Binding of SH2 Domains of Phospholipase Cγ1, GAP, and Src to Activated Growth Factor Receptors

Abstract

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.

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

“…Third, recombinant GST-Nck-SH2 and GST-GAP-SH2 fusion proteins were unable to precipitate GAP or Nck, respectively, from the EGF-stimulated cells. Since both Nck and GAP have previously been shown to associated with the activated EGFR (Anderson et al, 1990;Li et al, 1992;Margolis et al, 1990), the data from our experiments implied that Nck and GAP may not even associate stoichiometrically with the same EGFR. Otherwise, Nck and GAP would be in the same complex with activated EGFR and co-immunoprecipitable by using anti-Nck or anti-GAP antibody.…”

“…Guanine nucleotide exchange factors (GEFs) exchange Ras-bound GDP with GTP, whereas GTPase-activating proteins (GAPs) enhance the Ras intrinsic GTP-hydrolyzing activity, converting the active GTP-bound Ras to its inactive GDP-bound form. In EGF stimulated cells, GAP1 is tyrosine phosphorylated and associated, via its N-terminal SH2 domain, with the activated EGF receptor (Anderson et al, 1990;Margolis et al, 1990). As a result, GAP1 is translocated to the plasma membrane and deactivates Ras (reviewed by Boguski and McCormick).…”

Crk protein binds to PDGF receptor and insulin receptor substrate-1 with different modulating effects on PDGF- and insulin-dependent signaling pathways

“…As in the case of Crk-IRS-1 interaction overexpression of Crk did not cause enhanced association of Crk with PDGF receptor. Although the ability of SH2 domain of Crk to associate with PDGF receptor has been previously reported (Anderson et al, 1990) (Figure 5b). Here we show that PDGF receptor is a potent stimulator of cCrk phosphorylation (Figure 7).…”

Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells

“…The PAL cDNA encoding amino acids 11 ± 648 was cloned using XhoI restriction sites into the pGEX-4T3 vector (Pharmacia). Additional GST-fusion proteins; Shc PTB (Blaikie et al, 1994); Shc SH2 ; Grb2 SH2 (Rozakis-Adcock et al, 1992); Vav SH2 (Margolis et al, 1992); GAP-N SH2, PLCg-N SH2, PLCg-C SH2 (Anderson et al, 1990);and p85-N SH2, p85-C SH2 (McGlade et al, 1992b) have been previously described. Nck SH2 contains amino acids 281 ± 377 of human Nck (Lehman et al, 1990), and the GST Shc-CH1 consists of amino acids 212 ± 376 of human Shc.…”