kDa protein that cross-reacted with a cdc2 antibody to see if the p34 was complexed with other proteins.
Fractions containing the p34 were used for in vitro H1K studies. Kinase activity was highest in the higher
molecular mass fractions and was correlated with the presence of dephosphorylated p34 [97]. The protein
p13suc1is a protein that binds Cdks tightly enough to be used as a nonspecific Cdk affinity purification
reagent [21]. It was first identified in yeast and later found to be a component of the p34cdc2protein ki-
nase [131]. A human homologue of this protein forms a hexamer. It was suggested that this hexamer acts
as a hub for Cdk multimerization in vivo [132]. Protein fractions precipitated using p13suc1-Sepharose
beads also displayed H1K activity in maize, wheat, and pea [133–135]. H1K assays defined a minor peak
at G 2 /M and a much stronger peak at G 1 /S in extracts from synchronous pea root tips and alfalfa cell cul-
tures [21,136]. More recently, histone H1 kinase specifically associated with A-type Cdks has been ana-
lyzed in alfalfa [99,118], Arabidopsis, and tobacco [66]. Most results showed high kinase activity in S,
G 2 , and M phases and largely reduced activity in G 1. On the other hand, the B-type Cdks showed a peak
of H1 kinase activity in M phase [66,99]. A protein kinase that phosphorylated the heat shock protein
AtHSF1 in Arabidopsiswas identified as CDC2a, suggesting a possible regulatory interaction between
heat shock response and cell cycle control in plants [137].
The WEE1-type phosphorylation site sequence in the species for which it was reported is generally
conserved (GEGTYGV), the notable exception being AntirrhinumCDC2b, which has a nonconserved
substitution of Thr-14 by Ala [130]. The CAK phosphorylation site sequence has more variation, with
RTFTHEV being the most common sequence motif (Table 4). Evidence of the phosphorylation of these
sites is increasing. Multiple signals have been found around 34 kDa in immunoblots of plant proteins
probed with cdc2 antibody, which could be due to different phosphorylation states [97,98,122,138] but
could also be due to cross-reactivity with other members of the Cdk family. Yamaguchi et al. [36] found
that rice R2 could complement CAK deficiency in budding yeast and could phosphorylate rice CDC20s-
1 in vitro and the C-terminal domain of the large subunit of RNA polymerase II. Phosphorylation of RNA
polymerase II as well as Cdks by CAK has also been demonstrated in animals [139,140]. An Arabidop-
sisCdk (CAKAt) was isolated by complementation of a CAK-deficient yeast mutant [141]. It was able to
phosphorylate human Cdk2 but not RNA polymerase II. As phosphorylation by CAK of both Cdks and
RNA polymerase II has been shown in animals, CAKAt is unique in not phosphorylating RNA poly-
merase II. However, a CAK identified in Saccharomyces cerevisiaehas the same capability to phospho-
rylate the yeast Cdk, Cdc28p but not RNA polymerase II [35]. Evidence that plant Cdks are phosphory-
lated can also be seen from studies of cells arrested in G 2 by the absence of cytokinin [142]. The Cdk
complexes had reduced kinase activity and high phosphotyrosine content. A p34cdc2-like protein isolated
from cytokinin-depleted cells was treated in vitro with yeast CDC25 phosphatase, which led to its de-
phosphorylation and activation. The cytokinin requirement of N. plumbaginifoliacells could be alleviated
by expression of the cdc25 gene from yeast [143]. Mutations in the ArabidopsisCdks, CDC2aAt and
CDC2bAt, in which the Thr-14 and Tyr-15 were substituted for nonphosphorylatable residues, showed
an increase in histone H1 kinase activity, which supports the negative regulation of p34 kinase activity by
phosphorylation [144].
In plant cell division, plant cells form a preprophase band (PPB), a dense array of microtubules that
aggregate at the periphery of the location where the new cell wall will form at cytokinesis [145]. Two re-
ports have linked cdc2 homologues with this band [105,138]. In onion root tip cells, immunofluorescence
microscopy using an antibody raised against the PSTAIRE motif of cdc2 revealed general staining in the
cytoplasm throughout the cell cycle with more intense staining during prophase in an area reminiscent of
the PPB. Double staining in prophase cells with PSTAIR and tubulin antibodies showed that the cdc2 ho-
mologue band was always located with the microtubule band but the cdc2 homologue band was narrower
than the microtubule band [138]. Immunofluorescence microscopy studies in maize using similar anti-
bodies showed localization of maize cdc2-like protein to the nucleus during interphase and early prophase
[105]. Colocalization with the PPB was also found for some early prophase cells in both the root tip and
subsidiary cell mother cells that give rise to the subsidiary cells of the stomatal complex in leaves. Asso-
ciation studies indicate that the PPB is necessary for localization of the kinase rather than vice versa.
These two studies suggest a role for a cdc2-like kinase in establishing the division site of plant cells. An-
other microtubule-based structure, the spindle, was also shown to be affected by Cdk/cyclin function.
Blocking of Cdks in Vicia fabaroot tips by olomoucine resulted in cells with abnormally short and dense
kinetochore microtuble fibers that were randomly arranged in the vicinity of the kinetochores and chro-
mosome arms [117].
CELL CYCLE REGULATION IN PLANTS 237