Lck SH2 Domain Research Articles

A proline to glycine mutation in the Lck SH3-domain affects conformational sampling and increases ligand binding affinity

Loop flexibility is discussed as a factor that affects ligand binding affinity of SH3 domains. To test this hypothesis, we designed a mutant in which a proline in the RT-loop of the human Lck SH3-domain is replaced by glycine. The dynamics and ligand binding properties of wild-type and mutant LckSH3 were studied by fluorescence and NMR spectroscopy as well as molecular dynamics simulations. Although the mutated residue does not form direct contacts with the ligand, the mutation increases ligand affinity by a factor of eight. The mutant exhibits increased loop flexibility and enhanced sampling of binding-competent conformations. This effect is expected to facilitate ligand binding itself and might also allow formation of tighter contacts in the complex thus resulting in an increased binding affinity.

Lck SH3 Domain Function Is Required for T-Cell Receptor Signals Regulating Thymocyte Development

Thymocyte development is shaped by signals from the T-cell antigen receptor. The strength of receptor signaling determines developmental progression as well as deletion of self-reactive T cells. Receptor stimulation of the extracellular signal-regulated kinase (ERK) pathway plays an important regulatory role during thymocyte development. However, it is unclear how differences in receptor signaling are translated into distinctive activation of the ERK pathway. We have investigated the potential role of the Lck tyrosine kinase in regulating intracellular signaling during thymocyte development. While Lck is known to be critical for initial T-cell receptor signaling events, it may have an independent role in regulating intracellular signaling through the function of its SH3 domain. To determine whether such a regulatory mechanism functions during thymocyte development, we generated mice in which the normal lck allele is replaced with an lck SH3 domain mutant. Analysis of these mice revealed that both early thymocyte development and maturation of CD4(+) and CD8(+) lineages is impaired. Investigation of thymocyte responses to antigen receptor stimulation showed a significant reduction in proliferation and ERK pathway activation, although initial signaling events were intact. These findings indicate that Lck SH3 domain function may provide a means to independently couple receptor signaling to regulation of the ERK pathway during thymocyte development.

Crystal Structure Analysis and Solution Studies of Human Lck-SH3; Zinc-induced Homodimerization Competes with the Binding of Proline-rich Motifs

In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 Å. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn 2+ dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.

Growth Transformation of Human T Cells by Herpesvirus Saimiri Requires Multiple Tip-Lck Interaction Motifs

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

Development of Novel Small Chemical Inhibitors for Lck SH Domain with in vitro T-cell Inhibitory Activity

We investigated in vitro T-cell inhibitory activity and bioavailability of small chemical inhibitors for Lck SH2 domain, which had a different scaffold such as an amide bond, reduced amide bond, N-methyl amide bond, thioamide bond, and urethane bond. Each of these compounds, with its particular scaffold, showed a different logP value, stability against serum enzyme, stability in buffer solution, and in vitro T-cell inhibitory activity. Overall results indicated that the SH2 inhibitor containing urethane bond can be a new lead compound because of its superior bioavailability, potent in vitro T-cell inhibitory activity, and facile synthesis.

Structural orientation of the unique and protein binding domains of Lck

The structures of the SH3, SH2, and kinase domains of Lck, a member of the Src family of protein tyrosine kinases, have been published. Our project focuses on the structure and orientation of the unique domain of Lck because it is necessary for the association of CD4, contains two serine phosphorylation sites, and has the potential for a singular function not found in other Src family members. The phosphorylation of one serine, serine 59, causes both a reduced mobility of Lck of 4 kDa in SDS-PAGE gels and changes in the SH2 domain binding partners of Lck. We are observing structural characteristics in the N-terminus of Lck with respect to the phosphorylatyion status of serine 59 using NMR. Residues S7-P223 of Lck (S7) were observed. The 15N HSQC spectrum of S7 was very promising, suggesting the S7 protein is folded. The addition of an Lck SH2 domain-binding drug helped stabilize the SH2 domain. Structural studies of the S7 protein will provide information about the orientation of serine 59 with respect to the protein binding domains of Lck. Comparing these data to NMR analysis of S7 phosphorylated on serine 59 will provide data about the conformational changes that occur in the N-terminal half of Lck upon the phosphorylation of serine 59.

