From the Horticulture and Food Research Institute of
New Zealand, Private Bag, 92169 Auckland, New Zealand and the
§ Cancer Biology Program, Division of Hematology/Oncology,
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, Massachusetts 02215
Received for publication, August 3, 2000, and in revised form, September 21, 2000
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ABSTRACT |
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The phosphorylation-specific peptidyl
prolyl cis/trans isomerase (PPIase) Pin1 in
humans and its homologues in yeast and animal species play an important
role in cell cycle regulation. These PPIases consist of an
NH2-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset
of phosphoproteins and a COOH-terminal PPIase domain that specifically
isomerizes the phosphorylated serine/threonine-proline peptide bonds.
Here, we describe the isolation of MdPin1, a Pin1 homologue from the
plant species apple (Malus domestica) and show that it has
the same phosphorylation-specific substrate specificity and can be
inhibited by juglone in vitro, as is the case for Pin1. A
search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are
multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum).
Furthermore, all these plant Pin1-type PPIases, including AtPin1 in
Arabidopsis thaliana, do not have a WW domain, but all
contain a four-amino acid insertion next to the phospho-specific
recognition site of the active site. Interestingly, like Pin1, both
MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a
temperature-sensitive mutation in the Pin1 homologue
ESS1/PTF1 gene in Saccharomyces
cerevisiae. However, deleting the extra four amino acid
residues abolished the ability of AtPin1 to rescue the yeast mutation
under non-overexpression conditions, indicating that these extra amino
acids may be important for mediating the substrate interaction of plant
enzymes. Finally, expression of MdPin1 is tightly associated with cell
division both during apple fruit development in vivo and
during cell cultures in vitro. These results have
demonstrated that phosphorylation-specific PPIases are highly conserved
functionally in yeast, animal, and plant species. Furthermore, the
experiments suggest that although plant Pin1-type enzymes do not have a
WW domain, they may fulfill the same functions as Pin1 and its
homologues do in other organisms.
Peptidyl prolyl cis/trans isomerases
(PPIases)1 catalyze the
energetically unfavorable and intrinsically slow process of
cis/trans isomerization of peptide bonds
amino-terminal to a proline (1-3). The PPIases are ubiquitous enzymes
and have been shown to be involved in protein folding, protein
translocation through biological membranes, and signal transduction (1,
4, 5). Three structurally distinct families of PPIases have been
identified so far. Cyclophilins and FK506-binding proteins are
two well characterized families of PPIases, which are targets for the
immunosuppressive drugs cyclosporin A and FK506, respectively (2, 6,
7). However, inhibition of the PPIase activity of cyclophilins and
FK506-binding proteins is not required for the immunosuppressive
property of cyclosporin A and FK506 (8). Furthermore, despite their
high conservation in various organisms, no member of the cyclophilin or
FK506-binding protein families seems to be essential, because a
yeast strain with deletions in all these PPIases is still viable (9).
Thus, evidence for the biological importance of the isomerase activity
in these two PPIase families has been limited.
Recently, a third family of PPIases, with the Escherichia
coli parvulin being the prototype, has been described. Among this family are the highly conserved Pin1-type PPIases (10, 11), which are
the only PPIases that seem to be essential for cell survival, at least
in budding yeast and HeLa cells. The Pin1-type PPIases, such as the
human Pin1 (11), Saccharomyces
cerevisiae ESS1/Ptf1 (10, 12, 13),
Drosophila Dodo (14), Neurospora crassa Ssp1 (15), Xenopus Pin1 (16),
Aspergillus nidulans Pin1 (17), and
others, consist of an amino-terminal WW domain and a
carboxyl-terminal PPIase domain. WW domains, which are
characterized by two invariant tryptophans, are present in a variety of
signaling and regulatory proteins and were originally identified as
protein interaction modules that bind to proline-rich regions in
protein targets (18, 19). The WW domain of Pin1 has recently
been shown to function as a phosphoprotein-binding module that binds to
specific phosphoserine- and phosphothreonine-proline (pSer/pThr-Pro) motifs (20). Because these motifs are targets for some proline-directed kinases in the cell cycle, activation of these kinases is thought to
create the binding sites for the WW domain of Pin1 (21). The PPIase
domain of the Pin1-type proteins shows some homology to the E. coli parvulin (22) and the human parvulin homologue, hPar14 (23).
However, in contrast to other parvulin-type PPIases, all members of the
Pin1-type PPIases exhibit a very distinct substrate specificity. They
specifically isomerize only phosphorylated Ser/Thr-Pro bonds, but not
their nonphosphorylated counterparts (24, 25). As proposed earlier
(26), the phosphorylation-specific eukaryotic PPIases have been
designated Pin1-type PPIases for clarity throughout this article.
