Abstract
Activation of the platelet-derived growth factor (PDGF) alpha receptor (alphaPDGFR) leads to cell migration and DNA synthesis. These events are preceded by the ligand-induced 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-gamma1 (PLCgamma). In this study, we sought to systematically evaluate the relative roles of the signaling enzymes that are recruited to the alphaPDGFR for DNA synthesis and cell migration. Our approach was to generate and characterize tyrosine to phenylalanine alphaPDGFR 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 PLCgamma 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 alphaPDGFR, PI3K plays a major role in initiating DNA synthesis, whereas PI3K, PLCgamma, and especially Src are required for chemotaxis.
Highlights
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
We found that platelet-derived growth factor (PDGF)-AA-induced DNA synthesis required activation of only one signal relay enzyme, phosphatidylinositol 3-kinase (PI3K)
Eight major phosphorylation sites have been identified, and their phosphorylation leads to the recruitment of SH2 domain-containing signaling enzymes such as Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥1 (PLC␥)
Summary
Receptor tyrosine kinases elicit responses such as cell proliferation and migration via binding and activation of Src homology 2 (SH2) domain-containing signaling molecules. 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. At least some of the signaling enzymes that associate with the PDGFR and the fibroblast growth factor receptor have specific binding sites [1]. One of the consequences of phosphorylation of the ␣PDGFR is the selective recruitment of SH2-containing signaling enzymes such as Src family members (Src), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-␥1 (PLC␥). In transfected porcine aortic endothelial cells, Src family members did not contribute to ␣PDGFR-mediated chemotaxis [4]
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