Browsing by Author "Wang, Xidi"

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    Role of Cdk5rap2 in Cell Senescence and CENP-A-mediated Centromeric Chromatin Integrity
    (2020-04-22) Wang, Xidi; Lee, Kee-Young; Mains, Paul E.; Lees-Miller, Susan P.; Riabowol, Karl T.
    Cyclin-dependent kinase 5 (Cdk5) regulatory subunit-associated protein 2 (Cdk5rap2, also known as MCPH3) is one of the genes mutant in primary microcephaly (MCPH), an autosomal recessive neurodevelopmental disorder characterized by small brain and cognitive deficit. In fact, Cdk5rap2 is most abundant at the luminal surface of the brain’s ventricular zone, particularly in cells lining the ventricular wall where neural stem and progenitor cells reside. However, since Cdk5rap2 is also expressed in other tissues, it is not surprising that loss-of-function mutations in Cdk5rap2 are further associated with cochlear and retinal developmental defects as well as primordial dwarfism, a developmental disorder associated with small body size and other growth abnormalities. The molecular mechanisms by which Cdk5rap2 loss-of-function mutations cause these developmental disorders remain obscure. In this study, I show that loss of Cdk5rap2 induces cell senescence through activation of p53 and subsequent downregulation of the wild-type p53-induced phosphatase 1 (WIP1). Using BJ-5ta human foreskin fibroblast cells, I show that Cdk5rap2 loss causes increased (i) senescence-associated heterochromatin foci (SAHF), which colocalize with that SAHF marker, heterochromatin protein 1γ (HP1γ), (ii) SA-β-gal staining, (iii) p53 activation as measured by increased phosphorylation at Ser15, (iv) p16INK4a and p21CIP1, and (v) G0/G1 population, resulting in reduced cell proliferation. Interestingly, increased phosphorylation of p53 at Ser15 does not correlate with activation of the p53 Ser15 kinases, Ataxia Telangiectasia Mutated (ATM), checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), but correlates with decreased level of the p53 phosphatase, WIP1. Ectopic expression of WIP1 reverses the senescent phenotypes observed in cells depleted of Cdk5rap2, indicating that cell senescence due to loss of Cdk5rap2 is linked to down-regulation of WIP1. Since the WIP1 promoter contains an NF-κB binding site and β-catenin-associated NF-κB affects the expression of NF-κB target genes, I tested whether loss of Cdk5rap2 that reduces WIP1 level influences β-catenin expression. Indeed, I found that loss of Cdk5rap2 significantly reduces nuclear β-catenin level. Because β-catenin phosphorylation by GSK3β results in its degradation through the ubiquitin-proteasome pathway, I further examined whether Cdk5rap2 interacts with GSK3β and affects GSK3β activity. My studies show that Cdk5rap2 interacts with GSK3β and such interaction causes increased GSK3β phosphorylation and subsequent decrease in GSK3β activity. Consistently, Cdk5rap2-depleted cells exhibit decreased GSK3β phosphorylation and increased GSK3β activity. Depletion of GSK3β increases β-catenin and WIP1 levels while depletion of β-catenin inhibits WIP1 expression. These findings suggest that GSK3β activation due to Cdk5rap2 loss causes β-catenin phosphorylation and degradation, and subsequent downregulation of WIP1. The potent GSK3β inhibitor, TWS119, which increases β-catenin level and upregulates WIP1 expression, recues cell senescence due to Cdk5rap2 loss. TWS119 together with the potent WIP1 inhibitor, GSK2830371, causes recurrence of senescence due to Cdk5rap2 loss, suggesting that GSK3β activity controls the β-catenin/WIP1-mediated senescence due to Cdk5rap2 loss. Promoter bashing analysis by transfecting a luciferase reporter vector carrying wt WIP1 promoter (pGL3-WIP1) into cells depleted of β-catenin or Cdk5rap2 reveal reduced luciferase activity compared to cells that are not depleted of β-catenin or Cdk5rap2. Conversely, transfection with NF-κB binding site-deleted WIP1 (pGL3-WIP1-ΔκB) show no difference in luciferase activity in β-catenin- or Cdk5rap2-depleted and non-depleted cells. Reduced luciferase activity in Cdk5rap2-depleted cells transfected with pGL3-WIP1 is reversed by TWS119, suggesting that β-catenin/WIP1-mediated senescence due to Cdk5rap2 loss is controlled by GSK3β through regulation of the WIP1 promoter via β-catenin. Potentially, cell senescence contributes to the developmental disorders associated with loss-of-function of Cdk5rap2. In previous studies, loss of Cdk5rap2 or CENtromeric Protein A (CENP-A) was shown to trigger the occurrence of lagging chromosomes, leading me to investigate a possible link between these two proteins. I demonstrate that Cdk5rap2 loss causes reduced CENP-A expression while ectopic Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. In this regard, I found that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Loss of Cdk5rap2 particularly causes reduced centromeric CENP-A, which is known to induce kinetochore recruitment. I found that aside from manifesting lagging chromosomes, cells lacking Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation, indicating that loss of Cdk5rap2 compromises centromeric chromatin integrity through downregulation of CENP-A.