(Received for publication, March 7, 1995; and in revised form, June 23, 1995)
From the
Efficient translation of the mRNA encoding the 65-kDa regulatory
subunit (PR65) of protein phosphatase 2A (PP2A) is prevented by an
out of frame upstream AUG and a stable stem-loop structure (
G = -55.9 kcal/mol) in the 5`-untranslated region
(5`-UTR). Deletion of the 5`-UTR allows efficient translation of the
PR65
message in vitro and overexpression in COS-1 cells.
Insertion of the 5`-UTR into the
-galactosidase leader sequence
dramatically inhibits translation of the
-galactosidase message in vitro and in vivo, confirming that this sequence
functions as a potent translation regulatory sequence. Cells
transfected or microinjected with a PR65
expression vector lacking
the 5`-UTR, express high levels of PR65
, accumulating in both
nucleus and cytoplasm. PR65
overexpressing rat embryo fibroblasts
(REF-52 cells) become multinucleated. These data and previous results
(Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E.,
Baumgartner, S., Hemmings, B. A., and Glover, D. M.(1993) Cell 72, 621-633) suggest that PP2A participates in the
regulation of both mitosis and cytokinesis.
Protein phosphorylation/dephosphorylation is a crucial
intracellular control mechanism. The phosphorylation state of a protein
is the net result of the antagonistic activities of protein kinases and
protein phosphatases. Protein phosphatase 2A (PP2A), ()one
of the major Ser/Thr protein phosphatases, consists of a catalytic (C)
subunit of 36 kDa, complexed to a ``constant'' regulatory
subunit of 65 kDa (PR65). This core heterodimer further associates with
a single ``variable'' third subunit to form multiple trimeric
holoenzymes. To date several classes of variable subunits with
molecular sizes of 55 kDa (PR55), 72 kDa (PR72), 130 kDa (PR130), 54
kDa or 74 kDa have been identified (for reviews, see (1, 2, 3) ). Given this complexity of
regulatory subunits it can be anticipated that many different control
mechanisms are likely to operate to coordinate the synthesis and
assembly of the different holoenzymes. These controls will most
probably act at both transcriptional and post-transcriptional levels.
It appears that the level of PP2A catalytic subunit is tightly regulated because attempts to overexpress this protein have been unsuccessful even though it is possible to overexpress the mRNA(4, 5) . Recently, Wadzinski et al.(6) succeeded in expressing an amino-terminal tagged form of PP2A catalytic subunit, but this expression probably occurs at the expense of the genomically derived catalytic subunit. Taken together these results indicate that some form of translational or post-translational control mechanism operates to control the amount of the catalytic subunit. Whether such mechanisms operate to coordinate the translation of the PP2A regulatory subunits has not been investigated.
Translational control is often exerted at the level of
translation initiation. According to the scanning hypothesis of mRNA
translation initiation (reviewed in (7) ), the complex between
initiator Met-tRNA and the 40 S ribosomal subunit, binds at or near the
mGpppG cap structure at the 5` end of the mRNA and scans in
the 3` direction until an AUG codon is reached. The 60 S ribosomal
subunit is subsequently recruited to the complex and translation
starts. These steps are catalyzed by eukaryotic initiation factors. Two
elements within the 5`-UTR of a mRNA are known to inhibit translation
initiation. First, an AUG located upstream of the authentic start codon
can inhibit translation initiation. If this upstream AUG is followed by
a stop codon, the ribosome can resume scanning and reinitiate at the
correct AUG start codon. The longer the distance between this upstream
stop codon and the authentic start codon, the smaller the inhibitory
effect of the upstream AUG. If the stop codon is located within the
coding sequence (thus down stream of the authentic start AUG),
inhibitory effects of the upstream AUG are maximal, since only
ribosomes that ignore the upstream AUG will initiate at the correct
start codon(8) . Second, stable secondary structures in the
5`-UTR interfere with scanning of the ribosome and lead to inhibition
of translation. Kozak (9) showed that secondary structures in
the 5`-UTR must have a free energy of more than -30 kcal/mol to
inhibit translation in COS-1 cells, although Sagliocco et al.(10) reported that structures with a free energy of
-20 kcal/mol are able to inhibit translation in yeast. Besides
these inhibitory mechanisms, other mechanisms for control of
translation initiation exist, such as phosphorylation/dephosphorylation
of initiation factors(7) , but it is assumed that these have a
more general effect on translation and are less messenger-specific.
