A Carboxyl-terminal Mutation of the Epidermal Growth Factor Receptor Alters Tyrosine Kinase Activity and Substrate Specificity as Measured by a Fluorescence Polarization Assay

Abstract

The expression of certain COOH-terminal truncation mutants of the epidermal growth factor receptor (EGFR) can lead to cell transformation, and with ligand stimulation, a broader spectrum of phosphorylated proteins appears compared with EGF-treated cells expressing wild-type EGFR. Accordingly, it has been proposed that elements within the COOH terminus may determine substrate specificity of the EGFR tyrosine kinase (Decker, S. J., Alexander, C., and Habib, T. Our data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and the differences are substrate-dependent. These results support the concept that this oncogenic truncation/mutation alters EGFR substrate specificity, rather than causing a general alteration of activity. We performed the experiments using a non-radioactive fluorescence polarization assay that quantifies the degree of phosphorylation of peptide as well as natural substrates. The results are consistent with those from the traditional [␥-32 P]ATP/filtration assay.Binding of EGF 1 and other ligands to EGFR and its relatives (ErbB2, ErbB3, and ErbB4) triggers a highly complicated signaling network (1, 2). Overexpression as well as oncogenic deletions and truncations of EGFR have been observed in many cancer types, including glioblastomas and non-small cell lung, pancreatic, breast, head and neck, colon, prostate, ovarian, and cervical tumors (3-5). The most commonly found mutation (EGFRvIII) involves a deletion in the NH 2 -terminal portion of EGFR, from residues 6 to 273 (5-7). Several other types of rearrangements have been found, however, including even a tandem duplication of the tyrosine kinase and calcium internalization domains (4,5,8). Half of all glioblastomas exhibit EGFR amplification, and of these, 15% possess COOH-terminal truncated EGFR (at residue 958) (5). Furthermore, expressing certain COOH-terminal truncation mutants in cell culture results in dramatic EGF-induced increases in the variety of phosphorylated proteins present compared with those observed in cells expressing wild-type EGFR (9 -12).The cause of this increase in the assortment of phosphorylated proteins has not yet been completely explored; however, EGF-induced receptor internalization and degradation occurred at a slower rate with an oncogenic EGFR mutant possessing a COOH-terminal truncation at residue 973 rather than with wild-type EGFR (10, 13). Cells expressing this mutant develop a transformed phenotype when grown in the presence of low EGF concentrations (13). Moreover, data suggest that ⌬973-EGFR and ErbB2 heterodimers form in cells expressing this mutant. The ErbB2 in these cells is tyrosyl phosphorylated in the absence of added ligand (12). Interestingly, the oncogene product of the avian erythroblastosis virus (verbB) is an NH 2 -and COOH-terminal truncated version of EGFR (ErbB1) (14,15).Cellular changes in phosphotyrosine levels may occur not only as a result of changes in the catalytic properties of the enzyme, but may also reflect alteratio...

Computational delineation of tyrosyl-substrate recognition and catalytic landscapes by the epidermal growth factor receptor tyrosine kinase domain

“…This translates to a k cat value of r10 À6 s À1 or a reduction by at least a factor of 10 À6 relative to the experimental k cat value of the EGF activated receptor or a factor of 10 À5 relative to the experimental k cat value in the absence of EGF. 54,58,59,62,63 The reduction in k cat manifests itself as little or very weak residual activity through pathway II, which is consistent with recent experiments. 64,65 Given that the protonation states of titratable side-chains in the active site can have a considerable effect on the stability of the ground state as well as on the energies of the reaction landscape, we explored the different protonation states of the conserved catalytic residue D813 in EGFR.…”

“…Moreover, it has been suggested that protein tyrosine kinase (including EGFR) select substrates with glutamic acid at the P À 1 position and large hydrophobic amino acids at the P + 1 position. [22][23][24][25]54,58,59 Our predicted peptide bound ternary complex suggests that a highly conserved region, corresponding to Val852-Pro853-Ile854-Lys855-Trp856 in EGFR TKD is essential for substrate binding: Lys855 forms a salt bridge with the highly preferred glutamic acid residue at the P À 1 site, and the hydrophobic pocket formed by Val852, Pro853, Ile854 and Trp856 serves to anchor a large hydrophobic residue at the P + 1 position. This observed binding mode for our top-ranked configuration is consistent with the hypothesis that the substrate-binding surface formed through hydrophobic contacts and hydrogen bonds help orient the tyrosine residue for catalysis.…”

Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling

“…This evidence strongly suggests the contribution of some extrinsic factors that need to be modeled, such as unknown kinase inhibitors like Mig6 [53], [54], or the alteration of the substrate specificity of EGFR caused by the mutation [55]. An alternative possibility could be the involvement of SFKs that are highly associated with EGF-induced tyrosine phosphoproteome [45].…”

Glioblastoma-Derived Epidermal Growth Factor Receptor Carboxyl-Terminal Deletion Mutants Are Transforming and Are Sensitive to EGFR-Directed Therapies

“…Therefore, dysregulation of receptor degradation could be one of the possible oncogenic mechanisms of EGFR CTD deletion mutant, which is consistent with previous reports (42, 43). In addition, the CTD deletion mutants may have altered EGFR substrate specificity/receptor degradation or no longer bind regulatory proteins including Mig6 which has been found to negatively regulate EGFR kinase activity by blocking the activating dimer interface (44-46). Further experiments are needed to test these possibilities and to characterize a detailed mechanism of oncogenic activation of EGFR CTD deletion mutants.…”