MAP Kinase Signaling During M Phase Progression 235
2
The MAPK Cascade Regulating Plant Cytokinesis
2.1
NPK1 MAPKKK Is Required for Cell Plate Expansion
In yeast and animal cells, MAPK cascades are involved in various aspects of
the cell cycle, including entry into the cell cycle, transition from G2 to M
phase, and the spindle assembly checkpoint (Pages et al. 1993; Minshull et al.
1994; Takenaka et al. 1998; Wright et al. 1999). Some MAPKs have been re-
ported to localize to the midzone of the spindle and midbody (Shapiro et al.
1998; Zecevic et al. 1998; Willard and Crouch 2001) or to the phragmoplast
midzone (Calderini et al. 1998; Bögre et al. 1999). These findings suggest that
a MAPK cascade regulates cytokinesis.
TheNPK1gene from tobacco encodes a member of the MAPKKK fam-
ily, and the kinase domain of NPK1 can replace the functions of several
yeast MAPKKKs (Banno et al. 1993, Machida et al. 1998). TheNPK1gene
is transcribed in meristematic tissues and immature organs but not in ma-
ture organs (Nakashima et al. 1998).Arabidopsishomologs (ANP1, ANP2,
andANP3)ofNPK1are also preferentially transcribed in organs that contain
proliferating cells (Nishihama et al. 1997). These results suggest that NPK1
MAPKKKs play a role in the signaling pathway regulating plant cell division,
and further biochemical and immunological analysis in tobacco (see below)
have suggested that they are involved in formation of the cell plate (Nishi-
hama et al. 2001).
Structure of NPK1. Figure 2A shows a schematic representation of the do-
main and motif organization of NPK1. NPK1 consists of a carboxy-terminal
regulatory domain and an amino-terminal kinase domain related to that of
MAPKKKs. Deletion of the carboxy-terminal domain increases its kinase ac-
tivity, indicating that this domain is a negative regulator of NPK1 (Banno
et al. 1993; Machida et al. 1998). This domain also has a nuclear localization
signal, a coiled-coil structure, and consensus sequences for phosphorylation
by cyclin-dependent protein kinases (CDKs) (Nishihama et al. 1997). The
carboxy-terminal region was later identified as the binding site for the NACK1
KLP, which regulates the activity and localization of NPK1 (Nishihama et al.
2002; Ishikawa et al. 2002). Thus, it appears that phosphorylation of the reg-
ulatory domain by CDKs or its interaction with other factors mediates the
activation of NPK1.
Characterization of the kinase activity and the localization of NPK1 in
tobacco cells.InBY-2tobaccocells,thekinaseactivityofNPK1specifically
increases late in the M phase of the cell cycle (Fig. 3), following transcription
of theNPK1gene and accumulation of NPK1 protein at the initiation of the
M phase (Nishihama et al. 2001). NPK1 is localized in the nucleus at inter-
phase and prophase prior to breakdown of the nuclear envelope, whereas it