Identification Of MKK7Neddylation In Breast Cancer Cells And Its Biological Significance

The c-Jun N-terminal protein kinase (JNK) is a member of the MAPK superfamily. JNK is activated by sequential protein phosphorylation through a MAPK module; that is, MAPK kinase kinase (MAP3K)→MAPK kinase (MAP2K or MKK)→MAPK, in response to a variety of extracellular stimuli. It is phosphorylated by two upstream kinases known as MKK4and MKK7. MKK4activates both JNK and p38, whereas MKK7only activates JNK. Our previous studies suggest that targeting MKK7is associated with efficient and specific down-regulation of JNK activity in various cell context. Despite that MKK7has been demonstrated to undergo ubiquitination followed by degradation under certain circumstances, it remains largely unknown whether or not MKK7can also be modified by other ubiquitin-like proteins.NEDD8is one of the ubiquitin-like proteins, diaplays80%sequence homology with ubiquitin. The process of NEDD8activation, transfer, and covalent modification of target proteins (catalyzed by El, E2, and E3enzymes, respectively) is called neddylation. The heterodimeric E1-activating enzyme APPBP1(amyloid beta precursor protein binding protein-1)-UB A3(ubiquitin-like modifier activating enzyme3) determines specificity for NEDD8. Neddylation can enhance the activation of ubiquitin-protein ligases and promote cell cycle progression. Recent studies show that neddylation is essential in tumorigenesis. However, the underlying molecular mechanisms remain unclear.In this work, we set out to investigate whether MKK7undergoes neddylation in breast cancer cells and the possible biological significance. We chose the breast cancer cell lines MDA-MB-231and MCF-7as the primary research models. We observed the possible MKK7neddylation in breast cancer cells; the effects of UBA3/NEDD8knockdown on the phosphorylation of components of the MAPK pathways, the interaction of MKK7and JNK, and the kinase activity of MKK7. After examining the role of MKK7and JNK in the oncogenic growth of breast cancer cells, we explored the biological significance of MKK7neddylation and the possible E3. Our data show that MKK7undergoes neddylation in breast cancer cells and SUMOylation E3RanBP2is essential in this process. Neddylation does not affect the phosphorylation of MKK7by upstream kinases but suppresses the kinase activity of MKK7, which leads to decreased JNK phosphorylation. Further exploration revealed that MKK7plays a protumorigenic role and does not undergo neddylation in colon cancer cells, suggesting that MKK7neddylation occurs in a cell type-dependent manner. Since MKK7activity inhibits the oncogenic growth of breast cancer cells, our data collectively suggest that neddylation promotes the oncogenic growth of breast cancer cells partly through modifying MKK7and thereby suppressing MKK7activity.