs flooded the market but still, there is a huge demand for the creation of novel anticancer drugs. As the existing drugs are accompanied by several adverse effects and drug resistance due to rapid mutation in proteins. In this review, we have elaborated about the structure and activation of Src kinase, as well as the development of Src kinase inhibitors. Our group also provided a comprehensive overview of Src inhibitors throughout the last two decades, including their biological activity, structure-activity relationship, and Src kinase selectivity. The Src binding pocket has been investigated in detail to better comprehend the interaction of Src inhibitors with amino acid residues. We have strengthened the literature with our contribution in terms of molecular docking and ADMET studies of top compounds. We hope that the current analysis will be a useful resource for researchers and provide glimpse of direction toward the design and development of more specific, selective, and potent Src kinase inhibitors.

Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells.

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

Structural study of ponatinib in inhibiting SRC kinase.

Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.

Lipid chain-driven interaction of a lipidated Src-family kinase Lyn with the bilayer membrane.

N-Myristoylation is a process of ubiquitous protein modification, which promotes the interaction of lipidated proteins on cell surfaces, in conjunction with reversible S-palmitoylation. We report the cooperative lipid-lipid interaction of two acyl chains of proteins, which increases the protein-membrane interaction and facilitates selective targeting of membranes containing anionic lipids. Lyn is a member of the Src family kinases distributed on the membrane surface by N-myristoyl and neighbouring S-palmitoyl chain anchors at the unique N-terminus domain. We prepared N-terminal short segments of lipidated Lyn to investigate the behaviour of each acyl chain in the lipid composition-dependent membrane interaction by solid-state nuclear magnetic resonance (NMR) analysis. Solid-state 31P-NMR studies revealed that S-palmitoylation of N-myristoylated Lyn peptides increased the interaction between peptides and phospholipid head groups, particularly with the anionic phosphatidylserine-containing bilayers. The solid-state 2H-NMR of Lyn peptides with a perdeutero N-myristoyl chain indicated an increase (0.6-0.8 Å) in the extent of the N-myristoyl chain in the presence of nearby S-palmitoyl chains, probably through the interaction via the acyl chains. The cooperative hydrocarbon chain interaction of the two acyl chains of Lyn increased membrane binding by extending the hydrocarbon chains deeper into the membrane interior, thereby promoting the peptide-membrane surface interaction between the cationic peptide side chains and the anionic lipid head groups. This lipid-driven mechanism by S-palmitoylation promotes the partition of the lipidated proteins to the cytoplasmic surface of the cell membranes and may be involved in recruiting Lyn at the signalling domains rich in anionic lipids.

Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.

G protein-coupled receptors (GPCRs) initiate signaling cascades via G-proteins and beta-arrestins (ßarr). ßarr-dependent actions begin with recruitment of ßarr to the phosphorylated receptor tail and are followed by engagement with the receptor core. ßarrs are known to act as adaptor proteins binding receptors and various effectors, but it is unclear whether in addition to the scaffolding role ßarrs can allosterically activate their downstream targets. Here we demonstrate the direct allosteric activation of proto-oncogene kinase Src by GPCR-ßarr complexes in vitro and establish the conformational basis of the activation. Whereas free ßarr1 had no effect on Src activity, ßarr1 in complex with M2 muscarinic or ß2-adrenergic receptors reconstituted in lipid nanodiscs activate Src by reducing the lag phase in Src autophosphorylation. Interestingly, receptor-ßarr1 complexes formed with a ßarr1 mutant, in which the finger-loop, required to interact with the receptor core, has been deleted, fully retain the ability to activate Src. Similarly, ßarr1 in complex with only a phosphorylated C-terminal tail of the vasopressin 2 receptor activates Src as efficiently as GPCR-ßarr complexes. In contrast, ßarr1 and chimeric M2 receptor with nonphosphorylated C-terminal tail failed to activate Src. Taken together, these data demonstrate that the phosphorylated GPCR tail interaction with ßarr1 is necessary and sufficient to empower it to allosterically activate Src. Our findings may have implications for understanding more broadly the mechanisms of allosteric activation of downstream targets by ßarrs.

Src family kinases, adaptor proteins and the actin cytoskeleton in epithelial-to-mesenchymal transition.

Over a century of scientific inquiry since the discovery of v-SRC but still no final judgement on SRC function. However, a significant body of work has defined Src family kinases as key players in tumor progression, invasion and metastasis in human cancer. With the ever-growing evidence supporting the role of epithelial-mesenchymal transition (EMT) in invasion and metastasis, so does our understanding of the role SFKs play in mediating these processes. Here we describe some key mechanisms through which Src family kinases play critical role in epithelial homeostasis and how their function is essential for the propagation of invasive signals. Video abstract.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases.

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.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism.

