Arabidopsis PASTICCINO2 Is an Antiphosphatase Involved
in Regulation of Cyclin-Dependent Kinase A
Marco Da Costa,a,1Lie ˆn Bach,aIsabelle Landrieu,bYannick Bellec,aOlivier Catrice,cSpencer Brown,c
Lieven De Veylder,dGuy Lippens,bDirk Inze ´,dand Jean-Denis Faurea,2
aLaboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, F-78026 Versailles Cedex, France
bInstitut Pasteur de Lille, Centre National de la Recherche Scientifique, Unite ´ Mixte de Recherche 8525, Universite ´ de Lille,
F-59019 Lille Cedex, France
cDynamique de la Compartimentation Cellulaire, Institut des Sciences du Ve ´ge ´tal, Unite ´ Propre de Recherche 2355,
Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette, France
dDepartment of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B-9052
PASTICCINO2 (PAS2), a member of the protein Tyr phosphatase-like family, is conserved among all eukaryotes and is
characterized by a mutated catalytic site. The cellular functions of the Tyr phosphatase-like proteins are still unknown, even if
is phosphorylated on Tyr and not with its unphosphorylated isoform. Phosphorylation of the conserved regulatory Tyr-15 is
involved in the binding of CDK to PAS2. Loss of the PAS2 function dephosphorylated Arabidopsis thaliana CDKA;1 and
upregulated its kinase activity. In accordance with its role as a negative regulator of the cell cycle, overexpression of PAS2
slowed down cell division in suspension cell cultures at the G2-to-M transition and early mitosis and inhibited Arabidopsis
seedling growth. The latter was accompanied by altered leaf development and accelerated cotyledon senescence. PAS2 was
localized in the cytoplasm of dividing cells but moved into the nucleus upon cell differentiation, suggesting that the balance
between cell division and differentiation is regulated through the interaction between CDKA;1and the antiphosphatase PAS2.
In multicellular organisms, cell division and cell differentiation
have to be coordinated during development. This statement is
especially truefor plantsthatcarryon continuousorganogenesis
in the meristems, where cells have to maintain their proliferative
Cell cycle regulators are required for the control of cell cycle
transitions but seem also to be involved in coordinating transi-
tions between cell proliferation and cell differentiation (Gutierrez,
In eukaryotes, cell division is regulated by phosphorylation
events performed by cyclin-dependent kinases (CDKs) (Inze ´,
2005). The cell cycle machinery is conserved among eukaryotes,
and, in particular, several CDKs have been characterized in
bona fide CDK because of the presence of the conserved
PSTAIRE motif and by its ability to complement the yeast
Schizosaccharomyces pombe cdc2 mutant (Colasanti et al.,
1991; Ferreira et al., 1991). Of the plant-specific CDKs with the
PPTALRE and PPTTLRE motifs, CDKB can not complement the
CDKs, a large family of cyclins has been described in several
plant species (Inze ´, 2005). Correct cell cycle progression re-
quires a tight control of protein and activity levels of CDK/cyclin
complexes (De Veylder et al., 2003; Dewitte and Murray, 2003).
For instance, transcriptional regulation of the expression of
cyclins is important for the G1-to-S transition, but posttransla-
are essential for many cell cycle steps (Inze ´, 2005). Irreversible
inactivation of the CDK/cyclin complex results from ubiquitin-
mediated degradation of the cyclins (Genschik et al., 1998;
Koepp et al., 1999). One of the most important posttranslational
modifications of CDKs involves protein phosphorylation. CDKs
are initially activated by cyclin association and by phosphoryla-
tion on a Thr residue in the conserved T-loop (Connell-Crowley
et al., 1993; Solomon, 1993). CDK/cyclin complexes are revers-
ibly inactivated by phosphorylation of Thr-14 and Tyr-15 of CDK
(Morgan, 1995). Phosphorylation is mediated by the WEE1/
MYK1/MYT protein kinase, while dephosphorylation is caused
by the dual specificity protein Tyr phosphatase (dsPTP) CDC25.
1Current address: Cell Proliferation Group, Medical Research Council
Clinical Sciences Centre, Imperial College Faculty of Medicine,
Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
2To whom correspondence should be addressed. E-mail jean-denis.
firstname.lastname@example.org; fax 33-130-83-3099.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Jean-Denis Faure
WOnline version contains Web-only data.
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 18, 1426–1437, June 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
a rate-limiting step for CDK activation and, thus, for cell cycle
progression (Donzelli and Draetta, 2003).
The plant WEE1 regulatory kinase was identified in Arabidop-
sis thaliana and maize (Zea mays) (Sun et al., 1999; Sorrell et al.,
2002). Until recently, no gene with significant primary sequence
similarity to CDC25 could be detected in the Arabidopsis ge-
nome (Arabidopsis Genome Initiative, 2000), although the
CDC25-like activity was found to be involved in the G2-to-M
transition of Nicotiana plumbaginifolia cells (Zhang et al., 1996,
structure similar to that of the human CDC25 and able to activate
in vitro the CDK/cyclin complex has only recently been cloned
(Landrieu et al., 2004a, 2004b).
