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Joseph Schlessinger - One of the best experts on this subject based on the ideXlab platform.

  • GRB2 and Phospholipase C-Gamma 1 assoCiate with a 36- to 38-kilodalton phosphotyrosine protein after T-Cell reCeptor stimulation.
    Molecular and cellular biology, 1994
    Co-Authors: M. Sieh, Joseph Schlessinger, A Batzer, Arthur Weiss
    Abstract:

    GRB2, a 25-kDa protein Comprising a single SH2 domain flanked by two SH3 domains, has been impliCated in linking reCeptor protein tyrosine kinases (PTKs) to the Ras pathway by interaCting with the guanine nuCleotide exChange protein SOS. Previous studies have demonstrated that GRB2 direCtly interaCts with ShC, a proto-onCogene produCt that is tyrosine phosphorylated upon reCeptor and nonreCeptor PTK aCtivation. In this report, we deteCted low levels of tyrosine phosphorylation of ShC and induCed assoCiation with GRB2 upon T-Cell reCeptor (TCR) stimulation. Instead, a prominent 36- to 38-kDa tyrosine phosphoprotein (pp36-38) assoCiated with the SH2 domain of GRB2 and formed a stable Complex with GRB2/SOS upon TCR stimulation. Cellular fraCtionation studies showed that whereas both GRB2 and SOS partitioned to the soluble and partiCulate fraCtions, pp36-38 was present exClusively in the partiCulate fraCtion. This phosphoprotein had the same apparent mobility in sodium dodeCyl sulfate-polyaCrylamide gel eleCtrophoresis as the phosphoprotein that assoCiates with Phospholipase C-Gamma 1 (PLC-Gamma 1). Furthermore, following partial immunodepletion of GRB2 and of the assoCiated pp36-38, there was a signifiCant reduCtion in the amount of the 36-kDa phosphoprotein assoCiated with PLC-Gamma 1, suggesting that a trimeriC PLC-Gamma 1/pp36-38/GRB2 Complex Could form. In support of this notion, we have also been able to deteCt low levels of PLC-Gamma 1 in GRB2 immunopreCipitates. We suggest that pp36-38 may be a bridging protein, Coupling different signalling moleCules to CytoplasmiC PTKs regulated by the TCR.

  • Dynamin binds to SH3 domains of Phospholipase C Gamma and GRB-2.
    The Journal of biological chemistry, 1994
    Co-Authors: Klaus Seedorf, Joseph Schlessinger, Günter Kostka, Richard L. Lammers, P Bashkin, R Daly, W H Burgess, A M Van Der Bliek, Axel Ullrich
    Abstract:

    AbstraCt SrC homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduCtion or represent Components of the Cytoskeleton. These domains are thought to serve as modules that mediate speCifiC protein-protein interaCtions that inClude proline-riCh sequenCes on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryoniC fibroblasts that bind speCifiCally to the SH3 domain of Phospholipase C Gamma, a primary substrate of reCeptor tyrosine kinases, and CharaCterized the 110-kDa band as the miCrotubule-aCtivated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaCtion does not require the dynamin GTPase funCtion and involves a proline-riCh target sequenCe between residues 812 and 820 of dynamin.

  • Solution struCture of the SH3 domain of Phospholipase C-Gamma.
    Cell, 1993
    Co-Authors: Daisuke Kohda, Hideki Hatanaka, Masafumi Odaka, Valsan Mandiyan, Joseph Schlessinger, Axel Ullrich, Fuyuhiko Inagaki
    Abstract:

    : SH3 (SrC homology 3) domains are found in many signaling proteins and appear to funCtion as binding modules for CytoplasmiC target proteins. The solution struCture of the SH3 domain of human Phospholipase C-Gamma (PLC-Gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is Composed of eight antiparallel beta strands Consisting of two suCCessive "Greek key" motifs, whiCh form a barrel-like struCture. The Conserved aliphatiC and aromatiC residues form a hydrophobiC poCket on the moleCular surfaCe, and the Conserved CarboxyliC residues are loCalized to the periphery. The hydrophobiC poCket may serve as a binding site for target proteins. Analysis of the slowly exChanging amide protons by NMR measurements indiCates that despite Containing a high Content of beta struCture, the SH3 domain of PLC-Gamma is flexible.