In the present work we investigated whether expression of the PR65
subunit is controlled at the translational level. This subunit is
common to all forms of PP2A characterized and therefore forms the
scaffold for the assembly of the different trimeric holoenzymes. The
PR65 subunit is encoded by two genes(11) , termed PR65 and
PR65
. The
-isoform seems to be the most abundant in all
tissues and cells examined, except in Xenopus oocytes, where the
-isoform predominates (12) . We show that the 5`-UTR of
PR65
acts as a translational repressor, due to an upstream AUG and
to a stable stem-loop structure. We further show that release of this
translational inhibition leads to overexpression of PR65
and that
this apparently causes defects in cytokinesis resulting in
multinucleated cells.
The 5`-UTR regions of C,
PR55
, PR55
, and PR65
were deleted by introducing (using
PCR) a unique SalI site upstream of the ATG start codon (at
-28 bp for C
, -24 bp for PR65
), or a XbaI site at -18 for PR55
or a HindIII
site at -29 for PR55
. The resulting constructs were
subcloned into either pBluescript (C
, PR55
, PR55
) or
pGEM (PR65
) for in vitro transcriptions, or into pECE for
transfections.
The 5`-UTR of PR65 was isolated by PCR using a
full-length cDNA (11) as template and the following primers (HindIII sites underlined): ACAAGCTTCCGGTTCTCACTCTT (primer 1,
sense), ATAAGCTTCATGGGG GAGTCA (primer 2, sense), and
ATAAGCTTGGCTCCGTCCCTTT (primer 3, antisense), resulting in 138- and
62-bp fragments, respectively. In the 62-bp fragment, the upstream ATG
was mutated to ATT by PCR with primer 4 (ATAAGCTTCATTGGGGAGTCA, sense)
and primer 3 (antisense). To introduce an ATG to ATT mutation in the
138-bp fragment, we performed PCR with primer 1 and primer 5
(AGATGACTCCCCAATGGA, antisense, HinfI site underlined), and
introduced the product into the 138-bp 5`-UTR using an internal HinfI site. The PCR products are schematically presented in Fig. 4A. The resulting PR65
5`-UTR constructs were
subcloned in the 5`-UTR of the pSV-
-galactosidase vector (Promega)
using an HindIII site. An in-frame stop codon is present 11
residues downstream of the HindIII site. The orientation of
the 5`-UTR inserts was verified by dideoxy sequencing. Part of the
coding sequence of
-galactosidase was excised from the
pSV-
-galactosidase vector by digestion with HindIII and EcoRV (thus encoding a polypeptide with a predicted molecular
size of 49.8 kDa), and subcloned into the corresponding pBluescript
sites to create pBS.
-galactosidase. The PR65
5`-UTR PCR
fragments described above were then subcloned into the 5`-UTR using the HindIII site of pBS.
-galactosidase.
Figure 4:
The
5`-UTR of PR65 is a translational inhibitor. A, schematic
presentation of the various constructs of the 5`-UTR of PR65
. The
constructs correspond to either the entire 5`-UTR (stem/loop with
AUG), the entire 5`-UTR containing a point mutation in the upstream AUG
(stem/loop with AUU), the 5`-UTR truncated at residue -63 and
thus devoid of secondary structure (AUG), or the same construct
containing a point mutation in the upstream AUG (AUU). The stability of
each construct was calculated with the RNA-fold program (25, 26) . B, in vitro translation
of chimeric mRNA encoding a fragment of
-galactosidase fused to
various constructs of the 5`-UTR of PR65
(see panel A).
