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Protein-based therapeutics, including antibodies and antibody-like-proteins, have increasingly attracted attention due to their high specificity compared to small-molecular drugs. The Gγ recruitment system, one of the in vivo yeast two-hybrid systems for detecting protein–protein interactions, has been previously developed using yeast signal transduction machinery. In this study, we modified the Gγ recruitment system to screen the protein mutants that efficiently bind to the intracellular domain of the epidermal growth factor receptor L858R mutant (cytoEGFRL858R). Using the modified platform, we performed in vivo directed evolution for growth factor receptor-bound protein 2 (Grb2) and its truncated variant containing only the Src-homology 2 (SH2) domain, successfully identifying several mutants that more strongly bound to cytoEGFRL858R than their parental proteins. Some of them contained novel beneficial mutations (F108Y and Q144H) and specifically bound to the recombinant cytosolic phosphorylated EGFR in vitro , highlighting the utility of the evolutionary platform. [Display omitted] [ABSTRACT FROM AUTHOR]

Adaptor proteins play central roles in the assembly of molecular complexes and coordinated activation of specific pathways. Through their modular domain structure, the NCK family of adaptor proteins (NCK1 and NCK2) link protein targets via their single SRC Homology (SH) 2 and three SH3 domains. Classically, their SH2 domain binds to phosphotyrosine motif-containing receptors (e.g. receptor tyrosine kinases), while their SH3 domains bind polyproline motif-containing cytoplasmic effectors. Due to these functions being established for both NCK1 and NCK2, their roles were inaccurately assumed to be redundant. However, in contrast with this previously held view, NCK1 and NCK2 now have a growing list of paralog-specific functions, which underscores the need to further explore their differences. Here we review current evidence detailing how these two paralogs are unique, including differences in their gene/protein regulation, binding partners and overall contributions to cellular functions. To help explain these contrasting characteristics, we then discuss SH2/SH3 structural features, disordered interdomain linker regions and post-translational modifications. Together, this review seeks to highlight the importance of distinguishing NCK1 and NCK2 in research and to pave the way for investigations into the origins of their interaction specificity. [ABSTRACT FROM AUTHOR]

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve. Synopsis: How do off-target interactions between proteins inside crowded cells affect cellular fitness? Expression of human tyrosine kinases in yeast, which lacks general tyrosine phosphorylation, shows that many individual spurious pY sites have no impact, but also a strong negative correlation between phosphorylation and fitness. 20% of pY sites are predicted to destabilise the substrate. Native pS/pT signalling in yeast is likely dysregulated. Many Y sites are likely phosphorylated at low stoichiometry. Spurious phosphorylation is widespread and not restricted to homologs of native substrates. Evolutionary selection to avoid spurious Y phosphorylation appears unlikely. Expression of human tyrosine kinases in yeast shows that while there is a strong negative correlation between phosphorylation and fitness, many individual pY sites have negligible impact. [ABSTRACT FROM AUTHOR]

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphorylation plays a critical role in initiating upstream cellular signaling transduction. However, the challenge in biological samples is the variability in relative concentrations (0.1%) of site‐specific tyrosine phosphorylation on proteins. To navigate these fluctuations and accurately quantify the absolute levels of tyrosine phosphosites among different samples, we reported a hybrid data‐independent acquisition‐parallel reaction monitoring (DIA‐PRM) MS technique for the robust identification and quantification of the phosphoproteome, the establishment of a comprehensive library of tyrosine phosphosites, and the specific assessment of changes in tyrosine phosphorylation. In our model study on non‐small cell lung cancer cells, our PRM strategy accomplished by a spiked‐in synthetic heavy phosphopeptide demonstrated reliable targeted quantification of the pY1197 on EGFR, revealing levels of 2.5, 4.9, and 5.3 fmol in pervanadate (PV)‐treated cells at 0, 15, and 30 min, respectively. Additionally, DIA‐extensive phosphoproteomic analysis provided 2765 tyrosine phosphosites within 14,961 global phosphosites corresponding to 1536 phosphoproteins, contributing to the phospho‐library establishment and relative quantification of phosphorylation level, especially in the PV‐treated time‐dependent increase of ErbB signaling pathway. This hybrid DIA‐PRM approach will advance the application of precise measurement of changes in multiple phosphotyrosine residues and enhance our understanding of phosphoproteomic dynamics in drug‐resistant cascades. [ABSTRACT FROM AUTHOR]

