ABSTRACT
The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala-Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.
Subject(s)
Phosphopeptides/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Platelet-Derived Growth Factor , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Kinetics , Peptide Library , Phosphopeptides/chemical synthesis , Phosphopeptides/metabolism , Phosphorylation , Sequence Analysis , Substrate SpecificityABSTRACT
A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Quinazolines/antagonists & inhibitors , 3T3 Cells , Animals , Cell Transformation, Neoplastic/drug effects , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Gene Expression/drug effects , Humans , Kinetics , Mice , Mitosis/drug effects , Phosphorylation/drug effects , Platelet-Derived Growth Factor/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Tumor Cells, Cultured , Tyrosine/metabolismABSTRACT
High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.