Significantly, phosphorylation on Ser/Thr-Pro motifs further restrains
the already slow cis/trans prolyl isomerization of peptide bonds (25, 27). Moreover, phosphorylation also renders
pSer/pThr-Pro peptide bonds resistant to the catalytic action of
other PPIases characterized so far (25). Therefore, phosphorylation on
Ser/Thr-Pro leads to a distinct structural feature that cannot be
achieved by phosphorylation on other Ser/Thr sites that are not
followed by a proline, and Pin1-type PPIases are unique isomerases that
specifically isomerize phosphorylated Ser/Thr-Pro bonds (21).
As an essential mitotic regulator in budding yeast and HeLa cells, Pin1
binds to a defined subset of phosphoproteins, many of which are also
recognized by the mitosis- and phospho-specific monoclonal antibody
MPM-2, like Cdc25, tau, and the COOH-terminal domain of RNA
polymerase II (25, 28-30). Furthermore, Pin1 regulates the functions
of its binding proteins, including inhibiting the mitosis-promoting
activity of Cdc25C (28) and restoring the biological activity of
Alzheimer-associated tau protein (29). Depletion or mutations of
Pin1 induce premature mitotic entry and mitotic arrest in yeast, HeLa
cells, and Xenopus egg extracts (11, 13, 16, 17, 28). Pin1
is also required for the replication checkpoint in Xenopus
extracts (16). Furthermore, in neurons of Alzheimer's disease
patients, depletion of Pin1 is associated with hyperphosphorylated tau
and neuronal loss (29). In addition, ESS1/PTF1 seems to play an
important role in mRNA maturation, because its mutation leads to a
defect in 3'-end formation of pre-mRNAs (13). Recently, a link
between ESS1/PTF1 and the general transcription machinery via its
interaction with the phosphorylated COOH-terminal domain of RNA
polymerase II has been described (30). Recent results from our
laboratory indicate that the phosphatase PP2A is conformation-specific,
dephosphorylating only the trans isomer of its specific
pSer/pThr-Pro targets. Interestingly, the dephosphorylation of a
protein or peptide by PP2A can be dramatically influenced by the PPIase
activity of Pin1, which apparently converts cis
pSer/pThr-Pro bonds in the substrate into PP2A-accessible trans peptide bonds (31). Furthermore, a function in protein folding has been suggested for the N. crassa protein
Ssp1, because it is able to accelerate the refolding of
urea-denatured dihydrofolate reductase in vitro (15).
Recently, a new member of the Pin1-type PPIases, AtPin1, from the plant
Arabidopsis thaliana has been reported (26). AtPin1 lacks an
NH2-terminal WW domain but has significant homology to the
PPIase domain of Pin1 and as such a strong substrate specificity toward
phosphoserine-proline in a two-dimensional NMR spectroscopy PPIase
assay (26). However, it has not been described whether AtPin1 has any
role in cell cycle progression.
Here we describe the isolation and functional characterization of a
Malus domestica Pin1 homologue, MdPin1, which has the characteristic Pin1-type PPIase domain but lacks the
NH2-terminal WW domain. In the standard protease-coupled
PPIase assay, MdPin1 exhibits the same phosphorylation-specific
substrate specificity and can be inhibited by juglone, as is the case
for human Pin1. A search in the plant EST data bases reveals
that all plant Pin1-type PPIases do not contain a WW domain, but all
contain a four-amino acid insertion next to the phospho-specific
recognition site of the active site. Interestingly, like Pin1, both
MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a
temperature-sensitive mutation in the Pin1 homologue
ESS1/PTF1 gene in S. cerevisiae. In contrast,
deleting the extra four amino acid residues abolished the ability of
AtPin1 to rescue the yeast mutation. Finally, Northern blot analysis
indicates that expression of MdPin1 is tightly associated with cell
division of the plant cell in growing apple fruit in vivo
and in suspension cell cultures in vitro. These results have demonstrated for the first time that the function of
phosphorylation-specific prolyl isomerases is highly conserved in
plants and have further suggested that although plant Pin1-type
enzymes do not have a WW domain, they may fulfill the same functions as
Pin1 and its homologues do in other organisms.
Cloning MdPin1 using PCR--
The template cDNA was
synthesized from poly(A) mRNA extracted from apple (M. domestica cv. Granny Smith) fruitlets harvested 2 days after
pollination plus gibberellin treatment as described by Dong et
al. (32). DNA fragments were amplified from the cDNA using
the Expand High Fidelity PCR system (Roche Molecular Biochemicals) with the two degenerative primers MADS-4
(5'-CGTCTAGAATTCATGGCNMGNGGNAARAT) and MADS-5
(5'CGCTCGAGGATCCACRTTRTARTCNCCNYC) (M = AC).