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2. [ABSTRACT FROM AUTHOR]

Introduction: The synaptic adhesion molecule neuroligin-1 (NLGN1) is involved in the differentiation of excitatory synapses, but the precise underlying molecular mechanisms are still debated. Here, we explored the role of NLGN1 tyrosine phosphorylation in this process, focusing on a subset of receptor tyrosine kinases (RTKs), namely FGFR1 and Trks, that were previously described to phosphorylate NLGN1 at a unique intracellular residue (Y782). Methods: We used pharmacological inhibitors and genetic manipulation of those RTKs in dissociated hippocampal neurons, followed by biochemical measurement of NLGN1 phosphorylation and immunocytochemical staining of excitatory synaptic scaffolds. Results: This study shows that: (i) the accumulation of PSD-95 at de novo NLGN1 clusters induced by neurexin crosslinking is reduced by FGFR and Trk inhibitors; (ii) the increase in PSD-95 puncta caused by NLGN1 over-expression is impaired by FGFR and Trk inhibitors; (iii) TrkB activation by BDNF increases NLGN1 phosphorylation; and (iv) TrkB knock-down impairs the increase of PSD-95 puncta caused by NLGN1 over-expression, an effect which is not seen with the NLGN1 Y782A mutant. Discussion: Together, our data identify TrkB as one of the major RTKs responsible for NLGN1 tyrosine phosphorylation, and reveal that TrkB activity is necessary for the synaptogenic effects of NLGN1. [ABSTRACT FROM AUTHOR]

Endogenous stimuli-responsive injectable hydrogels hold significant promise for practical applications due to their spatio-temporal controllable drug delivery. Herein, we report a facile strategy to construct a series of in situ formation polypeptide hydrogels with thermal responsiveness and enzyme-triggered dynamic self-assembly. The thermo-responsive hydrogels are from the diblock random copolymer mPEG-b-P(Glu-co-Tyr). The L-glutamic acid (Glu) segments with different γ-alkyl groups, including methyl, ethyl, and n-butyl, offer specific secondary structure, facilitating the formation of hydrogel. The L-tyrosine (Tyr) residues not only provide hydrogen-bond interactions and thus adjust the sol–gel transition temperatures, but also endow polypeptide enzyme-responsive properties. The PTyr segments could be phosphorylated, and the phosphotyrosine copolymers were amphiphilies, which could readily self-assemble into spherical aggregates and transform into sheet-like structures upon dephosphorylation by alkaline phosphatase (ALP). P(MGlu-co-Tyr/P) and P(MGlu-co-Tyr) copolymers showed good compatibility with both MC3T3-E1 and Hela cells, with cell viability above 80% at concentrations up to 1000 μg/mL. The prepared injectable polypeptide hydrogel and its enzyme-triggered self-assemblies show particular potential for biomedical applications. [ABSTRACT FROM AUTHOR]

Purpose: To unravel the mechanism regulating the phosphorylation of glycogen synthase kinase 3 (GSK3) and the correlation between the inhibitory phosphorylation of GSK3α and sperm motility in human. Materials and Methods: The phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 were examined in human spermatozoa with various motility conditions. Results: In human spermatozoa, GSK3α/β was localized in the head, midpiece, and principal piece of tail and p-GSK3α(Ser21) was enriched in the midpiece. The ratio of p-GSK3α(Ser21)/GSK3α was positively coupled with normal sperm motility criteria of World Health Organization. In high-motility spermatozoa, p-GSK3α(Ser21) phosphotyrosine (p-Tyr) proteins but p-GSK3α(Tyr279) markedly increased together with decreased kinase activity of GSK3 after incubation in Ca2+ containing medium. In high-motility spermatozoa, p-GSK3α(Ser21) levels were negatively coupled with kinase activity of GSK3, and which was deregulated in low-motility spermatozoa. In high-motility spermatozoa, 6-bromo-indirubin-3’-oxime, an inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins. p-GSK3α(Ser21) and p-Tyr protein levels were decreased by inhibition of PKA and Akt. Calyculin A, a protein phosphatase-1/2A inhibitor, markedly increased the p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa. Conclusions: Down regulation of kinase activity of GSK3α by inhibitory phosphorylation was positively coupled with human sperm motility, and which was regulated by Ca2+, PKA, Akt, and PP1. Small-molecule inhibitors of GSK3 and PP1 can be considered to potentiate human sperm motility. [ABSTRACT FROM AUTHOR]

