Identification of the Receptor-associated Signaling Enzymes That Are Required for Platelet-derived Growth Factor-AA-dependent Chemotaxis and DNA Synthesis

Abstract

Activation of the platelet-derived growth factor (PDGF) ␣ receptor (␣PDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligandinduced tyrosine phosphorylation of the receptor and its association with SH2-containing signaling enzymes including Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥1 (PLC␥). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the ␣PDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine ␣PDGFR mutants that failed to associate with one or more of the above listed signaling enzymes. In a 3T3-like cell line (Ph cells), PDGF-dependent DNA synthesis was strictly dependent on only one of the receptor-associated proteins, PI3K. In contrast, multiple signaling enzymes were required for maximal chemotaxis, as receptors unable to associate with either Src, PI3K, or PLC␥ initiated chemotaxis to 4, 47, or 56% of the wild-type level, respectively. Furthermore, coexpression of mutant receptors revealed that these signaling enzymes do not need to be on the same receptor for a cell to respond chemotactically to PDGF. We conclude that for the ␣PDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLC␥, and especially Src are required for chemotaxis.Receptor tyrosine kinases elicit responses such as cell proliferation and migration via binding and activation of Src homology 2 (SH2) 1 domain-containing signaling molecules. Upon ligand binding, receptor tyrosine kinases dimerize and autophosphorylate, and the phosphorylated tyrosines serve as a key component of the docking sites for SH2 domain-containing signal relay enzymes. There are at least several different ways in which signaling enzymes associate with a tyrosinephosphorylated receptor tyrosine kinase. For the epidermal growth factor receptor, each of the signaling enzymes appears to bind to any one of the phosphorylation sites. The hepatocyte growth factor receptor contains a pair of tyrosine phosphorylation sites, which are required for stable recruitment of at least four signaling enzymes. In contrast, at least some of the signaling enzymes that associate with the ␤PDGFR and the fibroblast growth factor receptor have specific binding sites (1).Activation of the ␣PDGFR results in its phosphorylation at numerous tyrosine residues, and eight such phosphorylation sites have been identified (reviewed in Ref. 2

Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton

“…The involvement of PI-3K in PDGF-bb-stimulated chemotaxis has also been reported in CHO cells (Kundra et al, 1994), in human arterial SMC (Bornfeldt et al, 1994(Bornfeldt et al, , 1995, and in osteoblasts MC3T3-E1 (Godwin and Solto , 1997). Similar results were also reported for PDGF-aastimulated NIH3T3 cells (Rosenkranz et al, 1999). The action of PI-3K under these conditions is to connect the PDGFR to the Rac signaling pathway possibly by catalyzing the production of phosphatidylinositol 3,4,5 triphosphate, which binds and activates the GEF for Rac.…”

N-Acetylcysteine Suppresses Retinal Detachment in an Experimental Model of Proliferative Vitreoretinopathy

“…The left lane contains RCFs and is included as a positive control. C: F␣ (F cells expressing wild-type PDGFR␣ 18 ) or F31/42 (F cells expressing a PDGFR␣ mutant in which tyr at 731 and 742 are mutated to phe 22 ) were stimulated with PDGF-A (50 ng/ml) for 10 minutes and then lysed. In the top panel, cleared cell lysate was subjected to Western blot analysis using a phospho-Y742 PDGFR␣ antibody (upper half); the blot was stripped and subsequently probed with a pan-PDGFR␣ antibody (27P; lower half).…”

“…18,22 When normalized for the amount of PDGFR␣, there was 2.6-fold greater phosphorylation of PDGFR␣⌬X in membranes isolated from the control rabbits, as compared with those injected with NAC (arrowhead in the upper panel of Figure 6B). We conclude that although NAC did not compromise the viability of the injected cells, it suppressed both receptor phosphorylation/activation and retinal detachment.…”

Guidance of mesoderm cell migration in theXenopusgastrula requires PDGF signaling

“…Westermark et al, 1990;Kundra et al, 1994;Hansen et al, 1996;Ronnstrand et al, 1999), and PDGFRα promotes a chemotactic response at least in some cells (e.g. Hosang et al, 1989;Ferns et al, 1990;Rosenkranz et al, 1999;Yu et al, 2001), although probably through a different mechanism (for reviews, see Hayashi et al, 1995;Ronnstrand and Heldin, 2001). A great deal of work on PDGF-stimulated chemotaxis relates to cultured cells in vitro.…”