Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preferenc... more Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preference for manganese over magnesium for maximal activity. We have observed that peptides and proteins are nonenzymatically phosphorylated at tyrosine and serine by ATP when heated in the presence of MnC12 at neutral pH. The extent of the reaction is negligible below 50 "C but increases rapidly at higher temperatures. The reaction proceeds in the presence of sodium dode-cy1 sulfate but is blocked by EDTA. No reaction is observed in the absence of Mn2*, even if M 8 + is present. Manganese therefore acts as a catalyst for the nonenzymatic reaction, but magnesium does not. We propose that the preference for manganese shown by many protein tyrosine kinases is due at least in part to the intrinsic ability of Mn2+ to catalyze the transfer of phosphate from ATP to a phosphate acceptor such as tyrosine. The nonenzymatic phosphorylation reaction also offers a new synthetic pathway for the preparation of radiolabeled peptides containing phosphotyrosine and phosphoserine.
CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activa... more CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the µ2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The µ1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of µ2 and CTLA-4 were localized to residues, 161 TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165 YVKM, involved in CTLA-4 signaling. µ2 interacted preferentially with CTLA-4 when residue 165 Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165 Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 ( 165 Y and 182 Y) were phosphorylated by the T lymphocyteassociated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165 Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.
Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (F... more Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (Fc gamma RI) or Fc gamma receptor II (Fc gamma RII) gave rise to the rapid phosphorylation of multiple intracellular proteins. The pattern of proteins that were phosphorylated appeared to be identical. Analysis of these proteins by specific immunoprecipitation indicated that stimulation through either receptor did indeed give rise to phosphorylation of the same set of proteins. These included: Fc gamma RII, phospholipase C (PLC) gamma 1, PLC gamma 2, Vav, GAP, and a protein that co-precipitated with the Fc gamma receptors and migrated with a molecular weight of about 70,000. Co-cross-linking an F(ab')2 anti-CD45 monoclonal antibody together with monoclonal antibodies to either of the Fc gamma receptors inhibited phosphorylation of all these proteins. Analysis of the tyrosine kinases in the cells revealed that both receptors stimulated the phosphorylation and activation of a kinase rec...
Journal of immunology (Baltimore, Md. : 1950), 1994
Products of polyamine oxidase activity, at micromolar levels and during a period of 2 to 3 days, ... more Products of polyamine oxidase activity, at micromolar levels and during a period of 2 to 3 days, down-regulate IL-2 mRNA levels and activity in human lymphocytes. We studied whether this suppression was associated with signal transduction abnormalities. We found that polyamine oxidase activity suppresses both anti-CD3-induced IL-2 production and protein tyrosine phosphorylation. Polyamine oxidase activity also caused a reduction in intracellular calcium mobilization after mitogenic stimulation. The most distal step of CD3-mediated signal transduction is dependent upon transcription factors that regulate a set of genes, including IL-2. We found that polyamine oxidase-treated cells exhibited very low DNA binding activity of two such factors: NFAT and NF-kappa B. On the other hand, AP-1 DNA binding activity was enhanced in polyamine oxidase-treated cells, suggesting a possible role for AP-1 in the human lymphocyte stress response. In accordance with the oxidation dependence of this sup...
The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is high... more The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrate...
Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a ... more Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1991
Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is lig... more Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced s...
Journal of immunology (Baltimore, Md. : 1950), 1992
Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that h... more Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune c...
Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibr... more Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bo...
Tissire Ailtigens 1995: 46 14s-154 Prinred in Deiiriturk . All righrs reserved Copyrialif 0 M u n... more Tissire Ailtigens 1995: 46 14s-154 Prinred in Deiiriturk . All righrs reserved Copyrialif 0 M u n k s n a a r d 1995 . ZAP-70 and ~7 2 " ' are signaling response elements through M H C class I1 molecules.
