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???displayArticle.abstract??? Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C-terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and that Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development.
FIG. 1. Nucleotide and deduced amino acid sequences of Nek2A (A) and Nek2B (B). In (A), the kinase domain is boxed and the consensus
sequence for a spliced site is indicated by the small box. In (B), only a relevant region (or a 39-end region) is presented; the consensus
sequence for a splice donor site is boxed and polyadenylation signals are underlined. The nucleotide sequence data are deposited with the
DDBJ/EMBL/GenBank Data Libraries Under Accession Nos. AB019556 (for Nek2A) and AB019557 (for Nek2B).
FIG. 2. Comparison of the predicted amino acid sequences of Xenopus Nek2A (X1. A) and Nek2B (X1. B) with those of human (Schultz
et al., 1994) and mouse Nek2 (Rhee and Wolgemuth, 1997). Amino acid residues identical to those of Xenopus Nek2A are indicated by two
dots (:). The potential spliced site in Nek2A is indicated by the arrowhead. PEST-like sequences are underlined.
FIG. 3. Immunoblot analysis of in vitro-synthesized Nek2A and
Nek2B proteins. Either 1 mg of Nek2A mRNA or 1 mg of Nek2B
mRNA was translated in 50 ml of a reticulocyte lysate, and the total
lysate (5 ml) was subjected to Western blot analysis with affinitypurified
anti-Nek2B antibody. As a control, the same volume of a
reticulocyte lysate with no added mRNA was used. Positions of
Nek2A and Nek2B and sizes of protein markers (kDa) are indicated
on the right and left sides, respectively.
FIG. 4. Organ blot analysis of Nek2A and Nek2B. Total protein
(20 mg) from each of various Xenopus tissues was analyzed by
Western blot by using affinity-purified anti-Nek2B antibody. As
controls for molecular weight standards, in vitro-translated Nek2A
and Nek2B were used and are shown as A and B, respectively.
FIG. 5. Expression of Nek2A and Nek2B during oocyte maturation.
Prophase-I-arrested immature oocytes were treated with progesterone
(PG), and five oocytes each were sampled at the indicated
times and analyzed by Western blot by using affinity-purified
anti-Nek2B antibody. Each lane was loaded with proteins equivalent
to one oocyte. Germinal vesicle breakdown (GVBD) occurred
about 3 h after progesterone treatment. Periods of meiosis I (MI)
and meiosis II (MII) are indicated (cf. Furuno et al., 1994). Throughout
maturation, Nek2A was not detected at all.
FIG. 6. Expression of Nek2A and Nek2B during embryogenesis. After fertilization, five embryos each were sampled at the indicated stages
and analyzed as in Fig. 5. Each lane was loaded with proteins equivalent to one egg or embryo. UFE denotes an unfertilized egg.
NieuwkoopâFaber (N/F) stages are also indicated.
FIG. 7. Model for the function of Nek2A and Nek2B in Xenopus
development. The model is a speculative one and is presented in
the simplest form, based solely on the tissue-specific and developmental
expressions of Nek2A and Nek2B. (Though not shown,
Nek2B might also function, at least in part, for spermatogenesis,
since it was also detected, but at low levels, in the testis; see Fig. 4).
See text for details.