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10‐fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho‐Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine‐succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo‐hirudin and desulfo‐hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho‐hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo‐hirudin; succinylated hirudin was also prospective. Phospho‐hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho‐hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice. [ABSTRACT FROM AUTHOR]

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis. The Eyes Absent proteins (EYA1-4) are a group of tyrosine phosphatases. Here, the authors report a signalling pathway in which EYA4 and EYA1 dephosphorylate Polo-like kinase 1 (PLK1) at pY445 to support PLK1 activation and mitosis. [ABSTRACT FROM AUTHOR]

In this review, the structure, isoform, and physiological role of the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) are summarized. There are three isoforms of CAPON in humans, including long CAPON protein (CAPON-L), short CAPON protein (CAPON-S), and CAPON-S' protein. CAPON-L includes three functional regions: a C-terminal PDZ-binding motif, carboxypeptidase (CPE)-binding region, and N-terminal phosphotyrosine (PTB) structural domain. Both CAPON-S and CAPON-S' only contain the C-terminal PDZ-binding motif. The C-terminal PDZ-binding motif of CAPON can bind with neuronal nitric oxide synthase (nNOS) and participates in regulating NO production and neuronal development. An overview is given on the relationship between CAPON and heart diseases, diabetes, psychiatric disorders, and tumors. This review will clarify future research directions on the signal pathways related to CAPON, which will be helpful for studying the regulatory mechanism of CAPON. CAPON may be used as a drug target, which will provide new ideas and solutions for treating human diseases. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium pathogenicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic conditions in vivo , we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K 0.5 of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa 2 equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive inhibitors bearing a negatively charged group featuring p K a values lower than that of the substrate phosphate group. • At pH < 6.5 the activity of M. tuberculosis MptpA is negatively affected. • Acidic pH conditions trigger a considerable increase of the K m for phosphotyrosine. • Acidic pH conditions alter the tertiary structure of M. tuberculosis MptpA. • Competitive inhibition of MptpA is more effective under acidic conditions. [ABSTRACT FROM AUTHOR]

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.

Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Protein Tyrosine Phosphatase 1B (PTP1B) is the prototypical protein tyrosine phosphatase and plays an essential role in the regulation of several kinase-driven signalling pathways. PTP1B displays a preference for bisphosphorylated substrates. Here we identify PTP1B as an inhibitor of IL-6 and show that, in vitro, it can dephosphorylate all four members of the JAK family. In order to gain a detailed understanding of the molecular mechanism of JAK dephosphorylation, we undertook a structural and biochemical analysis of the dephosphorylation reaction. We identified a product-trapping PTP1B mutant that allowed visualisation of the tyrosine and phosphate products of the reaction and a substrate-trapping mutant with a vastly decreased off-rate compared to those previously described. The latter mutant was used to determine the structure of bisphosphorylated JAK peptides bound to the enzyme active site. These structures revealed that the downstream phosphotyrosine preferentially engaged the active site, in contrast to the analogous region of IRK. Biochemical analysis confirmed this preference. In this binding mode, the previously identified second aryl binding site remains unoccupied and the non-substrate phosphotyrosine engages Arg47. Mutation of this arginine disrupts the preference for the downstream phosphotyrosine. This study reveals a previously unappreciated plasticity in how PTP1B interacts with different substrates. Structural and biochemical characterization of the phosphastase and immune checkpoint PTP1B reveals the molecular basis of PTBP1B/JAK interaction and plasticity of substrate recognition by PTP1B. [ABSTRACT FROM AUTHOR]

Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains. [ABSTRACT FROM AUTHOR]

