Abstract
Progression through mitosis, the cell cycle phase deputed to segregate replicated chromosomes, is granted by a protein phosphorylation wave that follows an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1, the major mitosis-promoting enzyme. To ensure correct chromosome segregation, the safeguard mechanism spindle assembly checkpoint (SAC) delays Cdk1 inactivation by preventing cyclin B degradation until mitotic spindle assembly. At the end of mitosis, reversal of bulk mitotic protein phosphorylation, downstream Cdk1 inactivation, is required to complete mitosis and crucially relies on the activity of major protein phosphatases like PP2A. A role for PP2A, however, has also been suggested in spindle assembly and SAC-dependent control of Cdk1. Indeed, PP2A was found in complex with SAC proteins while small interfering RNAs (siRNAs)-mediated downregulation of PP2A holoenzyme components affected mitosis completion in mammalian cells. However, whether the SAC-dependent control of Cdk1 required the catalytic activity of PP2A has never been directly assessed. Here, using two PP2A inhibitors, okadaic acid and LB-100, we provide evidence that PP2A activity is dispensable for SAC control of Cdk1 in human cells.
Highlights
The PP2A holoenzyme is composed of one catalytic (C) subunit, one scaffold (A) subunit and one of many regulatory (B) subunits that provide substrate and subcellular localization specificity
Cell lysates were probed for cyclin B1 (Cyc B1) and Cdk1 to monitor Cdk1 inactivation kinetics during mitosis exit; for phosphorylated Cdk1 substrates (Cdk1 p-subs; with an anti phosphospecific antibody recognizing the sequence P-X-pS-P and pS‐P‐X‐K/R; where X = any residue and pS = phosphorylated Ser), PRC1 and phosphorylated PRC1 T481 to assess PP2A inhibition
In cells released into LB-100-containing medium, cyclin B1 was degraded with similar kinetics to control cells, dephosphorylation of Cdk1 p-subs and pT481PRC1 were substantially hampered for the duration of the experiment (Figure 1A, 1B)
Summary
The PP2A holoenzyme is composed of one catalytic (C) subunit, one scaffold (A) subunit and one of many regulatory (B) subunits that provide substrate and subcellular localization specificity. Different PP2A holoenzymes, containing different B subunits, have been involved in mitotic control [1]. Cdk, phosphorylated two closely related proteins, Arpp and Ensa, transforming them into potent interactors and inhibitors of PP2A-B55 [4]. Upon Cdk inactivation at the end of mitosis, Gwl activity downregulation allowed PP2A to dephosphorylate Arpp and Ensa and to autoactivate [4, 5, 6]. An important PP2A-B55 substrate is the Protein Required for Cytokinesis (PRC) 1, that is dephosphorylated at the end of mitosis at threonine 481 (pT481-PRC1), among several other Cdk substrates that are recognized by a commercially available anti phosphorylated Cdk1substrate (Cdk p-sub) [7]. SiRNAs-mediated downregulation of B56 and other www.impactjournals.com/oncotarget
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
