Abstract
Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.
Highlights
IntroductionThe exact execution of both the centrosome cycle and function lies behind the correct formation of bipolar spindles that, in turn, allows faithful chromosome segregation
With Polo-like kinase 1 (Plk1)—To determine whether ASAP interacts with Plk1, we first investigated ASAP spatiotemporal expression relative to Plk1 during the cell cycle in U-2 OS cells by immunofluorescence analysis
In contrast to the ASAPS289A mutant, ASAP-WT and the phosphomimetic mutant ASAP-S289E largely rescued the spindle pole defects due to ASAP depletion (Fig. 6D). These data show that the ASAP role in the maintenance of spindle pole integrity we describe in this paper, depends, at least in part, on its phosphorylation on Ser289 by Plk1
Summary
The exact execution of both the centrosome cycle and function lies behind the correct formation of bipolar spindles that, in turn, allows faithful chromosome segregation Deregulation of these events could impair accurate inheritance of genetic material. The Ser/Thr mitotic kinases Aurora A and Polo-like kinase 1 (Plk1) are considered key regulators of centrosome maturation in different organisms [2, 7,8,9]. Their deregulation has been linked to centrosome abnormalities, drug resistance, and oncogenesis [10]. Because ASAP is a mitotic substrate of Aurora A, we investigated whether ASAP could functionally interact with Plk as well
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