Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

Targeting the SH3 domain of human osteoclast‐stimulating factor with rationally designed peptoid inhibitors

Human osteoclast‐stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption. The protein contains a modular Src homology 3 (SH3) domain that mediates the intermolecular recognition and interaction of hOSF with its biological partners. Here, we proposed targeting the hOSF SH3 domain to disrupt hOSF–partner interactions for bone disease therapy by using SH3 inhibitors. In the procedure, the primary sequences of three known hOSF‐interacting proteins (c‐Src, SMN and Sam68) were parsed, from which totally 31 octapeptide segments that contain the core SH3‐binding motif PXXP were extracted, and their binding behavior to hOSF SH3 domain was investigated at structural level using a biomolecular modeling protocol. Several SH3‐binding candidates were identified theoretically and then determined to have high or moderate affinity for the domain using fluorescence spectroscopy assays. One potent peptide ⁴²⁵APP ARP VK⁴³² (K_d = 3.2 μM), which corresponds to the residues 425–432 of Sam68 protein, was used as template to derive N substitution of peptides (peptoids). Considering that proline is the only endogenous N‐substituted amino acid that plays a critical role in SH3–peptide binding, the substitution was addressed at the two key proline residues (Pro427 and Pro430) of the template peptide with nine N‐substituted amino acid types. By systematically evaluating the structural and energetic effects of different N‐substituted amino acids presenting at the two proline sites on peptide binding, we rationally designed five peptoid inhibitors and then determined in vitro their binding affinity to hOSF SH3 domain. Consequently, two designed peptoids APP AR( N ‐Clp) VK and APP AR( N ‐Ffa) VK with Pro430 replaced by N‐Clp and N‐Ffa were confirmed to have increased (K_d = 0.87 μM) and comparable (K_d = 2.9 μM) affinities relative to the template, respectively. In addition, we also found that the Pro427 residue plays an essential role in restricting peptide/peptoid conformations to polyproline II (PPII) helix as the basic requirement of SH3 binding so that the residue cannot be modified. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and evaluation of conformationally constrained peptide analogues as the Src SH3 domain binding ligands

Src kinase activity is regulated by the interaction of SH3 domain with protein sequences that are rich in proline residues. Identification of more potent SH3 domain binding ligands that can regulate Src kinase activity is a subject of major interest. Conformationally constrained peptides have been previously used for improving the binding potency of the Src SH2 domain binding peptide ligands and peptide substrates of the substrate-binding site of Src. A series of peptide analogues of Ac-VSLARRPLPPLP (1, Ac-VSL12, K_d = 0.34 μM) were synthesized by introducing conformational constraints to improve the binding affinity towards the Src SH3 domain. Peptides synthesized through cyclization between N-terminal to C-terminal [VSLARRPLPPLP] or N-terminal to side chain flanking residues (i.e., [^βAVS]LARRPLPPLP and [VSLE]RRPLPPLP) exhibited at least 6.4-fold less binding affinity (K_d = 2.19–4.85 μM) when compared to 1. The data suggest upon N-terminal cyclization with C-terminal or flanking residues, the interactions of the amino acids in the core RPLPPLP reduce significantly with the residues within the Src SH3 domain. Conformationally constrained peptide V[SLARRPLPPLP] (5) was synthesized through cyclization of C-terminal to the serine side chain and displayed a comparable binding affinity (K_d = 0.35 μM) towards the Src SH3 domain versus that of 1. Thus, this template may be used to optimize and generate more potent analogues with higher stability.     

Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein

The Shc (Src homology collagen-like) adaptor protein plays a crucial role in linking stimulated receptors to mitogen-activated protein kinase activation through the formation of dynamic signalling complexes. Shc comprises an N-terminal phosphotyrosine binding (PTB) domain, a C-terminal Src homology 2 (SH2) domain and a central proline-rich collagen homology 1 domain. The latter domain contains three tyrosine residues that are known to become phosphorylated. We have expressed and purified the human p52Shc isoform and characterised its binding to different ligands. CD spectra revealed that some parts of the Shc protein are not fully folded, remaining largely unaffected by the binding of ligands. The PTB domain binds peptide and Ins-1,4,5-P₃ (but not Ins-1,3,5-P₃) independently, suggesting two distinct sites of interaction. In the unphosphorylated Shc, the SH2 domain is non-functional. Ligand binding to the PTB domain does not affect this. However, phosphorylation of the three tyrosine residues promotes binding to the SH2 domain. Thus, Shc has an intrinsic phosphorylation-dependent gating mechanism where the SH2 domain adopts an open conformation only when tyrosine phosphorylation has occurred.     

Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

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 19^th 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.     

α‐Hemolysin enhances Staphylococcus aureus internalization and survival within mast cells by modulating the expression of β1 integrin

Mast cells (MCs) are important sentinels of the host defence against invading pathogens. We previously reported that Staphylococcus aureus evaded the extracellular antimicrobial activities of MCs by promoting its internalization within these cells via β1 integrins. Here, we investigated the molecular mechanisms governing this process. We found that S. aureus responded to the antimicrobial mediators released by MCs by up‐regulating the expression of α‐hemolysin (Hla), fibronectin‐binding protein A and several regulatory systems. We also found that S. aureus induced the up‐regulation of β1 integrin expression on MCs and that this effect was mediated by Hla‐ADAM10 (a disintegrin and metalloproteinase 10) interaction. Thus, deletion of Hla or inhibition of Hla‐ADAM10 interaction significantly impaired S. aureus internalization within MCs. Furthermore, purified Hla but not the inactive Hla_H35L induced up‐regulation of β1 integrin expression in MCs in a dose‐dependent manner. Our data support a model in which S. aureus counter‐reacts the extracellular microbicidal mechanisms of MCs by increasing expression of fibronectin‐binding proteins and by inducing Hla‐ADAM10‐mediated up‐regulation of β1 integrin in MCs. The up‐regulation of bacterial fibronectin‐binding proteins, concomitantly with the increased expression of its receptor β1 integrin on the MCs, resulted in enhanced S. aureus internalization through the binding of fibronectin‐binding proteins to integrin β1 via fibronectin.     

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2-tail dissociation. These results identify SH3-linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The Chlamydia trachomatis Ctad1 invasin exploits the human integrin β1 receptor for host cell entry

Infection of human cells by the obligate intracellular bacterium Chlamydia trachomatis requires adhesion and internalization of the infectious elementary body (EB). This highly complex process is poorly understood. Here, we characterize Ctad1 (CT017) as a new adhesin and invasin from C. trachomatis serovar E. Recombinant Ctad1 (rCtad1) binds to human cells via two bacterial SH3 domains located in its N‐terminal half. Pre‐incubation of host cells with rCtad1 reduces subsequent adhesion and infectivity of bacteria. Interestingly, protein‐coated latex beads revealed Ctad1 being an invasin. rCtad1 interacts with the integrin β1 subunit on human epithelial cells, and induces clustering of integrins at EB attachment sites. Receptor activation induces ERK1/2 phosphorylation. Accordingly, rCtad1 binding to integrin β1‐negative cells is significantly impaired, as is the chlamydial infection. Thus interaction of C. trachomatis Ctad1 with integrin β1 mediates EB adhesion and induces signaling processes that promote host‐cell invasion.     

SH3 interactome conserves general function over specific form

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two‐hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain‐mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.     

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.     

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphorylation control of the ubiquitin ligase Cbl is conserved in choanoflagellates

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine‐binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure‐function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation‐dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl‐recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.     

Alternative Splicing Modulates Autoinhibition and SH3 Accessibility in the Src Kinase Fyn

