Aurora A Regulates the Activity of HURP by Controlling the Accessibility of Its Microtubule-binding Domain

Abstract

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

The RanGTP gradient – a GPS for the mitotic spindle

“…Aurora A phosphorylation promotes spindle-pole formation by activating HURP binding to microtubules (Wong et al, 2008), by inhibiting the ubiquitylation activity of BRCA1 (Sankaran et al, 2007) and by enhancing the promotion of microtubule growth at spindle poles by TACC/maskin; (which occurs through its interaction with XMAP215) (reviewed in Barr and Gergely, 2007). The availability of TACC/maskin as a substrate also depends on its release from importins (Albee et al, 2006), augmenting the local effect of RanGTP.…”

FAM29A, a target of Plk1 regulation, controls the partitioning of NEDD1 between the mitotic spindle and the centrosomes

“…Rabbit anti-FAM29A and anti-HURP antibodies were described previously (Wong and Fang, 2006;Wong et al, 2008;Zhu et al, 2008). The rabbit anti-NEDD1 antibody was kindly provided by Jens Luders (Stanford University, Palo Alto, CA).…”

Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules

“…Human hepatoma upregulated protein (HURP) is an Aurora A substrate up-regulated in hepatomas (14,15). HURP stabilizes kinetochore fibers (Kfibers) and promotes nucleation and crosslinking of microtubules (16)(17)(18)(19). In Xenopus egg extract, anti-HURP antibodies disrupt the formation of chromosome-and centrosome-induced spindles (16), suggesting the involvement of HURP in both mechanisms.…”

“…In Xenopus egg extract, anti-HURP antibodies disrupt the formation of chromosome-and centrosome-induced spindles (16), suggesting the involvement of HURP in both mechanisms. HURP also has been characterized as a direct cargo of importin β, involved in RanGTP-regulated spindle (Ran spindle) assembly in the vicinity of chromosomes (17)(18)(19). Because the kinase activity of Aurora A is essential to the formation of Ran spindles (16), HURP has been proposed to be phosphorylated at the spindle poles by Aurora A, thereby allowing its translocation to RanGTP-dependent K-fibers (17).…”

“…Because HURP expression is cell-cycle dependent and limited to prophase through anaphase, investigating how HURP is temporally regulated by phosphorylation would require rapid inhibition of the kinase activity of Aurora A, which is not achievable using RNAi or other genetic methods (15,19). Here we use the Aurora kinase inhibitors we developed in house to dissect the Aurora-HURP pathway in the formation of spindles.…”