2493
Journal of Cell Science 112, 2493-2500 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0448
A hyperphosphorylated form of RNA polymerase II is the major interphase
antigen of the phosphoprotein antibody MPM-2 and interacts with the
peptidyl-prolyl isomerase Pin1
Alexandra Albert, Sébastien Lavoie and Michel Vincent*
Département de médecine and CREFSIP, Pavillon C.-E.-Marchand, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4
*Author for correspondence
Accepted 24 May; published on WWW 7 July 1999
SUMMARY
The monoclonal antibody MPM-2 recognizes a subset of M
phase phosphoproteins in a phosphorylation-dependent
manner. It is believed that phosphorylation at MPM-2
antigenic sites could regulate mitotic events since most of
the MPM-2 antigens identified to date have M phase
functions. In addition, many of these proteins are
substrates of the mitotic regulator Pin1, a peptidyl-prolyl
isomerase which is present throughout the cell cycle and
which is thought to alter its mitotic targets by changing
their conformation. In interphase cells, most MPM-2
reactivity is confined to nuclear speckles. We report here
that a hyperphosphorylated form of the RNA polymerase
II largest subunit is the major MPM-2 interphase antigen.
These findings were made possible by the availability of
another monoclonal antibody, CC-3, that was previously
used to identify a 255 kDa nuclear matrix protein
associated with spliceosomal components as a
hyperphosphorylated form of the RNA polymerase II
largest subunit. MPM-2 recognizes a phosphoepitope of the
large subunit that becomes hyperphosphorylated upon heat
shock in contrast to the phosphoepitope defined by CC-3,
whose reactivity is diminished by the heat treatment.
Therefore, these two antibodies may discriminate between
distinct functional forms of RNA polymerase II. We also
show that RNA polymerase II large subunit interacts with
Pin1 in HeLa cells. Pin1 may thus regulate transcriptional
and
post-transcriptional
events
by
catalyzing
phosphorylation-dependent conformational changes of the
large RNA polymerase II subunit.
INTRODUCTION
to the transcription complex (Cho et al., 1997; McCracken et
al., 1997; Steinmetz, 1997). It has been proposed that
modulation of the phosphorylation state of the CTD may
regulate its association with transcription and mRNA
processing factors, and may involve multiple kinases and
phosphatases (Archambault et al., 1998; Dahmus, 1994, 1996).
A few monoclonal antibodies (mAbs) have been produced
that recognize different phosphoepitopes on the CTD of the
RNAP II (Patturajan et al., 1998). One of these, mAb CC-3,
recognizes a hyperphosphorylated form of the enzyme which
associates with the nuclear matrix and is largely confined to
splicing factor-enriched nuclear speckles (Bisotto et al., 1995;
Chabot et al., 1995; Vincent et al., 1996). Besides its reactivity
with RNAP II largest subunit in interphase cells, mAb CC-3
recognizes a set of phosphorylated proteins in mitotic cells.
