Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

No abstract

Nek2, a mammalian structural homologue of Aspergillus protein kinase NIMA, is predominantly known as a centrosomal kinase that controls centriole-centriole linkage during the cell cycle. However, its dynamic subcellular localization during mitosis suggested that Nek2 might be involved in diverse cell cycle events in addition to the centrosomal cycle. In order to determine the importance of Nek2 during mammalian development, we investigated the expression and function of Nek2 in mouse early embryos. Our results show that both Nek2A and Nek2B were expressed throughout early embryogenesis. Unlike cultured human cells, however, embryonic Nek2A appeared not to be destroyed upon entry into mitosis, suggesting that the Nek2A protein level is controlled in a unique manner during mouse early embryogenesis. Suppression of Nek2 expression by RNAi resulted in developmental defects at the second mitosis. Many of the blastomeres in Nek2-suppressed embryos showed abnormality in nuclear morphology, including dumbbell-like nuclei, nuclear bridges and micronuclei. These results indicate the importance of Nek2 for proper chromosome segregation in embryonic mitoses.

Nek2 is a mitotic kinase with multiple cellular functions involving phosphorylation of diverse substrates. Suppression of Nek2 in early mouse embryos has been shown to arrest development at the 4-cell stage with defects in mitotic spindle assembly as well as in interphase nuclear morphology. In the present study, we suppressed expression of two Nek2 centrosomal substrates, Nip2 and C-Nap1, in early mouse embryos. The development of the Nip2-suppressed embryo was arrested at the 4-cell stage with mitotic defects in the blastomeres. In contrast, C-Nap1 suppression did not produce a visible phenotype. The phenotypic similarities of the Nip2- and Nek2-suppressed embryos suggest that Nip2 may be a substrate of Nek2 that is required for mitotic spindle assembly in early mouse embryos.

A typical centrosome consists of a pair of centrioles embedded in a proteinous matrix called pericentriolar material. However, the centrosomes in the mouse oocytes and early embryos lack centrioles, but consist of the γ-tubulin-enriched vesicle aggregates. We previously revealed that Nek2 and centrobin/Nip2, a centrosomal substrate of Nek2, is critical for the mouse early embryogenesis, especially at the step of spindle assembly during mitosis. In order to expand our understanding of the biological functions of Nek2, we examined expression and knockdown phenotypes of Nek2 and its substrates, centrobin and C-Nap1, in the mouse oocyte. Nek2, centrobin and C-Nap1 in the mouse oocytes were also centrosomal. Suppression of Nek2 and its substrates did not affect meiotic resumption of the oocytes. However, meiosis of the Nek2- and centrobin-suppressed oocytes was not completed, but arrested with defects in spindle assembly. No visible phenotype was observed in the C-Nap1-suppressed oocytes. These results indicate that Nek2 is critical for proper assembly of the meiotic spindles. Centrobin may be a possible substrate of Nek2 responsible for the meiotic spindle assembly in the mouse oocytes.