Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.

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Polymorphonuclear leukocytes-mediated lysis of A431 cells induced by IgG1 mouse anti-epidermal growth factor receptor monoclonal antibodies

In Vitro Cellular & Developmental Biology ◽

10.1007/bf02631068 ◽

1992 ◽

Vol 28 (11-12) ◽

pp. 782-786 ◽

Cited By ~ 6

Author(s):

Tomoyuki Kawamoto ◽

Kouji Kishimoto ◽

Kojiro Takahashi ◽

Tomohiro Matsumura ◽

J. Denry Sato ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Monoclonal Antibodies ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Polymorphonuclear Leukocytes ◽

Growth Factor Receptor ◽

A431 Cells ◽

Epidermal Growth

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Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42785-2 ◽

1984 ◽

Vol 259 (15) ◽

pp. 9906-9912 ◽

Cited By ~ 7

Author(s):

P J Parker ◽

S Young ◽

W J Gullick ◽

E L Mayes ◽

P Bennett ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Monoclonal Antibodies ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

A431 Cells ◽

Human Epidermal Growth Factor ◽

Epidermal Growth

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The carbohydrate specificities of the monoclonal antibodies 29.1, 445 and 3C1B12 to the epidermal growth factor receptor of A431 cells

Bioscience Reports ◽

10.1007/bf01117444 ◽

1985 ◽

Vol 5 (1) ◽

pp. 83-94 ◽

Cited By ~ 29

Author(s):

H. C. Gool ◽

E. F. Hounsell ◽

I. Lax ◽

R. M. Kris ◽

T. A. Libermann ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Monoclonal Antibodies ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Blood Group ◽

Cell Biology ◽

Structural Information ◽

Growth Factor Receptor ◽

A431 Cells ◽

Epidermal Growth

Sixteen hybridoma-derived antibodies to the epidermal growth factor receptor of A431 ceils were studied with respect to their reactions with blood group-related carbohydrate structures. Twelve of these were assessed as recognizing carbohydrate determinants on the basis of their immunostaining of reference blood group substances on nitrocellulose paper. Three of these antibodies were further investigated by inhibition of binding assays with giycoproteins and structurally defined oligosaccharides or by haemagglutination of erythrocytes before and after treatment with endo-β-galactosidase. Two of the antibodies, 29.1 and 455, were shown to have blood group A-related specificities which differed from one another and from those of monocional anti-A antibodies described previously. The third antibody, 3CIB12, which was shown to recognize a determinant based on αl+3 fucosylated Type 2 chains on linear and branched backbone sequences, also differs from previously described monoclonal antibodies of 3-fucosyl-N-acetyllactosamine type, such as anti-SSEA-1 (anti-mouse embryo) and several antibodies to human myeloid ceils. While these antibodies are invaluable in providing structural information on the carbohydrate chains of the receptor glycoprotein and should help to elucidate their functions, their use as ‘anti-receptor’ reagents in cell biology will be influenced by the knowledge that the determinants they recognize are shared by other glycoproteins and glycolipids of diverse cell types.

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Characterization of monoclonal antibodies generated to the 287–302 amino acid loop of the human epidermal growth factor receptor

Antibody Therapeutics ◽

10.1093/abt/tbz011 ◽

2019 ◽

Vol 2 (4) ◽

pp. 88-98 ◽

Cited By ~ 1

Author(s):

Eric Chun Hei Ho ◽

Antonella Antignani ◽

Robert Sarnovsky ◽

David FitzGerald

Keyword(s):

Epidermal Growth Factor Receptor ◽

Monoclonal Antibodies ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Enzyme Linked Immunosorbent Assay ◽

A431 Cells ◽

Binding Characteristics ◽

Recombinant Immunotoxin ◽

Epidermal Growth

Abstract Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop. Methods: The 286–303 peptide was coupled chemically to keyhole limpet hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V regions were cloned and sequenced. Receptor binding was characterized using enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines. Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be cytotoxic to MDA-MB-468, A431 and F98npEGFRvIII expressing cells. Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

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