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

All-atom adaptively biased path optimization of Src kinase conformational inactivation: Switched electrostatic network in the concerted motion of αC helix and the activation loop.

A method to optimize a conformational pathway through a space of well-chosen reduced variables is employed to advance our understanding of protein conformational equilibrium. The adaptively biased path optimization strategy utilizes unrestricted, enhanced sampling in the region of a path in the reduced-variable space to identify a broad path between two stable end-states. Application to the inactivation transition of the Src tyrosine kinase catalytic domain reveals new insight into this well studied conformational equilibrium. The mechanistic description gained from identifying the motions and structural features along the path includes details of the switched electrostatic network found to underpin the transition. The free energy barrier along the path results from rotation of a helix, αC, that is tightly correlated with motions in the activation loop (A-loop) as well as distal regions in the C-lobe. Path profiles of the reduced variables clearly demonstrate the strongly correlated motions. The exchange of electrostatic interactions among residues in the network is key to these interdependent motions. In addition, the increased resolution from an all-atom model in defining the path shows multiple components for the A-loop motion and that different parts of the A-loop contribute throughout the length of the path.

Phosphoproteomics Analysis Identifies Novel Candidate Substrates of the Nonreceptor Tyrosine Kinase, Src-related Kinase Lacking C-terminal Regulatory Tyrosine and N-terminal Myristoylation Sites (SRMS).

SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites), also known as PTK 70 (Protein tyrosine kinase 70), is a non-receptor tyrosine kinase that belongs to the BRK family of kinases (BFKs). To date less is known about the cellular role of SRMS primarily because of the unidentified substrates or signaling intermediates regulated by the kinase. In this study, we used phosphotyrosine antibody-based immunoaffinity purification in large-scale label-free quantitative phosphoproteomics to identify novel candidate substrates of SRMS. Our analyses led to the identification of 1258 tyrosine-phosphorylated peptides which mapped to 663 phosphoproteins, exclusively from SRMS-expressing cells. DOK1, a previously characterized SRMS substrate, was also identified in our analyses. Functional enrichment analyses revealed that the candidate SRMS substrates were enriched in various biological processes including protein ubiquitination, mitotic cell cycle, energy metabolism and RNA processing, as well as Wnt and TNF signaling. Analyses of the sequence surrounding the phospho-sites in these proteins revealed novel candidate SRMS consensus substrate motifs. We utilized customized high-throughput peptide arrays to validate a subset of the candidate SRMS substrates identified in our MS-based analyses. Finally, we independently validated Vimentin and Sam68, as bona fide SRMS substrates through in vitro and in vivo assays. Overall, our study identified a number of novel and biologically relevant SRMS candidate substrates, which suggests the involvement of the kinase in a vast array of unexplored cellular functions.

Structure, Function, and Regulation of the SRMS Tyrosine Kinase.

Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites (SRMS) is a tyrosine kinase that was discovered in 1994. It is a member of a family of nonreceptor tyrosine kinases that also includes Brk (PTK6) and Frk. Compared with other tyrosine kinases, there is relatively little information about the structure, function, and regulation of SRMS. In this review, we summarize the current state of knowledge regarding SRMS, including recent results aimed at identifying downstream signaling partners. We also present a structural model for the enzyme and discuss the potential involvement of SRMS in cancer cell signaling.

Nuclear Functions of the Tyrosine Kinase Src.

Src is the representative member of the Src-family kinases (SFKs), a group of tyrosine kinases involved in several cellular processes. Its main function has been for long confined to the plasma membrane/cytoplasm compartment, being a myristoylated protein anchored to the cell membrane and functioning downstream to receptors, most of them lacking intrinsic kinase activity. In the last decades, new roles for some SFKs have been described in the nuclear compartment, suggesting that these proteins can also be involved in directly regulating gene transcription or nucleoskeleton architecture. In this review, we focused on those nuclear functions specifically attributable to Src, by considering its function as both tyrosine kinase and adapting molecule. In particular, we addressed the Src involvement in physiological as well as in pathological conditions, especially in tumors.

Identification of Novel Src Inhibitors: Pharmacophore-Based Virtual Screening, Molecular Docking and Molecular Dynamics Simulations.

Src plays a crucial role in many signaling pathways and contributes to a variety of cancers. Therefore, Src has long been considered an attractive drug target in oncology. However, the development of Src inhibitors with selectivity and novelty has been challenging. In the present study, pharmacophore-based virtual screening and molecular docking were carried out to identify potential Src inhibitors. A total of 891 molecules were obtained after pharmacophore-based virtual screening, and 10 molecules with high docking scores and strong interactions were selected as potential active molecules for further study. Absorption, distribution, metabolism, elimination and toxicity (ADMET) property evaluation was used to ascertain the drug-like properties of the obtained molecules. The proposed inhibitor-protein complexes were further subjected to molecular dynamics (MD) simulations involving root-mean-square deviation and root-mean-square fluctuation to explore the binding mode stability inside active pockets. Finally, two molecules (ZINC3214460 and ZINC1380384) were obtained as potential lead compounds against Src kinase. All these analyses provide a reference for the further development of novel Src inhibitors.

