Preparation and characterization of antibodies to o‐phosphotyrosine and their use for identification of phosphotyrosine‐containing proteins

Abstract

To facilitate the identification of phosphotyrosine (P-tyr)-containing proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P-tyr were prepared by hyperimmunizing the animals with P-tyr-conjugated bovine serum albumin or poly-L-lysine. As determined by a solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay, the antibodies reacted with P-tyr-conjugated target antigens but not with those conjugated with phosphoserine (P-ser) or phosphothreonine (P-thr). This immune reaction was strongly blocked by 2 mM P-tyr and phenylphosphate but not by P-ser or P-thr. The antibodies were capable of isolating, as the major P-tyr-containing components, a 170kd protein (most likely the EGF receptor) from EGF-stimulated, 32P-labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)-transformed chick cell lysate which had been labelled in vitro with gamma-32P-ATP. Immunofluorescent staining of RSV-transformed cells and A431 cells showed specific localization of P-tyr-containing proteins in the cytoplasm, plasma membrane, and nucleolus-like structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P-tyr-containing proteins.

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

“…However, most of these proteins have low stoichiometries (1 to 6%) of phosphorylation (5,6,48). Moreover, many of these proteins are phosphorylated on tyrosine in nontransformed cells infected with RSV mutants that encode nonmyristoylated p60vsrc.Recently, a sensitive method to detect phosphotyrosine (ptyr)-containing proteins has been developed by using antiptyr antibodies (4,12,40,42,46,50). With this technique, a more comprehensive picture of overall changes has been obtained for cellular proteins phosphorylated in RSV transformation, and this has led to identification of some new ptyr-containing protein species whose phosphorylation correlates with transformation (16,18,26,36,37,45,52).…”

Evoidence that the Tyrosine Kinase Domain of a Small Fraction of Epidermal Growth Factor Receptor Molecules is Exposed on the Outer Surface of A431 Cells.

“…3D was extracted from the acrylamide gel by trypsinization. Tryptic phospho-peptides were hydrolyzed with 6 N HC1and two-dimensional phospho-amino acid determination was performed as described previously (14). The posi- composedof two components, a and b.…”

“…monoclonal antibody. Immunoprecipitates were bound to Protein A-Sepharose, eluted by boiling in sample buffer, and phosphorylated proteins were resolved by SDS-polyacrylamide (10%) gel electrophoresis (SDS-PAGE)and autoradiographed as described previously (14).…”

“…The tryptic phospho-peptides extracted from gels as described above were hydrolyzed with 6N HC1 for 2 h at 110°C. The lyophilized hydrolysate was mixed with cold phosphotyrosine (Ptyr), phosphoserine (Pser), and phosphothreonine (Pthr) as markers and subjected to two-dimensional separation, as described previously (14).…”

Induction of in vitro differentiation of mouse embryonal carcinoma (F9) and erythroleukemia (MEL) cells by herbimycin A, an inhibitor of protein phosphorylation.

“…For this, F9 cells were labeled in ES medium (with limited phosphate) with riP-phosphate (1 mCi/ml) in the presence of different concentrations of herbimycin A and the immunoprecipitates with phosphotyrosine specific antibody (Ohtsuka et al, 1984) from the cell extracts were analyzed by autoradiography and densitometry after SDS-PAGE. Consistent with the previous finding (Uehara et al, 1986), incorporation of riP-phosphate into proteins reacted with the antibody was uniformly reduced by >98% at 0.5 #g/ml of herbimycin A concentration when incorporation into the total protein was reduced by 25.5% (data not shown).…”