In animals, tumorigenesis is often preceded by the deregula-
tion of cell cycle components that triggers ectopic cell prolifer-
ation. Surprisingly, plants seem to be strikingly resistant to
unrestricted growth similar to mammalian hyperplasia or cancer
when cell cycle genes are deregulated (Cockcroft et al., 2000;
Gutierrez, 2005). Nonetheless, direct genetic screens for ectopic
cell proliferation and tumor-like development have identified
several mutants in Arabidopsis (Faure et al., 1998; Bouton et al.,
2002; Frank et al., 2002), among which the pasticcino (pas)
mutants (pas1, pas2, and pas3) have been characterized by
ectopic cell proliferation in the apical part, which is a cytokinin-
enhanced process (Faure et al., 1998). These pas mutants have
impaired embryo and seedling development associated with
modified cytokinin and auxin sensitivity (Harrar et al., 2003). The
three PAS genes have been identified as members of a con-
served gene family in eukaryotes (Vittorioso et al., 1998; Bellec
et al., 2002; Baud et al., 2004). PAS2 encodes the Arabidopsis
member of a new PTP family, the PTP-LIKE (PTPL) family,
originally described in mice and humans and characterized by
mutations in the active site that conferred phosphatase inactivity
(Uwanogho et al., 1999; Bellec et al., 2002; Pele ´ et al., 2005). The
cellular function of this protein family is still unknown but seems
to be critical because the absence of the PAS2 homolog in yeast
is lethal and in mammals leads to severe defects in myofibril
differentiation (Bellec et al., 2002; Pele ´ et al., 2005). The discov-
ery of a putative inactive PTP involved in plant cell proliferation
raised the possibility that the plant cell cycle might be regulated
by an antiphosphatase.
Here, we show that PAS2 is indeed a component of the
CDKA;1/cyclin complex by its interaction with phosphorylated
CDKA;1. PAS2 protects the phosphorylated CDKA;1 residues
from phosphatase activities and thus regulates CDKA;1 phos-
phorylation status and kinase activity; however, its absence
causes CDKA;1 dephosphorylation and increases cell compe-
tence to divide. A mechanism is proposed based on the com-
petitive action of an antiphosphatase and a dsPTP on CDKA;1 to
control cell competence for division.
PAS2 Interacts with CDKA;1 in Vivo
The sequence similarity between PAS2 and members of the
PTPL family suggested that PAS2 could be involved in protein
phosphorylation. The fact that PTPL members are characterized
bya mutatedcatalytic site ofPTPprompted ustoconsider PAS2
as a competitor of an active PTP for a phosphorylated substrate.
Arabidopsis CDKA;1 appeared to be a potential target of PAS2
for several reasons. First, this main regulator of the cell cycle is
one of the few plant proteins in which a regulatory Tyr phospho-
rylation site is conserved. Then, CDKA dephosphorylation by a
CDC25-like activity is required for cell cycle progression of N.
plumbaginifolia (Zhang et al., 2005). In particular, a CDC25-like
activity has been found to be necessary for cytokinin regulation
of the G2-to-M transition of the cell cycle. Finally, the pas2
mutants show cell dedifferentiation and proliferation, which are
specifically enhanced by cytokinins, suggesting that both PAS2
and cytokinins could regulate CDKA;1 phosphorylation by tar-
geting CDC25 activity.
We tested whether PAS2 and CDKA;1 interact in vivo by
purifying PAS2-interacting proteins by pull-down assays (Figure
1A). Total proteins from Arabidopsis cell cultures (lane 1) were
loaded on a column with PAS2 (lane 2) or on an empty control
column (lane3).The analysis of PAS2binding proteins byprotein
gel blots showed that CDKA;1 was specifically retained on the
PAS2 column (Figure 1B). The reverse experiment was per-
formed by affinity purification of the active CDKA;1 complexes
from Arabidopsis cell cultures with p10CKS1Atcolumns (Figure
1C, bottom). Protein gel blot analysis with anti-PAS2 serum
indicated that PAS2 copurified with CDKA;1 (Figure 1C, top).
Figure 1. Interaction of PAS2 with CDKA;1 in Vivo.
(A) and (B) Purification of CDKA;1 by recombinant PAS2 (PAS2:His) from
total protein extracts of Arabidopsis cells. The pull-down assay was
performed with a PAS2:His column. Proteins were detected by Coo-
massie staining (A) and by protein gel blots (B) from total protein extract
of Arabidopsis cells (1), protein extract after purification on PAS2:His
column (2), and protein extract after purification on empty column (3).
Protein gel blot analysis was performed with anti-PSTAIRE (PSTAIRE)
and antiphosphotyrosine (PTYR).
(C) Copurification of PAS2 with the CDK complex. Detection of PAS2
(anti-PAS2; PAS2) and CDKA;1 (PSTAIRE) after CDK purification by
p10CKS1Atbeads from protein extracts of Arabidopsis cells.
(D) Association of PAS2 with the CDK complex in Arabidopsis seedlings.
Detection of PAS2 and CDKA;1 (PSTAIRE) after CDK purification by
p10CKS1Atbeads from protein extracts of wild-type plants or pas2-1
Antiphosphatase Regulates Cell Division1427
A similar experiment was performed with wild-type Arabidopsis
seedlings, and, as expected, PAS2 was also associated with
CDKA;1 (Figure 1D). As anticipated, a low amount of PAS2 was
found in the complex with CDKA;1 in a weak pas2 allele, which
was characterized byastrongly reducedsteadystate levelof the
PAS2 mRNA (Bellec et al., 2002). Altogether, these data dem-
onstrate that PAS2 interacts with the CDKA;1 protein complex in
cell cultures as well as in seedlings.
PAS2 Interacts Only with Phosphorylated CDK and Inhibits
The PTP motif in the PAS2 sequence suggested that protein
phosphorylation was involved in the PAS2 function. The recom-
the protein could have still retained its ability to bind a phos-
phorylated substrate. We tested this hypothesis using an in vitro
binding assay with the recombinant PAS2:His and CDKA;1:mal-
tose binding protein (MBP). The recombinant CDKA;1:MBP was
purified from Escherichia coli and phosphorylated by Src Tyr
kinase in vitro. Phosphorylation was monitored by radioactive
incorporation of g32P-ATP (data not shown) and by protein gel
blots with an antiphosphotyrosine antibody (Figure 2A). We
verified that Tyr-15, which is an important regulatory residue of
CDKA;1 and a substrate of CDC25, was phosphorylated by Src
Tyr kinase. Both the unphosphorylated and phosphorylated
recombinant CDKA;1:MBP proteins were loaded onto a PAS2
gel blots (Figure2B). Onlythe phosphorylated CDKA;1:MBP was
retained on the PAS2 column, demonstrating that the PAS2-
CDKA;1 interaction requires CDKA;1 phosphorylation on Tyr
residues. Accordingly, a Tyr phosphorylated protein of a size
similar to that of CDKA;1 was also affinity purified by PAS2 from
Arabidopsis cell culture extract (Figure 1B).