The cytoplasmic domain of the CD45 leukocyte cell surface antigen has recently been shown to poss... more The cytoplasmic domain of the CD45 leukocyte cell surface antigen has recently been shown to possess protein tyrosine phosphatase (PTPase) activity. The existence of a cell membrane-bound PTPase may represent a mechanism by which an activation signal, initiated by ligand binding to a surface receptor, is down-regulated following delivery of the signal. Both the interleukin-2 (IL2) growth factor receptor and the CD3/Ti T-cell antigen receptor contain a subunit which is phosphorylated on tyrosine by an activated protein kinase (PTK) during T-cell activation. We compared the effect of CD45 ligation on signal transduction mediated by the binding of IL2 or anti-CD3 to these two receptors. Immunoblotting with anti-phosphotyrosine antiserum was used to investigate the effect of CD45 ligation on anti-CD3- or IL2-induced protein tyrosine phosphorylation. When CD3 and CD45 were triggered together, changes in the pattern of tyrosine phosphorylation of specific substrates was observed in comparison to the stimulus triggered through CD3 alone. In contrast, CD45 ligation did not alter the pattern of tyrosine-phosphorylated proteins in "resting" T-cell blasts responding to IL2, except for a mobility shift of a 55 kDa protein and increased phosphorylation of a 112 kDa substrate. The proliferative response of T cells to both anti-CD3 or IL2 was inhibited by ligating CD45. The CD45 molecule down-regulated CD3-induced T-cell activation when the CD45 and CD3 molecules were ligated simultaneously with immobilized antibodies. In contrast, immobilized CD45 mAb alone inhibited IL2-induced proliferation, and the inhibition was not potentiated by simultaneously using a CD25 mAb which was non-competitive for IL2-binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Proceedings of the National Academy of Sciences, 1993
Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydro... more Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) impairs Ca2+dependent transmembrane signaling in human and murine lymphocyts. The purpose of the present studies was to analyze potential mechanisms of immunosuppression by DMBA and to eamine effects on Ca2+ homeostasis and antigen-receptor signaling in human T cells. DMBA produced a rapid and
Proceedings of the National Academy of Sciences, 1990
The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transd... more The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been showu to cause increases in tyrosine phosphorylation of TCR-C and other substrates, sug-
Proceedings of the National Academy of Sciences, 1992
The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcin... more The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcinomas, has attracted attention as a therapeutic target for murine monoclonal antibodies and their humanized counterparts. Its molecular nature has, however, remained elusive. Here we describe the expression cloning of a cDNA encoding the L6 antigen. COS cells transfected with this cDNA direct the expression of an -24-kDa surface protein that reacts with the two anti-L6 monoclonal antibodies available. The predicted L6 peptide sequence is 202 amino acids long and contains three predicted NH2-terminal hydrophobic transmembrane regions, which are followed by a hydrophilic region containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. The L6 antigen is related to a number of cell surface proteins with similar predicted membrane topology that have been implicated in cell growth. Two other members of this family of proteins, CD63 (ME491) and CO-029, are also highly expressed on tumor cells. The present findings should make it possible to further study the role of the L6-defined antigen in normal and neoplastic cells and to construct animal models for development of improved agents for active and passive cancer immunotherapy.
Proceedings of the National Academy of Sciences, 1992
Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transducti... more Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways.
Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preferenc... more Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preference for manganese over magnesium for maximal activity. We have observed that peptides and proteins are nonenzymatically phosphorylated at tyrosine and serine by ATP when heated in the presence of MnC12 at neutral pH. The extent of the reaction is negligible below 50 "C but increases rapidly at higher temperatures. The reaction proceeds in the presence of sodium dode-cy1 sulfate but is blocked by EDTA. No reaction is observed in the absence of Mn2*, even if M 8 + is present. Manganese therefore acts as a catalyst for the nonenzymatic reaction, but magnesium does not. We propose that the preference for manganese shown by many protein tyrosine kinases is due at least in part to the intrinsic ability of Mn2+ to catalyze the transfer of phosphate from ATP to a phosphate acceptor such as tyrosine. The nonenzymatic phosphorylation reaction also offers a new synthetic pathway for the preparation of radiolabeled peptides containing phosphotyrosine and phosphoserine.
CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activa... more CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the µ2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The µ1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of µ2 and CTLA-4 were localized to residues, 161 TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165 YVKM, involved in CTLA-4 signaling. µ2 interacted preferentially with CTLA-4 when residue 165 Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165 Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 ( 165 Y and 182 Y) were phosphorylated by the T lymphocyteassociated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165 Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.
Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (F... more Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (Fc gamma RI) or Fc gamma receptor II (Fc gamma RII) gave rise to the rapid phosphorylation of multiple intracellular proteins. The pattern of proteins that were phosphorylated appeared to be identical. Analysis of these proteins by specific immunoprecipitation indicated that stimulation through either receptor did indeed give rise to phosphorylation of the same set of proteins. These included: Fc gamma RII, phospholipase C (PLC) gamma 1, PLC gamma 2, Vav, GAP, and a protein that co-precipitated with the Fc gamma receptors and migrated with a molecular weight of about 70,000. Co-cross-linking an F(ab')2 anti-CD45 monoclonal antibody together with monoclonal antibodies to either of the Fc gamma receptors inhibited phosphorylation of all these proteins. Analysis of the tyrosine kinases in the cells revealed that both receptors stimulated the phosphorylation and activation of a kinase rec...
