Abstract
ABSTRACTImportin-α serves as an adaptor linking importin-β to proteins carrying a nuclear localization sequence (NLS). During interphase, this interaction enables nuclear protein import, while in mitosis it regulates spindle assembly factors (SAFs) and controls microtubule nucleation, stabilization and spindle function. Here, we show that human importin-α1 is regulated during the cell cycle and is phosphorylated at two sites (threonine 9 and serine 62) during mitosis by the major mitotic protein kinase CDK1–cyclin B. Mutational analysis indicates that the mitotic phosphorylation of importin-α1 inhibits its binding to importin-β and promotes the release of TPX2 and KIFC1, which are then targeted like importin-β to the spindle. Loss of importin-α1 or expression of a non-phosphorylated mutant of importin-α1 results in the formation of shortened spindles with reduced microtubule density and induces a prolonged metaphase, whereas phosphorylation-mimicking mutants are functional in mitosis. We propose that phosphorylation of importin-α1 is a general mechanism for the spatial and temporal control of mitotic spindle assembly by CDK1–cyclin B1 that acts through the release of SAFs such as TPX2 and KIFC1 from inhibitory complexes that restrict spindle assembly.
Highlights
The mitotic spindle, composed mainly of microtubules (MTs) and MT-associated proteins (MAPs), functions to distribute the duplicated genome to daughter cells during cell division (Petry, 2016)
Importin-α1 is highly expressed in mitosis and is required for proper spindle assembly To investigate the functions of importin-α1 during the cell cycle, we first analyzed the expression levels of importin-α1 in human HeLa cells and we found that the protein is expressed 2- to 3-fold more highly in mitosis than in interphase (Fig. 1A)
When cells were synchronized at different stages of the cell cycle we found that, while importin-β was maintained throughout the cell cycle, the relatively high level of importin-α1 in early G1 was reduced in S and G2 phases and increased in mitosis (Fig. S1A)
Summary
The mitotic spindle, composed mainly of microtubules (MTs) and MT-associated proteins (MAPs), functions to distribute the duplicated genome to daughter cells during cell division (Petry, 2016). Importin-α contains an importin-β binding (IBB) domain in its N-terminus and two nuclear localization sequence (NLS)-binding sites (major and minor) in its C-terminus. It serves as a linker between importin-β and NLS-containing cargo to form a trimeric complex that is transported into the nucleus (Cingolani et al, 1999; Conti and Kuriyan, 2000; Conti et al, 1998; Giesecke and Stewart, 2010; Kobe, 1999). Once importin-β encounters Ran-GTP around chromosomes, it switches conformation, dissolving the trimer and releasing the SAF to function in mitotic spindle assembly (reviewed in Clarke and Zhang, 2008). While other importin-α isoforms recognize specific non-classical cargoes, the most conserved isoform, importin-α1 (encoded by KPNA2) recognizes classical NLSs for nuclear import and is likely to be the major regulator of SAFs in mitosis (Pumroy and Cingolani, 2015)
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