EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A

Abstract

EB1 (end-binding protein 1) is a key player in the regulation of microtubule dynamics. In concert with its binding partners, adenomatous polyposis coli and p150 glued , EB1 plays a crucial role in a variety of microtubule-based cellular processes. In this study we have identified in a yeast two-hybrid screen the mitotic kinase and chromosome passenger protein Aurora-B as a binding partner of EB1. GST pull-down and immunoprecipitation experiments reveal a specific interaction between Aurora-B and EB1 both in cells and in vitro . Immunofluorescence microscopy shows that these two proteins colocalize on the central spindle in anaphase and in the midbody during cytokinesis. Kinase assays using both immunoprecipitated and purified Aurora-B demonstrate that EB1 is not a substrate of Aurora-B. Rather, EB1 positively regulates Aurora-B kinase activity. EB1 overexpression remarkably enhances Aurora-B activity and knockdown of its expression impairs Aurora-B activity. Our data further show that EB1 is able to protect Aurora-B from dephosphorylation/inactivation by protein phosphatase 2A (PP2A) by blocking PP2A binding to Aurora-B. These findings establish Aurora-B as an EB1-interacting protein and suggest that EB1 stimulates Aurora-B activity through antagonizing its dephosphorylation/inactivation by PP2A.

A protein phosphatase 2A complex spatially controls plant cell division

“…One of these could be the cyclin-dependent kinase CDKA;1, previously shown to decorate the late PPB and to induce its disassembly [39][40][41][42] , and which co-purified with TON1 in TAP experiments 43 . Aurora kinases, which in animals are functional antagonists of PP2A at the centrosome and microtubule plus ends [44][45][46] , are also putative candidates as suggested by recent results pointing to a function for Aurora kinases in premitotic division plane orientation and cortical cytoskeleton organization 47,48 . Besides a role in PPB formation, the TTP complex likely has a role in interphase cortical array organization as well.…”

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors

“…The kinase activity of Aurora B is regulated by its phosphorylation level (25,26), and PP2A is a well-known negative regulator of Aurora B (26). Thus, depletion of PPP2R1A may enhance Aurora B activity by increasing its phosphorylation (33,34).…”

“…PP2A binds and dephosphorylates Aurora B (25,26). To determine whether Aurora B is involved in the PPP2R1A depletioninduced phenotype, control luciferase siRNA or PPP2R1A siRNA was transfected with or without Aurora B siRNA to HeLa and Mad2-overexpressing HeLa cells, and then a colony formation assay was performed.…”

Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

“…Using Lamin B1 and DAPI staining, we demonstrated that the nuclear envelope was closed and chromosomes were decondensed (Figure 1B); however, in CLL doublet cells the cytoplasm was interconnected (Figure 1A). When we performed immunofluorescence staining for Plk1 and Aurora B, proteins known to regulate cytokinesis,11, 12 we found that Plk1 localized at the cytokinesis contractile ring in CLL doublets (Figure 1C), which is a hallmark of cytokinesis 11. Taken together, our results indicate that CLL cell doublets were in the cytokinesis stage of the cell cycle, specifically, after reformation of the nucleus, but before abscission and physical separation of the cytoplasm 11, 13, 14, 15, 16…”