A Study On The Mechanism Of RNF19B Regulating PIK3CA Ubiquitination And Function In The Tumor Cells

PI3K/AKT pathway plays an important role in cell signal transduction,which is an important signaling pathway for tumor cells to possess immortalization ability and is highly activated in many tumors.PIK3CA is a catalytic subunit of PI3K,which plays an important role in tumor development and metastasis.However,the mechanism of protein homeostasis regulation is not clear.Our previous study found that RBR E3 ligase RNF19B interacts with PIK3CA and regulates ubiquitination and protein levels of the latter.In this study,we explored the molecular mechanism by which RNF19B regulates PIK3CA protein ubiquitination and degradation,thereby affecting tumor growth and iron death in tumor cells and animal models.Western blot and immunoprecipitation showed that overexpression of RNF19B could promote the ubiquitination and proteasome degradation of PIK3CA in tumor cells.DIRAS3 combines with RNF19B and PIK3CA to further enhance this ubiquitination modification.The binding of RNF19B to PIK3CA and the ubiquitination level of the latter were weakened after the mutation of the active site C302,indicating that RNF19B promoted PIK3CA ubiquitination and its E3 ubiquitin ligase activity.A series of mutant body granules were constructed according to the common PIK3CA mutation sites in tumor patients.Co-immunoprecipitation experiments showed that the binding of these mutants to RNF19B was weakened,and the ubiquitination level of PIK3CA mutants caused by overexpression of RNF19B was lower than that of the wild type.These results indicate that these sites and their domains play an important role in PIK3CA ubiquitination.C57BL/6 Rnf19b-/-mice were constructed using CRISPR-Cas9 technology,and MEFs were isolated for Western blot analysis.Both the PIK3CA level and the activation degree of PI3K/AKT signaling pathway protein in Rnf19b-/-MEF were higher than those of the wild type.The mouse lung cancer cell line LLC1 was used to construct the transplanted tumor model.It was confirmed that the microenvironment in Rnf19b-/-mice can promote the growth of transplanted tumor,and its molecular mechanism needs further study.Overexpression of RNF19B and/or iron death inducer RSL3 in human lung cancer cell lines resulted in increased intracellular lipid peroxidation levels,indicating increased ferroptosis.The level of lipid peroxidation in MEF cells of Rnf19b-/-mice was lower than that of wild-type mice.The rescue experiment of RNF19B enzyme activity mutation or overexpression of PIK3CA showed that RNF19B promoted the ubiquitination degradation of PIK3CA,increased the level of lipid peroxidation and promoted the ferroptosis.In conclusion,this study found that RNF19B is a possible E3 ubiquitin ligase of PIK3CA,promoting the ubiquitination of PIK3CA,and the process can be enhanced by DIRAS3.RNF19B promotes PIK3CA ubiquitination in association with its E3 ubiquitin ligase activity.Common PIK3CA mutations result in nonbinding to RNF19B and ubiquitination.RNF19B inhibits PI3K/AKT signaling by negatively regulating PIK3CA,and in mice,deletion of this gene promotes the growth of transplanted tumors through microenvironment.RNF19B can increase lipid peroxidation and iron death by promoting PIK3CA ubiquitination degradation.These findings provide a theoretical basis for understanding the basic mechanism of PI3K/AKT pathway and a new strategy for screening PIK3CA targeting drugs.