We then investigated the role of Tyr-15 phosphorylation in
PAS2 binding to CDK using the specific kinase WEE1. Because
the phosphorylation specificity of the Arabidopsis WEE1 had
never clearly been addressed, we used the well-characterized
human WEE1 kinase that phosphorylated specifically Tyr-15 of
CDK2 (Morgan, 1997). Human CDK2 and Arabidopsis CDKA;1
share 69% identity at the protein level and a complete identity
over 17 residues around Tyr-15. The glutathione S-transferase
Figure 2. Interaction of PAS2 with Phosphorylated CDKA;1.
(A) In vitro phosphorylation of CDKA;1:MBP. CDKA;1:MBP was incubated (þ) or not (?) with Src kinase and analyzed by protein gel blotting with
antiphosphotyrosine (PTYR), antiphosphotyrosine 15 (P-Y15), and anti-PSTAIRE antibodies.
(B) Tyr phosphorylation of CDKA;1 is required for the interaction with PAS2. Incubation of CDKA;1:MBP phosphorylated or not by Src kinase with
PAS2:His column. Interaction between PAS2 and CDKA;1:MBP or CDKA;1:MBP Y-P was monitored by protein gel blots with the anti-PSTAIRE
antibody. (1) CDKA;1:MBP, (2) elution of CDKA;1:MBP from PAS2:His column, and (3) elution of CDKA;1:MBP Y-P from PAS2:His column.
(C) In vitro phosphorylation of GST:CDK2 by WEE1. GST:CDK2 was coproduced in E. coli with (þ) or without (?) the WEE1 kinase and analyzed by
protein gel blotting with antiphosphotyrosine 15 (P-Y15) and anti-PSTAIRE antibodies.
(D) WEE1 phosphorylation of CDK2 is required for the interaction with PAS2. The GST:CDK2 fusion protein phosphorylated or not by WEE1 kinase was
incubated with PAS2:His column, and the interaction was monitored by protein gel blots with the anti-PSTAIRE antibody.
(E) The interaction of PAS2 with phosphorylated CDK2 is competed off with pTyr-15 peptide. The experiment was similar to (D) except that the
conserved peptide EKVEKIGEGTYGVVYK phosphorylated on Thr (pT14) or phosphorylated on Tyr (pT15) was included or not (?) in the binding
experiment, and the interaction was monitored by protein gel blots with the anti-PSTAIRE antibody.
1428 The Plant Cell
(GST):CDK2 fusion protein was produced in E. coli in the ab-
sence or in the presence of WEE1 as previously described
(Welburn and Endicott, 2004). The fusion proteins were purified,
and Tyr-15 was found to be phosphorylated only in the presence
of WEE1 (Figure 2C). Both fusion proteins were then incubated
with a PAS2:His column and an empty column. Only the
WEE1-treated GST:CDK2 fusion protein interacted with PAS2,
demonstrating that phosphorylation of the Tyr-15 residue was
necessary for PAS2 binding. Finally, to confirm the involvement
of pTyr-15, we performed competition experiments with the
conserved peptide EKVEKIGEGTYGVVYK overlapping Thr-14–
Tyr-15. Peptides phosphorylated on Tyr-15 but also on Thr-14
were used to displace WEE1-phosphorylated GST:CDK2 bind-
ing to PAS2. As shown in Figure 2E, both pTyr-15 and pThr-14
peptides competed off GST:CDK2 binding to PAS2 even if
phosphorylation of Tyr-15 seemed more efficient in the compe-
tition experiment. Altogether, our results showed that PAS2
binds only phosphorylated CDK and that the conserved Tyr-15
and Thr-14 are involved in the interaction.
We checked whether PAS2 binding could protect CDKA;1
from dephosphorylation. According to this hypothesis, loss of
PAS2 should improve the access of phosphatases to CDKA;1
and, in turn, decrease CDKA;1 Tyr phosphorylation. The CDKA;1
complex was purified from the wild type and the pas2-1 mutant,
and the CDKA;1 phosphorylation status and kinase activity were
measured. Compared with the wild type, the CDKA;1 Tyr phos-
phorylation level in the pas2-1 mutant had decreased severely,
and, accordingly, the associated CDKA;1 kinase activity had
increased (Figures 3A and 3B). In conclusion, PAS2 maintains
CDKA;1 in an inactive state by protecting CDKA;1 from dephos-
phorylation by phosphatases.