  • PresenCe of SH2 domains of Phospholipase C Gamma 1 enhanCes substrate phosphorylation by inCreasing the affinity toward the epidermal growth faCtor reCeptor.
    The Journal of biological chemistry, 1992
    Co-Authors: D Rotin, Annemarie Honegger, B Margolis, Axel Ullrich, Joseph Schlessinger
    Abstract:

    AbstraCt srC homology region 2 and 3 (SH2 and SH3) domains are Conserved nonCatalytiC regions originally desCribed in CytoplasmiC tyrosine kinases and subsequently identified in Phospholipase C Gamma 1 (PLC Gamma 1), GTPase-aCtivating protein of ras, and other signaling proteins. Although numerous studies indiCate that SH2 domains promote protein-protein interaCtions by speCifiC binding to tyrosine phosphorylated proteins, the funCtion of SH3 domains is not known. The SH2 domain of PLC Gamma 1 binds to Certain tyrosine-phosphorylated growth faCtor reCeptors, and following phosphorylation on Tyr783 the enzymatiC aCtivity of PLC Gamma 1 is enhanCed, leading to phosphatidylinositol hydrolysis. To determine the funCtional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in EsCheriChia Coli PLC Gamma 1 ConstruCts enCoding the region Containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjeCted to quantitative phosphorylation by the epidermal growth faCtor reCeptor (EGFR). Analysis of the kinetiCs of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanCed for substrates Containing SH2 domains: from S0.5 (average apparent Km) of 110 miCroM to S0.5 of 20 miCroM with the addition of a single SH2 domain and S0.5 of 3-4 miCroM for mutants Containing two SH2 domains. The presenCe of the SH3 domain did not influenCe the apparent Km of substrate phosphorylation. These results demonstrate that the presenCe of the SH2 domain in PLC Gamma 1 lowers the apparent Km (inCreases the affinity) of substrate phosphorylation by the EGFR, thereby faCilitating PLC Gamma 1 phosphorylation and aCtivation.

  • SH2 domains prevent tyrosine dephosphorylation of the EGF reCeptor: identifiCation of Tyr992 as the high-affinity binding site for SH2 domains of Phospholipase C Gamma.
    The EMBO journal, 1992
    Co-Authors: D Rotin, B Margolis, Axel Ullrich, W H Burgess, Moosa Mohammadi, Roger J. Daly, G. Daum, E.h. Fischer, Joseph Schlessinger
    Abstract:

    Several CytoplasmiC tyrosine kinases Contain a Conserved, non-CatalytiC stretCh of approximately 100 amino aCids Called the srC homology 2 (SH2) domain, and a region of approximately 50 amino aCids Called the SH3 domain. SH2/SH3 domains are also found in several other proteins, inCluding Phospholipase C-Gamma (PLC Gamma). ReCent studies indiCate that SH2 domains promote assoCiation between autophosphorylated growth faCtor reCeptors suCh as the epidermal growth faCtor (EGF) reCeptor and signal transduCing moleCules suCh as PLC Gamma. BeCause SH2 domains bind speCifiCally to protein sequenCes Containing phosphotyrosine, we examined their CapaCity to prevent tyrosine dephosphorylation of the EGF and other reCeptors with tyrosine kinase aCtivity. For this purpose, various SH2/SH3 ConstruCts of PLC Gamma were expressed in EsCheriChia Coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC Gamma are able to prevent tyrosine dephosphorylation of the EGF reCeptor and other reCeptors with tyrosine aCtivity. The inhibition of tyrosine dephosphorylation paralleled the CapaCity of various SH2-Containing ConstruCts to bind to the EGF reCeptor, suggesting that the tyrosine phosphatase and the SH2 domain Compete for the same tyrosine phosphorylation sites in the Carboxy-terminal tail of the EGF reCeptor. Analysis of the phosphorylation sites proteCted from dephosphorylation by PLC Gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 miCroM SH2. This indiCates that Tyr992 and its flanking sequenCe is the high-affinity binding site for SH2 domains of PLC Gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Axel Ullrich - One of the best experts on this subject based on the ideXlab platform.

