Mechanism of Keap1 cysteines mutated in cancer

Upregulation of the Nrf2 transcription factor increases the expression of cytoprotective enzymes, leading to reduced risk of diseases including cancer. Many botanical agents, including sulforaphane, upregulate Nrf2. The Keap1 protein represses Nrf2 by targeting it for ubiquitination and subsequent degradation. Keap1 C151 senses electrophiles, including sulforaphane, leading to loss of repression of Nrf2. Substitution of C151 with bulky amino acids can mimic modification of C151 by an electrophile. Two other Keap1 cysteines, 23 and 249, are mutated to the bulky residue Tyr in Keap1 from cancer cells and do not repress Nrf2. Our hypothesis was that the Tyr mutations were mimicking electrophilic modification at that position, similar to the Trp mutation at position 151, and therefore that C23 and C249 play a role in sensing electrophiles. Cells overexpressing Nrf2, Keap1 (wild type, C23S, C151S or C249S), and an ARE reporter were treated with sulforaphane. We find that the Keap1 C23S and Keap1 C249S were both able to suppress Nrf2 as well as wt Keap1, and that both responded to sulforaphane as well as wt. We conclude that the C23 and C249 are not sensing sulforaphane under the conditions tested. We also conclude that Ser does not substitute for Tyr at these positions, giving information about the mechanism of action of these mutations in cancer. Grant Funding Source : UIC College of Pharmacy and NIH P01 CA48112 and R03 CA128095