Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

Abstract

Ligand stimulation of the platelet-derived growth factor (PDGF) P-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 ,8-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF ,-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFRjY716F 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 ,8-receptors. The mutant PDGFRjIY716F 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 j-receptor.Platelet-derived growth factor (PDGF) is a connective tissue cell mitogen, consisting of dimers of disulfide-bonded A and B polypeptide chains, which combine to form the three isoforms, PDGF-AA, -AB, and -BB (for a review, see reference 21). Two types of PDGF tyrosine kinase receptors have been identified and cloned. The ox-receptor binds both A and B chains with high affinity, whereas the p-receptor binds only the B chain.Signal transduction through tyrosine kinase receptors follows a general scheme whereby ligand binding induces dimerization or oligomerization of receptor molecules, activation of the receptor tyrosine kinase, and autophosphorylation in trans on multiple tyrosine residues in the intracellular region (reviewed in reference 44). Thereby, binding sites are created for intracellular signal transduction molecules, containing one or two copies of Src homology 2 (SH2) domains. There are two principal groups of signal transduction molecules: those which are equipped with a catalytic domain, and those which lack such domains but which serve as adaptors and associate with catalytically active molecules (50). For the PDGF P-receptor, considerable information has accumulated regarding the positions of autophosphorylation sites as well as which signal transduction molecules interact with individual sites. Thus, members of the Src family, i.e., phospholipase C--y, the Ras GTPase-activating protein (GAP), phosphatidylinositol 3'-kinase (P13-kinase), the phosphotyrosine phosphatase PTP1D/ Syp, and the adaptor molecules Shc in the PDGF P-receptor (reviewed in reference 9). The specificity of these in...

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

“…To test if such predominant binding to p62 was real or some artifact resulting from the denaturation procedure, we tested the binding of GST-GRB2-SH2 to phosphotyrosine proteins in either Triton X-100 lysate or the heat and SDS denatured lysate of PDGF stimulated HER14 cells. It has been previously shown that GRB2 directly binds to two phosphotyrosine proteins in PDGF stimulated cells, the PDGF receptor and SHP-2 (Li et al, 1994;Arvidsson et al, 1994). Consistent with the previous reports, GST-GRB2-SH2 precipitated mainly the PDGF receptor and the p70 SHP-2 in the Triton X-100 cell extract (lane 6), and strongly to the SHP-2 but weakly to the PDGF receptor in heat and SDS denatured cell extract (lane 8).…”

“…This ®nding was not due to impaired, possibly caused by the denaturation procedure, ability of the other Nckassociated phosphotyrosine proteins to bind Nck, since under the same conditions GRB2 was able to bind the two previously reported proteins, SHP-2, and the PDGF receptor in response to PDGF (Li et al, 1994;Arvidsson et al, 1994). Thus, Nck coimmuoprecipitated with the other phosphotyrosine proteins through p62 and p62-associated proteins.…”

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…Activation of Ras and the MAP kinase cascade have been shown to be important in PDGF-induced mitogenicity (Arvidsson et al, 1994;Valius and Kazlauskas, 1993). However, no signi®cant alteration in ability to mediate cell growth as well as MAP kinase activation was seen in Y762F mutant receptor cells.…”

PDGF receptor as a specific in vivo target for low Mr phosphotyrosine protein phosphatase

“…PDGF-R activation leads to tyrosine phosphorylation of several receptor substrates, such as: PLC-y, PI-3kinase, PTP1D, Src, GAP, etc., [20]. Furthermore, protein interaction with PDGF-R can result in the positioning of an adaptor molecule (such as Grb2) leading to the activation of side signal pathways [21,22]. Finally, it is also possible that the generation of the mitogenic signal by PDGF-R is regulated via interactions modifying its kinase activity.…”