Neddylation controls basal MKK7 kinase activity in breast cancer cells

Abstract The c-Jun NH2-terminal protein kinase (JNK) pathway has been implicated in mammary tumor development. However, the molecular mechanisms regulating JNK activity in breast cancer cells remain unclear. Here, we report that the inhibition of ubiquitination-like post-translational modification neddylation through different strategies results in enhanced basal JNK phosphorylation in human breast cancer cells. The upregulation of basal JNK phosphorylation upon neddylation inhibition is independent of the deneddylation of Cullins, the well-characterized neddylation substrates. Since augmented basal JNK phosphorylation via ectopic MKK7 expression impedes proliferation and the epithelial-to-mesenchymal transition (EMT) phenotype, the neddylation system might contribute to mammary tumor development partially through limiting basal JNK phosphorylation. Further exploration reveals that MKK7, a JNK-specific MAP2K, undergoes neddylation in human breast cancer cells. MKK7 co-precipitates with a fragment of Ran-binding protein 2 (RanBP2), a large multimodular and pleiotropic protein that has been recognized as a SUMO E3 ligase. Knockdown of RanBP2 attenuates MKK7 neddylation and augments basal JNK phosphorylation without affecting the neddylation of Cullins, whereas ectopic expression of a RanBP2 fragment possessing SUMO E3 activity (RanBP2ΔFG) manifests the opposite effects. In vitro neddylation assays confirm that RanBP2ΔFG works as the neddylation E3 ligase for MKK7. The basal kinase activity of endogenous MKK7 increases upon RanBP2 knockdown but decreases upon the ectopic expression of RanBP2ΔFG. Furthermore, purified MKK7 shows reduced basal kinase activity after in vitro neddylation by RanBP2ΔFG. Consistently, RanBP2 knockdown leads to reduced proliferation and impaired EMT phenotype in human breast cancer cells and the effects of RanBP2 knockdown are reversed by simultaneous MKK7 knockdown. Taken together, our data suggest that MKK7 undergoes neddylation in human breast cancer cells, which limits its basal kinase activity.