From the Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, United Kingdom and the § Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom
Received for publication, October 4, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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D-type cyclins (CycD) play key roles in linking
the Arabidopsis cell cycle to extracellular and
developmental signals, but little is known of their regulation at the
post-transcriptional level or of their cyclin-dependent
kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we
demonstrate that the CDK partner of these Arabidopsis
cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has
shown that transcript levels of CycD2 and CycD3
are regulated in response to sucrose levels and that both their
mRNA levels and kinase activity are induced with different kinetics
during the G1 phase of cells reentering the division cycle
from quiescence (1). Here we analyze the protein levels and kinase
activity of CycD2 and CycD3. We show that CycD3 protein and kinase
activity parallel the abundance of its mRNA and that CycD3 protein
is rapidly lost from cells in stationary phase or following sucrose
removal. In contrast to both CycD3 and the regulation of its own
mRNA levels, CycD2 protein is present at constant levels. CycD2
kinase activity is regulated by sequestration of CycD2 protein in a
form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.
The cell cycle in eukaryotic organisms is primarily controlled by
cyclin-dependent kinases
(CDKs)1 in complexes with
their activating and substrate-specifying partners, cyclins. The action
of these protein complexes is perhaps best understood in yeast, in
which a single CDK interacts with different cyclins during the
G1 and G2/mitotic phases of the cell cycle (2).
In comparison with yeast, relatively little is known about the control
of the plant cell cycle, but complementation of yeast strains lacking
G1 cyclins has been used to identify the functionally equivalent proteins in plants (3, 4). These approaches identified plant
D-type (CycD) cyclins as less related to the yeast G1
cyclins than to mammalian and Drosophila D-type cyclins that
function during G1 phase to control cell cycle commitment
in response to growth and mitogenic signals (5, 6). CycD cyclins have
low overall similarity with animal D-type cyclins (7) but share key
features, including an LXCXE motif at the
N terminus, which in both animal and plant D-type cyclins is the
binding site for the retinoblastoma (Rb) protein (8-10). The
interaction between animal D-type cyclins and Rb results in
phosphorylation of Rb by the cyclin D-CDK complexes, and the resultant
phosphorylated Rb is unable to bind (and therefore inhibit) E2F
transcription factors involved in the G1/S transition and
progression through S phase (reviewed in Refs. 11 and 12). The
transcription of the mammalian D-type cyclins is dependent on
stimulation by serum growth factors, and thus D-type cyclins act as
mediators of external signals in the progression of the cell cycle (5,
6).
Higher organisms express multiple CDKs, which bind distinct cyclins,
and different CDKs are therefore characterized by sequence differences
in their cyclin interaction domain. In animals, mitotic cyclins (A- and
B-type) bind CDKs that contain the conserved cyclin-binding sequence
PSTAIRE, whereas D-type cyclins bind CDK4 and -6, which have the
sequence P(I/L)ST(V/I)RE (reviewed in Ref. 13). Plants express
two main types of cell cycle regulating CDKs: those containing the
PSTAIRE sequence (CDK-a or Cdc2a proteins) and a novel CDK class with
the variant sequence PPT(A/T)LRE, which shows cell cycle
regulation (reviewed in Refs. 14 and 15). Little is known of cyclin-CDK
partnerships in plants, although an alfalfa A-type cyclin CycA2 has
recently been shown to associate with the PSTAIRE-containing Cdc2a
(16), as has a tobacco CycD3 (17).
The isolation of plant Rb and E2F-like proteins and their interaction
in vitro therefore suggests overall similarities between G1/S control in animals and plants
(17-21).2 Evidence that an
in vitro assembled complex of tobacco CycD3 and the PSTAIRE
CDK Cdc2a can phosphorylate a tobacco Rb-related protein also
substantiates the broad parallels between plant and mammalian systems
(17). In addition, mRNA levels of the Arabidopsis cyclin
D2 (CycD2) and cyclin D3 (CycD3) genes are
controlled by external growth signals. In an Arabidopsis
cell culture, CycD2 and CycD3 mRNA levels are
induced by sucrose and CycD3 mRNA levels increase in
response to the presence of the plant hormone cytokinin (1, 22).