As a negative control no RNA was added. C, COS-1 cells were
transiently transfected with either pSV-
-galactosidase or with the
same vector containing various constructs of the 5`-UTR of PR65
(see panel A), inserted in the
-galactosidase leader
sequence. Cells transfected with the pECE vector without insert were
used as negative control. 48 h after transfection, cell extracts were
assayed for
-galactosidase activity. The activity is expressed per
microgram of protein in the cell extract. The figure shows means
± S.E. (n = 3), as indicated by the error
bars. Transfection efficiency was tested by cotransfection of a
pSV-chloramphenicol acetyltransferase plasmid. Variations in
chloramphenicol acetyltransferase activity were less than
10%.
Equal amounts of
RNA (1 µg), 10 µCi of [S]methionine
(Amersham), and 10 µl of cell-free rabbit reticulocyte lysate
(Stratagene) were incubated for 1 h at 30 °C with gentle shaking
every 15 min. Protein translation products were analyzed on 10%
SDS-PAGE followed by fluorography.
Antibodies were directed against recombinant PR65
(Ab65
) or against synthetic peptides of PR55
(Ab55
) and PR65
(Ab65
)(12, 13) . All of these
antisera have been extensively characterized and found to be specific
for the appropriate antigen(12, 13) .
For
chloramphenicol acetyltransferase and -galactosidase assays, cells
were scraped into 40 mM Tris-HCl buffer (pH 7.5) containing 1
mM EDTA and 150 mM NaCl, centrifuged for 10 min at
800
g and resuspended in 150 µl of 0.25 M Tris-HCl (pH 8). Cell pellets were freeze-thawed 3 times and
centrifuged for 10 min at 12,000
g at 4 °C.
-Galactosidase assays were carried out on the supernatant
following the instructions of the manufacturer (Promega). Prior to
chloramphenicol acetyltransferase assays(18) , the extracts
were heated for 10 min at 60 °C. Protein concentrations were
measured with the Protein Assay kit (Bio-Rad) using bovine serum
albumin as standard.
For Mono-Q FPLC analysis, cells were
homogenized in 50 mM Tris-HCl (pH 7.4), 1 mM EDTA,
10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine and centrifuged for 5 min at 12,000 g at 4 °C. 1 mg of soluble total protein was loaded on a Mono-Q
column and developed with a linear gradient from 100 to 600 mM NaCl in the same buffer. PP2A was assayed with a peptide substrate
(Leu-Arg-Arg-Ala-Ser-Val-Ala), phosphorylated with cAMP-dependent
protein kinase(19) .
Rat embryo fibroblasts (REF-52) cells
were cultured in Dulbecco's modified Eagle's medium,
supplemented with 10% fetal calf serum as described
previously(20) . Prior to microinjection, cells were
subcultured onto acid-washed glass coverslips. Microinjection was
performed with a normal Leitz micromanipulator, using 0.1 µM inner diameter micropipettes pulled on a linear horizontal puller.
The pECE.PR65 expression plasmids were injected at a concentration
of 0.5 mg/ml in a buffer containing 100 mM potassium
glutamate, 40 mM potassium citrate, 1 mM MgCl
(pH 7.2), and 1 mg/ml mouse IgG (to act as a marker for injected
cells). Cells were incubated for a further 24-48 h and then
processed for immunofluorescence. The results presented are the mean of
five independent microinjection experiments involving 15-30 cells
per experiment.
For immunofluorescence, cells were fixed in 3.7% formaldehyde and further treated as described previously(13, 20) . To mark the injected cells, cells were stained for the presence of mouse IgG using a fluorescein-conjugated anti-mouse IgG antibody (Organon Teknika).