The primers were designed according to the conserved amino acid
sequences MARGKI in the MADS-box domain and GGDYNV in the COOH-terminal region of an alignment of the three MADS-box genes APETALA3,
pMADS1, and Deficiens. The underlined
EcoRI and BamHI sites were used for cloning the
PCR product. The conditions for the PCR reaction were as follows:
initial denaturation at 94 °C for 4 min; then 35 cycles of 94 °C
for 1 min, 60 °C for 1 min, and 72 °C for 3 min; and a final
extension of 5 min at 72 °C. The amplified DNA fragments of ~600
bp were cloned into the Blueskript SK vector (Stratagene, San
Diego, CA) using EcoRI and BamHI and sequenced. Using the sequence information, the gene-specific primer PPI-1 (5'-GGCATCGTGGAAGGATCCTGAAGG) was designed. The 3' region
of the MdPin1 gene was amplified using PPI-1, and the
3' rapid amplification of cDNA ends primer
(5'-GAGAGAGAACTAGTCTCGAG), which anneals to the adapter used in the
cDNA library synthesis. The PCR conditions were the same as above.
The amplified fragments were cloned into the pGEM-T EASY vector
(Promega) and sequenced. A BamHI site in the primer PPI-1
was used to join the 5' and 3' clones to obtain a full-length cDNA.
AtPin1 was amplified from A. thaliana using PCR based on a
published sequence (26), and its deletion mutant was made using a
PCR-based mutagenesis technique and confirmed by sequencing, as
described previously (28, 29).
Northern Hybridization--
Total RNA was extracted from young
(1, 2, 3, and 4 weeks following hand pollination) and mature apple
fruits using the methods of Chang et al. (33) and from cell
suspension cultures using Trizol (Life Technologies, Inc.). The cell
suspension cultures were derived from the cortex of apple fruit
(34) and maintained in liquid medium with 4.5 µM
2,4-dichlorophenoxyacetic acid (35). Because the cell growth in the
liquid culture reaches the stationary phase after 7 days, 40 ml of
these cultures were transferred into 100 ml of fresh medium every week
to maintain growth. For RNA extraction, aliquots of the cell culture
were sampled at day 1, 2, 4, 5, 7, and 11. Northern blots were prepared
by transferring 10 µg of RNA from each time point to Hybond-N+ nylon
membranes (Amersham Pharmacia Biotech) following agarose gel
electrophoresis. The Northern blots were probed with
[32P]dCTP-labeled MdPin1 cDNA. Membranes were
hybridized in 0.5 M NaPO4 buffer (pH 7.2), 1 mM EDTA, 7% SDS at 65 °C and washed using 0.4 × SSC, 0.2% SDS at 65 °C. Autoradiographs were exposed for 2 days at
Peptidyl-Prolyl cis/trans Isomerase Activity
Assay--
Recombinant MdPin1 protein was produced by subcloning the
MdPin1 cDNA into the pGEX-2T vector. PPIase activity was assayed by
monitoring the absorbance of released 4-nitroaniline at 390 nm at
4 °C in triplicate, as described (25). Briefly, enzymes were
pre-incubated with substrate in a buffer containing 35 mM HEPES, pH 7.8, 1 mg/ml bovine serum albumin for 5 min. The reaction was
initiated by the addition of the protease stock solution. Measurement
of the bimolecular rate constants
kcat/Km for the
PPIase-catalyzed isomerization was performed by a protocol modified
from Fischer et al. (7). Because
[S]0 Expression of MdPin1 in the S. cerevisiae Strain YPM2--
The
coding sequence of MdPin1 was cloned into the vector pBC100, which
allows for galactose-induced expression of hemagglutinin-tagged proteins, as described previously (11). YPM2 cells were grown overnight
in YAPD medium (2% Difco Bacto-peptone, 1% Difco yeast extract, 2% glucose) at 23 °C and then transformed with
MdPin1/pBC100 using the polyethylene glycol method (20, 31). The
transformed cells were plated onto minimal medium containing 2%
glucose but lacking the amino acid leucine to select for transformants
and grown at 23 °C for 5-7 days. Colonies were then restreaked at least two times and incubated under the same conditions. For the spotting analysis, single yeast colonies were suspended in TE buffer 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA. The A600 was adjusted to 1, a 10-fold
series dilution in TE buffer was made, and 5 µl of each dilution were
spotted onto the plates. All plates were made of minimal media
without leucine and contained 2% glucose (no induction), a mixture of
2% glucose and 2% galactose (partial induction), or 2% galactose
(full induction). The plates were incubated for several days at
23 °C (permissive conditions) or 30 °C (restrictive conditions).
