HURP localization in metaphase is the result of a multi-step process requiring its phosphorylation at Ser627 residue

Abstract

Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin β, in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.

Molecular interplay between HURP and Kif18A in mitotic spindle regulation

“…In comparison, the 285-400 segment of HURP markedly reduces Kif18A’s velocity, which may be due to an interaction between this region and the 1-480 segment of Kif18A. Interestingly, a cross-talk between the N and C-termini of HURP has been described in previous studies, showing that HURP phosphorylation in the C-terminus by the Aurora A kinase can regulate accessibility of its N-terminus and contribute to HURP localization 29,41,42 . Therefore, the C-terminus of HURP may also regulate the activating role of the HURP N-terminus, resulting in fewer and slower Kif18A runs on the microtubule.…”

“…HURP-mediated activation could promote the enrichment of Kif18A in chromatin-proximal regions of the K-fibers, where HURP localizes during metaphase forming a comet-like gradient 15,42 . Yet, due to saturation of the binding sites by HURP, we observed that increased recruitment of motors to microtubules does not lead to productive motility at higher HURP concentrations.…”

“…The microtubule binding mechanism of HURP resembles that of another SAF and stabilizer, TPX2 36,51,52 . Both HURP and TPX2 are regulated by the Ran-GTP pathway 14,53 , both interact with mitotic kinesins (HURP with Kif18A and Kif11/Eg5 17,42 and TPX2 with Kif11/Eg5 and Kif15 54–56 ), and both nucleate and stabilize K-microtubules 14,41,57–60 . The structured binding motif in HURP stabilizes lateral interactions, while TPX2 establishes both lateral and longitudinal contacts.…”

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2

“…Apart from RanGTP, other studies have determined that the function of human HURP is also positively regulated by Aurora A phosphorylation. Phosphorylation of the C-terminus of human HURP is necessary for localization to spindle MTs 29,52 . Furthermore, this phosphorylation is hypothesized to contribute to its dynamic localization to the MTs near chromosomes during metaphase 29 .…”

“…Interestingly, in acentrosomal systems, such as Drosophila oocytes, mouse oocytes, and Xenopus egg extract, loss of HURP leads to spindle assembly defects and a reduction of spindle MT density 17,[26][27][28] . Additionally, HURP directly or indirectly interacts with TPX2 and augmin subunits in various species [28][29][30] , which prompted us to investigate HURP's role in branching microtubule nucleation.…”