Actually, this antibody was initially selected for its strong
general reactivity with mitotic cells of different species
(Thibodeau et al., 1989; Thibodeau and Vincent, 1991). When
eukaryotic cells divide, they undergo profound architectural
and functional modifications that have been correlated with a
dramatic increase in the level of protein phosphorylation
(Capony et al., 1986; Karsenti et al., 1987; Lohka et al., 1987;
RNA polymerase II (RNAP II) is a large multisubunit enzyme
found in two forms according to the level of phosphorylation
of its largest subunit, designated either IIa (unphosphorylated,
apparent
molecular
mass
210
kDa)
or
IIo
(multiphosphorylated, apparent molecular mass 240 kDa)
(Woychik and Young, 1990; Young, 1991). Phosphorylation
sites of IIo are located in its C-terminal domain (CTD)
composed of multiple conserved heptapeptide repeats of the
consensus sequence YSPTSPS whose number depends on the
complexity of the organism (Corden, 1990). Various studies
suggest that the IIa form interacts with the promoter to form a
stable preinitiation complex and that entry to initiation of
transcription is accompanied by CTD phosphorylation
(Corden, 1993; Dahmus, 1994; O’Brien et al., 1994; Payne et
al., 1989; Zawel and Reinberg, 1992). Upon completion of the
transcript, the IIo form must be dephosphorylated to regenerate
the IIa form and complete the cycle (Dahmus, 1996). In
addition, it has been recently proposed that the CTD acts as a
platform to couple mRNA processing to transcription as it was
shown to recruit splicing, polyadenylation and capping factors
Key words: RNA polymerase II, MPM-2, CC-3, Phosphoprotein,
Pin1, Heat shock
2494 A. Albert, S. Lavoie and M. Vincent
Nigg et al., 1996). This increase results from the activation of
a protein kinase cascade, at the head of which sits CDK1 kinase
which forms a protein complex with mitotic cyclin B (Nurse,
1990). Several laboratories have used mAbs recognizing
subsets of M phase phosphoproteins to identify mitotic
regulators and effectors (Davis et al., 1983; Butschak et al.,
1995; Kuriyama, 1989). The most characterized of these
antibodies, mAb MPM-2, was raised against mitotic HeLa cells
and selected for its preferential reactivity with mitotic versus
interphase cells (Davis et al., 1983; Vandré et al., 1984).
Because MPM-2 antigens are found in important mitotic
structures such as centrosomes, kinetochores, spindle fibers,
chromosomes scaffolds and the midbody (Taagepera et al.,
1993; Vandré et al., 1984, 1986, 1991), it is currently believed
that phosphorylation at MPM-2 antigenic sites could regulate
mitotic processes. The functional importance of the MPM-2
antigens is further suggested by the inhibition of entry into
(Kuang et al., 1989) or exit from mitosis (Davis and Rao, 1987)
when cells are microinjected with this antibody.
The strong general reactivity of mAbs MPM-2 and CC-3
with mitotic cells of different species and the fact that MPM2 staining of interphase cells is restricted to discrete
extranucleolar spots in the nucleus (Vandré et al., 1984)
prompted us to compare the reactivity of both reagents. In
this study, we report the identification of a
hyperphosphorylated form of RNAP II largest subunit as
the major MPM-2 antigen of interphase cells. The
immunoreactivity of MPM-2 against this protein is found in
both mitotic and interphase cells and is mediated by a
phosphoepitope different from that recognized by CC-3,
suggesting that these antibodies may react with functionally
distinct subsets of RNAP IIo. Incidentally, our results indicate
that phosphorylation of these CTD phosphoacceptor sites is
inversely modulated during a heat shock. As many MPM-2
antigens are known to be substrates of the phosphorylationdependent peptidyl-prolyl isomerase (PPIase) Pin1, a novel
human mitotic regulator present throughout the cell cycle (Lu
et al., 1996; Shen et al., 1998; Yaffe et al., 1997), we asked
whether the hyperphosphorylated form of RNAP II interacts
with Pin1. We show here that in HeLa cell extracts, both
MPM-2- and CC-3-reactive forms of RNAP II large subunit
specifically bind to Pin1. This isomerase may thus regulate
transcriptional and post-transcriptional events by catalyzing
phosphorylation-dependent conformational changes of
RNAP IIo.
MATERIALS AND METHODS
Cell cultures
HeLa and PtK2 cells (American Type Culture Collection, Rockville,
MD) were cultured in Iscove’s modified Dulbecco’s medium
(Canadian Life Technologies, Burlington, Ontario) supplemented
with 10% FBS (Medicorp, Montréal, Québec) and kept at 37°C in 5%
CO2. Mitotic HeLa cells were obtained by incubating cells with
nocodazole (Sigma-Aldrich, Oakville, Ontario) at a final
concentration of 0.04 µg/ml for 16 hours. Heat shock treatment was
carried out by incubating HeLa cells for 45 minutes at different
temperature (from 41°C to 47°C) in the same medium as above.