For ribonuclease T cleavage, RNA (1 µl) was
mixed with 3 µl of denaturing buffer (7 M urea, 1 mg/ml
tRNA, 1.5 mM EDTA, 40 mM sodium acetate (pH 5), 0.4%
xylene cyanol, 0.08% bromphenol blue), boiled for 1 min and cooled on
ice. Subsequently 1 µl of ribonuclease T
(100
milliunits, Pharmacia) was added and the mixture incubated for 15 min
at 55 °C. The reaction was terminated by the addition of 5 µl
of stop solution (10 mM EDTA, 7 M urea, 0.05% xylene
cyanol, 0.01% bromphenol blue) and frozen on dry ice.
For
renaturation, 15 µl of RNA was mixed with 45 µl of renaturation
buffer (15 mM Tris-HCl (pH 7), 80 mM NaCl, 10
mM MgCl, and 2 µg/µl tRNA), heated to 65
°C and allowed to cool slowly to room temperature. The mixture (3
µl) was incubated for 15 min at room temperature with distilled
water (control) or with the indicated concentrations of enzyme (see
below). Reactions were stopped by the addition of 7 µl of stop
solution, heated for 20 s at 80 °C, and frozen on dry ice. Reaction
products were analyzed on a denaturing 6% polyacrylamide sequencing
gel. Concentrations of the enzymes were as following: ribonuclease A,
0.2 microunits per reaction; nuclease S
(Pharmacia), 2.5,
5, 10 units per reaction; ribonuclease V
(Pharmacia), 2, 4,
8 milliunits per reaction; ribonuclease T
, 18, 37, 75
milliunits per reaction. Lead acetate (pH 5.5) was used at a final
concentration of 0.1, 0.25, or 0.5 mM.
Figure 1:
Upstream AUGs
are a common element in the 5`-UTR of PP2A subunit mRNAs. Schematic
representation of the 5`-UTR of different human PP2A subunit mRNAs:
C(23) ; PR65
(11) ; PR55
(15) ;
PR55
(43) ; PR72 and PR130(44) . The open reading
frame corresponding to the phosphatase subunit is shown as a solid
box, whereas the upstream open reading frames are shown as a hatched box. For the PR130 subunit only six of the eight
upstream AUGs are labeled.
We deleted the 5`-UTR of C,
PR55
, PR55
, and PR65
and compared in vitro translation efficiency of the full-length and 5`-UTR deleted
mRNAs. Deletion of the 5`-UTR had no significant effect on the in
vitro translation efficiency of C
and PR55
messages
because they lack upstream AUGs. In contrast, the translation
efficiency increased dramatically after deletion of the 5`-UTR of
PR65
(see below) and PR55
. These messages carry one and two
upstream AUGs in their 5`-UTR, respectively. These data prompted us to
further investigate the translational regulation of the PR65
subunit mRNA.
Figure 2:
Analysis of the secondary structure of the
5`-UTR of PR65. A, secondary structure of the 5`-UTR of
PR65
as predicted by the RNA-fold
program(25, 26) . A stem and two major loops
(
G = -55.9 kcal/mol) of more than 5
nucleotides (residues -76 to -71 and residues -52 to
-46) are predicted. The upstream AUG is indicated with bold
circles. Shaded symbols refer to residues that are cleaved with
ribonuclease A, nuclease S
, or lead acetate. The arrows point to the cleavage sites of ribonuclease T
, and the open arrow is used to indicate a weak cleavage site. B, the first 175 nucleotides of PR65
mRNA were labeled at
the 5` end and cleaved at single-stranded G residues with ribonuclease
T
. The RNA was either denatured (lane 1) or
renatured (lanes 2-4) before cleavage. Concentrations of
ribonulease T
were 100 (lane 1), 75 (lane
2), 37 (lane 3), and 18 (lane 4) milliunits. Arrows as in panel A.
