240 M.Sasabe·Y.Machida
(STD)/TETRASPORE(TES), respectively (Nishihama et al. 2002; Strompen
et al. 2002; Yang et al. 2003). Loss-of-function mutations inAtNACK1/HIK
andSTD/TES/AtNACK2result in the occasional failure of somatic and male-
meiotic cytokinesis, respectively (Hülskamp et al. 1997; Spielman et al. 1997;
Strompen et al. 2002; Nishihama et al. 2002; Yang et al. 2003). Recently, it has
been shown that these genes have redundant functions and are essential for
cytokinesis during both male and female gametogenesis (Tanaka et al. 2004).
2.3
Identification of Other Components of the MAPK Cascade
Activated by the NACK/NPK1 Complex
To isolate downstream factors of NPK1, we used a yeast genetic system that
is based on the yeast osmosensing MAPK cascade, which includes Ssk2/22p
and Ste11p MAPKKK, Pbs2p MAPKK, and Hog1p MAPK (Brewster et al.
1993; Maeda et al. 1994, 1995). Expression ofNQK1(Nicotiana kinase next
to NPK1)cDNAreplaced the function of the yeast MAPKK in the presence
of both NPK1 and NACK1 (Soyano et al. 2003). NRK1 (Nicotianakinase next
to NQK1) MAPK was isolated as a binding partner of NQK1 using a yeast
two-hybrid system (Soyano et al. 2003).
Characterization and function of NQK1 MAPKK. The cDNA cloned by the
yeast genetic system encoded a putative polypeptide that had high homology
to members of the MAPKK family, and the amino acid sequence of NQK1 was
identical to that of NtMEK1 (Calderini et al. 2001). Of the 10 MAPKKs inAra-
bidopsis, AtMKK6 (Ichimura et al. 2001) has the highest amino acid sequence
homology with NQK1/NtMEK1.
To examine the biochemical and biological function of NQK1/NtMEK1,
we prepared a DNA construct for expression of a kinase-defective mutant
of NQK1 in which the deduced ATP-binding site was mutated (NQK1:KW).
This kinase-defective NQK1 construct could not complement the osmosens-
ing MAPK of yeast cells that have thepbs2mutation, which was used for
the screening ofNQK1cDNA (Soyano et al. 2003), suggesting that NQK1
acts as a MAPKK. Recombinant NQK1 protein is phosphorylated by the ac-
tive form of NPK1 and is activated (Soyano et al. 2003). Although NPK1 and
NACK1 proteins rapidly disappear after M phase, NQK1 proteins accumu-
late throughout the cell cycle. The activity of NQK1 in tobacco cells, however,
increases at the late M phase of the cell cycle and decreases thereafter. The
pattern of NQK1 activity is similar to that of NPK1 as well as to the pattern
of NACK1 accumulation (Fig. 3; Nishihama et al. 2001, 2002; Soyano et al.
2003). Green fluorescent protein (GFP) fusions of NQK1 and AtMKK6 also
localize to the phragmoplast equator, similar to NPK1 and NACK1 (our un-
published results). These results suggest that activation of NPK1 MAPKKK by
NACK1 binding causes the activation of NQK1 MAPKK at the equator of the
phragmoplast during late M-phase.