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Results for “src Homology Domains”

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PubMed2003

Regulation of tensin-promoted cell migration by its focal adhesion binding and Src homology domain 2.

Tensin1 is an actin- and phosphotyrosine-binding protein that localizes to focal adhesions. Recently, we have shown that both tensin1 and a new family member, tensin2, promote cell migration [Chen, Duncan, Bozorgchami and Lo (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 733-738]. Since localization of proteins to particular intracellular compartments often regulates their functions, and Src homology domain 2 may mediate signals related to cell migration, we hypothesize that tensin-mediated cell migration is regulated by the focal adhesion localization and the Src homology domain 2 of tensin. To test this hypothesis, we have analysed the effects of a series of tensin1 mutants on cell migration. Our results have shown that (1) tensin1 contains two focal adhesion-binding sites, (2) the wild-type tensin1 significantly promotes cell migration, (3) mutants with one focal adhesion-binding site do not promote cell migration, (4) the non-focal adhesion localized mutant suppresses cell migration and (5) the mutant that is not able to bind to phosphotyrosine-containing proteins has no effect on cell migration. These results have indicated that focal adhesion localization of tensin1 and the phosphotyrosine-binding activity are two critical factors in regulating tensin-mediated cell migration.

3T3 Cells↗

PubMed1996

Src-homology domain 2 is responsible for transcriptional suppression induced by expression of Lck.

Overexpression of Lck was shown, by our previous study, to suppress gene transcription from various viral and cellular promoters. The suppression of transcription from human T-cell leukemia virus promoter by Lck was independent of the presence of the enhancer core sequences within the long terminal repeat. The suppression of transcription was observed with Lck mutants that had either diminished or enhanced tyrosine-kinase activity. A mutant lacking the myristylation site also suppressed transcription. From the analysis with various deletion mutants of Lck, it was suggested that Src-homology domain 2 (SH2) is both necessary and sufficient for the suppression of transcription. A similar effect was also observed with the SH2 domain of the v-src gene. Thus, overexpression of Lck could suppress gene expression through a unique function of the SH2 domain.

Amino Acid Sequence↗

PubMed2003

Dual role of SRC homology domain 2-containing inositol phosphatase 2 in the regulation of platelet-derived growth factor and insulin-like growth factor I signaling in rat vascular smooth muscle cells.

Src homology domain 2 (SH2)-containing inositol phosphatase 2 (SHIP2) possesses 5-phosphatase activity and an SH2 domain. The role of SHIP2 in platelet-derived growth factor (PDGF) and IGF-I signaling was studied by expressing wild-type (WT-) and a catalytically defective (Delta IP-) SHIP2 into rat aortic smooth muscle cells by adenovirus-mediated gene transfer. PDGF- and IGF-I-induced tyrosine phosphorylation of their respective receptors and phosphatidylinositol 3-kinase (PI3-kinase) activity were not affected by the expression of either WT- or Delta IP-SHIP2. SHIP2 possessed 5'-phosphatase activity to hydrolyze the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate in vivo. Akt and glycogen synthase kinase 3beta are known to be downstream molecules of PI3-kinase, leading to the antiapoptotic effect. Overexpression of WT-SHIP2 inhibited PDGF- and IGF-I-induced phosphorylation of these molecules and the protective effect of poly(ADP-ribose) polymerase degradation, whereas these phosphorylations and the protective effect were enhanced by the expression of Delta IP-SHIP2, which functions in a dominant negative fashion. Regarding the Ras-MAPK pathway, PDGF- and IGF-I-induced tyrosine phosphorylation of Shc was not affected by the expression of either WT- or Delta IP-SHIP2, whereas both expressed SHIP2 associated with Shc. Importantly, PDGF and IGF-I stimulation of Shc/Grb2 binding, MAPK activation, and 5-bromo-2'-deoxyuridine incorporation were all decreased in both WT- and Delta IP-SHIP2 expression. These results indicate that SHIP2 plays a negative regulatory role in PDGF and IGF-I signaling in vascular smooth muscle cells. As the bifunctional role, our results suggest that SHIP2 regulates PDGF- and IGF-I-mediated signaling downstream of PI3-kinase, leading to the antiapoptotic effect via 5-phosphatase activity, and that SHIP2 regulates the growth factor-induced Ras-MAPK pathway mainly via the SH2 domain.

Adaptor Proteins, Signal Transducing↗

PubMed1998

From Src Homology domains to other signaling modules: proposal of the 'protein recognition code'.

