pt7ht vector and over-expressed in E. coli as inclusion bodies. Cells were lysed in 6 M

Transcription

1 Supplementary Methods MIG6 production, purification, inhibition, and kinase assays MIG6 segment 1 (30mer, residues ) peptide was synthesized using standard solid-phase peptide synthesis as described previously 1. The inhibition assay of MIG6 seg 1 was identical to those of the IC 50 measurement for erlotinib, lapatinib and AMP-PNP. The concentrations of MIG6 seg 1 used ranged from 0 to 100 M. DNA encoding segment 1+2 of MIG6 (77aa, residue ) was cloned into the pt7ht vector and over-expressed in E. coli as inclusion bodies. Cells were lysed in 6 M guanidine hydrochloride and the soluble fraction collected after centrifugation was loaded on a Ni-NTA column. MIG6 seg 1+2 protein was eluted with 250 mm imidazole, 20 mm Tris ph 8.0, 500 mm NaCl and 8 M urea. After refolding by dialysis, MIG6 seg 1+2 was maintained in 250 mm imidazole, 20 mm Tris ph 8.0, 500 mm NaCl, 2 mm DTT and 10% (v/v) glycerol. The kinase inhibition assays with MIG6 seg 1+2 were performed identically to those of the IC 50 measurements for erlotinib, lapatinib, and AMP-PNP, except that streptavidin resin was used to isolate biotinylated peptide substrate from the reaction mixture and phosphorylated MIG6. The 25 L reactions were stopped after 10 min by adding 30 L 1% (w/v) SDS in PBS buffer and samples were incubated with streptavidin ultralink resin (Pierce) in a centrifuge column (Pierce) for 30 min at room temperature, then the resin was washed three times with 500 L wash buffer (1% (w/v) SDS, 1XPBS) before the radioactivity was measured by liquid scintillation counting (Beckman). Due to the limited solubility of MIG6 seg 1+2, the upper limit for the concentration range studied was 20 M. The WT EGFR kinase domain (Invitrogen,

2 PV3872) utilized in the MIG6 inhibition assay contains the entire EGFR intracellular domain (residues ) with an N-terminal GST tag. To verify whether MIG6 is a substrate of EGFR, 1 M MIG6 seg 1+2 was incubated with 10 M [ 32 P] ATP (10 Ci per reaction) and 25 nm WT EGF, L858R EGF, L858R Cetux, ( ) EGF, and ( ) Cetux tegfrs. The 25 L reactions were quenched after 10 min by adding 7 L SDS-PAGE loading dye (250 mm Tris ph 8.0, 10% (w/v) SDS, 0.5% (w/v) Bromophenol Blue, 50% (v/v) glycerol), boiled for 5 min, and loaded onto a 15% SDS-PAGE gel and run at 150 V for 66 min. Gels were stained with coomassie blue staining solution, dried, exposed in a Phosphorimager cassette for 16 h, and then analyzed using a Typhoon Scanner (GE Healthcare). The incorporation of radioactive phosphate ( 32 P) into tegfr and MIG6 was recorded simultaneously by the autoradiograph of a 15% SDS-PAGE gel. To characterize the kinetic parameters for MIG6 seg 1+2 phosphorylation by tegfrs, radiometric kinase assays were performed in buffer containing 50 mm HEPES (ph 7.4), 37.5 mm NaCl, 1 mm DTT, 5% (v/v) glycerol, 125 g ml -1 BSA, 2 mm MnCl 2, with fixed or varied concentrations of ATP and MIG6 seg 1+2. Linearity of kinase activity versus time was determined using 25 nm tegfr, 5 M MIG6 seg 1+2 and 10 M ATP. Reactions proved to be linear versus time over 16 min. The K app m for ATP and k cat values were obtained with fixed MIG6 concentration (5 M) and varying ATP concentrations (6.25, 12.5, 25, 50, 100, and 200 M). The K app m for MIG6 seg 1+2 values were obtained with fixed ATP concentration (10 M) and varying MIG6 concentrations (0.625, 1.25, 2.5, 5, 10 and 20 M). For specific kinase activity and K m measurements, the 25 L reactions were quenched after 10 min by adding 7 L SDS-PAGE loading dye, and loaded onto a 15%

