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A highly efficient peptide substrate for EGFR activates the kinase by inducing aggregation


Kate Engel, Tomoaki Sasaki, Qi Wang and John Kuriyan


Biochem. J. 2013 (local copy)

Abstract / Figures from the paper / Supplemental Material / References



Abstract

Formation of an asymmetric dimer by the epidermal growth factor receptor (EGFR) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C likely underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregation-mediated mechanism.


Figures from the paper

Click on the small image to get a bigger one.

First figure from paper

Figure 1 - Hill coefficient measurements for EGFR kinase domain catalysis with EGFR peptide substrates.
Hill coefficients for EGFR kinase domain catalysis with EGFR kinase peptide substrates (A) Peptide C, (B) Tail Peptide A, and (C) Tyrsub were derived by fitting the specific rates of catalysis at Log10 (peptide concentration) to the Hill equation using Prism software by GraphPad. Because the Tail Peptide A and Tyrsub measurements did not attain saturating concentrations, better fits for the Hill analysis were obtained when the Top (kcat) values were constrained to those derived from the Michaelis-Menten fits (Table 1 and Supplemental Figure 1). Specific rates for each of the peptides were measured using the enzyme-coupled kinase assay with either 2 or 8 uM EGFR kinase domain for Peptide C or Tail Peptide A and Tyrsub, respectively. Data points represent the means ± standard errors of mean from a minimum of three replicates, and nH values are ± standard errors of mean.






















Second figure from paper

Figure 2 - The effects of Peptide C on EGFR kinase domain phosphorylation of other substrates.
Autophosphorylation of the EGFR kinase domain on the activation loop at Tyr-845 and the C- terminal tail at Tyr-974 and Tyr-992 in the presence of 0-60 uM Peptide C was measured by immunoblot and is shown by (A) representative immunoblots and (B) quantified band densities. Autophosphorylation reactions were performed with 800 nM EGFR kinase domain for one minute and terminated by addition of EDTA prior to immunoblotting with antibodies specific to EGFR and each phosphotyrosine. Band densities were quantified using ImageJ and normalized to EGFR kinase domain levels [20]. Values are relative to the maximum levels of phosphorylation for each phosphotyrosine. Data points represent the averages ± standard errors of mean from a minimum of three replicates. (C) Phosphorylation of Tail Peptide A (gamma-P incorporated) was measured in a radiometric kinase assay in the presence of a Peptide C variant in which the substrate tyrosine was replaced by alanine and could not undergo phosphorylation. Reactions were performed for ten minutes with 500 nM EGFR kinase domain, 750 uM Tail Peptide A, Peptide C variant at indicated concentrations, and 25 uM ATP labeled at 8.325 uCi/ml [gamma-32P]ATP and terminated by addition of 0.5% v/v phosphoric acid. Reactions were spotted onto filters, and the radioactivity was quantified by liquid scintillation counting. Data points represent the means ± standard errors of mean from four replicate filters.
























Third figure from paper

Figure 3 - Catalytic activities of EGFR kinase domain dimerization mutants with Peptide C.
(A) The specific rates of catalysis of EGFR kinase domain dimerization mutants I682Q, which is on the N-lobe, and V924R, which is on the C-lobe, either alone or combined were measured over a titration of Peptide C. (B) The kinase inactivating mutation D813N was introduced to each of the I682Q and V924R dimerization mutants, and specific rates for each of the kinase domain pairs I682Q/D813N with V924R or I682Q with D813N/V924R over a titration of Peptide C were measured. (C) Hill coefficients for the pairs of I682Q and V924R or I682Q/D813N and V924R were derived by fitting the specific catalytic rates from (A) and (B) at Log10 (peptide concentration) to the Hill equation using Prism software by GraphPad. Specific rates at indicated Peptide C concentrations were measured using the enzyme-coupled kinase assay. All measurements were made using 2 uM of total kinase domain, and 1 uM of each of the mutant kinase domains when measured as a pair. Data points represent the means ± standard errors of mean from a minimum of three replicates, and nH values are ± standard errors of mean.





