  • Transforming potentials of epidermal growth faCtor and nerve growth faCtor reCeptors inversely Correlate with their Phospholipase C Gamma affinity and signal aCtivation.
    The EMBO journal, 1996
    Co-Authors: Axel Obermeier, I Tinhofer, H H Grunicke, Axel Ullrich
    Abstract:

    The exChange of nerve growth faCtor reCeptor/Trk and epidermal growth faCtor reCeptor (EGFR) Phospholipase C Gamma (PLC Gamma) binding sites resulted in the transfer of their distinCt affinities for this SrC homology 2 domain-Containing protein. Relative to wild-type EGFR, the PLC Gamma affinity inCrease of the EGFR switCh mutant EGFR.X enhanCed its inositol trisphosphate (IP3) and CalCium signals and resulted in a more sustained mitogen-aCtivated protein (MAP) kinase aCtivation and aCCelerated reCeptor dephosphorylation. In parallel, EGFR.X exhibited a signifiCantly deCreased mitogeniC and transforming potential in NIH 3T3 Cells. Conversely, the transfer of the EGFR PLC Gamma binding site into the Trk CytoplasmiC domain Context impaired the IP3/CalCium signal and attenuated the MAP kinase aCtivation and reCeptor dephosphorylation, but resulted in an enhanCement of the ETR.X exChange mutant mitogeniC and onCogeniC CapaCity. Our findings establish the signifiCanCe of PLC Gamma affinity for signal definition, the role of this reCeptor tyrosine kinase substrate as a negative feedbaCk regulator and the importanCe of this regulatory funCtion for mitogenesis and its disturbanCe in onCogeniC aberrations.

  • Dynamin binds to SH3 domains of Phospholipase C Gamma and GRB-2.
    The Journal of biological chemistry, 1994
    Co-Authors: Klaus Seedorf, Joseph Schlessinger, Günter Kostka, Richard L. Lammers, P Bashkin, R Daly, W H Burgess, A M Van Der Bliek, Axel Ullrich
    Abstract:

    AbstraCt SrC homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduCtion or represent Components of the Cytoskeleton. These domains are thought to serve as modules that mediate speCifiC protein-protein interaCtions that inClude proline-riCh sequenCes on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryoniC fibroblasts that bind speCifiCally to the SH3 domain of Phospholipase C Gamma, a primary substrate of reCeptor tyrosine kinases, and CharaCterized the 110-kDa band as the miCrotubule-aCtivated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaCtion does not require the dynamin GTPase funCtion and involves a proline-riCh target sequenCe between residues 812 and 820 of dynamin.

  • Solution struCture of the SH3 domain of Phospholipase C-Gamma.
    Cell, 1993
    Co-Authors: Daisuke Kohda, Hideki Hatanaka, Masafumi Odaka, Valsan Mandiyan, Joseph Schlessinger, Axel Ullrich, Fuyuhiko Inagaki
    Abstract:

    : SH3 (SrC homology 3) domains are found in many signaling proteins and appear to funCtion as binding modules for CytoplasmiC target proteins. The solution struCture of the SH3 domain of human Phospholipase C-Gamma (PLC-Gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is Composed of eight antiparallel beta strands Consisting of two suCCessive "Greek key" motifs, whiCh form a barrel-like struCture. The Conserved aliphatiC and aromatiC residues form a hydrophobiC poCket on the moleCular surfaCe, and the Conserved CarboxyliC residues are loCalized to the periphery. The hydrophobiC poCket may serve as a binding site for target proteins. Analysis of the slowly exChanging amide protons by NMR measurements indiCates that despite Containing a high Content of beta struCture, the SH3 domain of PLC-Gamma is flexible.

  • PresenCe of SH2 domains of Phospholipase C Gamma 1 enhanCes substrate phosphorylation by inCreasing the affinity toward the epidermal growth faCtor reCeptor.
    The Journal of biological chemistry, 1992
    Co-Authors: D Rotin, Annemarie Honegger, B Margolis, Axel Ullrich, Joseph Schlessinger
    Abstract:

    AbstraCt srC homology region 2 and 3 (SH2 and SH3) domains are Conserved nonCatalytiC regions originally desCribed in CytoplasmiC tyrosine kinases and subsequently identified in Phospholipase C Gamma 1 (PLC Gamma 1), GTPase-aCtivating protein of ras, and other signaling proteins. Although numerous studies indiCate that SH2 domains promote protein-protein interaCtions by speCifiC binding to tyrosine phosphorylated proteins, the funCtion of SH3 domains is not known. The SH2 domain of PLC Gamma 1 binds to Certain tyrosine-phosphorylated growth faCtor reCeptors, and following phosphorylation on Tyr783 the enzymatiC aCtivity of PLC Gamma 1 is enhanCed, leading to phosphatidylinositol hydrolysis. To determine the funCtional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in EsCheriChia Coli PLC Gamma 1 ConstruCts enCoding the region Containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjeCted to quantitative phosphorylation by the epidermal growth faCtor reCeptor (EGFR). Analysis of the kinetiCs of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanCed for substrates Containing SH2 domains: from S0.5 (average apparent Km) of 110 miCroM to S0.5 of 20 miCroM with the addition of a single SH2 domain and S0.5 of 3-4 miCroM for mutants Containing two SH2 domains. The presenCe of the SH3 domain did not influenCe the apparent Km of substrate phosphorylation. These results demonstrate that the presenCe of the SH2 domain in PLC Gamma 1 lowers the apparent Km (inCreases the affinity) of substrate phosphorylation by the EGFR, thereby faCilitating PLC Gamma 1 phosphorylation and aCtivation.

