PHOSPHORYLATION SITE-SPECIFIC INHIBITION OF PLATELET-DERIVED GROWTH-FACTOR BETA-RECEPTOR AUTOPHOSPHORYLATION BY THE RECEPTOR BLOCKING TYRPHOSTIN AG1296

The mechanism of action of AG1296, a potent and specific inhibitor of the platelet-derived growth factor (PDGF) receptor tyrosine kinase [Ko valenko, M., Gazit, A., Bohmer, A., Rorsman, Ch., Ronnstrand, L., Held in, C.-H., Waltenberger, J., Bohmer, F. D., & Levitzki, A. (1994) Canc er Res. 54, 6106-6114] was investigated. This quinoxalin-type tyrphost in neither interferes with PDGF-BB binding to the PDGF P-receptor nor has any effect on receptor dimerization. Kinetic analysis of the inhib ition was carried out using a synthetic peptide substrate (KY751) corr esponding to the sequence around tyrosine 751 autophosphorylation site of the PDGF receptor. It revealed purely competitive inhibition vis-a -vis ATP, mixed competitive inhibition vis-a-vis the peptide substrate for the non-activated receptor, and mixed competitive inhibition vis- a-vis both substrates for the activated receptor. Thus, the type of in hibition apparently changes upon receptor activation, indicating confo rmational changes at the ATP-binding site. The high degree of selectiv ity for the tyrphostin AG1296 might result from the complex type of in teraction with the active center of the receptor as revealed by the ki netic analysis. Dose-response curves for inhibition of the phosphoryla tion of individual autophosphorylation sites of the PDGF beta-receptor by AG1296 were different, phosphorylation of tyrosine 857 being the m ost susceptible to inhibition. Thus, phosphorylation of tyrosine 857 i n 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.