The kinase insert domain (KID) of RTK KIT is a key recruitment region for downstream signalling proteins (DSPs). KID, as a multisite phosphorylation region, provides alternative recognition sites for DSPs and activates them by binding a phosphotyrosine (pY) to their SH2 domains. Significant steric, biochemical, and biophysical requirements must be fulfilled by each pair of interacting proteins as the adaptation of their configurations is mandatory for the selective activation of DSPs. The accurate 3D atomistic models obtained by modelling and molecular dynamics (MD) simulations of phosphorylated KID (p-KID) have been delivered to describe KID INTERACTOME. By taking phosphorylated KIDpY721 and the N-terminal SH2 domain of phosphatidylinositol 3-kinase (PI3K), a physiological partner of KID, we showed the two proteins are intrinsically disordered. Using 3D models of both proteins, we probe alternative orientations of KIDpY721 relative to the SH2 binding pocket using automatic docking (HADDOCK) and intuitive user-guided docking. This modelling yields to two possible models of the functionally related non-covalent complex KIDpY721/SH2, where one can be regarded as the first precursor to probe PI3K activation via KIT KID. We suggest that such generation of a KID/SH2 complex is best suited for future studies of the post-transduction effects of RTK KIT. [ABSTRACT FROM AUTHOR]

The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr1510. Separately, Src homology 2 (SH2) domains mediate protein–protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling. [ABSTRACT FROM AUTHOR]

To study mechanisms involved in fertility, many experimental assays are conducted by incubating spermatozoa in the presence of molecules dissolved in solvents such as ethanol (EtOH) or dimethyl sulfoxide (DMSO). Although a vehicle control group is usually included in such studies, it does not allow to evaluate the intrinsic effect of the solvent on sperm parameters and its potential influence on the outcome of the experiment. In the present study, we incubated human spermatozoa for 4 h in a capacitation medium in the absence or the presence of different concentrations of EtOH and DMSO (0.1, 0.5, 1.0, and 2.0%) to assess the impact of these solvents on sperm motility, vitality, capacitation, and acrosome integrity. The presence of statistically significant relationships between increasing solvent concentrations and the investigated parameters was assessed using linear mixed models. A significant effect was observed with both solvents for total and progressive sperm motilities. We also evaluated the effect of time for these parameters and showed that the influence of the solvents was stable between 0 and 4 h, indicating an almost direct impact of the solvents. While EtOH did not influence sperm vitality and acrosome integrity, a significant effect of increasing DMSO concentrations was observed for these parameters. Finally, regarding capacitation, measured via phosphotyrosine content, although a dose-dependent effect was observed with both solvents, the statistical analysis did not allow to precisely evaluate the intensity of the effect. Based on the results obtained in the present study, and the corresponding linear mixed models, we calculated the concentration of both solvents which would result in a 5% decline in sperm parameters. For EtOH, these concentrations are 0.9, 0.7, and 0.3% for total motility, progressive motility, and capacitation, respectively, while for DMSO they are 1.5, 1.1, >2, 0.3 and >2% for total motility, progressive motility, vitality, capacitation, and acrosome integrity, respectively. We recommend using solvent concentrations below these values to dissolve molecules used to study sperm function in vitro, to limit side effects. [ABSTRACT FROM AUTHOR]

The Growth factor receptor-bound protein 2 (Grb2) participates in early signaling complexes and regulates tyrosine kinase-mediated signal transduction through a monomer-dimer equilibrium. Grb2 dimeric state inhibits signal transduction whereas the monomer promotes signaling downstream. Since Grb2 dimer K D is ∼0.8 μM, studies focused on the monomer are still challenging and require mutations or interaction with phosphotyrosine peptides. However, these mutants were never characterized considering their effects on protein structure and dynamics in solution. Here, we present the biophysical characterization of Grb2Y160F, the first Grb2 mutant to induce protein monomerization without disrupting its native behavior in solution due to net charge modifications or interaction with peptides. We also identified that Grb2Y160F exists in a monomer-dimer equilibrium. Grb2Y160F ability to dimerize implies that different dimerization interfaces might regulate signaling pathways in distinct ways and raises an important question about the role of the Y160 residue in other dimerization interfaces. • Grb2 is predominantly found in its auto-inhibited form (dimer) in solution. • Grb2 Y160F exhibits a monomer-dimer equilibrium that favors the monomer in solution. • Grb2 Y160F presents a slingshot like mechanism that induces dimer dissociation. • SAXS and MD were combined to determine the Grb2 Y160F monomer ensemble in solution. • The ability of Grb2 Y160F to dimerize suggests distinct dimerization interfaces. [ABSTRACT FROM AUTHOR]

