238 M.Sasabe·Y.Machida
negatively regulates NPK1 (Banno et al. 1993). Therefore we speculated that
there are activators of NPK1. Animals and yeast have several proteins that
can regulate the MAPKKKs via protein-protein interaction. For example, the
small G protein Ras (Farrar et al. 1996; Luo et al. 1996) and the 14-3-3 pro-
tein regulate Raf MAPKKK (Irie et al. 1994); TAB1 regulates TAK1 MAPKKK
(Shibuya et al. 1996); and SSK1 regulates SSK2 MAPKKK (Posas and Saito
1998).
To isolate the activators of NPK1 MAPKKK, we took advantage of a cloning
strategy using a functional yeast genetic system based on the mating
pheromone-responsive MAPK cascade, which consists of STE11 MAPKKK,
STE7 MAPKK, and FUS3 MAPK (Irie et al. 1994). This system is based on sup-
pression of a mutation in theSTE11gene for MAPKKK by a MAPKKK and
its potential activator introduced into the yeast cells from a heterologous or-
ganism. We transformed yeastste11mutant cells with tobaccoNPK1cDNA
and then introduced a tobacco cDNA library into the transformed cells. Using
this system, we isolated the tobacco cDNAs for two proteins that stimulate
the activity of NPK1 MAPKKK (Machida et al., 1998, Nishihama et al. 2002).
These cDNAs encoded two KLPs, which we designated NACK1 and NACK2
forNPK1-activatingkinesin-like proteins 1 and 2.
Structure and biochemical characterization of NACK proteins.Coexpres-
sion experiments in yeast cells revealed that NACK1 associates with NPK1
and increases the activity of NPK1 (Nishihama et al. 2002). In tobacco BY-2
cells, theNACK1andNACK2mRNAs and NACK1 protein accumulate only at
the M phase of the cell cycle, which is consistent with the increase of NPK1
kinase activity (Nishihama et al. 2002).
The amino-terminal halves of NACK1 and NACK2 contain sequences that
are similar to that of the MT-based motor domain, which are also conserved
among various KLPs, whereas the carboxy-terminal halves have typical stalk
domains with coiled-coil structures (Fig. 2A; Nishihama et al. 2002). Yeast
two-hybrid and in vitro immunoprecipitation assays using recombinant pro-
teins have shown that the stalk domain of NACK1 binds directly to the reg-
ulatory domain of NPK1 via these predicted coiled-coil structures (Ishikawa
et al. 2002). This direct interaction with NACK1 seems to regulate the sub-
cellular localization as well as the activity of NPK1 (Fig. 2C). During late
anaphase and telophase, NACK1 is consistently localized to the equatorial
zone of the phragmoplast, similar to NPK1 (Fig. 2B), whereas the deletion
of the regulatory domain of NPK1, which contains the NACK1-binding site,
eliminates its localization to the equator of the phragmoplast (Nishihama
et al. 2002). This suggests that NACK1 is responsible for the proper localiza-
tion and activation of NPK1 (Fig. 2C).
The motor and stalk domains of NACK1 also have several consensus se-
quences for phosphorylation by CDKs (Fig. 2A). Recently, Weingartner et al.
(2004) reported that overexpression of the constitutively active form cyclin
B1 disrupts the proper localization of NACK1 on phragmoplast MTs dur-