Aurora-A Phosphorylates Augmin Complex Component Hice1 Protein at an N-terminal Serine/Threonine Cluster to Modulate Its Microtubule Binding Activity during Spindle Assembly

Abstract

Proper assembly of mitotic spindles requires Hice1, a spindleassociated protein. Hice1 possesses direct microtubule binding activity at its N-terminal region and contributes to intraspindle microtubule nucleation as a subunit of the Augmin complex. However, whether microtubule binding activity of Hice1 is modulated by mitotic regulators remains unexplored. Here, we found that Aurora-A kinase, a major mitotic kinase, specifically binds to and phosphorylates Hice1. We identified four serine/ threonine clusters on Hice1 that can be phosphorylated by Aurora-A in vitro. Of the four clusters, the Ser/Thr-17-21 cluster was the most critical for bipolar spindle assembly, whereas other phospho-deficient point mutants had a minimal effect on spindle assembly. Immunostaining with a phospho-Ser-19/20 phospho-specific antibody revealed that phosphorylated Hice1 primarily localizes to spindle poles during prophase to metaphase but gradually diminishes after anaphase. Consistently, the phospho-mimic 17-21E mutant reduced microtubule binding activity in vitro and diminished localization to spindles in vivo. Furthermore, expression of the 17-21E mutant led to decreased association of Fam29a, an Augmin component, with spindles. On the other hand, expression of the phospho-deficient 17-21A mutant permitted intraspindle nucleation but delayed the separation of early mitotic spindle poles and the timely mitotic progression. Taken together, these results suggest that Aurora-A modulates the microtubule binding activity of Hice1 in a spatiotemporal manner for proper bipolar spindle assembly.

Microtubule assembly during mitosis – from distinct origins to distinct functions?

“…3). The role of the different Augmin complex subunits has not been entirely resolved, but FAM29A (also known as Dgt6 and HAUS6) binds the c-TuRC, and another component, HICE1 (also known as HAUS8), associates with MTs (Tsai et al, 2011;Zhu et al, 2008). These two activities might account for targeting of the c-TuRC to a pre-existing MT.…”

“…The mitotic kinases cyclin-dependent kinase 1 (CDK1), polo-like kinase 1 (PLK1) and Aurora A have been shown to be involved in c-tubulin recruitment to the centrosome (Blagden and Glover, 2003). PLK1 and Aurora A activities are involved in c-TuRC recruitment by the Augmin complex (Johmura et al, 2011;Tsai et al, 2011) (Tables 2, 3). Although the mechanism of c-tubulin targeting in the chromosomal pathway remains poorly understood, Aurora A kinase is also required for MT nucleation and stabilization around chromatin (Bayliss et al, 2003;Sardon et al, 2008) (Table 3).…”

“…This is fundamental for our understanding of how, in only a few minutes, the cell is able to build a machinery as complex as the mitotic spindle. (Gomez-Ferreria et al, 2007;Raynaud-Messina and Merdes, 2007) Augmin complex, FAM29A (Bucciarelli et al, 2009) TPX2, RHAMM (also known as HMMR) (Groen et al, 2004) Targeting regulators PLK1, Aurora A, CDK1 (Barr and Gergely, 2007) PLK1, Aurora A (Johmura et al, 2011;Tsai et al, 2011) Ran-GTP, Aurora A Tsai et al, 2003) In the upper part of the table, the proteins involved specifically in c-tubulin targeting to the MT nucleation sites are indicated for each of the three pathways. In the lower part of the table, the main regulators of c-tubulin recruitment for each MT assembly pathway are shown.…”

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

“…Seventy‐six genes showed a genetic aberration incidence of 10% or more in CIMP‐positive RCCs ( n  = 19), whereas only four genes did so in CIMP‐negative RCCs ( n  = 87) (Tables 2 and 3). Genes encoding microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10 ,31 RP1 32 and HAUS8 ,33, 34 those involved in histone modification, such as NCOA1 ,35 those involved in cell adhesion, such as CELSR1, CELSR2 ,36 CTNND1 ,37 LAMC2 38 and TJP1 ,39 and tumor‐related genes such as BAP1 40 and ATM ,41 were frequently mutated in CIMP‐positive RCCs (Table 3). As shown in Tables 2 and 3, 235 genetic aberrations (173 non‐synonymous single‐nucleotide mutations and 62 indels) revealed by whole‐exome analysis in the initial cohort were all successfully verified by Sanger sequencing.…”

“…Aberrations of genes involved in cell adhesion, such as CELSR1, CELSR2 ,36 CTNND1 ,37 LAMC2 38 and TJP1 ,39 may affect the invasiveness and metastatic potential of CIMP‐positive RCCs (CIMP‐positive RCCs show invasive growth and distant metastasis more frequently than CIMP‐negative RCCs13). Moreover, genetic aberrations of microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10 ,31 RP1 32 and HAUS8 ,33, 34 may be correlated with dysregulation of the spindle checkpoint in CIMP‐positive RCCs. DNAH2, DNAH5 and DNAH10 encode the heavy chains of axonal dynein 31.…”