Since the report of "DNA untwisting" activity in 1972, ∼50 years of research has revealed seven topoisomerases in humans (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α, TOP3β and Spo11). These conserved regulators of DNA topology catalyze controlled breakage to the DNA backbone to relieve the torsional stress that accumulates during essential DNA transactions including DNA replication, transcription, and DNA repair. Each topoisomerase-catalyzed reaction involves the formation of a topoisomerase cleavage complex (TOPcc), a covalent protein-DNA reaction intermediate formed between the DNA phosphodiester backbone and a topoisomerase catalytic tyrosine residue. A variety of perturbations to topoisomerase reaction cycles can trigger failure of the enzyme to re-ligate the broken DNA strand(s), thereby generating topoisomerase DNA-protein crosslinks (TOP-DPC). TOP-DPCs pose unique threats to genomic integrity. These complex lesions are comprised of structurally diverse protein components covalently linked to genomic DNA, which are bulky DNA adducts that can directly impact progression of the transcription and DNA replication apparatus. A variety of genome maintenance pathways have evolved to recognize and resolve TOP-DPCs. Eukaryotic cells harbor tyrosyl DNA phosphodiesterases (TDPs) that directly reverse 3′-phosphotyrosyl (TDP1) and 5′-phoshotyrosyl (TDP2) protein-DNA linkages. The broad specificity Mre11-Rad50-Nbs1 and APE2 nucleases are also critical for mitigating topoisomerase-generated DNA damage. These DNA-protein crosslink metabolizing enzymes are further enabled by proteolytic degradation, with the proteasome, Spartan, GCNA, Ddi2, and FAM111A proteases implicated thus far. Strategies to target, unfold, and degrade the protein component of TOP-DPCs have evolved as well. Here we survey mechanisms for addressing Topoisomerase 1 (TOP1) and Topoisomerase 2 (TOP2) DPCs, highlighting systems for which molecular structure information has illuminated function of these critical DNA damage response pathways. • TOP-DPCs are induced by cancer drugs, and diverse forms of DNA damage. • Repair of TOP-DPCs is mediated by nuclease-dependent and -independent pathways. • TOP-DPCs are degraded by multiple cellular proteases including the proteasome. [ABSTRACT FROM AUTHOR]

Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains.

Protein tyrosine phosphatases, together with protein tyrosine kinases, control many molecular signaling steps that control life at cellular and organismal levels. Impairing alterations in the genes encoding the involved proteins is expected to profoundly affect the quality of life—if compatible with life at all. Here, we review the current knowledge on the effects of germline variants that have been reported for genes encoding a subset of the protein tyrosine phosphatase superfamily; that of the thirty seven classical members. The conclusion must be that the newest genome research tools produced an avalanche of data that suggest ‘guilt by association’ for individual genes to specific disorders. Future research should face the challenge to investigate these accusations thoroughly and convincingly, to reach a mature genotype-phenotype map for this intriguing protein family.

Growth factor binding to EGFR drives conformational changes that promote homodimerization and transphosphorylation, followed by adaptor recruitment, oligomerization, and signaling through Ras. Whether specific receptor conformations and oligomerization states are necessary for efficient activation of Ras is unclear. We therefore evaluated the sufficiency of a phosphorylated EGFR dimer to activate Ras without growth factor by developing a chemical-genetic strategy to crosslink and "trap" full-length EGFR homodimers on cells. Trapped dimers become phosphorylated and recruit adaptor proteins at stoichiometry equivalent to that of EGF-stimulated receptors. Surprisingly, these phosphorylated dimers do not activate Ras, Erk, or Akt. In the absence of EGF, phosphorylated dimers do not further oligomerize or reorganize on cell membranes. These results suggest that a phosphorylated EGFR dimer loaded with core signaling adapters is not sufficient to activate Ras and that EGFR ligands contribute to conformational changes or receptor dynamics necessary for oligomerization and efficient signal propagation through the SOS-Ras-MAPK pathway.

SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here, we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine-phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function. [ABSTRACT FROM AUTHOR]

Tyrosine phosphorylation is an important post‐translational modification of proteins, and its accurate analysis is of vital importance. However, due to limited abundance of tyrosine phosphorylation as well as severe interference of serine/threonine phosphorylation and other phosphate‐containing species, approaches that can directly analyse tyrosine phosphorylation on the cell membrane still remain limited. Herein, we report the rational development of molecularly imprinted and cladded Raman nanotags and their successful application in surface enhanced Raman spectroscopy (SERS) imaging of tyrosine phosphorylation on cancerous cells and tissues. The prepared molecularly imprinted and cladded SERS nanotags could specifically recognize phosphotyrosine and thereby allowed for distinguishing phosphotyrosine from other phosphate‐containing species on cancerous cells and tissues by SERS imaging. Therefore, the molecularly imprinted and cladded nanotags‐based SERS imaging can be a promising tool for tyrosine phosphorylation analysis and tyrosine phosphorylation‐related studies, showing great potential for biomedical applications. [ABSTRACT FROM AUTHOR]

It has been demonstrated that APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) is involved in the regulation of several growth-related signaling pathways and thus closely associated with the development and progression of some cancers. Diallyl trisulfide (DAT), a garlic-derived bioactive compound, exerts selective cytotoxicity to various human cancer cells through interfering with pro-survival signaling pathways. However, whether and how DAT affects survival of human hepatocellular carcinoma (HCC) cells remain unclear. Herein, we tested the hypothesis of the involvement of APPL1 in DAT-induced cytotoxicity in HCC HepG2 cells. We found that Lys 63 (K63)-linked polyubiquitination of APPL1 was significantly decreased whereas phosphorylation of APPL1 at serine residues remained unchanged in DAT-treated HepG2 cells. Compared with wild-type APPL1, overexpression of APPL1 K63R mutant dramatically increased cell apoptosis and mitigated cell survival, along with a reduction of phosphorylation of STAT3, Akt, and Erk1/2. In addition, DAT administration markedly reduced protein levels of intracellular TNF receptor-associated factor 6 (TRAF6). Genetic inhibition of TRAF6 decreased K63-linked polyubiquitination of APPL1. Moreover, the cytotoxicity impacts of DAT on HepG2 cells were greatly attenuated by overexpression of wild-type APPL1. Taken together, these results suggest that APPL1 polyubiquitination probably mediates the inhibitory effects of DAT on survival of HepG2 cells by modulating STAT3, Akt, and Erk1/2 pathways. [ABSTRACT FROM AUTHOR]

SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2. [ABSTRACT FROM AUTHOR]

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance., Competing Interests: Competing interests statement:A.T. is a consultant for NovoNordisk Holdings, Inc. and receives funding support from BioHybrid Solutions. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals; is listed as an inventor on a patent (WO2019232403A1, Weill Cornell Medicine) for combination therapy for PI3K-associated disease or disorder, and the identification of therapeutic interventions to improve response to PI3K inhibitors for cancer treatment; is a co-founder and shareholder in Faeth Therapeutics; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y. is a co-founder of DeStroke. F.M.W. is a consultant and on the scientific advisory board for Crossbow Therapeutics and for Aethon Therapeutics.

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.

Biochemical signaling pathways often involve proteins with multiple, modular interaction domains. Signaling activates binding sites, such as by tyrosine phosphorylation, which enables protein recruitment and growth of networked protein assemblies. Although widely observed, the physical properties of the assemblies, as well as the mechanisms by which they function, remain largely unknown. Here we examine molecular mobility within LAT:Grb2:SOS assemblies on supported membranes by single-molecule tracking. Trajectory analysis reveals a discrete temporal transition to subdiffusive motion below a characteristic timescale, indicating that the LAT:Grb2:SOS assembly has the dynamical structure of a loosely entangled polymer. Such dynamical analysis is also applicable in living cells, where it offers another dimension on the characteristics of cellular signaling assemblies.

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination.

Protein phosphorylation is a reversible post-translational modification. Nine of the 20 natural amino acids in proteins can be phosphorylated, but most of what we know about the roles of protein phosphorylation has come from studies of serine, threonine, and tyrosine phosphorylation. Much less is understood about the phosphorylation of histidine, lysine, arginine, cysteine, aspartate, and glutamate, so-called non-canonical phosphorylations. Phosphohistidine (pHis) was discovered 60 years ago as a mitochondrial enzyme intermediate; since then, evidence for the existence of histidine kinases and phosphohistidine phosphatases has emerged, together with examples where protein function is regulated by reversible histidine phosphorylation. pHis is chemically unstable and has thus been challenging to study. However, the recent development of tools for studying pHis has accelerated our understanding of the multifaceted functions of histidine phosphorylation, revealing a large number of proteins that are phosphorylated on histidine and implicating pHis in a wide range of cellular processes. In this perspective, Hunter reviews the history of protein-histidine phosphorylation, the current status of the field, and the prospects for further advances in understanding the functions of histidine phosphorylation in regulating cellular processes. [ABSTRACT FROM AUTHOR]