Phenylpyrazalopyrimidines as Tyrosine Kinase Inhibitors: Synthesis, Antiproliferative Activity, and Molecular Simulations.

N1-(α,ß-Alkene)-substituted phenylpyrazolopyrimidine derivatives with acetyl and functionalized phenyl groups at α- and ß-positions, respectively, were synthesized by the reaction of 3-phenylpyrazolopyrimidine (PhPP) with bromoacetone, followed by a chalcone reaction with differently substituted aromatic aldehydes. The Src kinase enzyme assay revealed modest inhibitory activity (half maximal inhibitory concentration, IC50 = 21.7-192.1 µM) by a number of PhPP derivatives. Antiproliferative activity of the compounds was evaluated on human leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), breast carcinoma (MDA-MB-231), and colon adenocarcinoma (HT-29) cells in vitro. 4-Chlorophenyl carbo-enyl substituted 3-phenylpyrazolopyrimidine (10) inhibited the cell proliferation of HT-29 and SK-OV-3 by 90% and 79%, respectively, at a concentration of 50 µM after 96 h incubation. The compound showed modest inhibitory activity against c-Src (IC50 = 60.4 µM), Btk (IC50 = 90.5 µM), and Lck (IC50 = 110 µM), while it showed no activity against Abl1, Akt1, Alk, Braf, Cdk2, and PKCa. In combination with target selection and kinase profiling assay, extensive theoretical studies were carried out to explore the selectivity behavior of compound 10. Specific interactions were also explored by examining the changing trends of interactions of tyrosine kinases with the phenylpyrazolopyrimidine derivative. The results showed good agreement with the experimental selectivity pattern among c-Src, Btk, and Lck.

Site-Selective Lysine Reactions Guided by Protein-Peptide Interaction.

Site-selective lysine post-translational modifications such as acetylation, methylation, hydroxylation, and isopeptide formation mediate the precise control of important signaling events in cells with unmistakable accuracy. This unparalleled site selectivity (modification of a single lysine in a particular protein in the proteome) is still a challenge for non-enzymatic protein reactions; the difficulty lies in the differentiation of the lysine ε-amino group from other reactive groups and in the precise pinpointing of one particular lysine ε-amino group out of many other lysine ε-amino groups and the N-terminal amine of the protein that have similar chemical reactivity. Here, we have explored proximal lysine conjugation reactions through peptide-guided fluorodinitrobenzene, isothiocyanate, and phenyl ester reactions and have validated the site-specific targeting of the ε-amino group of one single lysine in natural proteins that contain multiple lysine residues. This precise site selectivity is a result of the proximity-induced reactivity guided by a specific protein-peptide interaction: the binding interaction preorganizes an amine-reactive group in the peptide and one of the lysine side chain ε-amino groups of the protein into close proximity, thereby confining the reactivity to a selected area of the target protein. The binding-guide lysine reactions were first examined on an SH3 domain and then tested on several ubiquitin-like proteins such as SUMO, Atg8 protein family, plant ATG8, and mammalian LC3 proteins that contain at least seven lysine residues on the surface. Exquisite site selectivity was confirmed in all of the proteins tested. A set of amine reactions were tested for their feasibility in the site-selective lysine reaction. Selected amine-reactive groups were optimized, and the reaction sites on the LC3 protein were confirmed by mass spectrometry.

Chemoproteomic Method for Profiling Inhibitor-Bound Kinase Complexes.

Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein's functions affects the state of the cell is not well-understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. Here, we describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src's cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src's interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src's localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets.

Integrating disorder in globular multidomain proteins: Fuzzy sensors and the role of SH3 domains.

Intrinsically disordered proteins represent about one third of eukaryotic proteins. An additional third correspond to proteins containing folded domains as well as large intrinsically disordered regions (IDR). While IDRs may represent functionally autonomous domains, in some instances it has become clear that they provide a new layer of regulation for the activity displayed by the folded domains. The sensitivity of the conformational ensembles defining the properties of IDR to small changes in the cellular environment and the capacity to modulate this response through post-translational modifications makes IDR ideal sensors enabling continuous, integrative responses to complex cellular inputs. Folded domains (FD), on the other hand, are ideal effectors, e.g. by catalyzing enzymatic reactions or participating in binary on/off switches. In this perspective review we discuss the possible role of intramolecular fuzzy complexes to integrate the very different dynamic scales of IDR and FD, inspired on the recent observations of such dynamic complexes in Src family kinases, and we explore the possible general role of the SH3 domains connecting IDRs and FD.