ARTICLE ABSTRACTUnder homeostatic conditions, Keap1 constitutively mediates the proteasomal degradation Nrf2. However, tertiary changes in Keap1 in response to the cellular environment allow for liberation of Nrf2 to transcriptionally regulate downstream cytoprotective genes that aid in cell survival. KEAP1/NRF2 somatic mutations causing constitutive NRF2 activation have been estimated to occur in approximately 25% of human lung tumors, with similar rates believed to exist in other tissue subtypes. As the stoichiometry between Keap1 and Nrf2 is 2:1, we hypothesized that heterozygous Keap1 mutations could suppress wild-type Keap1 (Keap1WT) activity by functioning as a dominant-negative protein through heterodimerization of mutant Keap1 (Keap1mutant) with Keap1WT. When Keap1G430C or Keap1G364C mutants were expressed in lieu of Keap1WT, premature juvenile mortality was observed. To test the hypothesis of a dominant-negative effect, Keap1-null mice were engineered to coexpress both Keap1WT and Keap1G430C transgenes, a phenotype analogous to that previously observed from a human lung tumor. These mice were viable, but phenotypically displayed hyperactivation of downstream Nrf2 target genes and moderate esophageal hyperkeratosis. Similar to the transgenic mouse Keap1G430C mutant, Keap1C273&288A substitutions also diminished Keap1WT activity in vivo. To further delineate involvement of the dominant-negative heterodimer, transgenic mice with a deletion or strategic mutation in Broad-complex, Tramtrack and Bric-a-Brac (BTB) domain that disallowed Keap1 dimerization were generated in the presence of Keap1WT, and failed to induce the dominant-negative effect in vivo. These results thus demonstrate that sequestration of Keap1WT into a Keap1mutant–Keap1WT heterodimer leads to a dominant-negative effect in vivo and gives rise to Nrf2 activation. Cancer Res; 71(5); 1700–9. ©2010 AACR.