Tyrosine sulfation plays a vital role in various biochemical reactions. Although sulfated tyrosine (sTyr) has a similar structure to phosphotyrosine (pTyr), the number of available sTyr sites is significantly less than that of pTyr sites, mainly because of the lack of effective sTyr probes. A few sTyr binders were identified on the basis of structural similarity by engineering the pTyr-binding pocket of an Src Homology 2 (SH2) domain through phage selections against sTyr peptides. Nevertheless, they still interact with pTyr peptides with comparable affinity. This study aims to identify sTyr superbinders using the SH2 domain as a template. We created a distinctive phage selection scheme that separately covered selections against sTyr and pTyr peptides, followed by next-generation sequencing (NGS). After selections, phage pools showed strong enzyme-linked immunosorbent assay (ELISA) signal intensities for both modified peptides, indicating that the variants evolved with a high affinity for these peptides, which causes difficulty in identifying sTyr-specific binders. In contrast, NGS data from selected pools showed significant differences, suggesting the enrichment of sTyr-specific variants during selections. Accordingly, we obtained the sTyr features based on NGS data analysis and prioritized a few potential sTyr binders. The variant SH2-4 showed a stronger affinity for sTyr than pTyr and was superior to previous sTyr binders as measured by the Biolayer Interferometry assay. In summary, we described the strategy of integrating NGS data mining with a novel selection scheme to identify sTyr superbinders. [ABSTRACT FROM AUTHOR]

Human microtubule-associated protein Tau (τ) is abundant in the axons of neurons where it stabilizes microtubule bundles; abnormally hyperphosphorylated τ is a hallmark of Alzheimer's disease (AD) and related tauopathies. The hyperphosphorylation events can be recognized by phosphotyrosine-recognition domain SH2 (Src homology 2) to elicit downstream τ signaling in AD pathology. In this study, a comprehensive binary interaction map (CBIM) of all the 6 τ phosphotyrosine sites with 120 SH2 domains in the human genome was systematically created at structural level using computational analyses and binding assays, from which we were able to identify those of strong and moderate binding pairs of sites to domains. It is found that the SH2-recognition specificity of different τ phosphotyrosine sites has been evolutionally optimized to become roughly orthogonal to each other, and thus these site phosphorylations would regulate different but probably partially overlapped biological functions in τ signaling. Some SH2 groups such as SRC, RIN, PLCG, SOCS and SH2D were revealed to have effective binding potency as compared to others; they could be regarded as potential τ-associated proteins to transduce the downstream signaling. We further determined the systematic binding affinities of 6 τ-phosphopeptides to the 11 SH2 domains in SRC group, from which the FYN-τ18 and YES-τ29 pairs were identified as strong binders. Subsequently, rational molecular design was performed on τ18 and τ29 to derive a number of τ-phosphopeptide mutants with increased affinity; they are self-inhibitory candidates to competitively target τ hyperphosphorylation events in AD. In addition, it is revealed that the primary anchor pY0 and secondary anchor X+3 of τ-phosphopeptides play an important role in SRC-group SH2 recognition, which confer stability and specificity to the SH2–phosphopeptide binding, respectively. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphorylation plays a critical role in signal transduction. We report the genetic incorporation of a phosphotyrosine (pTyr) analog, p-carboxymethyl- L -phenylalanine (CMF), into proteins in mammalian cells. This nonhydrolyzable pTyr analog can facilitate biological studies by removing complications caused by the dynamic interconversion between the phosphorylated and non-phosphorylated isoforms of a protein. [ABSTRACT FROM AUTHOR]