  • SH2 domains prevent tyrosine dephosphorylation of the EGF reCeptor: identifiCation of Tyr992 as the high-affinity binding site for SH2 domains of Phospholipase C Gamma.
    The EMBO journal, 1992
    Co-Authors: D Rotin, B Margolis, Axel Ullrich, W H Burgess, Moosa Mohammadi, Roger J. Daly, G. Daum, E.h. Fischer, Joseph Schlessinger
    Abstract:

    Several CytoplasmiC tyrosine kinases Contain a Conserved, non-CatalytiC stretCh of approximately 100 amino aCids Called the srC homology 2 (SH2) domain, and a region of approximately 50 amino aCids Called the SH3 domain. SH2/SH3 domains are also found in several other proteins, inCluding Phospholipase C-Gamma (PLC Gamma). ReCent studies indiCate that SH2 domains promote assoCiation between autophosphorylated growth faCtor reCeptors suCh as the epidermal growth faCtor (EGF) reCeptor and signal transduCing moleCules suCh as PLC Gamma. BeCause SH2 domains bind speCifiCally to protein sequenCes Containing phosphotyrosine, we examined their CapaCity to prevent tyrosine dephosphorylation of the EGF and other reCeptors with tyrosine kinase aCtivity. For this purpose, various SH2/SH3 ConstruCts of PLC Gamma were expressed in EsCheriChia Coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC Gamma are able to prevent tyrosine dephosphorylation of the EGF reCeptor and other reCeptors with tyrosine aCtivity. The inhibition of tyrosine dephosphorylation paralleled the CapaCity of various SH2-Containing ConstruCts to bind to the EGF reCeptor, suggesting that the tyrosine phosphatase and the SH2 domain Compete for the same tyrosine phosphorylation sites in the Carboxy-terminal tail of the EGF reCeptor. Analysis of the phosphorylation sites proteCted from dephosphorylation by PLC Gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 miCroM SH2. This indiCates that Tyr992 and its flanking sequenCe is the high-affinity binding site for SH2 domains of PLC Gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

D Rotin - One of the best experts on this subject based on the ideXlab platform.

  • PresenCe of SH2 domains of Phospholipase C Gamma 1 enhanCes substrate phosphorylation by inCreasing the affinity toward the epidermal growth faCtor reCeptor.
    The Journal of biological chemistry, 1992
    Co-Authors: D Rotin, Annemarie Honegger, B Margolis, Axel Ullrich, Joseph Schlessinger
    Abstract:

    AbstraCt srC homology region 2 and 3 (SH2 and SH3) domains are Conserved nonCatalytiC regions originally desCribed in CytoplasmiC tyrosine kinases and subsequently identified in Phospholipase C Gamma 1 (PLC Gamma 1), GTPase-aCtivating protein of ras, and other signaling proteins. Although numerous studies indiCate that SH2 domains promote protein-protein interaCtions by speCifiC binding to tyrosine phosphorylated proteins, the funCtion of SH3 domains is not known. The SH2 domain of PLC Gamma 1 binds to Certain tyrosine-phosphorylated growth faCtor reCeptors, and following phosphorylation on Tyr783 the enzymatiC aCtivity of PLC Gamma 1 is enhanCed, leading to phosphatidylinositol hydrolysis. To determine the funCtional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in EsCheriChia Coli PLC Gamma 1 ConstruCts enCoding the region Containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjeCted to quantitative phosphorylation by the epidermal growth faCtor reCeptor (EGFR). Analysis of the kinetiCs of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanCed for substrates Containing SH2 domains: from S0.5 (average apparent Km) of 110 miCroM to S0.5 of 20 miCroM with the addition of a single SH2 domain and S0.5 of 3-4 miCroM for mutants Containing two SH2 domains. The presenCe of the SH3 domain did not influenCe the apparent Km of substrate phosphorylation. These results demonstrate that the presenCe of the SH2 domain in PLC Gamma 1 lowers the apparent Km (inCreases the affinity) of substrate phosphorylation by the EGFR, thereby faCilitating PLC Gamma 1 phosphorylation and aCtivation.