Essential role of the T cell–specific adapter protein in the activation of LCK in peripheral T cells

T cell–specific adapter protein (TSAd) is a SRC-homology-2 (SH2) domain–containing intracellular signaling molecule that is required for T cell antigen receptor (TCR)–induced cytokine synthesis in T cells. How TSAd functions in TCR signal transduction is not clear. Previous work has suggested a nuclear role for this adapter. However, other evidence suggests that TSAd also functions in the cytoplasm. Using T cells from TSAd-deficient mice, we now show that the major role of TSAd in the cytoplasm is in activation of the LCK protein tyrosine kinase at the outset of TCR signal transduction. Consequently, TSAd regulates several downstream signaling events, including intracellular calcium mobilization and activation of the Ras–extracellular signal–regulated kinase signaling pathway. TSAd regulates LCK activity directly through physical interaction with LCK SH3 and SH2 domains. These studies reveal TSAd as a positive regulator of proximal TCR signal transduction and provide important new information on the mechanism of TCR-induced LCK activation.

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7

BackgroundThe activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd.ResultsTSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239–256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239–334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239–334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells.ConclusionThese data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239–334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.

Acidic Region Tyrosines Provide Access Points for Allosteric Activation of the Autoinhibited Vav1 Dbl Homology Domain

Autoinhibited proteins serve key roles in many signal transduction pathways, and therefore proper regulation of these proteins is critical for normal cellular function. Proto-oncogene Vav1 is an autoinhibited guanine nucleotide exchange factor (GEF) for Rho family GTPases. The core autoinhibitory module of Vav1 consists of the catalytic Dbl homology (DH) domain bound through its active site to an alpha helix centered about Tyr174 in the Acidic (Ac) region of the protein. Phosphorylation of Tyr174 and two other tyrosines in the Ac region, Tyr142 and Tyr160, relieves autoinhibition and activates the catalytic DH domain. In this study, we use biochemical and structural analyses of the Vav1 Ac and DH domains to examine the kinetic and thermodynamic properties of Vav1 activation by the Src family kinase, Lck, and the role of the Lck SH2 domain in this process. We find that in the Ac-DH fragment of Vav1, Tyr174, but not Tyr142 or Tyr160, is protected from phosphorylation by interactions with the DH domain. Binding of the Lck SH2 domain to phosphorylated Tyr142 increases kcat/KM for Tyr174 by 4-fold, likely because the kinase domain can act on the substrate effectively in an intramolecular fashion. These studies of the autoinhibited Ac-DH module provide the foundation for a quantitative structural and thermodynamic understanding of the regulation of full length Vav1. Moreover, kinetic pathways involving initial interactions with exposed sites or "access points", as observed here for Vav1, may be generally important in the regulation of many autoinhibited proteins.

Insights into Human Lck SH3 Domain Binding Specificity: Different Binding Modes of Artificial and Native Ligands

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lck SH3 ligands using phage display. In addition, we analyzed Lck SH3 binding sites within known natural Lck SH3 binding proteins using an Lck specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lck SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lck SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands-which are not due to significant K(D) value differences as shown by calculating Lck SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lck SH3 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions.

Insights into human Lck SH3 domain binding specificity: different binding of ligands from artificial and native sources

Insights into human Lck SH3 domain binding specificity: different binding of ligands from artificial and native sources

Phosphorylation-Driven Protein−Protein Interactions: A Protein Kinase Sensing System

A highly flexible protein kinase sensing system is described that furnishes severalfold changes in fluorescence in response to phosphorylation. A library of Src kinase peptide substrates was prepared that contained different environmentally sensitive fluorophores positioned at various sites on the active site directed sequence. Robust changes in fluorescent intensity were observed in the presence of a phosphotyrosine binding domain protein (Lck SH2 domain), which furnishes a hydrophobic environment for the fluorophore. This protein kinase sensing system has the advantages that the fluorescent indicator can be unobtrusively positioned on the peptide substrate, and that different environmentally sensitive fluorophores with distinct photophysical properties can be employed.

The C terminus of T cell‐specific adapter protein (TSAd) is necessary for TSAd‐mediated inhibition of Lck activity

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.

Tyrosine Phosphorylation of Adaptor Protein 3BP2 Induces T Cell Receptor-Mediated Activation of Transcription Factor

Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr(183), Tyr(446), or Arg(486) in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr(183) is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr(446) recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg(486) in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.