The study of oncogenes has illuminated many aspects of cellular signaling. The delineation and characterization of protein modules exemplified by Src Homology domains has revolutionized our understanding of the molecular events underlying signal transduction pathways. Several well characterized intracellular modules which mediate protein-protein interactions, namely SH2, SH3, PH, PTB, EH, PDZ, EVH1 and WW domains, are directly involved in the multitude of membrane, cytoplasmic and nuclear processes in multicellular and/or unicellular organisms. The modular character of these protein domains and their cognate motifs, the universality of their molecular function, their widespread occurrence, and the specificity as well as the degeneracy of their interactions have prompted us to propose the concept of the 'protein recognition code'. By a parallel analogy to the universal genetic code, we propose here that there will be a finite set of precise rules to govern and predict protein-protein interactions mediated by modules. Several rules of the 'protein recognition code' have already emerged.

Animals↗

PubMed1999

Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation.

The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55-60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation.

Animals↗

PubMed1996

PLC gamma 1 Src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts.

Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways.

3T3 Cells↗

PubMed1998

The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization.

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Delta myo5Delta cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A-induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37 degrees C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Delta myo5Delta cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Delta myo5Delta cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.

Actins↗

PubMed1995

Competitive binding assay of src homology domain 3 interactions between 5-lipoxygenase and growth factor receptor binding protein 2.

c-src homology 3 domains (SH3) modulate the formation of a number of protein complexes that are important in cell signaling and cytoskeletal organization. The SH3 domain is recognized by short conserved proline-rich motifs which adopt left-handed polyproline helices on binding. In order to examine molecular determinants of the proline motif:SH3 interaction, an enzyme-linked immunosorbent assay was developed to observe binding of 5-lipoxygenase to SH3 domains of growth factor receptor binding protein 2 (Grb2). The assay makes use of glutathione S-transferase fusion proteins of Grb2 and fragments of Grb2 immobilized onto wells of standard 96-well microtiter plates. Equilibrium binding is monitored colorimetrically and the measured absorbance is proportional to 5-LO concentration. The interactions is specific for the Grb2 portion of the fusion proteins, and 5-LO binds preferentially to Grb2 fragments containing an SH3 domain. Competitive binding assays with a synthetic peptide which mimicked the proline-rich region of 5-LO yielded results that are consistent with previous estimates. Binding was examined in the presence of a number of peptides containing the consensus sequence -PXXP-, in the presence of enzyme activity mediators and in the presence of plant lipoxygenases that lack the proline-rich binding motif. Results suggest that the specificity of the Grb2:5-LO interaction is high.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Titin as a giant scaffold for integrating stress and Src homology domain 3-mediated signaling pathways: the clustering of novel overlap ligand motifs in the elastic PEVK segment.

The richness of proline sequences in titins qualifies these giant proteins as the largest source of intrinsically disordered structures in nature. An extensive search and analysis for Src homology domain 3 (SH3) ligand motifs revealed a myriad of broadly distributed SH3 ligand motifs, with the highest density in the PEVK segments of human titin. Besides the canonical class I and II motifs with opposite orientations, novel overlapping motifs consisting of one or more of each canonical motif are abundant. Experimentally, the binding affinity and critical residues of these putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle and non-muscle cell extracts were validated with peptide array technology and by the sarcomere distribution of SH3-containing proteins. A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration. Molecular dynamics and molecular docking studies indicated that the overlapping motif can bind in opposite orientations with comparable energy and contact areas and predicts correctly orientation-specific contacts in NMR data. We propose that the overlap ligand motifs are a new class of ligands with innate ability to dictate SH3 domain orientation and to facilitate the rate, strength, and stereospecificity of receptor interactions. Proline-rich sequences of titins are candidates as major hubs of SH3-dependent signaling pathways. The interplay of elasticity and dense clustering of mixed receptor orientations in titin PEVK segment have important implications for the mechanical sensing, force sensitivity, and inter-adapter interactions in signaling pathways.

Amino Acid Sequence↗

PubMed2000

Effects of Src homology domain 2 (SH2)-containing inositol phosphatase (SHIP), SH2-containing phosphotyrosine phosphatase (SHP)-1, and SHP-2 SH2 decoy proteins on Fc gamma RIIB1-effector interactions and inhibitory functions.

Coaggregation of Fc gamma RIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate Fc gamma RIIB1 effects suggest that the receptor uses SH2-containing inositol phosphatase (SHIP) and SH2-containing phosphotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant Fc gamma RIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants' dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on Fc gamma RIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated Fc gamma RIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind Fc gamma RIIB1 and block Fc receptor function. The SHIP SH2, while not affecting Fc gamma RIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, Fc gamma RIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of Fc gamma RIIB1-mediated inhibitory signaling.

Animals↗

PubMed2003

Interaction of the growth hormone receptor with cytokine-induced Src homology domain 2 protein in rat adipocytes.

