LZTR1 interacts with AIFM3 protein and negatively regulates Ras signaling by downregulating SHOC2 and phosphorylated p90RSK protein levels in apoptosis

Objective: Leucine zipper like post translational regulator 1 (LZTR1) is a negative regulator of the canonical Ras pathway. LZTR1 mutations have been associated with several congenital diseases and cancer types. The identification of novel interaction partners of LZTR1 and an understanding of LZTR1’s roles in various cellular mechanisms are crucial for a better characterization of the molecular mechanisms underlying LZTR1-associated diseases. In this study, the interactions between LZTR1 and apoptosis inducing factor mitochondria associated 3 (AIFM3) protein, as well as their functional impacts on Ras signaling, were investigated in apoptosis-induced HEK293 cells. Method: FLAG-LZTR1 and Myc-AIFM3 expression plasmids were cloned for analyses. While stably FLAG-LZTR1-expressing HEK293 Flp-In cells were prepared, Myc-AIFM3 plasmids were transiently transfected. Interactions of LZTR1 and AIFM3 proteins were analyzed with immunofluorescence microscopy. Apoptosis was induced by treating cells with 0.4 mM hydrogen peroxide (H2O2) for 10 hours. Protein levels were detected with western blotting. Results: Interactions between LZTR1 and AIFM3 proteins were determined in the cytoplasm of HEK293 cells. Cleaved PARP1 proteins were detected in H2O2-treated cells, indicating that apoptosis was induced via H2O2 treatments. Elevated phosphorylated MEK1/2 and ERK1/2 protein levels were detected in apoptosis-induced cells, indicating that neither LZTR1 nor AIFM3 downregulated the Ras signaling in H2O2-induced apoptosis. Conversely, lower levels of SHOC2 and phosphorylated p90RSK levels were observed in the cells expressing LZTR1. Conclusion: During H2O2-induced apoptosis, the Ras signaling is activated, and LZTR1 downregulates this pathway not by inhibiting phosphorylated MEK1/2 and ERK1/2 levels but by diminishing the levels of SHOC2 and phosphorylated p90RSK in HEK293 cells.