  • SH2 domains prevent tyrosine dephosphorylation of the EGF reCeptor: identifiCation of Tyr992 as the high-affinity binding site for SH2 domains of Phospholipase C Gamma.
    The EMBO journal, 1992
    Co-Authors: D Rotin, B Margolis, Axel Ullrich, W H Burgess, Moosa Mohammadi, Roger J. Daly, G. Daum, E.h. Fischer, Joseph Schlessinger
    Abstract:

    Several CytoplasmiC tyrosine kinases Contain a Conserved, non-CatalytiC stretCh of approximately 100 amino aCids Called the srC homology 2 (SH2) domain, and a region of approximately 50 amino aCids Called the SH3 domain. SH2/SH3 domains are also found in several other proteins, inCluding Phospholipase C-Gamma (PLC Gamma). ReCent studies indiCate that SH2 domains promote assoCiation between autophosphorylated growth faCtor reCeptors suCh as the epidermal growth faCtor (EGF) reCeptor and signal transduCing moleCules suCh as PLC Gamma. BeCause SH2 domains bind speCifiCally to protein sequenCes Containing phosphotyrosine, we examined their CapaCity to prevent tyrosine dephosphorylation of the EGF and other reCeptors with tyrosine kinase aCtivity. For this purpose, various SH2/SH3 ConstruCts of PLC Gamma were expressed in EsCheriChia Coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC Gamma are able to prevent tyrosine dephosphorylation of the EGF reCeptor and other reCeptors with tyrosine aCtivity. The inhibition of tyrosine dephosphorylation paralleled the CapaCity of various SH2-Containing ConstruCts to bind to the EGF reCeptor, suggesting that the tyrosine phosphatase and the SH2 domain Compete for the same tyrosine phosphorylation sites in the Carboxy-terminal tail of the EGF reCeptor. Analysis of the phosphorylation sites proteCted from dephosphorylation by PLC Gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 miCroM SH2. This indiCates that Tyr992 and its flanking sequenCe is the high-affinity binding site for SH2 domains of PLC Gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Alan R Saltiel - One of the best experts on this subject based on the ideXlab platform.

  • IdentifiCation of amino aCids in the N-terminal SH2 domain of Phospholipase C Gamma 1 important in the interaCtion with epidermal growth faCtor reCeptor.
    Biochemistry, 1994
    Co-Authors: J R Gergel, Alan R Saltiel, Dennis Joseph Mcnamara, E M Dobrusin, Guochang Zhu, W T Miller
    Abstract:

    Photoaffinity labeling and site-direCted mutagenesis have been used to identify amino aCid residues of the Phospholipase C Gamma 1 (PLC Gamma 1) N-terminal SH2 domain involved in reCognition of the aCtivated epidermal growth faCtor reCeptor (EGFR). The photoaCtive amino aCid p-benzoylphenylalanine (Bpa) was inCorporated into phosphotyrosine-Containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presenCe of SH2 domains showed Cross-linking whiCh was time-dependent and speCifiC; labeling was inhibited with non-Bpa-Containing peptides from EGFR in molar exCess. The phosphotyrosine residue on the peptides was important for SH2 reCognition, as dephosphorylated peptides did not Cross-link. Radiolabeled peptides were used to identify sites of Cross-linking to the N-terminal SH2 of PLC Gamma 1. Bpa peptide-SH2 Complexes were digested with trypsin, and radioaCtive fragments were purified by HPLC and analyzed by Edman sequenCing. These experiments showed Arg562 and an additional site in the alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaCtion with the Tyr1068-derived photoaffinity label identified Leu653 as the Cross-linked site. Mutation of the neighboring residues of Glu587 deCreased photo-Cross-linking, emphasizing the importanCe of this region of the moleCule for reCognition. These results are Consistent with evidenCe from the v-SrC Crystal struCture and impliCate the loop spanning residues Gln640-Ser654 of PLC Gamma 1 in speCifiC reCognition of phosphopeptides.

