Application of two-dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors.
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 [gamma 32P]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 35S-labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well-recognized substrates (calpactin II, ezrin, EGF receptor) included beta-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.
Hansen
K
K
Institute of Medical Biochemistry, University of Aarhus, Denmark.
Møller
J V
JV
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D000099149:Ezrin
D000255:Adenosine Triphosphate; D003598:Cytoskeletal Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D008565:Membrane Proteins; D010750:Phosphoproteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D014443:Tyrosine; D000099149:Ezrin