Dimerization-induced inhibition of receptor protein tyrosine phosphatase function through an inhibitory wedge

Science

Volumn 279, Issue 5347, 1998, Pages 88-91

  Majeti, Ravindra ^a   Bilwes, Alexandrine M ^a   Noel, Joseph P ^a   Hunter, Tony ^a   Weiss, Arthur ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CD45 ANTIGEN; PROTEIN TYROSINE PHOSPHATASE; T LYMPHOCYTE RECEPTOR;

ARTICLE; CHIMERA; DIMERIZATION; ENZYME INHIBITION; ENZYME PHOSPHORYLATION; ESCHERICHIA COLI; NONHUMAN; PRIORITY JOURNAL; SIGNAL TRANSDUCTION; T LYMPHOCYTE ACTIVATION;

ANTIGENS, CD45; BINDING SITES; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CALCIUM; DIMERIZATION; EPIDERMAL GROWTH FACTOR; HUMANS; LIGANDS; LYMPHOCYTE ACTIVATION; MUTATION; PHOSPHORYLATION; PROTEIN-TYROSINE KINASES; PROTEIN-TYROSINE-PHOSPHATASE; RECEPTOR, EPIDERMAL GROWTH FACTOR; RECEPTORS, ANTIGEN, T-CELL; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; T-LYMPHOCYTES; TUMOR CELLS, CULTURED; ZAP-70 PROTEIN-TYROSINE KINASE;

ESCHERICHIA COLI;

EID: 0032472226     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5347.88     Document Type: Article

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26. Cells were stained with a control monoclonal antibody (mAb) [goat antibody to mouse immunoglobulin G (Caltag Laboratories)]; with LA22 (antibody to EGFR; Upstate Biotechnology, Lake Placid, NY) for the EGFR-CD45 chimera; with Hle-1 (Becton-Dickinson) for CD45; and with Leu4 (antibody to CD3ε; Becton-Dickinson) for the TCR. Cells were stained at 4°C with saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated primary antibody (control, Hle-1, and Leu4) or primary antibody (LA22) followed by FITC-conjugated goat antibody to mouse IgG. Cells were analyzed on a FACScan (Becton-Dickinson).
27. 5 per milliliter) were treated with antibody to CD3 (mAb 235 at a 1:3000 dilution of ascites) or EGF (100 ng/ml).
28. 8 per milliliter; 235 ascites (mAb to CD3) at 1:500 dilution; and EGF, 100 ng/ml. Cells were sedimented in a microfuge and lysed in 200 μl of lysis buffer [1% Triton X-100, 150 mM NaCl, and 10 mM tris (pH 8.0), supplemented with protease and phosphatase inhibitors as described (11)]. Lysates were incubated at 4°C for 30 min, followed by centrifugation at 13,000g for 10 min. Ninety percent of the lysate was subjected to immunoprecipitation with polyclonal rabbit antibody to ZAP-70 [1598 (17)] and protein A-Sepharose beads for 2 hours at 4°C, after which immune complexes were washed. Immune complexes and 10% of the untreated lysate were resolved separately by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). Immunoblotting was done with mAb to phosphotyrosine (4G10; Upstate Biotechnology) or anti-phospho-MAPK (New England Biolabs), followed by visualization by enhanced chemiluminescence (Amersham). The blots were stripped and reprobed with antibody to ZAP-70 (1598) or antibody to MAPK (Santa Cruz Biotechnology), respectively.
29. We thank S. M. Fu for mAb 235, G. Servant for help with equilibrium binding studies, members of the Weiss lab for discussions and assistance, and A. DeFranco for his critical reading of this manuscript. Supported in part by a grant from NIH (to A.W.). R.M. is supported by the NIH Medical Scientist Training Program.