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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 | 2021 Apr

REPOSITORIES: biostudies-literature

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

Gupta Kartik K Liu Bo B

iScience 20210317 4

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 ...[more]

PMID: 33870135

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Project description:Rationale: β-catenin is a component for cell adhesion and a transcriptional coactivator in epithelial-mesenchymal transition (EMT). Previously we found that catalytically active PLK1 drives EMT in non-small cell lung cancer (NSCLC), upregulating extracellular matrix factors including TSG6, laminin γ2, and CD44. To understand the underlying mechanism and clinical significance of PLK1 and β-catenin in NSCLC, their relationship and function in metastatic regulation were investigated. Methods: The clinical relevance between the survival rate of NSCLC patients and the expression of PLK1 and β-catenin was analyzed by a KM plot. Immunoprecipitation, kinase assay, LC-MS/MS spectrometry, and site-directed mutagenesis were performed to reveal their interaction and phosphorylation. A lentiviral doxycycline-inducible system, Transwell-based 3D culture, tail-vein injection model, confocal microscopy, and chromatin immunoprecipitation assays were used to elucidate the function of phosphorylated β-catenin in the EMT of NSCLC. Results: Clinical analysis revealed that the high expression of CTNNB1/PLK1 was inversely correlated with the survival rates of 1,292 NSCLC patients, especially in metastatic NSCLC. In TGF-β-induced or active PLK1-driven EMT, β-catenin, PLK1, TSG6, laminin γ2, and CD44 were concurrently upregulated. β-catenin is a binding partner of PLK1 in TGF-β-induced EMT and is phosphorylated at S311. Phosphomimetic β-catenin promotes cell motility, invasiveness of NSCLC cells, and metastasis in a tail-vein injection mouse model. Its upregulated stability by phosphorylation enhances transcriptional activity through nuclear translocation for the expression of laminin γ2, CD44, and c-Jun, therefore enhancing PLK1 expression by AP-1. Conclusions: Our findings provide evidence for the critical role of the PLK1/β-catenin/AP-1 axis in metastatic NSCLC, implying that β-catenin and PLK1 may serve as a molecular target and prognostic indicator of the therapeutic response in metastatic NSCLC patients.

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.

Dataset Information

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

Publications

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

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