Phosphorylation Site-Specific Inhibition of Platelet-Derived Growth Factor β-Receptor Autophosphorylation by the Receptor Blocking Tyrphostin AG1296

Abstract

The mechanism of action of AG1296, a potent and specific inhibitor of the platelet-derived growth factor (PDGF) receptor tyrosine kinase [Kovalenko, M., Gazit, A., Böhmer, A., Rorsman, Ch., Rönnstrand, L., Heldin, C.-H., Waltenberger, J., Böhmer, F. D., & Levitzki, A. (1994) Cancer Res. 54, 6106-6114] was investigated. This quinoxalin-type tyrphostin neither interferes with PDGF-BB binding to the PDGF beta-receptor nor has any effect on receptor dimerization. Kinetic analysis of the inhibition was carried out using a synthetic peptide substrate (KY751) corresponding to the sequence around tyrosine 751 autophosphorylation site of the PDGF receptor. It revealed purely competitive inhibition vis-à-vis ATP, mixed competitive inhibition vis-a-vis the peptide substrate for the non-activated receptor, and mixed competitive inhibition vis-à-vis both substrates for the activated receptor. Thus, the type of inhibition apparently changes upon receptor activation, indicating conformational changes at the ATP-binding site. The high degree of selectivity for the tyrphostin AG1296 might result from the complex type of interaction with the active center of the receptor as revealed by the kinetic analysis. Dose-response curves for inhibition of the phosphorylation of individual autophosphorylation sites of the PDGF beta-receptor by AG1296 were different, phosphorylation of tyrosine 857 being the most susceptible to inhibition. Thus, phosphorylation of tyrosine 857 in the PDGF receptor kinase domain seems dispensable for partial kinase activation. The findings are discussed in relation to current models of receptor tyrosine kinase activation.

The platelet-derived-growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells

“…In a previous study we investigated mesangial cells overexpressing the oncogene c-src and did not detect an enhanced, but rather a decreased, inducibility of PGHS-2 [33], rendering the possibility of the oncoprotein c-Src acting as an important mediator in this signal-transduction pathway rather unlikely. Activation of PDGF receptor-associated phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to be very sensitive to treatment with AG1296 [24], whereas PI 3-kinase γ has been implicated in G βγ -mediated activation of the p42\44 MAP kinase cascade [13]. PI 3-kinase may well play a role in LPA-mediated activation of mesangial cells, because inhibition of the enzyme by LY294002 decreased p42\44 MAP kinase activation and PGHS-2 induction (results not shown).…”

“…At higher concentrations, AG1296 has been shown to inhibit other kinases which might be involved in LPA signalling [24]. To confirm the involvement of the PDGF receptor, mesangial cells were treated with LPA, 5-HT or PDGF, and tyrosine phosphorylation was assessed by Western-blot analysis.…”

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3

“…Purification of the hPDGF␤R-mFlt3 and in vitro autophosphorylation were performed similarly as described previously. 39 The transfected cells were washed once with ice-cold PBS and subsequently lysed (1 ml lysis buffer per 10 cm dish). The cell extract was then incubated at 4°C with wheat germ agglutinin agarose (Pharmacia Biotech, Freiburg, Germany, 1 ml beads per cell extract of 5-10 dishes), equilibrated with 40 mM Hepes, pH 7.4, 0.5 M NaCl, 0.05% Triton X100, 10% glycerol, 1 mM EGTA and 1 mM DTT (WGA buffer) with end-over-end rotation for 90 min.…”

Specific Inhibitors of Platelet-Derived Growth Factor or Epidermal Growth Factor Receptor Tyrosine Kinase Reduce Pulmonary Fibrosis in Rats

“…AG1296 has been reported to specifically inhibit activation of PDGF receptors, 23 whereas AG1478 has been described as a selective blocker of EGF-R autophosphorylation. 22 To confirm the specificity of these tyrosine kinase inhibitors in our system, we stimulated rat lung myofibroblasts with PDGF-R ligands or EGF-R ligands after pretreating the cells with AG1296 or AG1478 to block autophosphorylation on tyrosine residues.…”

“…20 Although several studies have documented the expression of PDGF-R, EGF-R, and their respective ligands during fibroproliferative diseases, the relative contribution of the EGF-R and the PDGF-R system to pulmonary fibrosis in relation to other growth factor receptors remains unknown. Inhibitors of PDGF receptor and EGF receptor tyrosine kinase activity have been reported for low molecular weight compounds of the staurosporine, 21 tyrphostin, 22,23 and 2-phenylaminopyrimidine 24,25 classes of compounds. Specific tyrphostin analogs have been reported to block a variety of pathogenic effects.…”