Regulation of Cytokinesis by a MAP Kinase Pathway

The involvement of a MAP kinase pathway in the cytokinesis of plant cells has been studied intensively. In this section, we describe the MAP kinase path way that regulates cytokinesis in plant cells called the NACK-PQR pathway (Fig. 1).

NPK1 cDNA has been cloned from tobacco cells and has been shown to encode a member of the MAPKKK family (Banno et al. 1993). The promoter of the gene for NPK1 is active mainly in shoot and root apices in the tobacco plant, suggesting a role in cell proliferation (Nakashima et al. 1998). The NPK1 protein is localized to the nucleus during interphase; when the cells enter M-phase and the phragmoplast is formed from anaphase to cytokinesis, it is localized to the equatorial region of the phragmoplast (Nishi-hama et al. 2001; Ishikawa et al. 2002). The activity of the protein kinase is cell cycle-dependent and increases from anaphase to cytokinesis (Nishi-hama et al. 2001). The amount of protein and the activity of NPK1 decline markedly at the end of cytokinesis (Nishihama et al. 2001). Overexpression of the kinase-negative version of NPK1 in tobacco cells and plants results in multinucleate cells that contain stubs of cell walls but does not affect karyoki-nesis, suggesting that NPK1 plays an important role in the formation of cell walls (Nishihama et al. 2001).

Genetic studies of Arabidopsis homologs of NPK1 have provided direct evidence for a role of this MAPKKK in cytokinesis. Arabidopsis genome contains three genes for NPK1 homologs; ANP1, ANP2 and ANP3, (Nishihama et al.



Kinesin like protein NACK1, NACK2











MAP65-1 a

Fig. 1 MAP kinase pathways regulating cytokinesis in plants. All components of the MAP kinase pathways regulating phragmoplast expansion in tobacco and Arabidopsis are indicated. Each line indicates protein-protein binding between two components and each arrow indicates phosphorylation by the upper kinase. Pathways confirmed in biochemical experiments are shown as solid lines and arrows; unconfirmed pathways are shown as dashed lines and arrows

1997). Arabidopsis plants that have mutations in both ANP2 and ANP3 exhibit dwarfism of the plant body and weak defects in cytokinesis, and the presence of a mutation in all three genes, ANP1, ANP2 and ANP3, is gametophytic lethal (Krysan et al. 2002). These observations support the idea proposed by the study with tobacco cells (Nishihama et al. 2001).

To identify a downstream factor(s) of NPK1, a tobacco cDNA library was screened using mutant yeast cells that expressed NACK1 and NPK1 and lacked the intrinsic MAPKK, PBS2, which is involved in the osmosensing signal transduction pathway (Soyano et al. 2003). Using functional screening, a cDNA for a MAPKK was obtained and this type of MAPKK was designated NQK1. NPK1 binds and phosphorylates NQK1. Overexpression of the kinase-negative NQK1 in tobacco cells results in cytokinesis defects similar to those produced by the kinase-negative NPK1, the cells become larger than wildtype cells and contain multinuclei and cell wall stubs.

AtMKK6, one of the Arabidopsis MAPKKs, is most similar to NQK1 of tobacco, and mutants of the AtMKK6 gene also show multinucleation and stubs of cell walls resembling those found in atnack1 mutants (described below) and double mutants for ANPs (Soyano et al. 2003). These results suggest a role of NQK1 in the formation of the cell plates. However, unlike the anp1/anp2/anp3 triple mutant, the disruptions of AtMKK6 (AtMKK6-1 and AtMKK6-2) are not gametophytic lethal, suggesting the existence of another redundant gene (or genes) or that the currently studied disruptant is not a null mutant.

MAP kinase NRK1, which is located downstream of the NQK1 MAPKK, was identified using the yeast two-hybrid screening method (Soyano et al. 2003). NQK1 binds and phosphorylates NRK1 to activate the latter protein kinase in vitro. Both NQK1 and NRK1 are activated between anaphase and telophase, although the amounts of these proteins do not change throughout the cell cycle. NQK1 is very similar (only two amino acids are different) from the previously identified NtMEKl, which activates p43Ntf6 MAPK (Calderini et al. 2001). NQK1 and NRK1 are located in the equatorial region of the phrag-moplast (Calderini et al. 1998; Takahashi et al., unpublished data; Sasabe et al., unpublished data), implying that NPK1, NQK1 and NRK1 are phospho-rylated and activated in the equatorial plane of the phragmoplast. However, overexpression of kinase-negative NRK1 does not result in an abnormality of cytokinesis.

A MAP kinase that might function downstream of NQK1/AtMKK6 has been identified by the use of Arabidopsis mutants. Although the Arabidop-sis genome encodes 20 homologs of MAP kinase, only those belonging to the group B MAP kinases exhibit great structural similarity to NRK1. Our data show that recombinant NQK1 and AtMKK6 proteins phosphorylate only AtMPK4 (Takahashi et al., unpublished data). In addition, the mutant atmpk4-2 contains multinucleated cells with immature cell walls (Soyano et al., unpublished data). These results suggest that AtMPK4 is involved in cy tokinesis in Arabidopsis cells. Other MAP kinases in group B might also be involved in this cell cycle process.

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