Abstract
EG5 (KIF11) is a member of the kinesin-like protein family involved in centrosome separation and bipolar spindle formation. When a cell enters mitosis, CDK1 phosphorylates EG5 at Thr926 and promotes EG5 localization on the mitotic spindle which drives bipolar spindle formation. EG5 provides power for spindle movement and thus controls the dynamics of spindle assembly. However, little is known about EG5 regulation or how EG5 detaches from the spindle upon mitotic exit. In this study we identify EG5 as a novel substrate of PP2A phosphatase, and we show that the PP2A/B55α complex plays an important role in mitotic exit by a mechanism involving EG5. The PP2A/B55α complex physically associates with the EG5 C-terminal tail domain and dephosphorylates EG5 at Thr926 that enables mitotic exit. Conversely PP2A knockdown cells show a high level of phospho-EG5 in late metaphase, which is associated with a delay in mitotic exit. These phenotypic features are similar to those induced by EG5/T926D transfection that mimics phosphorylated EG5 status. Our results argue that PP2A controls mitotic exit through EG5 dephosphorylation. Lack of PP2A leads to abnormal EG5 activation, resulting in delay of mitotic exit.
Highlights
Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is ubiquitously expressed in eukaryotic cells which consists of a complex with three subunits including a scaffold subunit A, a catalytic subunit C, and a regulatory subunit B14, 15
To evaluate the effect of PP2A knock down, over 1000 cells were imaged and the mitotic index was calculated as the percentage of cells undergoing mitosis
We found HeLa cells with shPP2A/Cα-1 did not align at the metaphase plate, failed to complete mitosis, and underwent cell death (Fig. 1C, middle row, Fig. 1D), which is consistent with what has previously been reported[31]
Summary
Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is ubiquitously expressed in eukaryotic cells which consists of a complex with three subunits including a scaffold subunit A, a catalytic subunit C, and a regulatory subunit B14, 15. CDK1 is a key regulator of the mammalian cell cycle[21], and its degradation follows activation of the anaphase-promoting complex (APC). Sister kinetochores attach to opposite poles of the spindle and align at the metaphase plate, and APC promotes mitotic exit[23, 24]. Sister chromatids separate and move to opposite poles following the spindle fibers[25] This progressive movement of the spindle and chromosomes is a prominent morphologically identifiable change driven by motor proteins such as dynein and kinesins that generate the forces which result in chromosome separation. The motor domain at the head of EG5 hydrolyzes ATP to generate energy for the movement of EG5 along the microtubule, creating an outward force for spindle separation. We evaluate the timing and role of PP2A dephosphorylation of EG5 in mitosis and the consequences of failure of dephosphorylation
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