We checked whether a stem-loop structure
occurred in the 5`-UTR of the Drosophila PR65
mRNA(27) . Residues -194 to -47 have indeed the
potential to fold as a stem-loop, but this structure is less stable
(
G = -27.1 kcal/mol). The homology of this region
with the stem-loop of human PR65
is 37.6% (gap weight = 5,
length weight = 0.3), as tested with the GAP
program(28) . In contrast, no upstream AUGs are present in the
5`-UTR of Drosophila PR65(27) .
In order to
investigate whether the predicted stem-loop structure of the 5`-UTR of
human PR65 mRNA indeed occurred in solution, we probed the
structure of the in vitro transcribed 5`-UTR with several
ribonucleases and with lead acetate. The in vitro transcribed
RNA was 175 nucleotides long and comprised the 5`-UTR plus 31
nucleotides of the coding sequence. By comparing the effect of
ribonuclease T
(which cleaves single, but not double,
stranded RNA at G residues) on denatured and renatured RNA (Fig. 2B), we were able to elucidate the secondary
structure of the 5`-UTR in solution. A stretch of 5 G residues starting
at nucleotide -60 allowed us to locate the observed structures in
the sequence. The structure was then further analyzed (not shown) with
reagents that specifically cleave single-stranded RNA (lead acetate,
ribonuclease A, and S
nuclease) or double-stranded RNA
(ribonuclease V
). Our analysis confirmed the predicted loop
at residues -51 to -45. The predicted loop at position
-76 to -71 was not detected, but an additional loop at
postion -64 to -59 was observed. Surprisingly, in the
observed structure the upstream AUG (residue -62 to -60) is
exposed in this loop. We used the RNA-fold program to calculate the
stability (
G = -39.0 kcal/mol) of this
observed structure (using the ``prevent'' command to insert a
loop between residues -64 and -59). Taken together these
results demonstrate that a stem-loop structure is indeed formed by the
5`-UTR of PR65
, albeit somewhat less stable than predicted, but
within the range that is reported to inhibit mRNA scanning(9) .
Figure 3:
Deletion of the 5`-UTR of PR65 allows
efficient in vitro translation and overexpression in COS-1
cells. A, in vitro translation of 1 µg of either
full-length (FL) or 5`-UTR deleted (
5`) mRNA of PR65
in either the sense (S) or antisense (A) orientation.
A negative control (NC) lacking RNA was included. Protein
products were resolved by SDS-PAGE and autoradiographed. Molecular mass
markers were phosphorylase (97 kDa), bovine serum albumin (66 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21
kDa). B, COS-1 cells were transiently transfected with either
the full-length (FL) or the 5`-UTR deleted (
5`) PR65
cDNAs in the pECE expression vector. Control transfections (C)
were performed with the pECE vector without insert. 72 h after
transfection total cell extracts were analyzed by SDS-PAGE and Western
blotting with an affinity purified antibody against PR65
(Ab65
) and detected using enhanced chemiluminescence
detection. Molecular weight markers are as in A. C-F,
COS-1 cells were transfected with PR65
expression constructs as
described under ``Materials and Methods.'' 48 h later they
were fixed and immunostained with an affinity purified antibody against
PR65 (Ab65
). Immunofluorescent micrographs of
representative cells are shown after mock transfection (panel
C), after transfection with a full-length (panel D), and
with a 5`-UTR deleted PR65
expression construct (panels E and F). The images in panels C, D, and E were acquired at the same laser power, pinhole size, and
photomultiplier sensitivity settings and are therefore directly
comparable. Panel F shows the same field to that seen in panel E, but was collected with CLSM settings corresponding to
a 10-fold reduced exposure. Cells in panels E and F with lower immunostaining (i.e. not overexpressing the
PR65
) are indicated by arrowheads. Bar, 5
µm.
The
same full-length and 5`-UTR deleted cDNAs were subcloned in a mammalian
expression vector (pECE) and used to transiently transfect COS-1 cells.
Whereas transient transfection with both the full-length and the 5`-UTR
deleted construct resulted in efficient overexpression of the PR65
message (analyzed by Northern blot, data not shown), only the 5`-UTR
deleted construct resulted in efficient overexpression of PR65
protein as judged by immunoblotting (Fig. 3B).
