Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase

Abstract

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.

Cell cycle regulation by the NEK family of protein kinases

“…Thus, NEK2 might contribute to the release of HMGA2 from chromatin as cells progress from the pachytene stage of G2 into M phase. Interestingly, the NEK2C splice variant has a functional nuclear localisation signal that is absent from other splice variants, which supports the idea of a specific nuclear function for this isoform (Wu et al, 2007). However, the way in which NEK2 is activated by the MAPK pathway is not known.…”

Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint restricted Cdc20

“…Spindle checkpoint proteins typically prevent the binding of Cdc20 to a destruction motif such as the D-box or the KEN box. The Nek2 gene is spliced into three different isoforms of which Nek2A is the longest (Wu et al, 2007). This is the only isoform to contain an evolutionary conserved KEN box.…”

Nek2A phosphorylates and stabilizes SuFu: A new strategy of Gli2/Hedgehog signaling regulatory mechanism

“…The C terminus of Nek2A, but not Nek2B, contains both a binding site for protein phosphatase 1 and motifs targeting the protein for ubiquitin-mediated degradation after mitotic entry. The third splice variant, Nek2C, lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A and shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization [21].…”