Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR Mutants
Figure 3
Shc and STAT3 Signaling Pathways Are Constitutively Activated in Cells Expressing the Mutant EGFR
(A) Cells expressing the wild-type or L858R mutant EGFR were placed in 0.5% calf serum for 24 h and left unstimulated or stimulated with 20 ng/ml EGF (“+EGF”) for 8 min. EGFR was immunoprecipitated from 200 μg of cell lysate, and eluted immune complexes were separated by SDS-PAGE and immunoblotted with anti-Shc. Shc constitutively coimmunoprecipitates with the L858R EGFR but not the wild-type EGFR. IP, immunoprecipitation; NL, no-lysate control immunoprecipitation.
(B) Anti-phospho-Shc immunoblots (upper row of blots) and anti-Shc immunoblots (lower row) of whole cell lysates from the experiment in (A). All three Shc isoforms are constitutively phosphorylated in cells expressing the L858R EGFR.
(C) Immunoblots of whole cell lysates with anti-phospho-STAT3 Y705 (upper row), total STAT3 (middle row), or actin as a loading control (lower row). STAT3 is constitutively phosphorylated in cells expressing any of the four mutant EGFR proteins, representative of the four classes of EGFR mutations observed in lung adenocarcinoma tumor DNA. del, L747_E749del A750P; ins, D770_N771insNPG; pBp, pBabe-Puro vector control; wt, wild-type EGFR.
(D) M67 STAT3 luciferase reporter assay. NIH-3T3 cells expressing the indicated EGFR were transfected with a STAT-dependent reporter (m67-firefly luciferase) [25] and a control reporter expressing Renilla luciferase. STAT-dependent luciferase production was measured after 48 h and normalized to Renilla luciferase. The normalized luciferase values were divided by the values for cells expressing the wild-type EGFR to produce relative luciferase units. NIH-3T3 expressing mutant forms of EGFR exhibited elevated levels of STAT-dependent transcriptional activity relative to wild-type. ins, D770_N771insNPG EGFR; wt, wild-type EGFR.
(E) Immunoblots of whole cell lysates with anti-phospho-Akt S473 (upper row), total Akt (middle row), or actin as a loading control (lower row). Akt is constitutively phosphorylated in cells expressing mutant EGFR. del, L747_E749del A750P; ins, D770_N771insNPG; wt, wild-type EGFR.