Ligand Regulates Epidermal Growth Factor Receptor Kinase Specificity

Abstract

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K m and k cat , revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFRmediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k cat ϳ5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of ϳ5-fold in the specificity constants (k cat /K m ) for EGFR peptides, whereas ϳ15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins ϳ4 -6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.Protein-tyrosine kinases, which catalyze the transfer of ␥-phosphate from ATP to tyrosine side chains of peptide and protein substrates, play a central role in cellular signaling. Receptor tyrosine kinases (RTKs) 1 constitute one class of protein kinases that consist of an extracellular ligand binding, a transmembrane, an intracellular tyrosine kinase, and a Cterminal domain that contains tyrosine phosphorylation sites (1). The epidermal growth factor receptor (EGFR, ErbB1, and HER1) is the prototypical member of the ErbB family RTKs, which also includes ErbB2 (HER2, neu), ErbB3 (HER3), and ErbB4 (HER4). Binding of growth factors that are structurally related to EGF induces a process of the receptor dimerization, kinase activation and autophosphorylation (2). EGFR activation results in recruitment and phosphorylation of cytosolic downstream targets such as Grb2-associated binding protein 1 (Gab1) (3), Src homology 2 (SH2) domain, collagen-containing protein (Shc) (4), and phospholipase C␥-1 (PLC␥-1) (5, 6). EGFR functions in the proliferation, migration, survival, and differentiation of mammalian cells (2, 7), and dysregulation of signaling by EGFR ...

Identification of Critical Residues within the Conserved and Specificity Patches of Nerve Growth Factor Leading to Survival or Differentiation

“…The details of how the C-terminal tail tyrosines subsequently become phosphorylated are still unknown. However, kinetic studies of EGFR kinase activity have shown that the preference for Gab1-and Shc-docking proteins is different between the unliganded and EGF-liganded states (79). We hypothesized that the complex process of the formation of the signal transduction complex would depend upon the intricate relationship of the ICDs with docking proteins and thus be susceptible to manipulation by the manner in which the ligand, NGF in our case, brought two TrkA ECDs (and hence the ICDs and their docking partners) together, thereby altering the signal to the cell.…”

Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism

“…Geldanamycin was from Alexis Biochemicals, and heregulin-␤1 (HRG-␤1) was generously provided by Stewart Thompson at Berlex Biosciences. Peptides were prepared as described previously (21).…”

Identification of a Novel HSP70-binding Cochaperone Critical to HSP90-mediated Activation of Small Serine/Threonine Kinase

“…Protein Expression, Purification, and SIP-GST Binding AssaySSTKs and EGF receptor were expressed by baculoviral infection in Sf9 insect cells and purified as described previously (26), and HA-tagged SIP-glutathione S-transferase (SIP-GST) was expressed in E. coli BL21 and purified as described in Fan et al (27). SIP-GST binding was assessed by incubating 300 nM HSP70 or lysate from 5 ϫ 10 6 cells (1 ml) with 5 g of SIP-GST bound to 5 l of glutathione-agarose for 1 h at 4°C.…”

“…Briefly, the reaction was carried out in the kinase buffer that contained 25 mM HEPES (pH 7.4), 10 mM MgCl 2 , 2 mM EGTA, 60 M ATP, 10 Ci of [␥-32 P]ATP, and protein/peptide substrate at room temperature for 30 min. Histone 2A, GSK-3-GST, Gab1 627Y peptide (27), or myelin basic protein was used as substrate for SSTK, Akt, EGF receptor, and MAP4K4, respectively. In some assays, reactions were terminated by acidification and transferred to P30 filtermats, and phosphorylation was quantified with a 1450 MicroBeta TriLux liquid scintillation counter (PerkinElmer Life Sciences, Turku, Finland).…”