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The cell polarity kinase Par1b/MARK2 activation selects specific NF-kB transcripts via phosphorylation of core mediator Med17/TRAP80.

ABSTRACT: Par1b/MARK2 is a Ser/Thr kinase with pleiotropic effects that participates in the generation of apico-basal polarity in Caenorhabditis elegans. It is phosphorylated by atypical PKC(ι/λ) in Thr595 and inhibited. Because previous work showed a decrease in atypical protein kinase C (aPKC) activity under proinflammatory conditions, we analyzed the hypothesis that the resulting decrease in Thr595-MARK2 with increased kinase activity may also participate in innate immunity. We confirmed that pT595-MARK2 was decreased under inflammatory stimulation. The increase in MARK2 activity was verified by Par3 delocalization and rescue with a specific inhibitor. MARK2 overexpression significantly enhanced the transcriptional activity of NF-kB for a subset of transcripts. It also resulted in phosphorylation of a single band (∼Mr 80,000) coimmunoprecipitating with RelA, identified as Med17. In vitro phosphorylation showed direct phosphorylation of Med17 in Ser152 by recombinant MARK2. Expression of S152D-Med17 mimicked the effect of MARK2 activation on downstream transcriptional regulation, which was antagonized by S152A-Med17. The decrease in pThr595 phosphorylation was validated in aPKC-deficient mouse jejunal mucosae. The transcriptional effects were confirmed in transcriptome analysis and transcript enrichment determinations in cells expressing S152D-Med17. We conclude that theMARK2-Med17 axis represents a novel form of cross-talk between polarity signaling and transcriptional regulation including, but not restricted to, innate immunity responses.

SUBMITTER: Mashukova A

PROVIDER: S-EPMC8108508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicroglia, the resident immune cells of the brain, exhibit various morphologies that correlate with their functions under physiological and pathological conditions. In conditions such as aging and stress, microglia priming occurs, which leads to altered morphology and lower threshold for activation upon further insult. However, the molecular mechanisms that lead to microglia priming are unclear.MethodsTo understand the role of Par1b/MARK2 in microglia, we first expressed shRNA targeting luciferase or Par1b/MARK2 in primary microglial cells and imaged the cells using fluorescent microscopy to analyze for morphological changes. A phagocytosis assay was then used to assess functional changes. We then moved in vivo and used a Par1b/MARK2 knockout mouse model to assess for changes in microglia density, morphology, and phagocytosis using immunohistochemistry, confocal imaging, and 3D image reconstruction. Next, we used two-photon in vivo imaging in live Par1b/MARK2 deficient mice to examine microglia dynamics. In addition, a controlled-cortical impact injury was performed on wild-type and Par1b/MARK2-deficient mice and microglial response was determined by confocal imaging. Finally, to help rule out non-cell autonomous effects, we analyzed apoptosis by confocal imaging, cytokine levels by multiplex ELISA, and blood-brain barrier permeability using Evans Blue assay.ResultsHere, we show that loss of the cell polarity protein Par1b/MARK2 facilitates the activation of primary microglia in culture. We next found that microglia in Par1b/MARK2 deficient mice show increased density and a hypertrophic morphology. These morphological changes are accompanied with alterations in microglia functional responses including increased phagocytosis of neuronal particles early in development and decreased surveillance of the brain parenchyma, all reminiscent of a primed phenotype. Consistent with this, we found that microglia in Par1b/MARK2 deficient mice have a significantly lower threshold for activation upon injury.ConclusionsTogether, our studies show that loss of Par1b/MARK2 switches microglia from a surveillant to a primed state during development, resulting in an increased neuroinflammatory response to insults.

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The cell polarity kinase Par1b/MARK2 activation selects specific NF-kB transcripts via phosphorylation of core mediator Med17/TRAP80.

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