Characterization of centrosomal localization and dynamics of Cdc25C phosphatase in mitosis

Abstract

In mammalian cells, three Cdc25 phosphatases A, B, C coordinate cell cycle progression through activating dephosphorylation of Cyclin-dependent kinases. Whereas Cdc25B is believed to trigger entry into mitosis, Cdc25C is thought to act at a later stage of mitosis and in the nucleus. We report that a fraction of Cdc25C localises to centrosomes in a cell cycle-dependent fashion, as of late S phase and throughout G(2) and mitosis. Moreover, Cdc25C colocalises with Cyclin B1 at centrosomes in G(2) and in prophase and Fluorescence Recovery after Photobleaching experiments reveal that they are both in dynamic exchange between the centrosome and the cytoplasm. The centrosomal localisation of Cdc25C is essentially mediated by its catalytic C-terminal domain, but does not require catalytic activity. In fact phosphatase-dead and substrate-binding hotspot mutants of Cdc25C accumulate at centrosomes together with phosphoTyr15-Cdk1 and behave as dominant negative forms that impair entry into mitosis. Taken together, our data suggest an unexpected function for Cdc25C at the G(2)/M transition, in dephosphorylation of Cdk1. We propose that Cdc25C may participate in amplification of Cdk1-Cyclin B1 activity following initial activation by Cdc25B, and that this process is initiated at the centrosome, then further propagated throughout the cytoplasm thanks to the dynamic behavior of both Cdc25C and Cyclin B1.

Female infertility in PDE3A-/-mice

“…Taken together, results in Figure 4 are consistent with the recognized association of Cdc2 with centrosomes, which are thought to be a platform for dephosphorylation of Cdc2 and activation of MPF, 56,[71][72][73] as well as with the spindle apparatus. 45,74,75 Cdc2 phosphorylates the tumor suppressor WARTS, a protein which associates with mitotic spindles and spindle poles, and regulates cell cycle progression via its interaction with zyxin, a regulator of actin filament assembly.…”

The centrosomal FOP protein is required for cell cycle progression and survival

“…1,2,34 Apart from their involvement in microtubule cytoskeleton and mitotic spindle organization, they are proposed to have a role as signaling platforms. 16,35,36 Particularly, it was proposed that centrosome or centrosome integrity is required for G 1 /S transition in cells with a preserved p53 but not in tumor cells with compromized p53-dependent cellular response. 17,19 Recently, additional studies have challenged this view and shown that the G 1 arrest is related to a stress response and not to a putative centrosome integrity checkpoint.…”

“…Numerous studies involve the centrosome in the progression between different cell cycle phases (reviewed in refs. [10][11][12][13][14][15][16]. However, there is a debate concerning the role of the centrosome in the control of the G 1 to S phase transition.…”

Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis

“…Immunofluorescence study MDS-L cells were centrifuged onto cytospin slides and fixed for 30 s in 4% formalin/50% acetone, then permeabilized with lysis buffer including 0.2% Triton X-100, 25 mM HEPES, 60 mM PIPES, 10 mM EGTA and 2 mM MgCl 2 for 30 s. Antibody staining was performed using mouse monoclonal anti-aurora kinase A (BD biosciences, San Jose, CA, USA), rabbit polyclonal anti-CDC25C(Santa Cruz Biotechnology, Santa Cruz, CA, USA) to detect centrosomes, 23 and AlexaFluor488-or AlexaFluor594-conjugated anti-IgG antibodies (Molecular Probes, Eugene, OR, USA) as secondary antibodies. The nucleus was stained with DAPI (Dojindo, Kumamoto, Japan).…”