Antibodies
The mouse monoclonal antibody (mAb) MPM-2 (IgG1) was
purchased from DAKO (Carpinteria, CA). mAb CC-3 (IgG2a) was
obtained after immunization of a Balb/C mouse with pharyngeal
regions isolated from 3-day-old chick embryos in a search for
developmental markers (Thibodeau and Vincent, 1991). mAb POL3/3
(IgG1) recognizes RNAP II largest subunit in a conserved region
located outside the CTD and was kindly provided by E. K. F. Bautz
(Krämer et al., 1980).
Immunofluorescence
Cells were cultured overnight on glass coverslips, washed with
phosphate buffered saline (PBS) and fixed for 20 minutes at −20°C in
100% methanol, followed by rinses in PBS. After the incubation with
the primary antibodies MPM-2 and CC-3, cells were stained with a
Cy3-conjugated anti-mouse IgG1 antibody and a FITC-conjugated
anti-mouse IgG2a antibody (Caltag, San Francisco, CA). In some
experiments, cells were counterstained with 0.1 µg/ml 4′6-diamidino2-phenylindole (DAPI) to reveal DNA. All antibodies were incubated
for 1 hour at room temperature in 0.5% bovine serum albumin (BSA)
diluted in PBS. After a few PBS washes, slides were mounted with
p-phenylenediamine-PBS in glycerol and viewed with a DAS Leitz
microscope equipped with epifluorescence optics.
Sample preparation, immunoprecipitation and
immunoblotting
For whole cell extracts, mitotic or unsynchronized HeLa cells were
directly solubilized in SDS-sample buffer (63 mM Tris-HCl, pH 6.8,
2.3% SDS, 5% β-mercaptoethanol, 10% glycerol) and boiled for 6
minutes. For immunoprecipitation experiments, HeLa cells were
homogenized at 4°C with TD buffer (50 mM Tris-HCl, pH 7.55, 150
mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 0.5% sodium
deoxycholate, 0.5% Triton X-100, 150 nM okadaic acid, 2 mM PMSF,
2 mM sodium orthovanadate, 5 µg/ml each of leupeptin, pepstatin and
10 µg/ml of antipain). Extracts were spun at 16000 g for 5 minutes
for clarification and then incubated with the primary antibody for 90
minutes at room temperature followed by an additional 2-hour
incubation with goat anti-mouse IgG (whole molecule)-agarose
(Sigma-Aldrich, Oakville, Ontario). The beads were washed 3 times
using TD buffer and the immunoprecipitated complex was then
solublized in SDS-sample buffer as described above. For some
experiments (immunodepletion, Fig. 2C), the beads were preincubated with the primary antibody in PBS before incubation with
HeLa cell extracts for 90 minutes at room temperature. The depleted
supernatants and the immunocomplexes were solubilized with 1
volume of 2× SDS-sample buffer before boiling for 6 minutes.
Samples were resolved by SDS-PAGE on a 7% acrylamide or a 517% acrylamide gradient gel and transferred to nitrocellulose
membranes. The blots were blocked with 1% blocking reagent
(Boehringer Mannheim, Laval, Québec) diluted in Tris-buffered saline
(TBS: 10 mM Tris-HCl, pH 7.65, 150 mM NaCl) containing 0.05%
Tween-20 for 60 minutes at 37°C and probed overnight with the
primary antibody in the same solution at 4°C. The membranes were
washed 20 minutes in TBS-T. A peroxidase-conjugated anti-mouse
IgG secondary antibody (Jackson ImmunoResearch Laboratories,
West Grove, PA) was used at 1:10000 for chemiluminescence
detection (Boehringer Mannheim, Laval, Québec). The molecular
mass markers used were: α (240 kDa) and β (220 kDa) subunits of
spectrin, α2-macroglobulin (170 kDa), phosphorylase b (97 kDa),
bovine serum albumin (66 kDa), glutamate dehydrogenase (55 kDa)
and ovalbumin (45 kDa).
