Abstract
RIPK1/RIPK3-MLKL signaling molecules are fundamental in initiating necroptotic cell death, but their roles in the development of colon cancer are unclear. This study reports that RIPK3 interacted with SPOP, a component of the E3 ligase within the Cul3 complex. This interaction leads to K48-linked ubiquitination and subsequent proteasomal degradation of RIPK3. Two distinct degron motifs, PETST and SPTST, were identified within the linker domain of RIPK3 for SPOP. RIPK3 phosphorylations at Thr403 by PIM2 and at Thr412/Ser413 by ERK2 are essential to facilitate its interaction with SPOP. Computational docking studies and immunoprecipitation analyses showed that these PIM2 and ERK2 phosphorylations bolster the stability of the RIPK3-SPOP interaction. In particular, mutations of RIPK3 at the degron motifs extended the half-life of RIPK3 by preventing its phosphorylation and subsequent ubiquitination. The deletion of SPOP, which led to increased stability of the RIPK3 protein, intensified LPS/sMAC/zVAD-induced necroptotic cell death in colon cancer cells. These findings underscore the critical role of the SPOP-mediated RIPK3 stability regulation pathway in controlling necroptotic cell death.