Tyr-716 in the Platelet-Derived Growth Factor β-Receptor Kinase Insert Is Involved in GRB2 Binding and Ras Activation

Abstract

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

Protein kinase PKR is required for platelet-derived growth factor signaling of c-fos gene expression via Erks and Stat3

“…Growth factor‐mediated stimulation of receptor tyrosine kinases creates binding sites for Grb2, which in turn recruits the Grb2–Sos complex to the plasma membrane (Schlessinger, 1993; Arvidsson et al ., 1994; Claesson‐Welsh, 1994). This recruitment of Sos activates Ras, and the activation in turn triggers the Ras–MAPK kinase–Erk pathway.…”

Cell cycle control by the insulin-like growth factor signal: at the crossroad between cell growth and mitotic regulation

“…The canonical RAS-Raf-MEK-ERK-RSK signaling cascade has been found to be shared by both the Insulin/IGF ligand/receptor system as well as by other GFs/RTK systems with the unique difference being in the main upstream circuitry due to the almost exclusive use of the IRS adaptor proteins by the IR and IGF1R system (see also under chapter 4, Figure 2 and Table 4 ). These other GF/RTK systems (such as EGFR, PDGFR, and others) have been shown to directly recruit either Grb or Shc proteins towards triggering the SOS1-mediated activation of membrane-linked RAS [ 81 , 224 , 225 ]. The canonical RAS pathway has been further enriched by the demonstration of the direct RAS-induced activation of PIK3CA (the catalytic subunit of PI3K) [ 131 ] and very recently by mTORC2 [ 226 ] disclosing newer scenarios on the mechanisms by which RAS proteins trigger their established growth and proliferative effects (see Figure 2 and Table 4 ).…”