CycD1 expression is not detectable in this culture. Overexpression of the Arabidopsis CycD2 gene in tobacco has
also been shown to increase overall plant growth rate (23), and high level expression of CycD3 results in extopic cell divisions
and altered growth (22). Taken together, these results suggests that
plant CycD levels may serve to integrate such signals with commitment
of cells to division, paralleling the role of D-type cyclins of animals.
Despite the significance of CycD activity for cell cycle control,
development, and growth, almost nothing is known of the control of
protein levels or kinase activity of CycD kinases in plants. This is
particularly significant, since post-transcriptional and
post-translational controls are of considerable importance in
determining CDK-cyclin activity (24). Moreover, the CDK partner of the
Arabidopsis CycDs is unknown. Here we utilize antibodies that specifically detect CycD2 and CycD3 proteins of
Arabidopsis to analyze CycD protein levels in an
Arabidopsis cell culture and during plant growth, to
identify the CDK partner of these cyclins, and to analyze the kinase
activity and associations of the cyclin-CDK complexes in response to
external growth signals.
Arabidopsis Cell Suspension and Plant Growth--
Suspension
cultures of Arabidopsis thaliana ecotype Landsberg
erecta (25, 26) were maintained as previously described (1).
For cell cycle reentry experiments, quiescent cells were prepared from
cultures 7 days after the previous subculture; the cells were washed
and subcultured into MS medium with hormones and all supplements except
sucrose. After 24 h, sucrose was added to the culture, and time
point samples were taken as the cells reentered the cell cycle (1, 22).
Similarly, for cell cycle exit experiments, 3-day (exponential culture
growth phase) cells were harvested and replaced in MS medium containing
hormones and all supplements except sucrose, and samples were taken at
specified times. Arabidopsis seedlings (Landsberg
erecta) were grown in liquid medium as previously described
(1). Plants were grown to flowering at 22 °C in 16 h of light
in a growth room.
Protein Methods--
Polyclonal rabbit antisera were raised
against full length Arabidopsis CycD2 expressed with a
6-histidine tag in Escherichia coli and against
Arabidopsis CycD3 C-terminal peptide
MRGAEENEKKKPILHLPWAIVATP by the antibody facility at the Babraham
Institute. Antiserum against a common C-terminal peptide found
in Arabidopsis Cdc2b and in its tobacco homologue CdkB1 has
been described (data not shown) and
antiserum against Arabidopsis Cdc2a was produced using the
C-terminal peptide ARAALEHEYFKDLGGMP at the Babraham Institute. Procedures for protein extraction, SDS-polyacrylamide gel
electrophoresis, Western blot analysis, immunoprecipitations, and
histone H1 protein kinase assays have been described (1, 23). Antiserum
was used at 1:1000 dilution and incubated with Western blots overnight at room temperature. For competition of the antisera with CycD3 peptide
or CycD2 protein, the appropriate antiserum was incubated with CycD3
peptide (at a concentration of 1 µM) or CycD2 protein (40 µg of E. coli-expressed CycD2) for 1 h at 37 °C
before probing the Western blot.
In Vitro Translation--
CycD2 and CycD3
cDNAs were cloned into pET23 (Novagen, Madison, WI), and proteins
were produced using a T7 coupled transcription-translation rabbit
reticulocyte lysate expression system with incorporation of
[35S]methionine (Promega, Madison, WI).
Specific Antisera That Immunodetect and Immunoprecipitate
Arabidopsis CycD2 and CycD3 in Active Kinase Complexes--
For
analysis of Arabidopsis CycD cyclin proteins, rabbit
antibodies were generated against full-length CycD2 protein and against a CycD3 C-terminal peptide. To verify that these antisera specifically detect the intended proteins, they were used to probe Western blots of
Arabidopsis whole cell extract (WCE) and in vitro
translated (IVT) radiolabeled CycD2 and CycD3; the antibody probes were
used both directly and in competition with the relevant purified CycD2 protein or CycD3 peptide (Fig.