Overexpression was about 20-fold as judged from scanning of different
exposures of the Western blot. (The data in Fig. 3B show a longer exposure which underestimates the level of
overexpression relative to the control.) A small increase
(approximately 2-fold) in the amount of PR65
was also observed
with the full-length construct.
In a similar experiment COS-1 cells
were transiently transfected with PR65 expression constructs with
or without the 5`-UTR and subsequently analyzed by indirect
immunofluorescence with PR65 specific antibodies as described under
``Materials and Methods.'' When cells transfected with the
5`-UTR deleted PR65
were analyzed about one out of 5 cells
displayed an intense staining for PR65 (Fig. 3, E and F) which was approximately 10 times stronger than in the
surrounding cells. This low staining of the surrounding cells
corresponded to that observed in non-transfected (not shown) and mock
transfected cells (Fig. 3C). Therefore we concluded
that the intensely stained cells actively overexpressed PR65. COS-1
cells transfected with the full-length PR65
construct were
indistinguishable from untransfected cells (Fig. 3D),
again confirming that the 5`-UTR allowed only very low levels of
expression. The observed immunofluorescence signal was specific for
PR65
as it could be competed with the appropriate antigen (data
not shown; see also (13) ). In addition, we obtained identical
results with an antisera raised against the recombinant protein
(Ab65
). The distribution of the overexpressed PR65 was
found to change slightly when different time points after transfection
were analyzed. In about 30% of the transfected cells (harvested 48 h
after transfection), the overexpressed PR65
was almost exclusively
nuclear, whereas in about 70% of the transfected cells, PR65
was
present in both nucleus and cytoplasm. The reasons for this almost
exclusive nuclear staining remain to be further investigated. By 72 h
most if not all transfected cells stained homogeneously for PR65
in the cytoplasm and the nuclear compartment. This distribution
corresponds to that found for endogenous PR65 (Fig. 3C and (13) ).
The same set of PR65 5`-UTR sequences (see Fig. 4A) were ligated into the 5`-UTR of the
-galactosidase gene (in the pSV vector) and used to transiently
transfect COS-1 cells (Fig. 4C). We first demonstrated
by Northern blotting that insertion of the 5`-UTR in the expression
plasmid did not influence transcriptional efficiency (not shown). The
5`-UTR of PR65
inhibits translation of the
-galactosidase
message dramatically (79 ± 4%). The secondary structure alone is
responsible for a moderate inhibition of translation (17 ± 5%),
whereas the AUG alone inhibits translation by 57% (±5%). In
contrast to the in vitro data, mutation of the upstream AUG
and disruption of the secondary structure, resulted in a slight
stimulation of translation by 15% (±7%).
Taken together, the
results demonstrate that the 5`-UTR of PR65 is a strong
translational inhibitor, mainly due to the presence of an upstream AUG,
and to a lesser (but significant) extent to the presence of a stem-loop
structure. Apparently, the inhibitory effects of secondary structure
and upstream AUG are additive.
COS-1 cells overexpressing PR65 show no
apparent phenotype. This might be explained by the observation that the
cells are essentially non-dividing in the 48-72-h period after
DEAE-dextran transfection. (The presence of more than one nucleus or
fragmentated nuclei in the COS-1 cell transfection experiment did not
correlate with PR65 overexpression. Indeed non-transfected, mock
transfected, and cells transfected with the full-length PR65
expression construct (Fig. 3D) showed the same
proportion of abnormal nuclei compared to COS-1 cells overexpressing
the PR65.)
