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.
Highlights
Tin, breakdown of the nuclear envelope, and reorganization of the microtubule cytoskeleton into a spindle capable of bipolar segregation. These events are known to be regulated in large part by cell cycle-dependent protein kinases, most notably members of the Cdk, Plk, Aurora, and Nek (NIMA-related kinase) families [1]
NIMA was first identified in a screen for conditional loss-of-function mutants that were unable to enter mitosis [3]. nimA mutants remain blocked in G2 despite activation of cyclin-dependent kinase 1 [4], whereas overexpression of wild-type NIMA drives cells into mitosis from any point in the cell cycle with premature condensation of chromosomes and spindle formation [5]
Upon overexpression, there is a significant increase in accumulation of Nek2C in the nucleus as compared with Nek2A or Nek2B, and through mutagenesis we show that this is the result of the eight-amino acid deletion creating a strong nuclear localization sequence (NLS) in Nek2C that is weaker in Nek2A and absent in Nek2B
Summary
Plasmid Construction—pCMV-mycNek2A, pCMV-mycNek2AK37R, and pCMV-mycNek2B have been previously described [15, 19]. Immunoprecipitation (IP) Kinase Assays—For IP of proteins generated by IVT, 10 l of pre-washed protein G-Sepharose beads (Sigma) were added to 490 l of Neb buffer [30] containing 9 l of nonradioactive IVT reaction and mixed on a rotating wheel for 30 min at 4 °C before centrifugation at room temperature for 10 s. 40 l of protein G-Sepharose beads were blocked with 5 l of rabbit reticulocyte lysate in 500 l of Neb buffer on a rotating wheel for 30 min. B, agarose gel analysis of reverse transcription-PCR reactions performed on RNA extracted from cell lines or Nek2-containing plasmids. Immune complexes generated by IP were Invitrogen), mouse anti-phospho-H3
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