1A). The CycD2 antibody
detected IVT CycD2 protein of ~46 kDa and a protein of the same size
in WCE. Neither were detected when the antibody was preincubated with
purified CycD2 protein. Similarly, the CycD3 antibody detected IVT
CycD3 and a protein of the same size (~60 kDa) from the WCE, and
these bands were not detected after preincubation of the antibody with
purified CycD3 peptide. No cross-reactivity was observed between the
CycD2 and CycD3 antisera (not shown). These results were confirmed by
immunoblots of plants overexpressing CycD2 or CycD3, in which a
stronger signal was observed (not shown).
The quantitative immunoprecipitation of CycD2 and CycD3 by these
antibodies was demonstrated by the recovery of IVT CycD2 and CycD3
(Fig. 1B). Native CycD2 and CycD3 were then
immunoprecipitated from WCE of an exponential phase culture. The
resultant immunoprecipitates were assayed for histone H1 kinase
activity to verify the presence of active CycD-CDK complexes. Both
CycD2 and CycD3 immunoprecipitates phosphorylate histone H1 (Fig.
1C). The phosphorylation of 2-4 other proteins of 90-120
kDa present in WCE was also observed, and these may represent
endogenous substrates that have coimmunoprecipitated with CycD2 and
CycD3. In addition, a protein of about 60 kDa is phosphorylated in
CycD3 immunoprecipitates, which may be a result of
autophosphorylation of CycD3 protein (27, 28).
Arabidopsis CycD2 and CycD3 Proteins Interact with the PSTAIRE CDK
Cdc2a--
In mammalian and Drosophila cells, D-type
cyclins interact with the non-PSTAIRE CDK4 and (in mammals) CDK6.
However, no direct plant homologues of these CDKs have been identified,
although plants contain two main groups of CDKs (14, 15). These are represented by the Arabidopsis PSTAIRE CDK Cdc2a and the
PPTALRE CDK Cdc2b. To establish the CDK partners of
Arabidopsis CycD2 and CycD3, the cyclins were
immunoprecipitated from exponentially growing cells, and eluants were
analyzed by protein gel blotting using antisera raised against
Arabidopsis Cdc2a and Cdc2b (Fig. 2). Cdc2a was detected in eluants from
both CycD2 and CycD3 immunoprepitates, but Cdc2b did not
coimmunoprecipitate with either cyclin. We conclude that Cdc2a is an
in vivo partner of both CycD2 and CycD3 in
Arabidopsis cells, but Cdc2b is not.
CycD2 and CycD3 Proteins Accumulate Differentially during
Arabidopsis Development--
CycD proteins probably play key roles in
activating cell division during development, so the presence of CycD2
and CycD3 was examined during Arabidopsis vegetative
development (Fig. 3A). CycD2
levels are lower in 4-day seedlings but are subsequently present at a
constant level in whole seedling/plant extracts (5-17 days), whereas
CycD3 is present at similar levels from 4 to 14 days but subsequently
declines at 17 days. In contrast, the kinase partner Cdc2a is present
at a constant level in these protein extracts. The distribution of the
protein between roots and shoots was examined in 9-day seedlings. CycD2
is primarily expressed in the shoot, although a weak signal was
detected in root extract (Fig. 3B), while CycD3 was detected
less readily than D2 and primarily in the root. In flowering
Arabidopsis plants, CycD2 was more abundant in leaves and
stem and detectable at a lower level in flowers but appeared to be
absent from root extracts. In contrast, CycD3 was present only in the
root, where it was detected weakly.
CycD3 but Not CycD2 Protein Level Is Higher in Actively Dividing
Cells--
Levels of CycD2 and CycD3 were examined throughout the
growth cycle from samples taken on consecutive days after subculture. This culture reaches maximum cell density after 6 days (1). The level
of CycD2 remained fairly constant throughout the growth cycle, compared
with CycD3 which decreased dramatically after 6 days, corresponding to
the onset of stationary phase (Fig.
4A). CycD3 is therefore
present only in cells from actively dividing cultures.