GH stimulates the phosphorylation of tyrosine residues in the GH receptor (GHR), Janus kinase 2 (JAK2), and other signaling proteins in a transient manner that subsides within 1 h. To assess the possible roles of cytokine-induced Src homology domain 2 (SH2) (CIS/SOCS) proteins in these transient responses, we studied the expression and disposition of CIS/SOCS proteins in rat adipocytes, a physiological target of GH action. A tyrosine-phosphorylated protein that appears to be the GHR was coprecipitated from extracts of GH-treated adipocytes with alpha-CIS. In contrast, no tyrosine-phosphorylated adipocyte proteins were recovered after immunoprecipitation with alpha-SOCS3, although coprecipitation of GHR with SOCS3 was readily detected in extracts of 3T3-F442A fibroblasts. Interaction of GHR with CIS peaked between 2 and 10 min after adipocytes were treated with GH, when tyrosine phosphorylation of the GHR was maximal. By 60 min after GH, tyrosine phosphorylation of the GHR declined to very low levels, and its interaction with CIS was reduced correspondingly. Proteasome inhibitors prevented the decline in tyrosine-phosphorylated GHR and prolonged interaction of GHR and CIS for at least 1 h. These findings demonstrate the interaction of CIS with the GHR in vivo and suggest that CIS may enhance degradation of the receptor by a proteasomal pathway.

Adipocytes↗

PubMed1999

The Src-homology domain 2-bearing protein tyrosine phosphatase-1 inhibits antigen receptor-induced apoptosis of activated peripheral T cells.

Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.

Animals↗

PubMed2000

Src homology domain 2-containing tyrosine phosphatase 2 associates with intercellular adhesion molecule 1 to regulate cell survival.

Intercellular adhesion molecule-1 (ICAM-1) binds to the plasma protein fibrinogen (Fg) to mediate leukocyte/endothelial cell interactions. In our studies, the ligation of Fg to ICAM-1 on tumor necrosis factor-alpha-stimulated endothelial cells resulted in the tyrosine phosphorylation of Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). The ICAM-1 cytoplasmic sequence IKKYRLQ conforms poorly to the concensus immunoreceptor tyrosine-based inhibition motifs found in receptors that bind SHP-2. Nevertheless, the tyrosine phosphorylated sequence (IKKpYRLQ) bound specifically to the SH2 domain proximal to the NH(2)-terminal of SHP-2 (SHP-2-N) but not to the SH2 domain proximal on the COOH-terminal side (SHP-2-C). Phosphorylated ICAM-1 bound SHP-2-N. In immunoprecipitation experiments, SHP-2 associated with phosphorylated ICAM-1. Cells expressing truncated ICAM-1 that lacked the cytoplasmic sequence (ICAM-1(TR)) failed to associate with SHP-2. ICAM-1 containing the tyrosine to alanine substitution at position 485 (ICAM-1(Y485A)) associated weakly with SHP-2. Cells expressing ICAM-1(TR) and ICAM-1(Y485A) underwent apoptosis upon adhesion to Fg, whereas the wild type ICAM-1 maintained cell survival. These results indicate that ICAM-1 interactions with SHP-2 allow better cellular survival mediated through Fg-ICAM-1 ligation.

Amino Acid Motifs↗

PubMed2004

The critical role of SRC homology domain 2-containing tyrosine phosphatase-1 in recombinant human erythropoietin hyporesponsive anemia in chronic hemodialysis patients.

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.

Anemia↗

PubMed2001

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase.

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.

Abatacept↗

PubMed1994

Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).

The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation.

Adult↗

PubMed2004

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals.

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.

Cytotoxicity, Immunologic↗

PubMed1991

Nerve growth factor binds to the 140 kd trk proto-oncogene product and stimulates its association with the src homology domain of phospholipase C gamma 1.

The cellular actions of nerve growth factor (NGF) involve regulation of protein phosphorylation. In PC-12 pheochromocytoma cells, exposure of [125I]NGF followed by crosslinking indicates that the ligand binds to two discreet receptors, the previously described 75 kd protein, as well as the trk proto-oncogene product pp140c-trk. Competition experiments reveal that of the two, pp 140c-trk binds to NGF with higher affinity. Following exposure to NGF, pp140c-trk undergoes a rapid autophosphorylation on tyrosine residues, and concomitantly phosphorylates and associates with phospholipase C gamma 1 (PLC gamma 1), through interaction with its src homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent activation of its tyrosine kinase activity and the specific association with the effector molecule, PLC gamma 1, suggests that this is the biologically relevant signaling receptor for NGF.

Adrenal Gland Neoplasms↗