To investigate possible effects of PR65
overexpression on cell division, we microinjected REF-52 cells with
PR65
expression constructs. When cells were microinjected with a
5`-UTR deleted PR65
expression construct this resulted in a
dramatic increase of PR65
protein detected in both the nucleus and
the cytoplasm (data not shown). As determined by indirect
immunofluorescence and confocal laser scanning microscopy this increase
of PR65 protein was 10-20-fold compared to control uninjected
cells. In contrast, microinjection of the full-length PR65
expression construct did not alter PR65 protein levels (data not
shown). These results agree with those obtained using COS-1 cells (Fig. 3). Analysis of PR65
overexpressing cells 24 h after
microinjection revealed major phenotypic changes during mitosis. The
most marked effect observed was the formation of cells containing
multiple nuclei (Fig. 5, A and B). Whereas
only 3% of the non-injected control cells were binucleated, 65% of the
PR65
overexpressing cells contained two or more nuclei. The
remaining 35% of the overexpressing cells did not divide in the 24 h
after microinjection. REF-52 cells microinjected with a full-length
PR65
expression plasmid did not show any phenotypic changes (Fig. 5, C and D). These results indicate that
cytokinesis is blocked in PR65 overexpressing cells without apparently
affecting nuclear division.
Figure 5:
PR65 overexpression leads to
multinucleated cells. REF-52 cells were microinjected with the
5`-UTR deleted (panels A and B) or the full-length
PR65
pECE expression vector (panels C and D)
diluted in injection buffer (containing mouse IgG as a marker for
injected cells). 48 h after microinjection, cells were fixed, and
stained. Panels A and C, immunostained for marker
IgG; panels B and D, DNA counterstained with Hoechst
dye. All cells injected with the 5`-UTR deletion construct
overexpressed the PR65 (data not shown). Bars, 5
µm.
In the present work we show that deregulation of the
translational control of PR65 mRNA leads to overexpression of
PR65
, which apparently disrupts cytokinesis and leads to bi- and
multinucleated cells. This translational control is mediated by the
5`-UTR of PR65
, which is a strong translational inhibitor, due to
the presence of an upstream AUG and a stable stem-loop structure.
Translation of mRNAs with stable secondary structures in the 5`-UTR
may be highly dependent on the helicase activities of eIF-4B and
eIF-4F. The latter factor is a complex between eIF-4A, eIF-4E, and p220 (7) . Phosphorylation of eIF-4E and eIF-4B increases after
insulin treatment of fibroblasts (30) and this coincides with
an increase of the translation of ornithine decarboxylase mRNA. Under
basal conditions translation of this message is inhibited by a stable
stem-loop structure (G = -68.2 kcal/mol) in
the 5`-UTR. Moreover, overexpression of eIF-4E in NIH 3T3 cells
overcomes the translational inhibition of cyclin D1(31) . These
data suggest that phosphorylation of initiation factors may lead to a
more efficient unwinding of 5`-UTRs, and thereby up-regulate the
translation of certain mRNAs. Furthermore, an eIF-4E interacting
protein (4E-BP1 or PHAS-I) has been identified, which inhibits
translation(32, 33) . Phosphorylation of 4E-BP1 in
response to insulin causes its dissociation from eIF-4E, and relieves
the inhibition.
In the case of PR65, however, unwinding of the
5`-UTR may not be sufficient, since the upstream AUG alone inhibits
translation (Fig. 4). The use of an alternative promoter, or
alternative splicing, might produce messages that lack the inhibitory
5`-UTR and would therefore be efficiently translated(34) .
However, none of the PR65
cDNA clones isolated so far contain
different 5`-UTR sequences, in fact the majority of the isolated cDNAs
starts just downstream of the start ATG.
This probably
reflects the inability of reverse transcriptase to proceed through a
stable stem-loop structure.
In addition to a stem-loop structure,
nucleotides -134 to -40 of the 5`-UTR of PR65 have the
potential to base pair with nucleotides 517-612 in the coding
sequence. This potential base pairing and the stem-loop structure in
the 5`-UTR are mutually exclusive. The ability of the 5`-UTR to base
pair with an internal coding sequence is not a unique characteristic of
PR65
, but is also found in the c-myc proto-oncogene
transcripts. Saito et al.(35) suggest that a
translocated c-myc gene, which lacks the exon encoding the
5`-UTR, is no longer under translational control and becomes oncogenic.
