Select Heterozygous Keap1 Mutations Have a Dominant-Negative Effect on Wild-Type Keap1 In Vivo

Abstract

Under 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 (Keap1(WT)) activity by functioning as a dominant-negative protein through heterodimerization of mutant Keap1 (Keap1(mutant)) with Keap1(WT). When Keap1(G430C) or Keap1(G364C) mutants were expressed in lieu of Keap1(WT), premature juvenile mortality was observed. To test the hypothesis of a dominant-negative effect, Keap1-null mice were engineered to coexpress both Keap1(WT) and Keap1(G430C) 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 Keap1(G430C) mutant, Keap1(C273&288A) substitutions also diminished Keap1(WT) 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 Keap1(WT), and failed to induce the dominant-negative effect in vivo. These results thus demonstrate that sequestration of Keap1(WT) into a Keap1(mutant)-Keap1(WT) heterodimer leads to a dominant-negative effect in vivo and gives rise to Nrf2 activation.