In vitro dephosphorylation
Immunoprecipitation was carried out as described above and the
resulting pellet washed three times with alkaline phosphatase buffer
(100 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 2 mM PMSF, 10 µg/ml
antipain and 5 µg/ml each of leupeptin and pepstatin). After
centrifugation, the immunoprecipitates were incubated with 50 U/ml
of alkaline phosphatase (Boehringer Mannheim, Laval, Québec) at
RNAP II large subunit interacts with Pin1 2495
37°C for 0 and 90 minutes in the same buffer. The dephosphorylation
was stopped by adding an equal volume of 2× SDS-sample buffer
before boiling for 6 minutes.
Cloning, expression and purification of histidine-tagged
Pin1 protein
Pin1 was cloned by RT-PCR (buffers and M-MLV from Canadian Life
Technologies, Burlington, Ontario) using total mRNA extracted from
HeLa cells with Trizol (Canadian Life Technologies, Burlington,
Ontario). The primers used for the PCR amplification (5′-CGCGGATCCGAAGATGGCGGACGAGGA-3′ and 3′-GACTCACTCCCACCCCTCCTTAAGCCTAGGCGC-5′) were derived form the Pin1
coding sequence (GenBank accession number U49070; see Lu et al.,
1996). The Pin1 PCR product was cloned in a pET19b+ vector
(Novagen, Madison, WI), sequenced using T7 promoter and T7
terminator primers and transformed into the E. coli strain Novablue
(DE3) for expression (Novagen, Madison, WI). The expression of the
His-tagged Pin1 fusion protein was induced at 37°C in LB liquid
medium using 0.4 mM isoprolyl-β-D-thiolgalactopyranoside (IPTG).
The fusion protein was purified using a His-bind resin (Novagen,
Madison, WI) according to the pET system manual (Novagen).
Interaction between Pin1 and RNAP IIo
These experiments were performed using 80 µg of His-tagged Pin1
immobilized on 20 µl of His-bind resin (Novagen). Irrelevant pbp5
His-tagged fusion protein (120 µg) immobilized on the same amount
of resin was used as a negative control in the same conditions. Pbp5
is a penicillin binding protein from Pseudomonas aeruginosa
(PA01) kindly provided by L. Gagnon and A. Huletsky
(Département de Biologie médicale, Université Laval). Resins
saturated with fusion proteins were washed twice with 400 µl of
resin buffer (25 mM Tris, 150 mM NaCl and 0.21% NaF, pH 7.5).
HeLa cells, grown at 37°C or heat shocked 1 hour at 45°C, were
homogenized in a CHAPS buffer (resin buffer with 0.5% CHAPS,
60 nM microcystin-LR (Sigma), 2 mM PMSF and 1× of the
CompleteTM protease inhibitor cocktail tablets (Boehringer
Mannheim, Laval, Québec)). After centrifuging 5 minutes at 16,000
g, the homogenates were incubated for 2 hours at 4°C with either
the Pin1-resin or the pbp5-resin followed by six sequential washes
with 65 µl of the CHAPS buffer containing 10 mM of imidazole.
The resins were eluted twice with 65 µl of SDS-sample buffer.
Samples obtained from the initial homogenate, the flowthrough, the
washes and the eluted fractions were subjected to SDS-PAGE and
immunoblotting.
RESULTS
Comparison of MPM-2 and CC-3 immunoreactivity
mAb MPM-2 was raised against mitotic cells and was shown
to react with a set of phosphorylated proteins which are
abundant during mitosis (Davis et al., 1983). We have selected
mAb CC-3 for its strong immunoreactivity with mitotic cells
and this antibody was also shown to react with a family of
mitotic phosphoproteins with apparent molecular mass ranging
from 55 to >250 kDa (Thibodeau and Vincent, 1991). The
immunoblot reactivities of MPM-2 and CC-3 on mitotic
proteins show rather different patterns (Fig. 1A), although the
existence of common reactive species could not be excluded.