The same procedure was used to isolate YPM2 cells expressing human
Pin1, its PPIase domain AtPin1, or its deletion mutant. As
controls, cells carrying either the pBC100 vector without insert or the
construct yESS1/Yep451, which allows for constitutive expression of
ESS1, were used. To check for protein expression, YPM2 cells with
different pBC100 constructs were grown at 23 °C in minimal
media without leucine but with 2% galactose to induce protein
expression. The cells were harvested and lysed with glass beads, and 50 µg of total protein extract were separated on a 17.5%
SDS-polyacrylamide gel electrophoresis gel. After Western blotting, the
expressed proteins were detected using 12CA5 monoclonal antibody.
Isolation of MdPin1--
The full-length cDNA of M. domestica Pin1 (MdPin1) and the deduced amino acid sequence are
shown in Fig. 1. The cDNA was cloned in two steps. First, DNA fragments of ~600 bp were amplified from an
apple fruitlet cDNA library using the two degenerated PCR primers MADS-4 and MADS-5. The primers were originally designed to detect APETALA3-like MADS-box genes. After sequencing nine
random clones, we found that they contained the same DNA sequence,
which had significant sequence homology to a part of the human Pin1
(11). Two clones represent an independent sequence that had an 8-bp deletion in the 5' untranslated region when compared with the other
seven sequences (Fig. 1). This indicates that at least two alleles of
MdPin1 were expressed in apple fruit tissues. The missing 3' region of
MdPin1 was amplified in a second step using the specific primer PPI-1
and a 3' rapid amplification of cDNA ends primer. Four independent
PCR fragments were analyzed and found to contain the same sequence that
overlapped with the original 600-bp fragments. The full-length cDNA
was then assembled by joining the 5' and 3' clones at the
BamHI site in the overlapping region.
Existence of Multiple Pin1-type PPIase Genes in Plants--
An
alignment of the MdPin1 amino acid sequence with homologues of Pin1
from selected organisms is shown in Fig.
2. Pin1 and ESS1 play significant roles
in the cell cycle and consist of two functional domains, the WW domain
and the PPIase domain. The Pin1 WW domain is a
pSer/pThr-Pro-binding module (20), and the PPIase domain
catalyzes sequence-specific and phosphorylationdependent prolyl
isomerization (25). MdPin1, however, lacks the WW domain and consists
only of the Pin1-type PPIase domain (Fig. 2). The absence of the
NH2-terminal WW domain in the MdPin1 cDNA is
probably not due to a partial sequence, because an in-frame
stop codon was identified in the 5' untranslated region.
Furthermore, homologues of Pin1-type PPIases, which also lack the
NH2-terminal WW domain, have recently been
identified in the plants A. thaliana (26) (Fig. 2) and
Digitalis lanata (41). MdPin1 has an amino acid sequence identity of 79% to the A. thaliana homologue and
of 55% to the PPIase domain of Pin1. A search in the plant EST
data bases reveals that the Pin1-type PPIases are present in various
plants. Furthermore, there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon
esculentum), which were also noted by Metzner et al.
(41). These observations suggest that multiple Pin1-type proteins might
also be present in mammalian cells. Interestingly, we also found that
all plant enzymes do not have a WW domain but contain a
four-amino acid insertion next to the phospho-specific recognition site
of the active site (Fig. 2). These four amino acid residues are
important for the in vivo function of proteins, as
shown below (see Fig. 4).
Phosphorylation-specific PPIase Activity of MdPin1 and Its
Inhibition by Juglone--
Because of the high amino acid homology of
MdPin1 to the PPIase domain of human Pin1, which includes the highly
conserved amino acids Lys-63, Arg-68, and Arg-69 that have been shown
to be critical for the unique substrate specificity of Pin1 (24), we
wanted to know if MdPin1 also shows a phosphorylation
specificity in a PPIase assay. The results in Table
I show that MdPin1 exhibited the same
substrate specificity in the standard protease-coupled PPIase assay as
Pin1. Namely, MdPin1 prefers acidic residues like glutamate
NH2-terminal to the proline. Furthermore, like Pin1, MdPin1
showed an even higher isomerase activity, when a pSer-Pro-containing peptide was used as a substrate, with a specific activity similar to
that of Pin1 (Table I). In addition, because juglone has been shown to
inhibit the enzymatic activity of many enzymes, including Pin1-type
PPIases, by covalently inactivating their active site cysteine residue
(36), we examined the effect of juglone on MdPin1. Not surprisingly,
juglone inhibited the PPIase activity of MdPin1 in a
concentration-dependent manner (Fig.
