Ligand Stimulation Reduces Platelet-derived Growth Factor β-Receptor Susceptibility to Tyrosine Dephosphorylation

Akira Shimizu, Camilla Persson, Carl-Henrik Heldin, Arne Östman
2001 Journal of Biological Chemistry  
Ligand binding to the platelet-derived growth factor (PDGF) ␤-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerizationinduced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligandstimulated PDGF ␤-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and nonstimulated
more » ... d and nonstimulated forms of tyrosine-phosphorylated PDGF ␤-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligandstimulated PDGF ␤-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligandstimulated forms of the PDGF ␤-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF ␤-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation. Receptor tyrosine kinases (RTKs) 1 are critical components of signaling pathways that control cellular processes like proliferation, differentiation, migration, and metabolism. Ligand binding of RTKs often leads to dimerization and subsequent increases in autophosphorylation of tyrosine residues in the intracellular portion of the receptors (reviewed in Refs. 1 and 2). Autophosphorylation of intracellular receptor tyrosine residues controls the intrinsic tyrosine kinase activity and creates binding sites to recruit downstream signaling molecules (3, 4). The mechanism whereby ligand-induced dimerization stimulates these phosphorylation events is incompletely understood but may involve a conformational change of the receptor or a proximity effect. RTK net tyrosine phosphorylation is not only controlled by the receptor kinase activity but is also determined by the action of protein-tyrosine phosphatases (PTPs). Accumulating evidence suggest that PTPs are regulatory components of RTK signaling pathways. Antisense studies have demonstrated increased signaling via receptors for insulin, epidermal growth factor (EGF), and hepatocyte growth factor after attenuation of expression of the receptor-like PTP LAR (5-7), and disruption of PTP1B in mice results in enhanced insulin sensitivity (8, 9) . Furthermore, genetic studies in Caenorhabditis elegans have identified the receptor-like PTP CLR-1 as a negative regulator of signaling through the fibroblast growth factor receptor ortholog EGL-15 (10). Physical association between the insulin receptor and the receptor-like PTP LAR, as well as between PDGF ␤-receptor and the receptor-like PTP DEP-1, have also been demonstrated (11, 12) . In this study we set out to investigate the possibility that reduced susceptibility to PTP action contributes to ligand-induced increases in net tyrosine phosphorylation of RTKs. The well characterized PDGF ␤-receptor was chosen as a prototype dimerization-activated receptor tyrosine kinase. The autophosphorylation sites of the PDGF ␤-receptor have been extensively studied and include a regulatory site, Tyr 857 , as well as numerous sites, which in their phosphorylated form act as binding sites for SH2 domain-containing proteins including c-Src, phospholipase C-␥, and phosphatidylinositol 3Ј-kinase (PI3-kinase) (reviewed in Ref. 13). To study the effects of ligand stimulation on PTP sensitivity, preparations of tyrosine-phosphorylated ligand-stimulated and unstimulated PDGF ␤-receptors were obtained. Using these preparations we demonstrate that ligand-stimulated forms of the PDGF ␤-receptor display a reduced susceptibility to dephosphorylation, as compared with unstimulated forms. EXPERIMENTAL PROCEDURES Cell Culture and Antibodies-Parental PAE cells and PAE cells stably transfected with PDGF ␤-receptor (14) were cultured in Ham's F-12 medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum (Life Technologies, Inc.), 100 unit/ml penicillin, and 100 g/ml streptomycin. Analysis of Receptor Dimerization-After overnight incubation in serum-free Ham's F-12, supplemented with 1 mg/ml BSA, PAE/ PDGF␤R cells were left unstimulated or treated with 100 ng/ml PDGF-BB for 60 min on ice, with 100 M pervanadate for 30 min at 37°C and 60 min on ice, or with pervanadate for 30 min at 37°C and then stimulated with 100 ng/ml PDGF-BB, in the presence of pervanadate, for 60 min on ice. After stimulation, cells were washed with ice-cold PBS. Ligand-receptor complexes were cross-linked by incuba-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address:
doi:10.1074/jbc.c100286200 pmid:11390370 fatcat:xoeohyemcvgdbat7aicw6gq4km