In mitotic HeLa cells, the CC-3 immunoreactive material is
concentrated in the high molecular mass region of the gel
whereas the major MPM-2 reactive bands are found in a lower
molecular mass range. Immunofluorescence microscopy of
metaphase PtK2 cells, which do not round up when entering
mitosis, allows the visualization of staining associated with the
mitotic structures (Fig. 1B). Both MPM-2 and CC-3 react with
the centrosomes and the mitotic spindle. The contour of the
chromosomes is labelled but this is more evident with CC-3.
Some diffuse staining is also observed throughout the
cytoplasm with both antibodies.
In interphase cells, MPM-2 staining is concentrated in
discrete spots in the nucleus (Fig. 1D and see Vandré et al.,
1984). Some diffuse staining is also observed in the
nucleoplasm apart from the nucleoli. A very similar
fluorescent pattern is obtained with CC-3 (Fig. 1D) and was
previously shown to colocalize with the non-snRNP splicing
factor SC-35 in the so-called nuclear speckles (Bisotto et al.,
1995). Double-labelling interphase HeLa cells with CC-3 and
MPM-2 shows that the nuclear colocalization is almost
perfect (Fig. 1D). Longer exposures of MPM-2-mediated
fluorescence are however required to compensate for its
relatively weak strength in cells grown at 37°C (see below).
As shown on the merged images, slight differences are
observed in some cells into which some MPM-2 staining is
still present in the cytoplasm. Presumably, these cells have
just quit telophase for interphase as suggested by the stronger
MPM-2 staining in late telophase cells (data not shown). In
addition, occasionnal MPM-2 reactive dots are not labelled
by CC-3 (arrows in Fig. 1D). The immunoblot reactivities of
both antibodies on unsynchronized cell extracts show a major
reactive species around 240 kDa (Fig. 1C). The CC-3-reactive
peptide, formerly named p255 (Bisotto et al., 1995), was
identified as a hyperphosphorylated form of the largest
subunit of RNA polymerase II (Vincent et al., 1996). Both
antibodies also react with a minor species: an unidentified
180 kDa protein in the case of CC-3 (see below) and a faint
116 kDa protein in the case of MPM-2.
A hyperphosphorylated form of the RNA polymerase
II largest subunit is the major protein recognized by
MPM-2 during interphase
To investigate the possibility that the major MPM-2 antigen is
also RNAP II large subunit, immunoprecipitations of HeLa cell
homogenates were carried out with both antibodies and the
immunoprecipitated fractions were reciprocally revealed by
immunoblotting (Fig. 2A). mAbs CC-3 and MPM-2
immunoprecipitate the same 240 kDa protein which is also
recognized by mAb POL3/3, an antibody specific to the largest
subunit of RNAP II whose epitope is located in a conserved
region outside the phosphorylated CTD (Krämer et al., 1980).
POL3/3 is a phospho-independent antibody and can thus
recognize the two forms of the RNAP II largest subunit: the
210 kDa hypophosphorylated form (IIa) and the 240 kDa
hyperphosphorylated form (IIo) (Krämer et al., 1980; Dubois
et al., 1994a). The minor IIa bands seen in both CC-3 and
MPM-2 immunoprecipitated fractions on the POL3/3
immunoblot is most probably due to slight dephosphorylation
of the IIo form after immunoprecipitation (Fig. 2A). The 180
kDa band recognized by CC-3 on immunoblots (Fig. 1C) is
also immunoprecipitated by CC-3. The identity of this protein
is not yet known, but it is not thought to be a degradation
fragment since it is neither recognized by POL3/3 nor by any
other antibody against the RNAP II large subunit (Fig. 2A; see
also Vincent et al., 1996).