PAS2 Overexpression Alters Cell Division in Tobacco Cells
Loss-of-function of PAS2 leads to ectopic cell proliferation. This
feature is consistent with its role as a CDKA;1 inhibitor. By
contrast, an increase in the PAS2 protein in the cell should alter
cell division and, eventually, impair development. To assess
whether PAS2 is involved in the regulation of cell division, we
overexpressed PAS2 in tobacco (Nicotiana tabacum) Bright
Yellow-2 (BY-2) suspension cells. Because the PAS2 effect
could be rapidly counterselected after several rounds of subcul-
tures, we expressed PAS2 under the control of an inducible Cre/
gene is cloned under the control of the constitutive cauliflower
spacer with a transcription termination site prevents its expres-
sion. The GFP spacer isflanked with the Lox recombination sites
and can be excised in the presence of the CRE recombinase. A
ligand-dependent CRE-GR construct expressed under a heat
shock promoter was used to tightly control the recombinase
activity. Thus, GFP excision and PAS2 expression are induced
by the expression of the construct pHSP-CRE:GR after heat
shock (HS) and dexamethasone (Dex) treatment (Figures 4A and
4B). The HS/Dex treatment did not affect the BY-2 cell cultures
(Figure4C;Joube `setal.,2004).WithoutHS/Dextreatment, 90to
95% of the BY-2 cells harboring the 35S-lox:GFP:lox:PAS2
construct (PAS2loxGFPlox) were GFP positive, and, correspond-
ingly, only a low basal level of PAS2 expression was detected in
the culture (Figures 4A and 4B). By contrast, upon HS/Dex
treatment, >80% of the cells became GFP negative after 3 d and
accumulated PAS2 transcripts as early as 7 h after treatment
(Figures 4A and 4B). The effect of the PAS2 expression could be
(Figure 4C). To check whether the effect of PAS2 on culture
growth was related to cell division, we analyzed the cell cycle
progression in synchronized PAS2loxGFPloxcells. BY-2 cells were
first treated with HS/Dex and 24 h later blocked at the G1-to-S
transition by aphidicolin treatment. Aphidicolin reversibly arrests
cells in early S phase by inhibiting DNA polymerase a and d (Sala
et al., 1983; Nagata et al., 1992). Cells were released from the
Figure 3. Inhibition of CDKA;1 Phosphorylation and Activity by PAS2.
(A) Decreased Tyr phosphorylation in pas2-1 mutant by CDKA;1. CDK
complexes were purified on p10CKS1Atfrom wild-type and pas2-1 mutant
plants. Phosphorylated CDKA;1 was revealed by anti-PTYR and anti-
PSTAIRE protein gel blots.
(B) Increase of the CDK-associated kinase activity in pas2-1 mutant
plants. The Arabidopsis CDK complexes were purified on p10CKS1At, and
the kinase activity was quantified by monitoring the histone H1 phos-
phorylation. Data were normalized according to the CDKA;1 protein level
as quantified by the protein gel blots with anti-PSTAIRE antibody. The
kinase activity represents the result of three independent experiments.
Error bars represent SE.
Antiphosphatase Regulates Cell Division 1429
Figure 4. Inducible PAS2 Overexpression in BY-2 Cells.
(A) Extensive GFP activity in BY-2 cells transformed with PAS2loxGFPlox(left). Three days after HS and Dex induction, no GFP fluorescence could be
detected (right). For each condition, BY-2 cells were analyzed in light transmission and for GFP fluorescence. Bars ¼ 30 mm.
(B) Increase in PAS2 expression after HS/Dex treatment. Time course of PAS2 expression was measured by PCR on reverse transcription of RNA from
PAS2loxGFPloxBY-2 cells induced (þ) or not (?).
(C) Growth inhibition of induced (pink) versus uninduced (blue) PAS2loxGFPloxcell cultures. HS/Dex treatment did not affect growthof the control GFP cell
culture (blue dashed line) compared with the untreated condition (pink dashed line).
1430 The Plant Cell
aphidicolin block, and cell cycle progression was monitored by
measuring the mitotic index. The aphidicolin block associated
with HS and Dex did not modify cell cycle progression because
control 35S-GFP transgenic BY-2 cells entered mitosis at the
same time whether they had been treated with HS/Dex or not
(Figure 4D). Untreated PAS2loxGFPloxshowed, like the control cell
lines, a peak of mitosis at 12 h and then at 28 h after aphidicolin
release. By contrast, HS/Dex-treated PAS2loxGFPloxcells reached
mitosis 13 and 30 h after aphidicolin release, corresponding to a
1- and 2-h delay compared with untreated cells, respectively
(Figure 4E). This delay in mitosis was not caused by a stress
response from the combination of HS and aphidicolin treatment
because a similar delay was observed in the second mitosis. The
lag in mitosis entry of PAS2loxGFPlox-treated cells was on average
treated or not always entered into mitosis simultaneously. The
fact that entry into mitosis had already been delayed during the
first cycle after aphidicolin block suggested that PAS2 acts in S
or G2 phases or at the transition between G2 and mitosis. To
discriminate between these hypotheses, the cell cycle phases
were measured by flow cytometry for the first 25 h of the
synchronization experiment (Figure 4F). Treated PAS2loxGFPlox
cells were arrested earlier in S phase than untreated cells after
aphidicolin block, but time of entry into G2 phase was similar for
both cell lines (Figure 4G, top and middle). Thus, the delay
observed in S phase could not explain the difference in mitosis
timing between treated and untreated cell cultures. While both
types of cells entered the G2 phase synchronously, HS/Dex-
treated cells had a shorter G2 phase because cells exited G2 1 h
earlier than control cells (Figure 4G, top). The same pattern of
early entry in G2 of HS/Dex-treated PAS2loxGFPloxcells was
observed in two independent experiments. The delay in mitosis
of the HS/Dex cell population must then originate from an
inhibition of the cell progression through mitosis. Thus, we
checked whether PAS2 expression had an effect on specific
mitotic phases by scoring the frequency of the different mitotic
figures. Treated cells had an unbalanced prophase:metaphase
ratio (Figure 4H), suggesting that the PAS2 overexpression
inhibited early mitosis. While the cell cycle study on BY-2
cultures demonstrated that PAS2 has a direct negative effect
on cell division, this effect is probably not the only cause of
growth inhibition. The reduction of PAS2loxGFPloxcell culture
growth rate after induction could also result from an inhibition of
cell expansion. The measurements of PAS2loxGFPloxcell length
48 h after HS/Dex induction showed that PAS2 induction led to a
shift of the cell length distribution toward smaller cells (Figure 4I).
In summary, the induction of PAS2 in BY-2 cells reduced the
growth of cell culture, which was caused by a combined inhibi-
tion of cell cycle progression and cell expansion.