Differential Abundance and Kinase Activity of CycD2 and CycD3
Proteins after Removal of Growth Stimulation Signals--
Previous
analysis has shown the dependence of CycD2 and
CycD3 gene expression on sucrose availability (1). To
measure the response of CycD2 and CycD3 protein levels and kinase
activity to the removal of such growth stimulation signals, sucrose was removed from an exponentially growing (3-day) cell culture, and samples
were taken at specific times (Fig. 4B). CycD2 protein levels
remained relatively constant, but CycD3 protein levels decreased
rapidly with ~90% of CycD3 being degraded within 2 h of sucrose
removal. Longer exposures of this blot show that low levels of CycD3
persist for at least 7 h, but by 24 h CycD3 is almost
undetectable (Fig. 4B). In contrast to CycD2
mRNA, CycD2 protein levels are constant following sucrose removal,
whereas CycD3 protein rapidly declines in abundance. The abundance of Cdc2a and Cdc2b proteins after removal of sucrose remained constant for
at least 24 h (Fig. 4B).
When the kinase activity of immunoprecipitations was examined in a
similar experiment, a somewhat different pattern was observed. In
contrast to the constant abundance of CycD2 protein, CycD2-associated kinase activity started to decline within 4 h of sucrose removal and by 24 h was reduced by more than 50%. CycD3 kinase activity decreased to 50% of its original level within 4 h and then
declined more gradually up to 24 h. However, although CycD3
protein was almost undetectable after 24 h, there was still
one-third of the kinase activity observed in the exponentially growing
cells. This may suggest that much of the early decline in CycD3 levels
may be due to a reduction of cyclin not associated with active kinase complexes.
Differential Accumulation and Activity of CycD2 and CycD3 in Cells
Reentering the Division Cycle--
Arabidopsis cells
deprived of sucrose for 24 h resume division and show a relatively
synchronous entry into S phase when sucrose is readded (1, 22).
CycD2 and CycD3 mRNA levels decline after sucrose removal and increase after readdition. CycD2
mRNA increases within 1 h of sucrose addition, whereas
CycD3 mRNA accumulates only after 4 h,
corresponding to late G1 phase. To examine the abundance of
CycD2 and CycD3 proteins and their associated kinase activity as cells
reenter the cell cycle, early stationary phase (7-day) cells were
prepared by removing sucrose from the growth medium for 24 h, and
then the levels of D-type cyclins (Fig.
5A) and kinase activity of
their immunoprecipitations (Fig. 5B) were observed after
sucrose was readded. CycD2 was present at relatively high levels in
stationary phase cells before (data not shown) and after sucrose
removal. CycD3 was present at a low level before (data not shown)
sucrose removal and absent after 24 h without sucrose, consistent
with results observed after sucrose removal from an exponentially
growing cell culture (Fig. 4B). Fig. 5A shows
that in samples taken every hour after sucrose readdition, CycD2
remains at a constant level. During this time, cells progress through
G1, reaching S phase after 6 h (1, 22). CycD3 protein was absent in early G1 cells but started to accumulate
rapidly at 4-5 h in late G1, coincident with the increase
in CycD3 mRNA (1, 22). The abundance of Cdc2a, the CDK
partner of CycD2 and CycD3, was constant during this experiment.
The kinase activities of CycD2 and CycD3 were examined in a separate
experiment (Fig. 5B) and showed that despite the constant protein abundance of CycD2, its associated kinase activity is strongly
regulated, being very low after sucrose removal and increasing within
2 h of sucrose readdition, as previously reported (1). CycD2
kinase activity continues to increase up to 24 h after sucrose readdition, despite no change in protein abundance (Fig.
5B). In contrast, CycD3 kinase activity starts to increase
only after 4 h and largely reflects mRNA levels and protein
abundance during G1/S phase, although a further increase in
kinase activity is seen in cells 24 h after sucrose addition (Fig.
5B). We conclude that CycD2 is subject to strong
post-translational regulation.
CycD2 Does Not Interact with Cdc2a in Quiescent Cells--
The
experiments described above show that CycD2 protein abundance remains
constant in actively dividing and nondividing Arabidopsis suspension cells, although CycD2-associated kinase activity is only
found in actively dividing cells. To analyze the nature of this
post-translational regulation, we first examined whether the CDK
partner is present. Both CycD2 and Cdc2a were found in quiescent cells
(prepared as for the cell cycle reentry experiment) as well as
exponentially growing cells, although Cdc2a levels were lower than in
exponentially growing cells (Fig.
6A). However, the differences
in the kinase activity of the CycD2-Cdc2a complex were marked between
the two growth phases, and almost no activity was present in quiescent
cells (Fig. 6A).