Introduction: Osteosarcoma (OS) is the most common primary bone tumor, and the main affected population is adolescents. The survival of OS patients was 10-20% when surgery was used as a single treatment. There is less basic research on OS than other tumors, and we need more ways to improve the survival rate. Phosphotyrosine picked threonine kinase (TTK) has been widely reported as an oncogene in multiple types of cancers, and it is also known as a clinical therapeutic target. This study aims to assess TTK expression levels in human OS tissues and its link with the clinical characteristics of OS patients, and to evaluate the potential role in OS development.Material and methods: Immunohistochemical (IHC) assays were conducted to detect the expression levels of TTK in a total of 74 OS tissues and the corresponding adjacent tissues. Furthermore, according to the staining intensity of TTK in tumor tissues, patients were divided into TTK high and low expression groups. The possible correlation between TTK expression levels and clinical features were analyzed, and the effects of TTK on OS cell proliferation were detected through colony formation and cell counting kit-8 (CCK8) assays. The effects of TTK on tumor growth were detected using an animal model.Results: Phosphotyrosine picked threonine kinase was abnormally highly expressed in human OS tissues. Meanwhile, TTK was significantly correlated with the clinical characteristics such as tumor size (p = 0.004*) and clinical stage (p = 0.014*) of OS patients. Our results also revealed that the inhibition of TTK dramatically suppressed the proliferation of OS cells in vitro and blocked tumor growth in mice.Conclusions: We demonstrated the involvement of TTK in the development of OS, and therefore we suggest that TTK should be considered as a promising therapy target for OS. [ABSTRACT FROM AUTHOR]

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters. We initially found that organic cation transporters (OCTs), uptake carriers of metformin and oxaliplatin, were inhibited by several clinically used TKIs. Mechanistic studies showed that these TKIs inhibit the Src family kinase Yes1, which was found to be essential for OCT2 tyrosine phosphorylation and function. Yes1 inhibition in vivo diminished OCT2 activity, significantly mitigating oxaliplatin-induced acute sensory neuropathy. Along with OCT2, other SLC-family drug transporters are potentially part of an extensive 'transporter-phosphoproteome' with unique susceptibility to TKIs. On the basis of these findings we propose that TKIs, an important and rapidly expanding class of therapeutics, can functionally modulate pharmacologically important proteins by inhibiting protein kinases essential for their post-translational regulation.

Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic β-cells in young vs. adult animals, and its effects on DNA synthesis, β-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse β-cells, especially within immature β-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-β/ζ, were also expressed in the proliferative INS1E β-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with β-cells. The percentage of β-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during β-cell regeneration in young mice following depletion of β-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse β-cells in young and adult life and could potentially contribute to adaptive increases in β-cell mass during early life or pregnancy. [ABSTRACT FROM AUTHOR]

The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr1510. Separately, Src homology 2 (SH2) domains mediate protein–protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.

Phosphotyrosine (pTyr) recognition coordinates the assembly of protein complexes, thus controlling key events of cell cycle, cell development and programmed cell death. Although many aspects of membrane receptor function and intracellular signal transduction have been deciphered in the last decades, the details of how phosphorylation alters protein-protein interaction and creates regulating switches of protein activity and localization often remains unclear. We developed a synthetic route to a protected phophotyrosine building block with isolated 13 C- 1 H spins in the aromatic ring. The compound can be used for solid phase peptide synthesis (SPPS) and readily applied to study affinity, dynamics and interactions on an atomic level using NMR spectroscopy. As a first example, we prepared an isotopologue of a pTyr containing 12mer peptide (pY1021) as part of the platelet-derived growth factor to analyze the binding to the phospholipase C-γ (PLCγ-1) SH2 domain., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Dual Specificity Phosphatase 5 (DUSP5) is a human protein that targets specific kinases and dephosphorylates phosphoserine/threonine and phosphotyrosine residues. DUSP5 is found to be involved in cardiovascular diseases and many cancer pathways, including skin and breast cancer. For this reason, availability of an efficient protocol of expression and purification of DUSP proteins can play a crucial role in their studies towards better understanding of the disease process and development of better therapeutic approaches. For example, purification of DUSP5 could be used for the in vitro assays of the inhibitors of DUSP5 identified from the in-silico studies. This report provides the full procedure for protein purification thereby allowing the collection of desired amounts of DUSP5 using Glutathione S-transferase (GST) tag. The described method shows an efficient way to solubilize and purify DUSP5 for further protein studies. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling. SOCS2 is a key regulator of growth hormone and cytokine signaling, which recognizes phosphotyrosine (pTyr)-modified targets via a central SH2 domain. Here, the authors discover and characterize an exosite on this SH2 domain that can bind a non-phosphorylated peptide to enhance SOCS2:pTyr affinity. [ABSTRACT FROM AUTHOR]