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.The Src family of nonreceptor protein tyrosine kinases comprises nine members, including Src, Blk, Fgr, Fyn, Hck, Lyn, Lck, Yes, and Yrk. These kinases play crucial roles in a variety of cellular processes, such as cell cycle control, cell adhesion, cell motility, cell proliferation, and cell differentiation (41). Extensive studies indicate that the complexity of functional roles of Src kinases derives mainly from their ability to communicate with a large number of upstream receptors and downstream effectors, which vary by cell type (31). Given their critical role, diverse mechanisms of autoregulation have evolved, and their importance is highlighted by the implication of elevated Src expression levels and/or activity in epithelial cancers (for a review, see reference 48). The autoregulatory mechanisms depend on the composition and order of various domains and on posttranslational modification sites in the linker segments that connect the domains (35). From the N to C terminus, Src contains a myristoyl group attached to a unique domain, an Src homology 3 (SH3) domain that typically binds left-handed polyproline type II sequence motifs, an SH2 domain that binds to tyrosine-phosphorylated protein motifs, a protein-tyrosine kinase domain (SH1), and a C-terminal regulatory segment. Early biochemical studies suggested that these domains were critical for keeping Src catalytic activity under control (4, 23, 39, 40). The validation of the autoinhibitory role of these regulatory moieties came from the structures of Src and Hck kinases (36, 37, 43, 46, 47). The structures showed how interdomain interactions, stabilized by the binding of the SH2 domain to the tyrosine-phosphorylated C terminus (pTyr528), lock the molecule in a closed conformation. They further showed the unanticipated finding that residues in the linker region between the SH2 domain and the kinase domain, the SH2-kinase linker, make direct contact with the catalytic domain and adopts a polyproline type II helix conformation that docks onto the SH3 domain. This intramolecular interaction hinders the formation of a salt bridge that is crucial for the kinase activity, thereby eliciting an inhibitory effect. However, these interactions are suboptimal, and other phosphotyrosine- or polyproline-containing sequences can compete favorably with Src''s own sequences for SH2 or SH3 domain binding (3, 25). These binding events lead to the stimulation of Src kinase activity by disrupting the intramolecular constraints imposed on the kinase domain. Once released from the repressed state, the autophosphorylation of tyrosine residue Tyr416 (pTyr416) in the activation loop rapidly occurs, resulting in a conformational change that releases a fully active kinase.Remarkably, recent advances have highlighted the crucial role of linker regions in establishing the structural and functional assembly of multimodular proteins in signal transduction, and Src kinases are influential in our understanding of these mechanisms (13). The nine Src family members are very similar in terms of sequence identity, with, for example, the strong conservation of the SH3 binding surface and the cores of the kinase domain (44). Nevertheless, high sequence variability is noted in the SH2-kinase linker segment, except that the overall hydrophobicity is conserved. The interactions that this linker makes with both the SH3 domain and the back of the kinase domain probably result in a high-specificity binding. Indeed, the activities of chimeras in which the SH3 domain of Src kinases have been swapped show altered regulation (12, 14, 16). Furthermore, in contrast to deletion or point mutations in the SH3 domain, Src mutants in the linker segment or in the linker-interacting surface on the catalytic domain can transform fibroblast, suggesting specific function(s) (14).Src kinases originated by the duplication and diversification of the same ancestral gene with an original 10-intron structure before the separations of Teleostei from Tetrapoda (6). One of the Src-related kinases, Fyn, possesses two kinds of exon 7, exon 7A and exon 7B, essentially encoding for the SH2-kinase linker segment and the N terminus of the SH1 domain. The alternative splicing of exon 7A or 7B yields two major Fyn isoforms, FynB (exon 7A) and FynT (exon 7B) (7). Exon 7A shows a different evolutionary pattern from that of the other parts of the gene, suggesting that it is derived by a recombinatorial event with another gene (33). The newly captured exon, encoding FynB, was coopted by the central nervous system and possibly other tissues, while the ancestral isoform, encoding FynT, is expressed mainly in the hematopoietic system (7, 32). Whether this diversification process generated intrinsic biochemical functional novelty in addition to the differential tissue distribution and related functional divergence currently is unknown. Since the alternatively spliced exon that distinguishes the two isoforms essentially encodes for the SH2-kinase linker segment, it is possible that it confers distinct regulatory features. Thus, this evolutionary divergence in the SH2-linker segment of FynT and FynB, which maintain identical SH2, SH3, and kinase domains, offers the unique opportunity to explore the specific functions that this linker segment may impose on Src kinase function and/or regulation.Here, we have investigated how exons 7A and 7B affect the functional interaction of Fyn with the RNA-binding protein Sam68. Sam68 is known to activate Fyn by binding to its SH3 domain and also to serve as a substrate for phosphorylation by Fyn. We show that FynT and FynB display a distinct capacity to bind and phosphorylate Sam68. This differential interaction with a substrate is functionally relevant, because it allows the specific phosphorylation-mediated regulation of the Sam68-dependent alternative splicing of Bcl-x by FynT and results in the differential regulation of apoptosis in epithelial cells. Swapping experiments identify core residues of the exon 7A- or 7B-encoded SH2-kinase linker segment as both required and sufficient to confer this distinct function. In agreement with structural models, our data show that exon 7B reinforces the autoinhibitory lock that the SH2-linker region imposes onto the kinase domain and on SH3 domain accessibility. These results uncover a novel specific function that the SH2-kinase linker segment can play in Src biology and highlight the importance of alternative splicing for the acquisition of fine-tuning regulatory functions during evolution.     

Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.