To investigate whether the lack of kinase activity in quiescent cells
was due to the inability of CycD2 and Cdc2a to interact, CycD2 was
immunoprecipitated from both quiescent and exponentially growing cells
to identify if similar amounts of Cdc2a were coimmunoprecipitated. The
amount of Cdc2a coimmunoprecipitated in quiescent cell extract was much
lower (Fig. 6B, c), suggesting that the
interaction between Cdc2a and CycD2 was inhibited in the quiescent
state. However, reprobing these blots with CycD2 antibody (Fig.
6B, g) showed that the quantity of CycD2 protein
immunoprecipitated from quiescent cell extract was much lower than from
exponential cell extract although CycD2 was equally abundant in
quiescent cells (Fig. 6A). Thus, anti-CycD2 antiserum
efficiently immunoprecipitates CycD2 from exponential cell extract but
not from quiescent cells, a result that was observed in several
repeated experiments. From this experiment, it is therefore impossible
to determine whether CycD2 and Cdc2a are associated in quiescent cells,
since CycD2 is not immunoprecipitated.
To determine whether this inefficient immunoprecipitation is a feature
of quiescent cell extract or particular to CycD2, Cdc2a was
immunoprecipitated directly from quiescent and exponentially growing
cells, and the eluates were immunoblotted with the same antiserum (Fig.
6C, c and d). Cdc2a was
immunoprecipitated in proportion to its abundance (Fig. 6A)
from cells in both phases of growth, suggesting that the inefficient
immunoprecipitation of CycD2 in quiescent cells was a behavior specific
to CycD2. The eluted Cdc2a immunoprecipitates were immunoblotted with
anti-CycD2 antiserum (Fig. 6C, a and
b). CycD2 was efficiently coimmunoprecipitated by the
anti-Cdc2a antiserum from exponential cells (Fig. 6C,
a) but not from quiescent cells (Fig. 6C,
b). This demonstrates that the majority of CycD2 present in
quiescent cells is not associated with Cdc2a; nor, as we show in Fig.
6B, is it accessible for immunoprecipitation by the CycD2 antiserum.
We also noted that the small amount of CycD2 immunoprecipitated from
quiescent cells by the CycD2 antiserum (Fig. 6B,
g) and the amount of CycD2 coimmunoprecipitated by the Cdc2a
antiserum (Fig. 6C, b) is a similar proportion of
the equivalent immunoprecipitations from exponential cells (compare
Fig. 6B (g) with Fig. 6B
(f), and compare Fig. 6C (b) with Fig.
6C (a)). Moreover, the ratio between the amount
of Cdc2a coimmunoprecipitated by the CycD2 antiserum from quiescent and
dividing cells (Fig. 6B, c and b) is
similar to the ratio of the CycD2 directly immunoprecipitated from the
same extracts (Fig. 6B, g and f).
Taken together, this suggests that the CycD2 that was
immunoprecipitated was interacting with Cdc2a and therefore that there
is a correlation between the availability of CycD2 for
immunoprecipitation and its ability to associate with Cdc2a.
Plant D-type (CycD) cyclins play important roles in controlling
the cell cycle in development and in response to external signals (1,
3, 4, 22, 23, 29). However, despite the importance of
post-transcriptional mechanisms in regulating cyclin-CDK activity (24,
30), previous studies have examined either the consequences of CycD
misexpression (22, 23), the developmental regulation of their gene
expression, or the response of mRNA levels to external signals (1,
3, 4, 22, 29).
Here we show that the development of specific antisera to CycD2 and
CycD3 of Arabidopsis allows the identification of the CDK
partner of these cyclins and analysis of their protein abundance and
kinase activity during development and during the response to sucrose
removal and addition. We show that both CycD2 and CycD3 associate with
the Arabidopsis PSTAIRE CDK Cdc2a and that CycD2 and CycD3
show strikingly different modes of regulation at the protein level.