3), with a potency similar to that of
inhibiting Pin1 or parvulin, as shown previously (36). These results
show that MdPin1, although it lacks an NH2-terminal WW
domain, belongs to the Pin1-type subfamily of PPIases, which require a
phosphorylated amino acid NH2-terminal to the proline in
their substrate to efficiently catalyze the isomerase reaction.
Functional Rescue of the Essential ESS1/PTF1 in Yeast by MdPin1 or
AtPin1--
In light of the same phosphorylation-specific substrate
specificity of Pin1 and MdPin1, we asked whether MdPin1 could perform the function of Pin1 in vivo. To answer this question, we
examined whether expression of MdPin1 in the yeast strain YPM2 (12,
13), which carries a temperature-sensitive mutation in the Pin1
homologue ESS1/PTF1, could support growth of YPM2 at the restrictive
temperature. The coding sequences of Pin1, the Pin1 PPIase domain, and
MdPin1 were cloned into the vector pBC100 under the control of the
inducible galactose promoter. The activity of the galactose
promoter depends on the carbon source in the medium. The promoter is
repressed when glucose is used as the carbon source but induced when
galactose is used instead. To examine the relative levels of the
expressed proteins, a hemagglutinin epitope tag, which does not
affect the function of Pin1 in vivo (11), was
inserted at the NH2 termini of all constructs.
Immunoblotting analysis of lysates from YPM2 cells transformed with the
different constructs using anti-hemagglutinin antibodies showed
that all proteins were expressed at similar levels after galactose
induction (Fig. 4C). As a
positive control, we used YPM2 cells carrying a construct containing
the functional ESS1/PTF1 gene, which grew
independently on the carbon source at both the permissive and the
nonpermissive temperature. As expected, YPM2 cells harboring the
different plasmids were able to grow at the permissive temperature on
glucose (Fig. 4A) or galactose media (data not shown). All
YPM2 cells, with the exception of the positive control, failed to grow
on glucose medium when incubated at the restrictive temperature. At the
restrictive temperature, however, the same cells were able to grow on
glucose/galactose or galactose medium, which allows partial or full
induction of the transgene, respectively. These results indicate that
MdPin1 is able to perform the essential function in yeast.
We have demonstrated that the WW domain is normally required for Pin1
to perform its essential function in yeast (20) and that the Pin1
PPIase domain alone is able to rescue the yeast mutation only when it
is overexpressed (31), as shown in Fig. 4A. These results
indicate that the phosphoserine/phosphothreonine-binding module
WW domain is important for targeting Pin1 to its substrates. However,
the finding that MdPin1 was able to support the growth of YPM2 cells to
the same extent as full-length Pin1 either at partially or fully
induced conditions was surprising. To confirm this result, we asked
whether other plant Pin1-type PPIases would have the similar function
and chose AtPin1. As shown in Fig. 4B, AtPin1 was able to
support the growth of YPM2 cells in a manner similar to full-length
Pin1 on glucose/galactose or galactose media. These results
confirm that plant Pin1-type PPIases are indeed able to function like
full-length Pin1 in yeast, although they do not have a WW domain. After
comparing sequences of Pin1-type genes from plants and other organisms,
we found that the plant Pin1s contain an extra four-amino acid
insertion in their PPIase domains. To examine the importance of this
insertion, we generated a mutant AtPin1 Cell Division Regulated Expression of MdPin1 in
Plants--
Because MdPin1 is able to rescue the temperature-sensitive
phenotype of YPM2, the above results suggest that MdPin1 may also be
involved in the regulation of cell division in apples. To examine this
possibility, we analyzed the expression of MdPin1 in apple fruit
tissues at progressive development stages and cell suspension cultures
to determine whether the expression of MdPin1 is correlated to plant
cell division. Northern blot analysis revealed that MdPin1 was
expressed in 1-3-week-old fruit but not in 4-week-old or mature fruit
(Fig. 5A). Previous research
into apple fruit development suggested that fruit growth in the first
3-4 weeks resulted primarily from cell division, whereas the
subsequent growth is mainly based on cell enlargement (37). It was also
recently shown that the major cell division period in "Braeburn"
apple fruit occurs in the first 3-4 weeks after pollination (38). In
this respect, the Northern data indicates that the MdPin1 expression is
associated with cell division in apple fruit development. To further
support this observation, we examined the MdPin1 expression in cultured apple cells. Significant levels of MdPin1 transcripts were detected in
2-5-day-old cultures but not in 7-11-day-old cultures (Fig. 5B). It is important to note that under our conditions, the
growth in the suspension cell cultures reaches a stationary level after 7 days (data not shown), i.e. cell division stops. Similar
to the experiments using the apple fruit, MdPin1 expression in
suspension-cultured cells was only detected in dividing cells. These
data therefore further support the conclusion that MdPin1 expression is
tightly associated with cell division of the plant cell.