As expected from their phosphodependence, incubation of
the immunoprecipitated fractions with alkaline phosphatase
2496 A. Albert, S. Lavoie and M. Vincent
Fig. 1. Comparison of the
immunoreactivities of the
phosphodependent mAbs MPM-2
and CC-3. (A) Total mitotic HeLa
cell extracts were separated on a 517% gradient SDS-PAGE gel and
transferred to nitrocellulose. The
membranes were probed with
MPM-2 and CC-3 as described in
Materials and Methods. (B) Mitotic
structures labelled by CC-3 and
MPM-2. PtK2 cells were doublelabelled with CC-3 (green), MPM-2
(red), and the chromosomes were
revealed by DAPI (blue)
counterstaining.
Immunofluorescence micrograph
showing a metaphase cell in which
the centrosomes and the mitotic
spindle are labelled by both
antibodies, as illustrated by their
yellow colour on the merged image.
The contour of the chromosomes is
also labelled but this is more
evident with CC-3. Some diffuse
staining is observed throughout the
cytoplasm. (C) Unsynchronized
HeLa cell extracts were separated
on a 7% acrylamide gel, transferred
to nitrocellulose and probed with
MPM-2 and CC-3. (D) Comparison
of CC-3 and MPM-2 nuclear
fluorescence in interphase cells.
HeLa cells grown on coverslips
were subjected to indirect
immunofluorescence with CC-3 and
MPM-2. The same nuclear speckles
are labelled by both antibodies, as
illustrated by their yellow colour on the merged images. A faint cytoplasmic MPM-2
labelling can be detected in some cells (arrowheads) and a few MPM-2-reactive dots
(arrows) are not labelled by CC-3. Bar, 10 µm. IB, immunoblotting mAb. The positions
of the molecular mass standards are indicated.
abolished both CC-3 and MPM-2 reactivities on their own
immunoprecipitate (Fig. 2B). POL3/3 in turn, clearly reveals
the conversion from the IIo to the IIa form in each
immunoprecipitated fraction. In the POL3/3 immunoblots of
the dephosphorylated fractions, a faint band (arrow in Fig. 2B)
migrating faster than the IIa band may correspond to isoform
IIb, a breakdown product of RNAP II largest subunit not found
in vivo (Corden, 1990; Kim and Dahmus, 1986). To further
substantiate the identity of the antigens, both antibodies were
shown to immunodeplete a lysate of its POL3/3-reactive RNAP
IIo (Fig. 2C). These results confirm that the major MPM-2
interphase antigen is a hyperphosphorylated form of the RNAP
II largest subunit localized to the nuclear speckles, as is the
CC-3 antigen.
The MPM-2 and CC-3 epitopes of RNAP IIo are
different
As it was previously observed that the CC-3 phosphoepitope
of RNAP IIo diminished markedly in HeLa cells subjected to
a heat shock (Dubois et al., 1997), we analyzed the reactivity
of both antibodies at different temperatures to compare the
D
behaviour of their respective epitopes. Surprisingly, MPM-2
immunoblot signal increases at higher temperatures as the CC3 signal decreases (Fig. 3). The 180 kDa CC-3 reactive band
is also diminished in heat-shocked HeLa cells. This experiment
demonstrates that MPM-2 and CC-3 recognize different
subsets of phosphorylated RNAP II large subunit in defining
two distinct epitopes that are inversely modulated during heat
shock.
