Supplementary Figure 1 from Threonine 2609 Phosphorylation of the DNA-Dependent Protein Kinase Is a Critical Prerequisite for Epidermal Growth Factor Receptor–Mediated Radiation Resistance

PDF file - 286K, Alterations in ATM or DNA-PKcs do not affect IR-induced nuclear import. Deficiency or inhibition of either ATM or DNA-PKcs is associated with an absence of EGFR-DNA-PKcs binding. The data in S1A eliminate the possibility that this is due to a potential abrogation of IR-induced nuclear translocation of EGFR which could prevent access to the predominantly nuclear DNA-PKcs. (A) WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from V3, V3-WT DNA-PKCS and V3-7A cells(A) or AT5 and 1BR3 fibroblast cells (B) expressing wild type, L858R or gammaE746-E750 EGFR showing EGFR, cytoplasmic marker, calnexin, and nuclear marker, lamin B. With exposure to 4 Gy, wild type but not L858R or gammaE746-E750 EGFR is seen in nuclear fractions of cells that lack DNA-PKcs or ATM. (C) DNA-PKcs or ATM inhibition does not affect EGFR nuclear import. WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from HBEC cells expressing wild type EGFR that were pretreated for 2 hours with vehicle or DNA-PKcs inhibitor, NU7441, (top panel) or ATM inhibitor, KU55933, (bottom panel). (D) Mutant forms of EGFR are also defective in binding DNA-PKcs. The data in S1A and S1B show that IR-induced nuclear translocation is completely abrogated in L858R or gammaE746-E750 forms of EGFR, which could prevent interactions with the predominantly nuclear DNA-PKcs. Here we use in vitro binding assay to demonstrate that relative to wild type EGFR, the L858R or gammaE746-E750 are also intrinsically defective in physical association with purified DNA-PKcs. In vitro binding assay: wild type, L858R or gammaE746-E750 forms of EGFR were immuno-precipitated with anti-V5 antibody from whole cell lysates from un-irradiated HBEC cells. Lane 1 from left shows no non-specific binding to beads. Antibody-bound EGFR was thoroughly washed. Lane 2 from left shows no evidence of endogenous bound protein. Antibody bound EGFR was incubated with purified human DNA-PKcs in the presence or absence of calf thymus DNA and immuno-precipitated. DNA-PKcs was detected by western blot assay. Lane 3 and 4 show that purified DNA-PKcs physically bound wild type but not the L858R or gammaE746-E750 forms of EGFR and the binding was slightly enhanced with presence of sheared calf thymus DNA.