This paper describes the isolation and characterization of MdPin1,
a new member of the Pin1-type PPIases from M. domestica, and also demonstrates the functional conservation of
phosphorylation-specific PPIases in plants. MdPin1 consists only of a
PPIase domain, which shows a high homology to other members of the
Pin1-type PPIases and exhibits phosphorylation-specific PPIase activity
similar to Pin1. Interestingly, there are multiple Pin1-type PPIases
present in plant EST data bases, even within one organism, and
all do not have a WW domain but contain a four-amino acid insertion
next to the phospho-specific recognition site of the active site in their PPIase domains. Importantly, both MdPin1 and AtPin1 are able to
rescue the temperature-sensitive mutant phenotype of the yeast
ESS1/PTF1 gene under partially induced conditions, as is the
case for Pin1. However, deleting the extra four amino acid residues
abolished the ability of AtPin1 to rescue the yeast mutation under the
same conditions, indicating the critical role of these extra amino
acids for the plant enzyme in vivo. Finally, expression of
MdPin1 is tightly associated with cell division during apple fruit
development in vivo and during cell cultures in
vitro. These results have demonstrated that
phosphorylation-specific PPIases are highly conserved functionally from
plants to humans and also suggest that despite the absence of the WW
domain, plant Pin1-type enzymes may fulfill the same functions as Pin1
and its homologues do in other organisms.
MdPin1 does not have a WW domain at the NH2 terminus,
in contrast to human Pin1 and its homologues from various other
organisms (Fig. 2). Furthermore, two other Pin1-type PPIases from
plants, AtPin1 (26) and DlPin1 (DlPar13) (41), also lack the WW domain. The WW domain of Pin1 specifically interacts with phosphoserine- or
phosphothreonine-proline motifs and is normally essential for human
Pin1 to perform its biological function (20). Furthermore, Pin1 also
binds Alzheimer's associated phosphorylated tau protein via its WW
domain and restores its biological activity, which also requires the
PPIase activity of Pin1 (29). Moreover, neither the WW domain nor the
PPIase domain alone is sufficient to rescue the yeast mitotic phenotype
under normal expression conditions. However, when overexpressed, only
the PPIase domain, but not the WW domain, can rescue the yeast mutation
(31). These results indicate that although the WW domain of Pin1 is
normally required for targeting Pin1 to its substrates, the PPIase
domain performs the essential function (31). Although neither MdPin1
nor AtPin1 contains a WW domain (Fig. 2), both enzymes are able to
carry out the essential mitotic function in yeast (Fig. 4). These
results further support the notion that it is the PPIase domain that
carries out the essential function of Pin1 in vivo.
MdPin1 seems to play a role in the plant cell cycle similar to that of
hPin1 and its homologues in other eukaryotic cells. Our results have
shown that the presence of MdPin1 transcripts in apple cells is tightly
associated with cell division. Developing fruits show MdPin1 expression
only for up to 3 weeks, when the division of the fruit cells stops
(Fig. 5A), and the continuing growth of the fruits is
achieved primarily by cell enlargement (37). This result is consistent
with the absence of a mitotic regulator like MdPin1, whose RNA
transcripts are no longer detected at this later time point. Further
support for this model comes from our studies using apple suspension
cell cultures. After transferring the cells to fresh medium, cell
growth starts, and the cell number increases gradually because of cell
division. After 7 days the culture reaches a stationary state without
cell division. As can be seen in the Northern blots of these cultures
(Fig. 5B), the appearance of the MdPin1 transcript exactly
reflects the growth stages. MdPin1 mRNA can be detected as long as
the cells divide but not when the culture reaches the stationary state.
These results suggest that MdPin1 probably plays an important role in
cell cycle progression in apples, as does Pin1 in vertebrate and yeast cells.
Progression through the cell cycle is regulated by conserved
cyclin-dependent kinases in both plant and mammalian cells.
Cyclin-dependent kinases phosphorylate proteins on
Ser/Thr-Pro motifs and probably create binding sites for Pin1 (21). The
phosphorylated proteins that Pin1 binds to are also recognized by the
MPM2 antibody (25), which was developed using total mitotic extract
from HeLa cells (39). Interestingly, MPM2 antibody has also been shown
to interact with a similar subset of mitosis-specific phosphoproteins
both in mammals and plants (40). The conservation of
cyclin-dependent kinases and MPM2 antigens in mammals and
plants indicates that the basic cell cycle regulation is similar and
that the target proteins for Pin1 are probably conserved between humans
and plants. We have now shown that two different plant Pin1-type
PPIases, MdPin1 and AtPin1, like human Pin1, can rescue the yeast
strain YPM2 at the nonpermissive temperature (Fig. 3A). YPM2
has a temperature-sensitive mutation in the
ESS1/PTF1 gene that arrests cells in mitosis at the nonpermissive temperature. Together with the findings that expression of MdPin1 is cell division-dependent, these
results have demonstrated for the first time that
phosphorylation-specific PPIases are functionally conserved in yeast,
mammalian, and plant cells.