Tobacco CycD3 has previously been shown to associate with a CDK of the
PSTAIRE type in vitro and in BY-2 cell extract (17), and
Arabidopsis CycD1 was identified in a yeast two-hybrid
screen using Cdc2a as a bait (31). Moreover, Arabidopsis
CycD2 expressed in tobacco forms functional kinase complexes with the
PSTAIRE tobacco CDK Cdc2a (23). Here we present the first evidence that endogenous Arabidopsis CycD2 and CycD3 interact with and
form functional kinase complexes with Cdc2a in vivo, whereas
these cyclins do not interact with the non-PSTAIRE CDK Cdc2b. These results are in contrast to mammalian and Drosophila D-type
cyclins, which do not form functional complexes with PSTAIRE CDKs but
rather associate with the non-PSTAIRE-containing CDK4 and CDK6 (2, 32).
Immunoprecipitates of CycD2 and CycD3 were found to exhibit in
vitro protein kinase activity against added histone H1 as
previously reported (1, 23) and against coimmunoprecipitated proteins from the Arabidopsis cell extract. The identity of these
proteins is currently unknown, but since CycD2 and CycD3 interact with maize Rb in vitro and in yeast two-hybrid assays (10), one
candidate is the Arabidopsis homologue of the Rb protein.
The suitability of histone H1 as a substrate for plant CycD kinases (1,
23) is further confirmed, highlighting a further difference from animal cyclin D-CDK4 complexes for which histone H1 is a poor substrate. Since
Arabidopsis Rb was unpublished at the time of this work (33), we have been unable to confirm its suitability as an in vitro substrate of Arabidopsis CycD kinases, although
this would be predicted from previous analysis in vitro (10,
17).
Mammalian D-type cyclins are highly unstable proteins whose synthesis
is linked to the presence of external growth signals (5, 6, 34-36).
Previous analysis has shown that mRNA levels of plant
CycD respond to external signals of hormones and sucrose levels (1, 3, 22, 29, 37). Arabidopsis CycD2 and
CycD3 mRNA levels decline on sucrose removal and are
induced on its readdition, although the magnitude of the CycD3 response
is twice that of CycD2. Here we examined the protein levels and kinase activity of CycD2 and CycD3 and found strikingly different results.
When sucrose-starved cells were induced to reenter the cell cycle,
CycD3 protein was detectable 4-5 h after the readdition of sucrose, at
the same time as CycD3 mRNA and kinase activity are
induced (1). Since we found that Cdc2a is present throughout the
sucrose starvation, this suggests that CycD3 kinase activity is
regulated by CycD3 mRNA abundance during this
experiment. The importance of transcriptional regulation of CycD3
activity is supported by the strong phenotypes produced in
vivo from its overexpression (22).
When sucrose was removed from cells in a midexponential phase culture,
the majority of CycD3 was no longer present after 1-2 h, suggesting
that in this situation it is rapidly turned over. A residual level of
CycD3 persists for several hours, and the reduction in kinase activity
is less abrupt than the loss of CycD3 protein, suggesting that CycD3
not present in active kinase complexes is turned over more rapidly. In
this regard, it is interesting to note the low amount of Cdc2a
coimmunoprecipitated with CycD3 (Fig. 2). We note that the response of
CycD3 to sucrose removal appears to be an immediate and specific
response to loss of this signal, since the levels of other cell cycle
proteins remain constant for 24 h after sucrose removal, for
example the kinases Cdc2a and Cdc2b (Fig. 4B), although
Cdc2b abundance is cell cycle-regulated and the protein is only present
from S to M phases (14). We conclude that changes in CycD3 level are an
immediate response to sucrose removal and not an indirect consequence
of a cessation of cell division caused by declining intracellular
carbohydrate levels.
CycD2 regulation is strikingly different from CycD3 and D-type cyclins
in other organisms. Despite the regulation of its mRNA during
sucrose removal and resupply (1), CycD2 protein remains almost constant
in abundance through sucrose starvation and reentry into the cell
cycle, although its kinase activity is strongly regulated, being
activated more rapidly than that of CycD3. Further investigation showed
that CycD2 present in quiescent or sucrose-starved cells is not
associated with Cdc2a and is not accessible for immunoprecipitation. This suggests regulation of CycD2-associated kinase activity by a novel
post-translational mechanism involving its sequestration in an inactive
form. It is unknown whether the reappearance of CycD2-Cdc2a kinase
activity within 2 h of sucrose readdition is a result of release
and activation of existing CycD2 or the rising levels of CycD2 activity
result from de novo synthesis. The increase in
CycD2 mRNA shortly after sucrose readdition may be
consistent with the latter explanation. We conclude that the kinase
activity of CycD2-containing complexes is not dependent on the level of CycD2 protein, in contrast to the situation observed with CycD3.