Another example where abrogation of translation control leads to
proto-oncogene activation is the lymphocyte-specific tyrosine kinase lck(36) . The 5`-UTR of lck contains three
upstream AUGs. Substitution of the 5`-UTR for retroviral sequences
results in malignant transformation, as is observed in some murine
lymphomas(36) .
Another characteristic of certain
proto-oncogenes transcripts, such as the c-myc transcript, is
the presence of potential RNA-destabilizing sequences in the
3`-UTR(37) . The AUUUA sequence, which is thought to mediate
rapid mRNA turnover, is also found in most PP2A subunit messages. Five
copies of this motif are present in the 3`-UTR of C, six in
C
, one in PR65
, four in PR55
, seven in PR55
, one in
PR72, and four in PR130. The striking exception is PR65
itself,
which seems to be devoid of this motif. The absence of rapid turnover
signals might explain why translation of the PR65
signal is
tightly controlled. It therefore appears that the cell controls PP2A
subunit transcripts in much the same way as mRNAs that encode proteins
involved in growth control, such as proto-oncogenes.
Overexpression
of PR65 leads to defects in cytokinesis and multinucleated cells.
Cytokinesis is brought about by the contraction of actin-myosin fibers
in the cleavage furrow. Injection of myosin antibodies specifically
blocks cytokinesis, without affecting chromosome movement, and
ultimately leads to multinucleated cells(38) . Although
speculative at the moment, one possible mechanism to explain our
results is that overexpression of PR65
results in inefficient
dephosphorylation and activation of myosin light chain kinase, a
presumed PP2A substrate(1) , resulting in hypophosphorylated
myosin light chains which in turn could inhibit the contractile force
in the cleavage furrow. The question that emerges from this study is,
how does the overexpression of PR65
disrupt the normal regulation
of PP2A? As judged from its elution on a Mono-Q column most of the
overexpressed PR65
seems to be present as a free protein, i.e. not complexed with the catalytic or other regulatory subunits.
Furthermore, neither the elution profile, nor the amount, of the PP2A
trimer (C/PR65/PR55) is influenced by overexpression of PR65
in
COS-1 cells. Also the total PP2A activity (as measured with a peptide
substrate) remains unchanged. Two possibilities exist to explain the
results described in this article. First, the overexpression of
PR65
could disrupt a PP2A holoenzyme which plays a specific role
in cytokinesis. PR65
could do so by sequestering the catalytic or
variable subunit. Although we do not detect such dimers in a Mono-Q
profile, we cannot exclude that a low abundance PP2A trimer is
dissociated upon PR65
overexpression. Second, the excess PR65
could act as an inhibitor of the catalytic subunit of PP2A, as
predicted from in vitro data(39) . In this model it is
necessary to suggest that the catalytic subunit (or the complex of the
catalytic subunit with a variable subunit) is released from the PR65
subunit at a certain point in the cell cycle to dephosphorylate
specific targets. Free PR65
in overexpressing cells would in this
model immediately capture the released catalytic subunit and suppress
its activity. We have recently obtained evidence that indicates that
PP2A undergoes subunit
rearrangements(12, 13, 40) . Interestingly, a
very similar phenotype (defects in cytokinesis and multinucleated
cells) is observed in budding yeast when the TPD3 gene,
encoding the PR65 homologue, is mutated(41) .
In summary, the data presented in this paper, and in other recent publications (19, 40, 41, 42) demonstrate the importance of PP2A in cell cycle regulation. In this context it should be pointed out that substrates of the cyclin-dependent protein kinase family need to be dephosphorylated prior to the subsequent round of cell division. This dephosphorylation is most likely not a stoichastic process, but stringently regulated by the action of PP2A and other protein phosphatases.