PAS2 Overexpression Alters Plant Growth
The effect of PAS2 overexpression in Arabidopsis was analyzed
with transgenic plants overexpressing PAS2 or PAS2:GFP fu-
sions. All the constructs were functional because they could
complement the pas2-1 mutant (see Supplemental Figure 1 on-
line). High GFP-producing plants were retained for further anal-
ysis because PAS2 accumulation could be monitored easily in
the whole seedlings throughout development (Figures 5A and
in 35S-PAS2:GFP seedlings compared with the wild type
(Columbia-0) (Figure 5C). The transgenic plants accumulated
30-fold more PAS2 transcripts than untransformed wild type.
Several independent transgenic lines with high GFP staining had
obvious developmental defects with slowed growth and stunted
phenotype (Figure 5A). Cotyledons were smaller in 35S-PAS2:
to have an accelerated senescence. The development of first
leaves was often altered: either the first leaves were absent or
had a delayed growth with primordia-like shapes or reduced leaf
blades (Figure 5D). Epidermal cells of young cotyledons of
35S-PAS2:GFP seedlings were similar to those of the wild
type, suggesting that the smaller size of the cotyledons was
caused by a reduced number of cells (Figure 5E).
PAS2 Subcellular Distribution
The intracellular distribution of PAS2 protein was investigated in
the root cells of transgenic Arabidopsis expressing the construct
35S-PAS2:GFP (Figures 5F and 5G). PAS2:GFP was excluded
from the nucleus in the root meristem (Figure 5F) and in the
elongation zone as well (data not shown). On the contrary, most
of the cells in the differentiated zone displayed cytosolic but also
nuclear distribution of the PAS2:GFP protein fusion (Figure 5G).
The presence of PAS2 in the nucleus could also be seen in root
hairs (see Supplemental Figure 2 online).
Figure 4. (continued).
(D) and (E) Delay in cell cycle progression induced by PAS2. HS/Dex treatments did not modify cell cycle progression. Mitotic index was measured,
after release from aphidicolin block, in 35S-GFP and PAS2loxGFPloxcell cultures that were induced (pink line) or not (blue line). While HS/Dex treatment
did not affect growth of the control GFP cell culture (D), it delayed PAS2loxGFPloxcell mitosis (E).
(F) Cytometric analysis of the cell cycle of synchronized cells uninduced (top) and induced (bottom) from the experiment presented in (E).
(G) Time course progression through the cell cycle of uninduced (blue) and induced (pink) PAS2loxGFPloxcells in G2 (top panel), S (middle panel), and G1
phases (bottom panel), respectively.
(H) Distribution of prophase, metaphase, and anaphase plus telophase during mitosis of uninduced (blue) and induced (pink) PAS2loxGFPloxcells. Mitotic
figures were sampled in five time points during the first peak of mitosis of synchronized cells described in (E). Approximately 200 mitotic figures were
counted for each time point. Error bars represent SE.
(I) Cell length distribution of uninduced (blue) and induced (pink) PAS2loxGFPloxcells. Cell length was measured 48 h after induction on 439 uninduced
and 575 induced cells.
Antiphosphatase Regulates Cell Division1431
Figure 5. PAS2:GFP Expression in Arabidopsis.
(A) Growth inhibition by stable expression of 35S-PAS2:GFP in 10-d-old seedlings.
(B) GFP activity in seedling meristem and primary root of transgenic seedlings compared with control (Columbia-0).
(C) Expression level of PAS2 in 35S-PAS2:GFP seedlings compared with the wild type. Real time RT-PCR amplification of PAS2 transcript was
normalized against EF1a. Error bars represent SE.
(D) Altered development of young leaves in 10-d-old transgenic 35S-PAS2:GFP seedlings with fused (left), finger-shaped (right), or missing leaf
(E) Cotyledon epidermis cells of 35S-PAS2:GFP plants compared with the wild type.
(F) and (G) Subcellular distribution of PAS2:GFP changed according to cell type. In root meristem with actively dividing cells, PAS2:GFP fusion is mainly
present in the cytosol (F). In differentiated cells in the root hair initiation zone, PAS2:GFP shows cytosolic and nuclear localization (G). For each panel,
the GFP signal, the DRAQ5 nuclear staining, and merged image are shown in the left, middle, and right panels, respectively.
(H) to (L) Subcellular distribution of PAS2:GFP in BY-2 cells. Most of the interphasic cells showed PAS2:GFP accumulation outside the nucleus (H) but
could also be detected strongly accumulated inside the nucleus in a minority of cells (I). PAS2:GFP fusion was found to embrace chromosomes during
the different phases of mitosis as shown for metaphase (J), anaphase (K), and telophase (L). For each panel, the GFP signal, the DRAQ5 nuclear
staining, and merged image are shown in the top, middle, and bottom panels, respectively.
Bars ¼ 1 mm ([A], [B], and [D]), 25 mm (E), 5 mm ([F] and [G]), and 30 mM ([H] to [L]).
1432 The Plant Cell
transgenic BY-2 cell lines producing the PAS2:GFP fusion pro-
tein. As observed in Arabidopsis seedlings, PAS2:GFP fusion
protein was mainly found outside the nucleus with a strong
accumulation in the perinuclear region (Figure 5H). Nonetheless,
a regular, but limited, occurrence of nucleus-localized GFP
signal could be found in BY-2 cells, similar to that observed in
Arabidopsis (Figure 5I). During mitosis, PAS2:GFP was tightly
associated with the chromosomes capping their distal side
during metaphase (Figure 5J), anaphase (Figure 5K), and late
telophase (Figure 5L).
PAS2 is a member of the PTPL family that is conserved among
eukaryotes. The PTPL family most probably fulfills important
functions because PAS2 is not only essential in Arabidopsis but
also in yeast and mammals (Bellec et al., 2002; Pele ´ et al., 2005).
