Abstract
Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2+/+ and Cdk5rap2+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.
Highlights
CDK5RAP2 was identified based on its ability to interact with the Cdk5 regulatory subunit, p35 [1]
Our findings demonstrate that premature cell senescence due to CDK5RAP2 loss-of-function occurs via elevation of glycogen synthase kinase-3β (GSK3β) activity that causes β-catenin-mediated downregulation of WIP1 and subsequent upregulation of p53 Ser15 phosphorylation
Loss of CDK5RAP2 induces senescence To investigate how CDK5RAP2 loss-of-function causes proliferation defects associated with developmental disorders, we examined embryos with reduced body weight display increased SA-β-gal staining compared to Cdk5rap2+/+ and Cdk5rap2+/an embryos
Summary
CDK5RAP2 was identified based on its ability to interact with the Cdk regulatory subunit, p35 [1]. CDK5RAP2 localizes to the centrosome in a dynein-dependent manner [2] and regulates centriole engagement and centrosome cohesion during the cell cycle [3,4,5]. CDK5RAP2 moved into the spotlight as loss-offunction mutations cause primary microcephaly, an autosomal recessive neurodevelopmental disorder characterized by the small brain and cognitive deficit [6, 7]. Since CDK5RAP2 is expressed in other tissues, it is not surprising that loss-of-function mutations are further associated with other developmental disorders [8]; e.g., primordial dwarfism [9, 10] and Seckel syndrome [11]. The molecular mechanisms by which CDK5RAP2 loss-of-function mutations cause these developmental disorders to remain elusive
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