The peptidyl-prolylisomerase Pin1 associates with
RNAP IIo
PPIases are ubiquitous enzymes catalyzing rotation about the
peptide bond preceding a proline residue and are thought to be
important in protein folding, activity, assembly and/or transport
(Schmid, 1995). Three distinct families of PPIases are known:
the cyclophilins, the FKBPs and the parvulins (Dolinski and
Heitman, 1997). Pin1, a member of the parvulin family, has
been identified in a yeast two-hybrid screen as a protein that
interacts with the essential NIMA protein kinase and
suppresses its mitosis-promoting activity (Lu et al., 1996). Pin1
preferentially isomerises proline residues preceded by
RNAP II large subunit interacts with Pin1 2497
Fig. 3. The MPM-2 and CC-3 epitopes of RNAP IIo are inversely
modulated during a heat shock. HeLa cells were heat shocked at
different temperatures for 45 minutes and lysed in sample buffer. The
proteins extracted from 3x104 cells were loaded in each well and
resolved by 7% PAGE-SDS before immunoblotting with either CC-3
or MPM-2. The MPM-2 signal is weak at lower temperatures but
increases as the heat shock temperature reaches 43°C. When probed
with CC-3, the reactivity of subunit IIo and of the 180 kDa
progressively decreases with increasing temperature.
Fig. 2. The major MPM-2 interphase antigen is a
hyperphosphorylated form of RNAP II largest subunit.
(A) Unsynchronized HeLa cell extracts were immunoprecipitated
with MPM-2 and CC-3 and the washed immunoprecipitates were
separated on a 5-17% gradient polyacrylamide gel, transferred to
nitrocellulose and immunoblotted with MPM-2, CC-3 or the RNAP
II large subunit specific monoclonal antibody POL3/3. (B) Mobility
shift of the CC-3 and MPM-2 antigens after in vitro
dephosphorylation. HeLa cell extracts were immunoprecipitated with
MPM-2 or CC-3 and the resulting pellets were dephosphorylated for
0 and 90 minutes as described in Materials and Methods. The
dephosphorylation was stopped by adding 2× sample buffer and the
samples were resolved by 7% SDS-PAGE and transferred to
nitrocellulose. Membranes were immunoblotted with POL3/3 in
addition to MPM-2 and CC-3. The arrows indicate a putative
degradation fragment that may correspond to RNAP IIb (see text).
(C) Immunodepletion of the POL3/3-reactive RNAP IIo by MPM-2
and CC-3. HeLa cell extracts were immunoprecipitated with goat
anti-mouse IgG-agarose beads alone (control) or with beads
preincubated with either MPM-2 or CC-3 as described in Materials
and Methods. The samples were submitted to 7% SDS-PAGE and
immunoblotted with POL3/3. IB, immunoblotting mAbs. IP,
immunoprecipitating mAbs.
phosphorylated serine or threonine (Yaffe et al., 1997). It
appears to play an important role in regulation of mitotic
progression by directly interacting with different mitosisspecific phosphoproteins including NIMA kinase, Cdc25
phosphatase, Wee1 and Plk1 (Lu et al., 1996; Shen et al., 1998;
Crenshaw et al., 1998). Many of these Pin1-binding proteins
are also recognized by mAb MPM-2 (Shen et al., 1998). For
this reason, and because Pin1 localizes to nuclear speckles in
interphase cells (Lu et al., 1996), we investigated whether it
interacts with RNAP II. Moreover, the CTD of RNAP II
contains multiple pS/pT-P sites which may be targets for this
isomerase. Pin1 cDNA was obtained by RT-PCR and cloned
into the pET19b+ vector allowing the expression of a Nterminal His-tagged protein. Once immobilized on a His-bind
resin (Ni2+), His-tagged Pin1 was incubated with a HeLa cell
homogenate solubilized in a CHAPS-containing buffer. As
seen on the CC-3 and MPM-2 immunoblots of Fig. 4, the IIo
form of the RNAP II was retained on the Pin1 column and the
POL3/3 immunoblot revealed that most, if not all, Pin1binding species were of the hyperphosphorylated form. The
unidentified CC-3-reactive p180 also interacts with the PPIase.
Controls were carried out by saturating a His-bind resin with
an irrelevant bacterial His-tagged protein and incubating with
a HeLa cell homogenate as for Pin1. Samples from the initial
homogenate, flow through, washes and eluted fractions were
subjected to immunoblot with mAb POL3/3. The specificity of
the association between Pin1 and the RNAP II was confirmed
since neither IIo nor IIa forms were retained on the control
resin.