The function of PAS2 is also conserved because the Arabidopsis
PAS2 gene is able to complement yeast lethality (Bellec et al.,
2002). Thefact thatPAS2has anessential functionthathas been
maintained from yeast to plants suggests that PAS2 is involved
in a highly evolutionarily conserved cellular process.
Here, we provide evidences that PAS2, as the first plant
member of the PTPL family, is involved in the regulation of cell
division. PAS2 has been found to bind specifically to Tyr phos-
phorylated CDKA;1, suggesting that it has lost the PTP catalytic
activity but not its substrate binding affinity. In animals, the dual
dephosphorylation of Thr-14 and Tyr-15 of CDK by CDC25 is
responsible for CDK2 activation. We demonstrated that PAS2 is
also able to bind to WEE1-phosphorylatedCDK2, confirming not
only the biochemical conservation of PAS2 function through
evolution from yeast to human but also the importance of the
regulatory Tyr-15 for PAS2 interaction. Nonetheless, Thr-14
phosphorylation is also probably involved in PAS2 CDK interac-
tion, which is consistent with the conservation of Thr-14 among
plant CDKAs and its known regulatory role in CDK activity. The
combined mutation of Thr-14A and Tyr-15F in Arabidopsis
CDKA;1 led to constitutively active CDKA;1, as demonstrated
in yeast by premature cell division (Hemerly et al., 1995). Over-
expression of this mutant form of CDKA;1 in Arabidopsis pro-
vokes a mild phenotype with loss of apical dominance (Hemerly
et al., 1995), which is reminiscent of cytokinin-overproducing
plants (Howell et al., 2003). Interestingly, cytokinins promote cell
division by upregulating a CDC25-like activity in N. plumbagini-
folia cells, suggesting that the positive effect of cytokinins on cell
division is caused by CDKA;1 dephosphorylation and activation
(Zhang et al., 1996, 2005). In the pas2 mutant, the dephosphor-
ylation and activation of CDKA is thus consistent with the
increase in cell proliferation induced by cytokinin application
(Faure et al., 1998). PAS2 and cytokinins have antagonistic
effects on CDC25-like activity, the activation of CDKA;1, and
consequently on cell division.
Our work suggests also that Tyr phosphorylation is important
for regulating cell proliferation. Phosphorylated Tyr is not a
common posttranslational modification in plants compared with
animals. Nonetheless, several PTPs and kinases have been
described in Arabidopsis (Gupta et al., 1998; Xu et al., 1998;
Fordham-Skelton et al., 1999, 2002; Ulm et al., 2001, 2002). Tyr
phosphorylation has also been involved in several physiological
responses, such as bending or phytohormone-stimulated cell
proliferation (Kameyama et al., 2000; Huang et al., 2003). We
show here that the level of CDKA;1 Tyr phosphorylation is
controlled by PAS2. The mode of action of PAS2 is reminiscent
of PTP inhibitors or antiphosphatases, such as STYX and Sbf1,
which are inactive dsPTPs involved in suppression of cell trans-
1998). First, PAS2 associates only with phosphorylated CDKA;1.
Then, the absence of PAS2 leads to a decrease in Tyr phosphor-
ylation levels of CDKA;1 probably by directly inhibiting CDKA;1
activation by a CDC25-like activity. The previously identified
plant CDC25, Arabidopsis CDC25;1, might not be a bona fide
Figure 6. Model of PAS2 Action as an Antiphosphatase.
During the cell cycle, CDKA;1 activity is regulated by inhibitory phosphorylation (P). WEE1 is the kinase that most probably inactivates CDKA;1 after
mitosis; thereafter, CDKA;1 is progressively dephosphorylated to the onset of mitosis by phosphatase(s). Cells could continue cycling or could be
committed to the quiescent cycling state (G0) or a differentiated state. By maintaining CDKA;1 in a hyperphosphorylated inactive form, PAS2 could
directly inhibit cell division and induce the cell to differentiate. Alternatively, once cells have exited from the cell cycle, PAS2 would be required to hold
the cells committed to their differentiation state
Antiphosphatase Regulates Cell Division 1433
CDC25 because it is probably involved in arsenate reduction
(Bleeker et al., 2006). An attractive model would be that plants
have nonspecific phosphatases that dephosphorylate CDKA
unless it is protected by proteins such as PAS2. In such a case,
PAS2 overexpression would antagonize several phosphatase(s)
contradictory results like the acceleration of G2 exit and delay in
Thus, we propose the following model where the cell cycle
progression and the entry into mitosis is controlled by CDKA;1
phosphorylation (Figure 6). Premature mitosis is prevented by
the WEE1 kinase that phosphorylates CDKA;1 after mitosis.
PAS2 would then maintain CDKA;1 in a phosphorylated and inac-
tive state,preventing the premature action of phosphatases. The
next cell division would then be initiated upon the action of a
CDC25-like activity by signals such as cytokinins. The dissoci-
ation of PAS2 from CDKA;1 might have two explanations: an
increase of the CDC25-like phosphatase activity at the onset of
mitosis sufficient to dephosphorylate CDKA and thus release
PAS2 or an active mechanism removing PAS2 from the CDKA
complex and allowing access to the CDC25-like phosphatase. A
precise analysis of CDC25-like activity during the cell cycle
would discriminate between the two mechanisms.