  • Phospholipase C Gamma 1 direCtly assoCiates with the p70 trk onCogene produCt through its srC homology domains
    Journal of Biological Chemistry, 1991
    Co-Authors: M Ohmichi, Stuart J Decker, Long Pang, Alan R Saltiel
    Abstract:

    AbstraCt Tyrosine phosphorylation of proteins was examined in NIH3T3 Cells transformed by an onCogeniC form of the trk protein. Proteins of 148, 140, 70, and 55 kDa were phosphorylated on tyrosine residues in trk-transformed Cells but not Control NIH3T3 Cells. The 70-kDa protein may represent the trk onCogene protein itself whiCh was shown to be tyrosine-phosphorylated in vivo using trk-speCifiC antiserum. Phospholipase C-Gamma 1 (PLC-Gamma 1) was also found to be Constitutively tyrosine-phosphorylated in trk-transformed Cells and the trk protein Co-immunopreCipitated with PLC-Gamma. The GTPase-aCtivating protein of ras (GAP) and the 62-kDa GAP-assoCiated protein were tyrosine-phosphorylated in trk-transformed Cells, and a lesser amount of trk Co-immunopreCipitated with GAP relative to with PLC-Gamma. The trk onCogene produCt bound speCifiCally to a baCterially expressed fusion protein Containing the srC homology domains of PLC-Gamma. The data suggest a signifiCant role for PLC-Gamma in intraCellular signaling by the trk onCogene.

Jeffrey A. Ledbetter - One of the best experts on this subject based on the ideXlab platform.

  • AssoCiation of tyrosine and serine kinases with the B Cell surfaCe antigen CD20. InduCtion via CD20 of tyrosine phosphorylation and aCtivation of Phospholipase C-Gamma 1 and PLC Phospholipase C-Gamma 2.
    Journal of immunology (Baltimore Md. : 1950), 1993
    Co-Authors: Julie P. Deans, Gary L. Schieven, Geraldine L. Shu, Mary A. Valentine, Lisa A. Gilliland, Alejandro Aruffo, Edward A. Clark, Jeffrey A. Ledbetter
    Abstract:

    CD20 is a B Cell-speCifiC 35/37 kDa integral membrane protein whiCh modulates proliferation and differentiation of normal resting B Cells when stimulated by CD20 antibodies. An inCrease in C-myC mRNA levels oCCurs within hours after treatment of resting B Cells with CD20 mAb; however earlier events in the CD20 signal transduCtion pathway have not been desCribed. Here we demonstrate that anti-CD20 mediated induCtion of C-myC mRNA is inhibited by the tyrosine kinase inhibitor herbimyCin A, that CD20 is assoCiated with both tyrosine and serine kinase aCtivity, and that tyrosine phosphorylation of multiple substrates is induCed within minutes upon ligation of CD20 with mAb. AssoCiation of the tyrosine and serine kinases with CD20 was stable in lysis buffer Containing 1% NP40 and 0.25% deoxyCholate. Under the same Conditions, antibodies against several other B Cell surfaCe moleCules failed to Co-preCipitate tyrosine kinase aCtivity, however, a serine kinase was preCipitated by the anti-CD19 mAb, B43. Both Phospholipase C-Gamma 1 and -Gamma 2 were phosphorylated on tyrosine after Cross-linking of CD20-bound mAb, and this Correlated with inCreases in intraCellular CalCium that were partially resistant to depletion of extraCellular CalCium with EGTA. The pattern of tyrosine phosphorylated proteins observed in whole Cell lysates after anti-CD20 Cross-linking appeared to be a subset of those induCed by anti-IgM; however, differenCes in phosphoproteins induCed by anti-IgM and anti-CD20 were deteCted using a fynSH2-fusion protein.

  • Beta 2-integrin LFA-1 signaling through Phospholipase C-Gamma 1 aCtivation.
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Steven B. Kanner, Laura Sue Grosmaire, Jeffrey A. Ledbetter, Nitin K. Damle
    Abstract:

