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Application of two‐dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors
Author(s) -
Hansen Klaus,
Møller Jesper V.
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150140120
Subject(s) - protein tyrosine phosphatase , cytoskeleton , tyrosine phosphorylation , epidermal growth factor , phosphorylation , receptor tyrosine kinase , tyrosine kinase , biochemistry , tyrosine , biology , glycoprotein , microbiology and biotechnology , chemistry , receptor , cell
The technique of two‐dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)‐induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [γ 32 P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine‐specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X‐114 fractionation, immunoreaction with specific antibodies, and comigration with 35 S‐labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well‐recognized substrates (calpactin II, ezrin, EGF receptor) included β‐tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two‐dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.