intracellularly with its target. Contrary to Arabidopsis CDC25;1,
the subcellular localization of CDKs has been described in sev-
are mainly located in the nucleus in interphase and up to early
prophase, and during mitosis, they are associated with chroma-
tin, the preprophase band, the mitotic spindle, and the phrag-
moplast (Colasanti et al., 1993; Bo ¨gre et al., 1997; Stals et al.,
1997). CDKA was also seen in the cytosol of Medicago sativa
cells during the G2 phase (Bo ¨gre et al., 1997). These different
localizations were confirmed by dynamic analysis of a Medicago
CDKA:GFP fusion protein (Weingartner et al., 2001). The pres-
ence of PAS2 closely associated with mitotic chromosomes is
consistent with its effect during cell division and supports the
hypothesis of aninteraction between PAS2andCDKA;1atG2/M
or early mitosis to prevent premature cytokinesis. In actively
dividing cells,such as in the rootmeristem or in BY-2 cells, PAS2
is principally excluded from the nucleus, and it remains to be
determined whether PAS2 interacts with the cytosolic fraction of
phosphorylated CDKA;1 or if the low amount of PAS2 remaining
in the nucleus throughout the cell cycle could be sufficient for
of PAS2 in interphasic BY-2 cells could be interpreted either as a
premitotic mark or as a sign of resting or quiescent cells. The
latter interpretation is supported by the fact that PAS2:GFP is
clearly not excluded anymore from the nucleus in differentiated
Arabidopsis root cells, probably downregulating CDKA;1 and
preventing the cells from reentering mitosis (Figure 6). Alterna-
tively, PAS2 might fulfill different functions in the nucleus and in
the cytosol. Inboth cellular compartments, PAS2 might actasan
antiphosphatase on CDKA but toward different phosphatases:
one involved in the nucleus controlling the G2/M transition and
or differentiated cellular state. While in the absence of PAS2,
cells are more prone or competent for cell division, and PAS2
and, in the strongest case, the development of leaf primordia.
The fact that cotyledons of transgenic PAS2:GFP have a phe-
notype similar to early onset of senescence could be a sign of
accelerated cell differentiation. Thus, it is likely that PAS2 has a
distinct function in dividing and differentiated cells (i.e., in divid-
ing cells, PAS2 would interact transiently or at low levels with
increased and stabilized. The role of PAS2 at the interface of cell
division and differentiation is also illustrated by its expression
pattern during early embryo development (Casson et al., 2005).
PAS2 is one of the most differentially expressed genes in the
apical zone of the globular embryo, and its expression precedes
and marks the onset of cotyledon initiation. This developmental
stageischaracterized byhighproliferative activityofcellsandby
the main differentiation step, leading from radial to bilateral
symmetry during embryo development.
Inconclusion, PAS2 might represent a clue to understand how
plants maintain a high cellular plasticity with coordinated cell
differentiation. The conservation of sequences and functions in
the PTPL family would also suggest that PAS2 might represent a
general regulatory mechanism involved in the control of cell
proliferation. Deciphering the precise role of PAS2 during cell
differentiation and in particular how its intracellular localization is
regulated will be necessary to fully understand the function of its
The protein fusions PAS2:GFP and PAS2:His were constructed by
cloning the full-length cDNA of PAS2 (666 bp) into the Gateway-modified
vector pK7WG2D (Karimi et al., 2005) and into the expression vector
pIVEX2.4 (Roche Diagnostics), respectively. The CDKA;1 gene was
cloned into an MBP tag-containing pMALC2X vector and resulted in the
MBP:CDKA;1 expression construct.
The peptide CMLGQRKRALSKSKRE-amide from the PAS2 sequence
was synthesized and used as antigen in rabbits to produce the antiserum
(anti-PAS2) (Biogenes). The anti-PSTAIRE (anti-CDKA) is a mouse IgG
monoclonal antibody (Sigma-Aldrich; reference P7962). For detection of
Tyr phosphorylated CDKA, a mouse IgG polyclonal antibody anti-PTYR
was used (Santa Cruz Biotechnology; reference Sc7020). Phosphor-
Technology; reference 9111).
Protein–Protein Interaction and Phosphorylation in Vitro
Production of recombinant CDKA;1 was performed after induction of
BL21(DE3)pLysE cells carrying the MBP:CDKA;1 construct by 2 mM
isopropyl-b-D-thiogalactopyranoside for 2.5 h at 378C. After one wash in
1 mM DTT, 1 mM vanadate, and protease inhibitor), cells were resus-
pended in 1 mL lysis buffer with 4 mg/mL lysozyme and incubated 15 min
at room temperature. The cells were sonicated on ice twice (15 pulses of
1 s) and centrifuged (25 min at 48C; 6000g). The supernatant was incu-
bated for 30 min at room temperature with 50 mL amylose matrix (50%)
and washed twice with binding buffer (10 mM Tris, pH 7.5, 75 mM NaCl,
1 mM DTT, 1 mM vanadate, 1 mM EDTA, and protease inhibitor). In vitro
Tyr phosphorylation of MBP:CDKA;1 with the Src kinase (Upstate
1434 The Plant Cell
Biotechnology; reference 14-117) was performed by incubation of the
MBP:CDKA;1 maltose matrix with 25 units of Src and ATP (or [g-32P]ATP)
and detected with the BAS 1500 imaging analyzer (Fuji) in phosphor-
ylation buffer (100 mM Tris, pH 7.5, 125 mM MgCl2, 25 mM MnCl2, 2 mM
EGTA, 1 mM vanadate, and protease inhibitor) for 1 h at 308C. The beads
were washed three times with binding buffer, and MBP:CDKA;1 was
RTS100 Escherichia coli kit according to the manufacturer’s instructions
(Roche Diagnostics). PAS2:His waspurified onnickel beadsblocked with
3% BSA, 0.1% Tween 20, and 1% Triton X-100 in binding buffer for 30
min at room temperature and washed with 1% Triton X-100 in PBS. An
RTS100 reaction containing PAS2:His was incubated with the nickel
Sepharose matrix for 30 min and washed three times with binding buffer.
MBP:CDKA;1 phosphorylated or not by Src kinase was incubated with
buffer, and finally eluted by boiling the matrix in Laemmli buffer. The
gel blotting with an anti-PSTAIRE antibody.
was performed as previously described (Welburn and Endicott, 2004).