Fourth figure from paper

Figure 4 - Precipitation of proteins by Peptide C.
Co-sedimentation of (A) wild type EGFR kinase domain, (B) I682Q EGFR kinase domain, and (C) BSA in the presence of Peptide C is visualized by representative SDS-PAGE gels, and (D) band densities of the precipitated proteins are quantified. Each protein (2 uM) was incubated with 0-175 uM Peptide C for ten minutes and centrifuged, and the resuspended pellets were resolved by SDS-PAGE. Band densities were quantified using ImageJ [20]. Data points represent the means ± standard errors of mean from a minimum of three replicates.














Fifth figure from paper

Figure 5 - TEM images of Peptide C with and without EGFR kinase domain.
Representative TEM images of (A) 20 uM, (B) 40 uM, and (C) 80 uM samples of Peptide C show Peptide C forms fibrils as well as globular aggregates, which are indicated by the arrows. Representative TEM images upon addition of 2 uM EGFR kinase domain to (D) 0 uM, (E) 20 uM, and (F) 80 uM Peptide C indicate that Peptide C forms fibrils in the presence of EGFR. There appears to be lower levels of EGFR in solution at high Peptide C concentration, and the fibril edges are less defined. Samples were incubated for about 10 minutes prior to being deposited on Formvar carbon grids. The grids were negatively stained with 1% uranyl acetate and viewed using a JEOL 1200 EX transmission electron microscope. Scale bars are 100 nm.




Supplemental Material


First supplimental figure
Supplemental Figure 1 - Michaelis-Menten fits for Tail Peptide A and Tyrsub. The specific rates of EGFR catalysis as a function of peptide concentration for the peptide substrates Tail Peptide A and Tyrsub are fit to the Michaelis-Menten equation to derive the catalytic parameters of kcat and KM (Table 1) using Prism software by GraphPad. Specific rates for each of the peptides at the indicated peptide concentrations were measured using the enzyme-coupled kinase assay with 8 uM EGFR kinase domain. Data points represent the means ± standard errors of mean from a minimum of three replicates.











First supplimental figure
Supplemental Figure 2 - Effect of activation loop Tyr-845 phosphorylation on EGFR kinase domain catalytic activity. A) EGFR kinase domain was incubated with Src kinase domain for the times indicated to increase phosphorylation on the EGFR kinase domain activation loop at Tyr-845, and the specific rates for EGFR phosphorylation of Tail Peptide A by the pre-phosphorylated EGFR kinase domain were measured. (B) A representative immunoblot for phosphorylation of Tyr-845 indicates levels of EGFR kinase domain activation loop phosphorylation following incubation with Src and performance of the enzyme-coupled kinase reactions. Both Src-mediated phosphorylation and EGFR autophosphorylation contribute to phosphorylation of Tyr-845 in vitro. Src phosphorylation of EGFR was performed by incubating 200 nM Src kinase domain, 63.52 uM EGFR kinase domain, 500 uM ATP, 20 mM Tris, pH. 7.5, and 10 mM MgCl2 for the indicated amounts of time at room temperature, and the reactions were inhibited by addition of 3.75 uM Dasatinib. The Src-treated EGFR mixture was diluted into the enzyme-coupled kinase assay, and EGFR activity was measured with 500 uM Tail Peptide A and 8 uM EGFR at room temperature. Though the prior incubation of EGFR with Src results in addition of ~25 nM Src to the enzyme-coupled kinase assay, the 500 nM Dasatinib in the solution is sufficient to inhibit Src, and the assay only measures EGFR catalysis. The enzyme-coupled kinase reactions proceeded for approximately 20 minutes, and the reaction mixtures were subject to immunoblot analysis with an antibody specific to phosphorylation on Tyr-845. Data points represent the means ± standard errors of mean from three replicates.