Journal of immunology (Baltimore, Md. : 1950), 1994
Products of polyamine oxidase activity, at micromolar levels and during a period of 2 to 3 days, ... more Products of polyamine oxidase activity, at micromolar levels and during a period of 2 to 3 days, down-regulate IL-2 mRNA levels and activity in human lymphocytes. We studied whether this suppression was associated with signal transduction abnormalities. We found that polyamine oxidase activity suppresses both anti-CD3-induced IL-2 production and protein tyrosine phosphorylation. Polyamine oxidase activity also caused a reduction in intracellular calcium mobilization after mitogenic stimulation. The most distal step of CD3-mediated signal transduction is dependent upon transcription factors that regulate a set of genes, including IL-2. We found that polyamine oxidase-treated cells exhibited very low DNA binding activity of two such factors: NFAT and NF-kappa B. On the other hand, AP-1 DNA binding activity was enhanced in polyamine oxidase-treated cells, suggesting a possible role for AP-1 in the human lymphocyte stress response. In accordance with the oxidation dependence of this sup...
The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is high... more The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrate...
Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a ... more Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1991
Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is lig... more Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced s...
Journal of immunology (Baltimore, Md. : 1950), 1992
Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that h... more Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune c...
Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibr... more Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bo...
Tissire Ailtigens 1995: 46 14s-154 Prinred in Deiiriturk . All righrs reserved Copyrialif 0 M u n... more Tissire Ailtigens 1995: 46 14s-154 Prinred in Deiiriturk . All righrs reserved Copyrialif 0 M u n k s n a a r d 1995 . ZAP-70 and ~7 2 " ' are signaling response elements through M H C class I1 molecules.
The cytoplasmic domain of the CD45 leukocyte cell surface antigen has recently been shown to poss... more The cytoplasmic domain of the CD45 leukocyte cell surface antigen has recently been shown to possess protein tyrosine phosphatase (PTPase) activity. The existence of a cell membrane-bound PTPase may represent a mechanism by which an activation signal, initiated by ligand binding to a surface receptor, is down-regulated following delivery of the signal. Both the interleukin-2 (IL2) growth factor receptor and the CD3/Ti T-cell antigen receptor contain a subunit which is phosphorylated on tyrosine by an activated protein kinase (PTK) during T-cell activation. We compared the effect of CD45 ligation on signal transduction mediated by the binding of IL2 or anti-CD3 to these two receptors. Immunoblotting with anti-phosphotyrosine antiserum was used to investigate the effect of CD45 ligation on anti-CD3- or IL2-induced protein tyrosine phosphorylation. When CD3 and CD45 were triggered together, changes in the pattern of tyrosine phosphorylation of specific substrates was observed in comparison to the stimulus triggered through CD3 alone. In contrast, CD45 ligation did not alter the pattern of tyrosine-phosphorylated proteins in "resting" T-cell blasts responding to IL2, except for a mobility shift of a 55 kDa protein and increased phosphorylation of a 112 kDa substrate. The proliferative response of T cells to both anti-CD3 or IL2 was inhibited by ligating CD45. The CD45 molecule down-regulated CD3-induced T-cell activation when the CD45 and CD3 molecules were ligated simultaneously with immobilized antibodies. In contrast, immobilized CD45 mAb alone inhibited IL2-induced proliferation, and the inhibition was not potentiated by simultaneously using a CD25 mAb which was non-competitive for IL2-binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Proceedings of the National Academy of Sciences, 1993
Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydro... more Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) impairs Ca2+dependent transmembrane signaling in human and murine lymphocyts. The purpose of the present studies was to analyze potential mechanisms of immunosuppression by DMBA and to eamine effects on Ca2+ homeostasis and antigen-receptor signaling in human T cells. DMBA produced a rapid and
Proceedings of the National Academy of Sciences, 1990
The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transd... more The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been showu to cause increases in tyrosine phosphorylation of TCR-C and other substrates, sug-
Proceedings of the National Academy of Sciences, 1992
The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcin... more The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcinomas, has attracted attention as a therapeutic target for murine monoclonal antibodies and their humanized counterparts. Its molecular nature has, however, remained elusive. Here we describe the expression cloning of a cDNA encoding the L6 antigen. COS cells transfected with this cDNA direct the expression of an -24-kDa surface protein that reacts with the two anti-L6 monoclonal antibodies available. The predicted L6 peptide sequence is 202 amino acids long and contains three predicted NH2-terminal hydrophobic transmembrane regions, which are followed by a hydrophilic region containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. The L6 antigen is related to a number of cell surface proteins with similar predicted membrane topology that have been implicated in cell growth. Two other members of this family of proteins, CD63 (ME491) and CO-029, are also highly expressed on tumor cells. The present findings should make it possible to further study the role of the L6-defined antigen in normal and neoplastic cells and to construct animal models for development of improved agents for active and passive cancer immunotherapy.
Proceedings of the National Academy of Sciences, 1992
Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transducti... more Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways.
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Papers by Gary Schieven