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PLK1-mediated S369 phosphorylation of RIPK3 during G2 and M phases enables its ripoptosome incorporation and activity.

ABSTRACT: Receptor-interacting protein kinase 3 executes a form of regulated necrosis called necroptosis. Upon induction of an altered conformation by chemical inhibitors or via mutations in its kinase site, RIPK3 associates with a multiprotein complex called the ripoptosome-a signaling platform containing FADD, RIPK1, caspase 8, and cFLIP-and becomes decisive in the execution of apoptosis. Surprisingly, in contexts not completely understood, the ripoptosome itself cleaves RIPK3, highlighting an apparent conundrum on how RIPK3 fulfills its role via the complex responsible for its own degradation. Recently, ripoptosome assembly was found to occur in mitosis where we found elevated RIPK3 levels. We now report that PLK1 directly associates with RIPK3 and phosphorylates it at S369 as cells enter mitosis. G2/M phase RIPK3 has pro-apoptotic activity but upon release from ripoptosome, can trigger necroptosis. Taken together, phosphorylation of RIPK3 at S369 prevents its ripoptosome-mediated cleavage thereby retaining its pro-death activity during mitosis.

SUBMITTER: Gupta K

PROVIDER: S-EPMC8040267 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMore than 90% of the mortality of triple-negative breast cancer (TNBC) patients is attributed to cancer metastasis with organotropism. The lung is a frequent site of TNBC metastasis. However, the precise molecular mechanism for lung-specific metastasis of TNBC is not well understood.MethodsRNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, MBA-MB-231-LM3, and their parental cells (4T1-P, MBA-MB-231-P). Expressions of RGCC, called regulator of cell cycle or response gene to complement 32 protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting, and immunohistochemistry. Kinase activity assay was performed to evaluate PLK1 kinase activity. The amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2) was detected by immunoblotting. RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay was conducted to measure the status of RGCC-mediated fatty acid oxidation. NADPH and ROS levels were detected by well-established assays. The chemical sensitivity of cells was evaluated by CCK8 assay.ResultsRGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis in comparison with other organ metastases. RGCC can effectively promote kinase activity of PLK1, and the activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, the RGCC/PLK1/AMPKα2 signal axis increases oxidative phosphorylation of mitochondria to generate more energy, and promotes fatty acid oxidation to produce abundant NADPH. These metabolic changes contribute to sustaining redox homeostasis and preventing excessive accumulation of potentially detrimental ROS in metastatic tumor cells, thereby supporting TNBC cell survival and colonization during metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary TNBC lung metastasis in a mouse model.ConclusionsRGCC overexpression is significantly associated with lung-specific metastasis of TNBC. RGCC activates AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis. The study provides implications for RGCC-driven OXPHOS and fatty acid oxidation as important therapeutic targets for TNBC treatment.

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PLK1-mediated S369 phosphorylation of RIPK3 during G2 and M phases enables its ripoptosome incorporation and activity.

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