First supplimental figure
Supplemental Figure 3 - Hill coefficient measurement for Peptide C variant stimulation of EGFR kinase domain phosphorylation of Tail Peptide A. The phosphorylation of Tail Peptide A by EGFR kinase domain in the presence of a non-phosphorylatable Peptide C variant data that are presented in Figure 2C are plotted as gamma-phosphoryl (gamma-P) incorporated as a function of Log10 (Peptide C variant concentration) and fit to the Hill equation. Reactions were performed for ten minutes with 500 nM EGFR kinase domain, 750 uM Tail Peptide A, Peptide C variant at indicated concentrations, and 25 uM ATP labeled with 8.325 uCi/ml [gamma-32P]ATP and terminated by addition of 0.5% v/v phosphoric acid. Reactions were spotted onto filters, and the radioactivity was quantified by liquid scintillation counting. The Hill coefficient was derived by fitting the gamma-P incorporated at Log10 (Peptide C variant) to the Hill equation using Prism software by GraphPad. Data points represent the means ± standard errors of mean from four replicate filters, and the value of nH is ± standard error of mean.











First supplimental figure
Supplemental Figure 4 - Dynamic light scattering autocorrelation curves for Peptide C samples. The autocorrelation curves for 1 mM samples of Peptide C in water, 20 mM sodium chloride, and 15 mM Tris, pH 7.5, 0.02% v/v polysorbate 20, and 20 mM sodium chloride derived from dynamic light scattering measurements indicate Peptide C aggregation occurs in the presence of other solutes. Samples were measured at 25°C using 15% of the 60 mW laser power at a 90° angle on a DynaPro Titan instrument from Wyatt.











First supplimental figure
Supplemental Figure 5 - TEM images of Peptide C with I682Q EGFR kinase domain. Representative TEM images of 2 uM I682Q kinase domain with (A) 0 uM, (B) 20 uM, and (C) 80 uM Peptide C indicate the samples appear similar in morphology to those that contain wild type EGFR with Peptide C. Samples were incubated for about 10 minutes in solution prior to being deposited on Formvar carbon grids. The grids were negatively stained with 1% uranyl acetate and viewed using a JEOL 1200 EX transmission electron microscope. Scale bars are 100 nm.












References


Zhang, X., Gureasko, J., Shen, K., Cole, P. A. and Kuriyan, J. (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137-1149.

Jura, N., Endres, N. F., Engel, K., Deindl, S., Das, R., Lamers, M. H., Wemmer, D. E., Zhang, X. and Kuriyan, J. (2009) Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell 137, 1293-1307.

Red Brewer, M., Choi, S. H., Alvarado, D., Moravcevic, K., Pozzi, A., Lemmon, M. A. and Carpenter, G. (2009) The juxtamembrane region of the EGF receptor functions as an activation domain. Mol. Cell 34, 641-651.

Jorissen, R. N., Walker, F., Pouliot, N., Garrett, T. P. J., Ward, C. W. and Burgess, A. W. (2003) Epidermal growth factor receptor: mechanisms of activation and signalling. Exp. Cell Res. 284, 31-53.

Fan, Y.-X., Wong, L. and Johnson, G. R. (2005) EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity. Biochem. J. 392, 417-423.

Muthuswamy, S. K., Gilman, M. and Brugge, J. S. (1999) Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol. Cell. Biol. 19, 6845-6857.

Clayton, A. H. A., Walker, F., Orchard, S. G., Henderson, C., Fuchs, D., Rothacker, J., Nice, E. C. and Burgess, A. W. (2005) Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-a multidimensional microscopy analysis. J. Biol. Chem. 280, 30392-30399.

Blackburn, R. K., Bramson, H. N., Moyer, M. B. and Stuart, J. D. (2003) Protein kinase peptide substrate determination using peptide libraries, US Patent Office, US Patent Application 2003.0113711.