Based on the data presented here, we present a model for activation of
cell division in Arabidopsis cells (Table
I and Fig. 7). In quiescent cells, CycD2 protein is
sequestered and presumably inactive, since it is not associated with
its CDK partner. CycD3 protein is absent. After stimulation of
division, CycD2 mRNA accumulates, CycD2 protein
associates in an active form with Cdc2a, and kinase activity starts to
accumulate within 2 h in early G1 phase. The amount of
CycD2 present in this active form is unknown. After 4 h, in late
G1, CycD3 transcript levels increase sharply, accompanied by an increase in CycD3 protein levels and kinase activity. This is
followed by entry into S phase after ~6 h.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Protein gel (Western) blots of CycD2 and
CycD3 demonstrating specificity and sensitivity of antibodies developed
against CycD2 and CycD3. A, whole cell extracts and IVT
products of CycD2 (left) and CycD3 (right) probed
with CycD2 antiserum (left) and CycD3 antiserum
(right). The left halves of blots were
probed with relevant antibody; the right halves
were probed with the antibody after precompetition with purified
protein/peptide (comp). The center
lane containing IVT CycD2/CycD3 protein was cut in half.
CycD2 antibody was competed with purified CycD2 protein; CycD3 was
competed with CycD3 C-terminal peptide. B,
immunoprecipitations of CycD2 and CycD3 IVT proteins. From
left to right, IVT protein; immunoprecipitations
(ip) of 1, 2, and 4 µl of IVT protein. C,
protein kinase assays of immunoprecipitates for the CycD2-CDK
(left) and CycD3-CDK (right) complexes using
histone H1 as an added substrate; the left lane
of each blot contains an immunoprecipitation using preimmune
sera (PI), and the right lane
contains an immunoprecipitation using immune sera specific for each
D-type cyclin protein (I). Dots indicate
coimmunoprecipitated bands from WCE that are phosphorylated. The
free standing arrow indicates a band
corresponding to potential autophosphorylation of CycD3.
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Fig. 2.
The PSTAIRE CDK Cdc2a coimmunoprecipitates
with CycD2 and CycD3 from Arabidopsis cell extracts,
but the non-PSTAIRE Cdc2b does not. Protein gel (Western) blots of
immunoprecipitations from WCE using CycD2 and CycD3 antisera were
probed with Cdc2a and Cdc2b antisera. WCE, 40 µg of whole
cell extract; PI, immunoprecipitation with preimmune serum;
I, immunoprecipitation of protein with immune serum;
ip, immunoprecipitation.
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Fig. 3.
CycD2 and CycD3 protein levels during
vegetative development and in mature tissues. A, levels
of CycD2, CycD3, and Cdc2a in seedlings/plants of 4, 5, 7, 9, 14, and
17 days grown in vitro. B, levels of CycD2 and
CycD3 in shoots and roots of 9-day seedlings. C, presence of
CycD2 and CycD3 in leaf, stem, root, and flower of mature soil-grown
Arabidopsis.
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Fig. 4.
Levels of CycD proteins during the growth
cycle of suspension-cultured cells and in response to sucrose
removal. A, protein gel (Western) blots showing levels
of CycD2 and CycD3 from the lag phase (days 1 and
2) through exponential growth (days
3-6) to stationary phase (day 7) (1).
The upper band visible in some CycD2 blots is
nonspecific and not competed by preincubation of the CycD2 antiserum
with purified CycD2 protein (data not shown). B, protein gel
blots showing levels of CycD2, CycD3, Cdc2a, and Cdc2b in response to
removal of sucrose from medium of 3-day (exponential phase)
suspension-cultured cells. 3d, WCE before sucrose removal;
further lanes are extracts at times (in hours)
after replacing cells in fresh medium lacking sucrose. Short
(above) and longer (below) exposures of CycD3 gel
blots are shown to demonstrate the continued presence of low levels of
CycD3 for at least 7 h. C, protein kinase activity
(measured in arbitrary units) of CycD2 and CycD3 immunoprecipitates
against histone H1 at the times indicated. Background phosphorylation
observed using preimmune serum for immunoprecipitation has been
subtracted from the data. Corresponding protein gel (Western) blots for
the extracts used for kinase assays are shown above the
graphs.