    AbstraCt One of the beta 2-integrins found on hematopoietiC Cells is lymphoCyte funCtion-assoCiated antigen 1 (LFA-1), a lymphoCyte/myeloid Cell-speCifiC reCeptor that binds to members of the interCellular adhesion moleCule (ICAM) family on antigen-presenting Cells. Stimulation of LFA-1 with antibodies or purified ICAMs induCes augmentation of T-Cell antigen reCeptor (TCR)-direCted T-Cell responsiveness. In the present study, LFA-1 was shown to be linked to the tyrosine kinase signaling pathway that stimulates tyrosine phosphorylation and aCtivation of Phospholipase C-Gamma 1 (PLC-Gamma 1). Integrin beta-Chain (CD18) Crosslinking independently induCed downstream mobilization of intraCellular Ca2+ and potently Costimulated TCR-induCed Ca2+ flux with an inCrease in both amplitude and kinetiCs. beta 2-Integrin signaling through this pathway was Completely inhibited by herbimyCin A and was prevented by TCR modulation. Coligation of the TCR via antibody and LFA-1 with a Counter-reCeptor in the form of a soluble ICAM-1/Rg fusion protein resulted in prolonged tyrosine phosphorylation of PLC-Gamma 1. MonoClonal antibodies to both the alpha Chain (CD11a) and the beta Chain (CD18) of LFA-1 induCed Ca2+ mobilization to different levels, suggesting epitope speCifiCity for aCtivation potential. In addition to PLC-Gamma 1, tyrosine phosphorylation of an 80-kDa protein substrate was augmented following CD18 Crosslinking but was not TCR-dependent. The beta 2-integrin LFA-1 on T Cells is therefore direCtly linked to a tyrosine kinase pathway that stimulates signaling by phosphatidylinositol-speCifiC PLC-Gamma 1.

  • 7,12-Dimethylbenz[a]anthraCene aCtivates protein-tyrosine kinases Fyn and LCk in the HPB-ALL human T-Cell line and inCreases tyrosine phosphorylation of Phospholipase C-Gamma 1, formation of inositol 1,4,5-trisphosphate, and mobilization of intraCell
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Melecita M. Archuleta, Jeffrey A. Ledbetter, Gary L. Schieven, G G Deanin, Scott W. Burchiel
    Abstract:

    AbstraCt Previous studies have shown that the immunosuppressive and CarCinogeniC polyCyCliC aromatiC hydroCarbon 7,12-dimethylbenz(a)anthraCene (DMBA) impairs Ca(2+)-dependent transmembrane signaling in human and murine lymphoCytes. The purpose of the present studies was to analyze potential meChanisms of immunosuppression by DMBA and to examine effeCts on Ca2+ homeostasis and antigen-reCeptor signaling in human T Cells. DMBA produCed a rapid and sustained inCrease in Ca2+ levels in HPB-ALL Cells by release of CytoplasmiC Ca2+. DMBA also inhibited anti-CD3/CD4 mobilization of Ca2+ in HPB-ALL Cells, with half-maximal inhibition oCCurring at approximately 4 hr. Thus, the kinetiCs for initial Ca2+ mobilization and inhibition of the anti-CD3/CD4 response differed. The rapid rise in intraCellular Ca2+ induCed by DMBA alone was aCCompanied by a rapid but transient inCrease in inositol 1,4,5-trisphosphate and tyrosine phosphorylation of Phospholipase C-Gamma 1. The pattern of tyrosine phosphorylation induCed by DMBA in HPB-ALL Cells was remarkably similar to that induCed by anti-CD3/CD4 aCtivation. Thus, DMBA-induCed phosphorylation may mimiC antigen-reCeptor aCtivation in T Cells, whiCh may lead to alterations in antigen responsiveness. The meChanism of DMBA-induCed tyrosine phosphorylation of Phospholipase C-Gamma 1 may have been due to an inCrease in protein-tyrosine kinase aCtivity, sinCe it was found that DMBA produCed a > 2-fold inCrease in the aCtivity of the T-Cell reCeptor-assoCiated SrC-family kinases Fyn and LCk. The kinetiCs of aCtivation of protein-tyrosine kinases demonstrated that Fyn aCtivity was inCreased within 10 min of exposure to DMBA, whereas maximal LCk aCtivation required 30 min. Thus, it is likely that the Fyn kinase or other protein-tyrosine kinases may be responsible for the early tyrosine phosphorylation of Phospholipase C-Gamma 1, whiCh results in inositol 1,4,5-trisphosphate release and mobilization of intraCellular Ca2+.