3 SDS-PAGE gel. Radioactivity in bands corresponding to phosphorylated MIG6 seg 1+2 was quantified both by Image Quant and liquid scintillation counting. MIG6 anti-phosphotyrosine western blots Four micrograms of MIG6 seg 1+2 was incubated with 25 M tegfr (WT tegfr EGF, ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, and L858R tegfr Cetux) in 50 L reaction buffer (50 mm HEPES ph 7.4, 37.5 mm NaCl, 1 mm DTT, 5% (v/v) glycerol, 125 g ml -1 BSA, 2 mm MnCl 2, 1 mm ATP and 1 mm Na 3 VO 4 ) for 30 min at room temperature. Reactions were stopped by adding SDS gel loading dye and the reaction mixtures were divided equally to two SDS-PAGE gels. Resolved proteins on one gel were transferred to PVDF membranes, and probed with the antiphosphotyrosine antibody 4G10 (Millipore). Resolved proteins on the other gel were stained with coomassie blue solution to verify that the same amount of MIG6 seg 1+2 was utilized among various reactions.

4 Supplementary Figures Supplementary Figure 1. The kinase activity of both and L858R in their EGF and cetuximab bound forms is linear versus time as well as enzyme concentration. Assays for the linear range were performed with 100 μmm peptide substrate, 100 μm ATP, and 2 mm Mn 2+. (a) P/E versus time for ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, and L858R tegfr Cetux, with varying reaction times (4, 8, 10, 12, and 16 min). (b) Activity versus enzyme concentration for ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, and L858R tegfr Cetux, with varying tegfr concentrations (1.25, 2.5, 5, 10, and 25 nm).

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6 Supplementary Figure 2. Steady-state kinetic analysis of ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, L858R tegfr Cetux, and L858R I706Q tegfr EGF. (a) Peptide K app m values were obtained with varying concentrations of peptide substrate (1.88, 3.75, 7.5, 15, 30, 60, and 120 µm). (b) ATP K app m and k cat values were obtained with varying concentrations of ATP (12.5, 25, 50, 100, 200, 400, 600, and 800 µm).

7 Supplementary Figure 3. Inhibitory effects of AMP-PNP on WT tegfr EGF, L858R tegfr EGF and ( ) tegfr EGF. IC 50 values for AMP-PNP were measured with varying concentrations of AMP-PNP (6.25, 12.5, 25, 50, 100, 200, 400, and 800 µm).

8 Supplementary Figure 4. Comparison of erlotinib and lapatinib inhibition potency on various tegfr mutant forms ( ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, L858R tegfr Cetux, and L858R I706Q tegfr EGF). (a) IC 50 values for erlotinib were measured with varying concentrations of erlotinib (0. 039, 0.078, 0.156, 0.313, 0.625, 2..5, and 10 µm). (b) IC 50 values for lapatinib were measured with varying concentrations of lapatinib (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, and 40.0 µ M).

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10 Supplementary Figure 5. Steady-state kinetic analysis of MIG6 seg 1+2 phosphorylation by tegfr. (a) The MIG6 seg 1+2 phosphorylation by tegfrs is linear with time. Assays for the linear range were performed with varying reaction times (4, 8, 10, 12, and 16 min). (b) MIG6 K app m values for WT tegfr EGF, ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, and L858R tegfr Cetux. Assays were run with varying concentrations of MIG6 seg 1+2 (0.625, 1.25, 2.5, 5, 10, and 20 µm). (c) ATP K app m and k cat values for WT tegfr EGF, ( ) tegfr EGF, ( ) tegfr Cetux, L858R tegfr EGF, and L858R tegfr Cetux. Assays were run with varying ATP concentrations (6.25, 12.5, 25, 50, 100, and 200 µm).

11 Supplementary Figure 6. Inhibitory effects of MIG6 seg 1 on WT tegfr EGF, L858R tegfr EGF and ( ) tegfr EGF. Inhibition assayss were performed with varying concentrations of MIG6 seg 1( (12.5, 25, 50, and 100 μm). Supplementary Reference 1. Zhang, X. et al. Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface. Nature 450, (2007).