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[in a new window]
Fig. 5.
CycD2 and CycD3 levels and protein kinase
activity during cell cycle reentry. Stationary phase cells were
resuspended in fresh medium lacking sucrose for 24 h, and sucrose
was then added. Samples were taken for protein gel analysis at the time
of sucrose addition (0) and at later times indicated in hours.
A, protein gel blots of CycD2, CycD3, and Cdc2a levels.
B, in a similar experiment, protein kinase levels (measured
in arbitrary units) of CycD2 and CycD3 immunoprecipitates against
histone H1 were determined from the time of sucrose addition (0).
Background obtained using preimmune serum has been subtracted.
Corresponding protein gel blots for extracts used for kinase assays are
shown above the graphs.
View larger version (21K):
[in a new window]
Fig. 6.
CycD2 is present in stationary phase cells
but is not associated with Cdc2a and is inaccessible for
immunoprecipitation. A (top), protein gel
blots of WCE from exponentially growing (E) and quiescent
(Q) cells (prepared as for the cell cycle reentry
experiments) probed with antisera against CycD2 and Cdc2a. A
(bottom), histone H1 kinase activity of CycD2
immunoprecipitates from exponentially growing and quiescent cells.
PI, histone H1 kinase assays following immunoprecipitation
with preimmune sera; I, histone H1 kinase assays following
immunoprecipitation with immune sera. B, immunoprecipitates
from WCE of exponentially growing and quiescent Arabidopsis
cells using preimmune (PI) or immune CycD2 (I)
antisera were subject to protein gel blotting and probed with Cdc2a or
CycD2 antisera. CycD2 is efficiently immunoprecipitated only from
exponentially growing cell extract. C, immunoprecipitates
from WCE of exponentially growing and quiescent cells using Cdc2a
antiserum were blotted and probed with CycD2 and Cdc2a antisera. Note
that CycD2 only coimmunoprecipitates efficiently from exponentially
growing cells, whereas Cdc2a is immunoprecipitated in equal proportions
to its presence in exponentially growing and quiescent cells
(A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary model of CycD2 and CycD3 mRNA levels (1), protein
abundance, association with Cdc2a, and kinase activity levels in an
Arabidopsis cell culture during the transition from exponential growth
to stationary phase and during reentry into the cell cycle.
View larger version (24K):
[in a new window]
Fig. 7.
Summary model of CycD2 and CycD3 protein
associations with Cdc2a in an Arabidopsis cell culture
during the transition from quiescent cells, through cell cycle reentry
to active division. See "Discussion" and Table I for
details. CycD2 is shown complexed with an unknown protein X in
quiescent (nondividing) cells; the rising CycD2-associated kinase
activity in early G1 may result from the liberation of
CycD2 from these complexes; alternatively, CycD2 may be synthesized
de novo (not illustrated).
The work presented here and elsewhere (reviewed in Ref. 14) suggests
that the overall similarities between plant and mammalian controls of
the cell cycle are overlaid by complex and important differences in the
control, interactions, and targets of cell cycle regulators in plants.
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ACKNOWLEDGEMENTS |
---|
We thank Bart den Boer, Marc de Block, and Masami Sekine for useful discussions.
![]() |
FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grants G02552 and P09509, a BBSRC studentship (to M. M.), and Aventis CropScience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dipartimento di Genetica, IV Piano, Torre A,
Università di Milano, 20133 Milan, Italy.
¶ To whom correspondence should be addressed: Institute of Biotechnology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QT, United Kingdom. Tel.: 44-1223-334166; Fax: 44-1223-334162; E-mail: j.murray@biotech.cam.ac.uk.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009074200
2 S. de Jager, M. Menges, U.-M. Bauer, and J. A. H. Murray, submitted for publication.
3
D. A. Sorrell, , submitted for publication.
![]() |
ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; WCE, whole cell extract; IVT, in vitro translated.
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REFERENCES |
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