  • Superantigen and HLA-DR ligation induCe Phospholipase-C Gamma 1 aCtivation in Class II+ T Cells.
    Journal of immunology (Baltimore Md. : 1950), 1992
    Co-Authors: Steven B. Kanner, Laura Sue Grosmaire, Niels Ødum, Susan Masewicz, Arne Svejgaard, Jeffrey A. Ledbetter
    Abstract:

    BaCterial enterotoxin superantigens bind direCtly to HLA Class II moleCules (HLA-DR) expressed on both APC and aCtivated human T Cells, and simultaneously bind to Certain V beta Chains of the TCR. In this report, we Compared early T Cell signaling events in human alloantigen-stimulated T Cells when aCtivated by HLA-DR ligation through antibody Cross-linking or by direCt enterotoxin superantigen binding. Both types of stimuli induCed tyrosine phosphorylation of phosphatidylinositol-speCifiC Phospholipase C Gamma 1 (PLC Gamma 1) and an inCrease in intraCellular CalCium ConCentration; however, superantigen-induCed signaling was stronger than Class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC Gamma 1 aCtivation and inCreased CalCium mobilization, Consistent with a meChanism of superantigen aCtivity through a Combination of Class II and CD3/Ti signals. In addition, down-modulation of CD3 reCeptors with antibody demonstrated that superantigen-induCed signaling events were CD3-dependent. Superantigen signaling was also Class II-dependent, in that resting T Cells were not responsive to direCt enterotoxin stimulation. To address how early signal transduCing aCtivity Correlated with T Cell responsiveness, alloantigen-primed T Cells were aCtivated with immobilized Class II-speCifiC mAb or soluble superantigen. Both HLA-DR mAb-stimulated T Cells and enterotoxin-treated T Cells proliferated strongly in response to Co-stimulation by a Combination of CD28 reCeptor engagement and PMA addition. In addition, superantigen-induCed growth was induCed by CD28 reCeptor ligation with antibody or the B7 Counter-reCeptor expressed on Chinese hamster ovary Cells. Taken together, these results indiCate that Class II moleCules expressed on aCtivated T Cells are direCtly Coupled to the PLC Gamma 1 signal transduCtion pathway, and that Coligation of HLA-DR with CD3 augments T Cell signaling Comparable to that induCed by enterotoxin superantigen. Thus, we suggest that superantigen-induCed early signaling responses in aCtivated T Cells may be due in part to Class II transmembrane signals induCed when HLA-DR and V beta are ligated in Cis.

  • Sulfhydryl oxidation down-regulates T-Cell signaling and inhibits tyrosine phosphorylation of Phospholipase C Gamma 1.
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Steven B. Kanner, Terrance J. Kavanagh, Angelika Grossmann, Joseph B. Bolen, Peter S. Rabinovitch, Jeffrey A. Ledbetter
    Abstract:

    AbstraCt Early events in both T-Cell reCeptor (CD3)- and CD4-induCed signal transduCtion pathways inClude tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown produCts, and the mobilization of intraCellular Ca2+. Oxidative stress in T Cells mediated by sulfhydryl-reaCtive nonpolar maleimides was shown previously to down-regulate both reCeptor-mediated Ca2+ mobilization and interleukin 2 produCtion. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induCed Ca2+ responses in human T Cells Correlating with a reduCtion in the level of Phospholipase C Gamma 1 (PLC Gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC Gamma 1 and additional protein substrates was observed at ConCentrations of N-ethylmaleimide above 20 miCroM, whereas lower ConCentrations of oxidant appeared to inCrease tyrosine kinase aCtivity following Cell stimulation. Sulfhydryl oxidation did not direCtly affeCt the CatalytiC aCtivity of PLC Gamma 1, sinCe immunopurified enzyme from N-ethylmaleimide-treated T Cells was fully aCtive. Although N-ethylmaleimide treatment of T Cells did not Cause a direCt effeCt on total pp56lCk kinase aCtivity measured in vitro, the interaCtion between CD4 and pp56lCk was oxidation-sensitive in vivo. However, CD3-induCed signaling was inhibited at N-ethylmaleimide ConCentrations lower than that required for CD4/pp56lCk dissoCiation, suggesting that CD3-assoCiated tyrosine kinase aCtivity involves aCutely sensitive regulatory thiols. In addition to ChemiCally induCed sulfhydryl oxidation, naturally regulated Cellular redox states appear to diCtate the potential for T-Cell responsiveness, sinCe degranulating human peripheral blood neutrophils inhibited CD3-induCed Ca2+ mobilization in T lymphoCytes. These data indiCate that signal transduCtion in T Cells involves the aCtivation of PLC Gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surfaCe reCeptors and tyrosine kinases, perhaps inCluding the interaCtion between CD4 and pp56lCk.