After purification of CDK2 or CDK2-WEE1 on glutathione beads, proteins
were eluted using reduced glutathione and incubated 2 h at 48C with
nickel columns containing either PAS:His or His tag alone. The nickel
of CDK2 was analyzed by protein gel blotting using the PSTAIRE an-
by protein gel blotting with the antiphosphorylated Tyr-15. The compe-
tition experiments were performed by washing CDK2-WEE1 bound to
PAS:HIS columns with 1 mM of the following peptides: EKVEKIGEGp-
TYGVVYK (pThr-14), EKVEKIGEGTYGVVYK (control peptide), and
EKVEKIGEGTpYGVVYK (pTyr-15). The presence of CDK2-WEE1 was
Whole-Plant Extracts and Pull-Down Assays
Approximately 3 g (fresh weight) of cells of Arabidopsis thaliana were
ground in liquid nitrogen, and ;7 mg/mL of proteins were extracted in
5 mLofextraction buffer(25mMTris,pH7.6,15mMMgCl2, 15mMEGTA,
85 mM NaCl, 15 mM pNO2PhePO4, 60 mM B-glycerophosphate, 1 mM
DTT, 0.1% Nonidet P-40, 1 mM vanadate, 1 mM NaF, and protease
inhibitor). For pull-down assays, 30 mg of PAS2:His recombinant protein
produced from a RTS500 reaction (Roche Diagnostics) was adsorbed on
nickel Sepharose as described above. Proteins from Arabidopsis were
incubated with an empty sepharose or with PAS2:His nickel Sepharose
eluted by boiling in Laemmli buffer. The Tyr-phosphorylated form of
CDKA;1 was detected using the anti-PTYR antibody.
extract was incubated with 50 mL of p10CKS1Atbeads prepared as
described byBrizuela et al.(1987) andLandrieuet al. (1999) for 2hat 48C.
NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM vanadate, and
protease inhibitor). Protein extracts bound to p10CKS1Atbeads were
analyzed by protein gel blotting with the anti-PSTAIRE and anti-PAS2
Kinase Activity of CDKA;1
CDKA;1 from Arabidopsis seedlings was purified as described above.
The p10CKS1At beads were washed twice with bead buffer and once
with kinase buffer (50 mM Tris, pH 7.8, 15 mM MgCl2, 5 mM EGTA, and
1 mM DTT). In vitro histone H1 kinase was assayed as described by
Magyar et al. (1997). The samples were analyzed by 12% SDS-PAGE,
stained with Coomassie Brilliant Blue R 250, and analyzed with BAS1500
to detect histone H1 phosphorylation. Samples were normalized accord-
ing to PSTAIRE labeling. For competition assays between PAS2 and
p10CKS1At as described above. The beads were washed twice with
bead buffer and once with kinase buffer. The histone H1 kinase activity
was assayed in vitro as described above.
RNA were extracted and treated with DNase according to the RNeasy
plant kit (Qiagen). Reverse transcriptions were performed from total
DNase-treated RNA with superscript II enzyme (Qiagen) according to
standard protocols. PCRs were performed with primers for Arabidopsis
PAS2 (59-CCATGAAGAATCTCGAGAACG-39 and 59-TCTATGACGC-
CATTGAGAAGC-39) and Nicotiana sylvestris atp2 as control (59-GTGAA-
GAGGCGCGTGAAG-39 and 59-GTCTAATTTCCCGATCGTTAGGA-39).
Nicotiana tabacum BY-2 cells were grown in Murashige and Skoog
medium in a growth chamber under constant darkness and 258C (Joube `s
et al., 2004). For Cre/Lox induction, a 3-d-old culture was agitated for 2 h
at 378C and cooled down for 5 h at 258C. Finally, 10 mM Dex (Sigma-
Aldrich) was added. For synchronization, 5 mg/L aphidicolin was added
24 h after HS, and cells were blocked for 24 h. Cells were washed
extensively with fresh medium, resuspended in conditioned medium
subculture, and sampled at different times. Cells were washed, fixed in
0.1 M citric acid and 1% Triton X-100, and chopped with a razor blade.
49,6-diamidino-2-phenylindole (DAPI) and analyzed with a UV flow cy-
tometer (EPS Elite; Beckman-Coulter). The mitotic index was monitored
on DAPI-stained cells. Confocal microscopy was performed on an
inverted TCS-SP2-AOBS spectral confocal laser scanning microscope
(Leica Microsystems) with an HCX PL APO 363/1.2w long-working-
distance (220 mm) water immersion objective (Leica). Samples were ex-
cited with a 488-nm argon laser with an emission band of 500 to 510 nm
684 to 735 nm for the detection of the vital DNA marker DRAQ5 (100 mM).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: AY047708 (PAS2),
DQ158862 (CDKA;1), and NM120425 (Arabidopsis CDC25).
The following materials are available in the online version of this article.
Supplemental Figure 1. Complementation of the pas2.1 Mutant
Phenotype by the 35S-PAS2 Construct.
Supplemental Figure 2. Nuclear Accumulation of PAS2:GFP in the
Root Hair Cell.
We thank Hilde Stals for the generous gift of the p10CKS1Atprotein and
Martine De Cock for help preparing the manuscript. We also thank Jane
Endicott for providing CDK2 and CDK2-WEE1 constructs and Jesus Gil
for his help in the CDK2/PAS2 binding experiments. M.D.C. was
Antiphosphatase Regulates Cell Division1435
supported by a grant from the Ministe `re de la Recherche Franc ¸aise and
by a short-term Marie Curie fellowship. L.D.V. is a postdoctoral fellow of
the Research Foundation-Flanders.
Received December 19, 2005; revised April 12, 2006; accepted April 22,
2006; published May 12, 2006.
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