The Relationship Between the Py3 Amino Acid in the Phosphopeptide Ligands and the Specificity for Various Sh2 Domains

After consideration of the tertiary structure of the pY+3 binding pocket in SH2 domains, isoleucine of Ac-pYEEIE was replaced with various unnatural aliphatic amino acids and the binding affinities for Lck, Src, and Fyn SH2 domains were measured. We determined the characteristics of the amino acid at the pY+3 position in the phosphopeptide ligands for the specificity for the SH2 domains.

Characterization of Lck-Binding Elements in the Herpesviral Regulatory Tip Protein

Herpesvirus saimiri encodes a tyrosine kinase interacting protein (Tip) that binds to T-cell-specific tyrosine kinase Lck via multiple sequence motifs and controls its activity. The regulation of Lck by Tip represents a key mechanism in the transformation of human T-lymphocytes during herpesviral infection. In this study, the interaction of Tip with the regulatory SH3 and SH2 domains of Lck was investigated by biophysical and computational techniques. NMR spectroscopy of isotopically labeled Tip(140-191) revealed that the interaction with the LckSH3 domain is not restricted to the classical proline-rich motif, but also involves the C-terminally adjacent residues which pack into a hydrophobic pocket on the surface of the SH3 domain, thus playing a likely role in mediating binding specificity. Fluorescence binding studies of Tip further demonstrate that Tyr127 in its phosphorylated form represents a strong ligand of the LckSH2 domain, indicating the presence of an additional Lck interaction motif. In contrast, Tyr114, known to be essential for STAT-3 binding, does not interact with the LckSH2 domain, showing that the tyrosines in Tip exhibit distinct binding specificity. The existence of numerous interaction sites between Tip and the regulatory domains of Lck implies a complex regulatory mechanism and may have evolved to allow a gradual regulation of Lck activity in different pathogenic states.

Inhibitors for Proteins Endowed with Catalytic and Non-Catalytic Activity which Recognize pTyr

Reversible phosphorylation of Tyr residues in proteins plays a central role in the transduction of signals. For both SH2 domains and for protein tyrosine phosphatases (PTPs) the phosphate group of phosphotyrosine (pTyr) of peptides provides a key affinity element, but its highly charged nature and its hydrolytic lability render it unsuitable in inhibitor design. The research in the recent years has been addressed to find pTyr bioisosters devoid of the phenylphosphate moiety and more potent inhibitors with less peptidic character. Several derivatives were prepared as pTyr bioisosters, and their activity appears to depend on the nature of the substrate, peptidic or low-molecular weight compounds, in which they are placed. In the field of PTPs, the research was mainly focused on new and selective PTP1B inhibitors, possibly useful in the treatment of Type 2 diabetes. The discovery of non-peptidic low molecular weight compounds able to inhibit PTP1B, by means of docking procedures and HTS screening, and the presence of secondary binding sites on PTP1B afforded new potent and selective inhibitors; several leads devoid of negative charges were also found. To date, however, few compounds have been tested In vivo and found to show a significant activity in diabetic mouse models. Other neutral compounds, mainly quinones, were found to inhibit CD45 and Cdc25. Several papers have appeared in recent years on the discovery of new Grb2, Src, Syk, and Lck SH2 domains binding antagonists. In this field very good inhibitors derived from high affinity peptides were found, with less peptidic character and with a reduced number of negative charges; however the presence of some negative charges, especially the one present on the pTyr bioisoster moiety, seems to be indispensable. As regards Grb2, Src and Lck SH2 domains, rigidification of the starting high affinity binding peptides afforded derivatives with improved affinity; cellular activity was achieved by modification of the side chains of these inhibitors.

Design and Synthesis of Small Chemical Inhibitors Containing Different Scaffolds for lck SH2 Domain.

On the basis of the structure of (R)-rosmarinic acid, a series of small chemical compounds with a different scaffold was synthesized as inhibitors for lck SH2 domain. From ELISA results, most of all chemical compounds showed a similar or a little lower binding activity for lck SH2 domain compared to the lead compound, (R)-rosmarinic acid. It was characterized that the backbone rigidity between two catechol substructures was required for the full activity and acid substructure of the lead compound was important for the activity. We successfully identified novel lead compounds that did not contain phosphotyrosine moiety and might have an improved bioavailability as inhibitor for lck SH2 domain.

Rosmarinic acid induces p56lck-dependent apoptosis in Jurkat and peripheral T cells via mitochondrial pathway independent from Fas/Fas ligand interaction.

Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56(lck) (Lck)-dependent manner; Lck(+) Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck(-) Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck(+) subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA's ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.