Brignola, P. S., Lackey, K., Kadwell, S. H., Hoffman, C., Horne, E., Carter, H. L., Stuart, J. D., Blackburn, K., Moyer, M. B., Alligood, K. J., et al. (2002) Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains. J. Biol. Chem. 277, 1576-1585.

Guyer, C. A., Woltjer, R. L., Coker, K. J. and Staros, J. V. (1994) Peptide substrate recognition by the epidermal growth factor receptor. Arch. Biochem. Biophys. 312, 573- 578.

Songyang, Z., Carraway, K. L., Eck, M. J., Harrison, S. C., Feldman, R. A., Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C., et al. (1995) Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature 373, 536-539.

Wood, E. R., Truesdale, A. T., McDonald, O. B., Yuan, D., Hassell, A., Dickerson, S. H., Ellis, B., Pennisi, C., Horne, E., Lackey, K., et al. (2004) A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res. 64, 6652- 6659.

Yun, C-H., Boggon, T. J., Li, Y., Woo, M. S., Greulich, H., Meyerson, M. and Eck, M. J. (2007) Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11, 217-227.

Wang, Z., Longo, P. A., Tarrant, M. K., Kim, K., Head, S., Leahy, D. J. and Cole, P. A. (2011) Mechanistic insights into the activation of oncogenic forms of EGF receptor. Nat. Struct. Mol. Biol. 18, 1388-1393.

Hubler, L., Kher, U. and Bertics, P. J. (1992) Potentiation of epidermal growth factor receptor protein-tyrosine kinase activity by sulfate. Biochim. Biophys. Acta 1133, 307-315.

Hubler, L., Leventhal, P. S. and Bertics, P. J. (1992) Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins. Biochem. J. 281, 107-114.

Mohammadi, M., Honegger, A., Sorokin, A., Ullrich, A., Schlessinger, J. and Hurwitz, D. R. (1993) Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase. Biochemistry 32, 8742-8748.

Downward, J., Waterfield, M. D. and Parker, P. J. (1985) Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J. Biol. Chem. 260, 14538-14546.

Barker, S. C., Kassel, D. B., Weigl, D., Huang, X., Luther, M. A. and Knight, W. B. (1995) Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry 34, 14843-14851.

Abramoff, M. D., Magalhães, P. J. and Ram, S. J. (2004) Image processing with ImageJ. Biophotonics Int. 11, 36-42.

Khan, E. M., Lanir, R., Danielson, A. R. and Goldkorn, T. (2008) Epidermal growth factor receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking. FASEB J. 22, 910-917.

Ge, G., Wu, J., Wang, Y. and Lin, Q. (2002) Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase. Biochem. Biophys. Res. Commun. 290, 914-920.

Stamos, J., Sliwkowski, M. X. and Eigenbrot, C. (2002) Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 277, 46265-46272.

Feng, B. Y., Simeonov, A., Jadhav, A., Babaoglu, K., Inglese, J., Shoichet, B. K. and Austin, C. P. (2007) A high-throughput screen for aggregation-based inhibition in a large compound library. J. Med. Chem. 50, 2385-2390.

Shan, Y., Eastwood, M. P., Zhang, X., Kim, E. T., Arkhipov, A., Dror, R. O., Jumper, J., Kuriyan, J. and Shaw, D. E. (2012) Oncogenic mutations counteract intrinsic disorder in the EGFR kinase and promote receptor dimerization. Cell 149, 860-870.

Zorn, J. A., Wille, H., Wolan, D. W. and Wells, J. A. (2011) Self-assembling small molecules form nanofibrils that bind procaspase-3 to promote activation. J. Am. Chem. Soc. 133, 19630-19633.

Monsey, J., Shen, W., Schlesinger, P. and Bose, R. (2010) Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system. J. Biol. Chem. 285, 7035-7044.

Lu, C., Mi, L.-Z., Schürpf, T., Walz, T. and Springer, T. A. (2012) Mechanisms for kinase- mediated dimerization of the epidermal growth factor receptor. J. Biol. Chem. 287, 38244- 38253.