An interesting question is why all the known members of the Pin1-type
PPIases in plants do not have a WW domain. The simplest explanation
would be that the WW domain-containing Pin1-type PPIases have not yet
been identified, which may be clarified when the Arabidopsis
genome is fully sequenced. However, the more likely explanation is that
the PPIase domain of plant Pin1-type enzymes has a much higher affinity
to its substrates than does the PPIase domain of Pin1, due to some
sequence differences. Indeed, there are four additional amino acids
inserted at the phosphorylation-specific substrate recognition site in
the catalytic domain of all known plant Pin1-type PPIases (Fig. 2)
(24). More importantly, although AtPin1 can rescue the yeast mutation
either under partially induced or fully induced conditions, as is the
case for Pin1, when these extra four amino acids are deleted, the
mutant AtPin1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
Km was valid,
absorbance data points were fitted to a first-order kinetics. The
kcat/Km values were
calculated according to the equation
kcat/Km = (kobs - ku)/[PPIase], where
ku is the first-order rate constant for spontaneous cis/trans isomerization, and
kobs is the pseudo-first-order rate constant for
cis/trans isomerization in the presence of
PPIase. Inhibition of PPIase activity by juglone was performed by
pre-incubating enzymes with different concentrations of juglone for
4 h, followed by PPIase assay, as described previously (36).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (64K):
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Fig. 1.
cDNA and deduced amino acid sequence of
MdPin1. Arrows indicate the position of the PCR primers
used for the isolation of MdPin1. The 8-bp deletion found in the
untranslated 5' region of two of the nine sequences is
boxed. The BamHI site used to assemble the
full-length cDNA is indicated. The in-frame stop codon
upstream of the reading frame is in bold. The mRNA
sequence of MdPin1 has been submitted to GenBankTM with the
accession number AF209200.
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Fig. 2.
Structural conservation of Pin1-type PPIases
from various eukaryotic organisms. MdPin1, M. domestica; AtPin1, A. thaliana; LePin1-1
(GenBankTM accession number AW621939);
LePin1-2 (GenBankTM accession number AW945046), L. esculentum; GmPin1-1 (GenBankTM accession number
AW308915); GmPin1-2 (GenBankTM accession number AW397670),
G. max; HsPin1, human; ScESS1, S. cerevisiae; NcSsp1, N. crassa; AnPin1, A. nidulans. The dashed line indicates the WW domain, and
the solid line indicates the PPIase domain. The four-amino
acid insertion in the plant proteins is boxed. The
arrowhead points to the active site Cys conserved in all
Pin1-type PPIases.
Comparison of the substrate specificity of MdPin1, AtPin1, and hPin1
-chymotrypsin
(succinyl-Ala-Ala-Pro-Phe-p-nitroanilide,
succinyl-Ala-Glu-Pro-Phe-p-nitroanilide) and
trypsin (acetyl-Ala-Ala-pSer-Pro-Phe-p-nitro-anilide),
were used.
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Fig. 3.
Inhibition of the PPIase activity of
MdPin1 by juglone. 100 nM MdPin1
was incubated with different concentrations of juglone, and the PPIase
activity toward the peptide
succinyl-Ala-Glu-Pro-Phe-p-nitroanilide was
measured using the protease-coupled PPIase assay. A,
reaction curve of MdPin1 PPIase assays in the absence or presence of
juglone. The curves were fitted to a first-order reaction kinetics,
with the maximal absorbance at 390 nm being defined as 1.0. B, concentration-dependent inhibition of MdPin1
by juglone. The kcat/Km of a
PPIase reaction without juglone was set at 100%. The relative PPIase
activity is plotted against the amount of juglone used in the
reaction.
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Fig. 4.
Functional rescue of the yeast ESS1/PTF1
mutation by plant Pin1s and the critical role of the four-amino acid
insertion in the PPIase domain of plant Pin1s. A,
functional rescue of the yeast ESS1/PTF1 mutation by MdPin1. YPM2 cells
transformed with the MdPin1/pBC100 and hPin1-PPI/pBC100 expression
constructs were spotted in a series 10× dilution onto agar plates with
glucose, glucose/galactose, or galactose as carbon source. The plates
were incubated at 23 °C (permissive) or 30 °C (restrictive).
Galactose allows for overexpression of the transgene. Growth of the
cells indicates the complementation of the YPM2 mutation. The control
was a strain that constitutively expresses ESS1 (yESS1/Yep451) and the
empty vector pBC100. B, functional rescue of the yeast
ESS1/PTF1 mutation by AtPin1 and its deletion mutant. YPM2 cells
transformed with Pin1, AtPin1, and AtPin1 expression constructs were
spotted onto agar plates with glucose/galactose or galactose as carbon
source. The plates were incubated at the permissive or restrictive
temperature. AtPin1
contains a deletion of four amino acid residues
that are present in plant Pin1s but not other Pin1s with a WW domain,
as shown in Fig. 2. C, similar expression of transgenes in
yeast cells. YPM2 strains stably transformed with various Pin1
constructs were subjected to immunoblotting analysis using 12CA5
antibody specific to the hemagglutinin tag inserted at the
NH2 terminus of all transgenes. HA,
hemagglutinin.
, from which the four amino
acid residues were deleted. When AtPin1
was expressed in the YPM2
strain, it was expressed at a level similar to that of wild type AtPin1
protein (Fig. 4C). However, AtPin1
failed to support the
growth of YPM2 cells at partially induced conditions (Glu/Gal
media) but was able to fully support cell growth at fully
induced conditions (Gal media) (Fig. 4B), a
phenotype almost identical to that of the Pin1 PPIase domain alone
(Fig. 4A). These results indicate that these extra four
amino acids in AtPin1 are apparently able to substitute for the
function of the WW domain, at least in this yeast functional assay.
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Fig. 5.
Association of MdPin1 expression with cell
division during apple fruit development in vivo and in
cell cultures in vitro. 10 µg of total RNA was
used for each lane of the Northern blot. As a probe,
[32P]dCTP-labeled MdPin1 cDNA was used. A,
RNA extracted from fruitlets in different stages of their development
(1-4 weeks and mature (Ma) fruit). B, RNA
extracted from cell suspension cultures after 1, 2, 4, 5, 7, and 11 days of growth.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can rescue the yeast mutation only under fully
induced, but not under partially induced, conditions (Fig.
4B). These phenotypes of AtPin1
are almost identical to
those of the Pin1 PPIase domain alone, as shown previously (31; also
see Fig. 4A). These results indicate that these extra four
amino acids somehow convert AtPin1 to function like full-length Pin1 at
least in yeast, suggesting that they may substitute for the function of
the WW domain. Because these four amino acid residues are next to the
phospho-specific recognition site of the active site, it is possible
that they play an important role in mediating the substrate interaction
for plant Pin1-type PPIases. Further studies on identifying the plant
Pin1 substrates and elucidating their biological functions will help to
understand the role of phosphorylation-dependent prolyl
isomerization in controlling cell growth in plants.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Ian Ferguson for providing the apple cell suspension cultures.
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Note Added in Proof |
---|
Due to some recent publications (Chao, S. H., Greenleaf, A. L., and Price, D. H. (2001) Nucleic Acids Res. 29, 767-773; Rippmann, J. F., Hobbie, S., Daiber, C., Guilliard, B., Bauer, M., Birk, J., Nar, H., Garin-Chesa, P., Rettig, W. J., and Schnapp, A. (2000) Cell Growth Differ. 11, 409-416), it is necessary to clarify the role of juglone as an inhibitor of Pin1. Juglone nonspecifically and irreversibly modifies sulfhydryl groups in proteins. In contrast to cyclophilins and FK506-binding proteins, members of the Pin1 subfamily have a cysteine in their active site, so that their activity can be inhibited by juglone in vitro. However, juglone also inhibits many other proteins and enzymes, some even with several hundred-fold lower Ki values than that of Pin1. Therefore, juglone is unlikely to be a Pin1-specific inhibitor in vivo.
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FOOTNOTES |
---|
* This research was supported by the New Zealand Foundation for Research, Science, and Technology (C06411) and by National Institutes of Health Grants R01GM56230 and GM58556 (to K. P. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF209200.
¶ An Ernst Schering Research Foundation Fellow.
A Leukemia and Lymphoma Society Fellow.
** A Pew Scholar and a Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, HIM 1047, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-4143; Fax: 617-667-0610; E-mail: klu@caregroup.harvard.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M007006200
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ABBREVIATIONS |
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The abbreviations used are: PPIase, peptidyl prolyl cis/trans isomerase; p, phospho; PCR, polymerase chain reaction; bp, base pair(s); EST, expressed sequence tag.
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REFERENCES |
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