The same experiments were carried out with homogenates
from heat shocked HeLa cells (Fig. 4). The heat treatment
increases the overall phosphorylation levels of IIo (Dubois et
al., 1997) as well as its MPM-2 antigenicity (Fig. 3). The
interaction of Pin1 with RNAP II was maintained, but the
MPM-2 / CC-3 signal ratio of the fraction retained on the
column (E1) was greater than that obtained in normal growth
conditions, consistent with the reactivities observed during the
heat treatment.
2498 A. Albert, S. Lavoie and M. Vincent
Fig. 4. Pin1 interacts with the
hyperphosphorylated form of the
RNAP II largest subunit.
Unsynchronized (>90%
interphase) or heat-shocked HeLa
cell homogenates were incubated
with a His-binding resin satured
with the His-tagged fusion
protein Pin1, washed and eluted.
Samples obtained from the initial
homogenates, the flowthrough,
the washes and the eluted
fractions were subjected to SDSPAGE and immunoblotting with
MPM-2, CC-3 and POL3/3. The
RNAP IIo form was specifically
retained on the Pin1 column (E1).
Samples from the eluted fractions
contained 4 times the number of
cell equivalents as the other fractions. No RNAP II species was retained by an irrelevant His-tagged fusion protein (Control). The p180 also
seems to interact with Pin1 as revealed by CC-3 in the eluted fractions. IB Abs: immunoblotting antibodies, +: total HeLa cell homogenate, -:
flowthrough, W: washes, E: eluted fractions.
DISCUSSION
In the last decade, the study of mitosis has been mainly directed
to the identification and characterization of kinases and
phosphatases modulating the mitotic process. However, less is
known about the targets of these regulatory enzymes and the
way they perform cell division. Monoclonal antibodies that
recognize phosphoepitopes appearing during the G2/M
transition have been proposed to represent remarkable probes
to identify and characterize these targets, as the
phosphoproteins bearing the antigenic determinants are likely
to contain a common site phosphorylated by a single or related
kinases. mAb MPM-2 was originally prepared against mitotic
HeLa cell extracts and was shown to react with a large family
of mitotic proteins in a phosphorylation-dependent manner
(Davis et al., 1986). A few of these phosphoproteins have been
identified to date and comprise mitotic regulators and effectors
(see the Introduction for detailed references).
A key finding of the present study is that a
hyperphosphorylated form of RNAP II largest subunit represents
the major interphase protein reactive with MPM-2. From the
observation that mAbs MPM-2 and CC-3 showed a similar
punctate staining of interphase nuclei and a strong increase in
the immunofluorescence of mitotic cells, we hypothesized that
both monoclonals may react with related antigens. The identity
of the major interphase MPM-2 antigen as RNAP IIo was
demonstrated by reciprocal immunoprecipitation with CC-3,
previously shown to react with a hyperphosphorylated form of
RNAP II in interphase cells (Vincent et al., 1996) and by
immunodepletion by MPM-2. The results was corroborated by
immunoblotting with the RNA polymerase II large subunit
specific mAb POL3/3. We have also observed that MPM-2
reacts with RNAP IIo in CC-3-immunoprecipitates prepared
from mitotic cells (data not shown).
Results from many laboratories favored a broad epitope
definition for MPM-2, suggesting that the MPM-2 epitope
overlaps with the phosphorylation consensus sequences of many
kinases (Taagepera et al., 1994; Westendorf et al., 1994; Ding et
al., 1997). Several protein kinases including MAPK (Kuang and
Ashorn, 1993), MEK (Taagepera et al., 1994), cdc2 (Westendorf
et al., 1994) and ME-kinase-H, a kinase activated by cdc2
(Kuang and Ashorn, 1993), have been implicated in the
phosphorylation of the MPM-2 epitope. In an attempt to identify
a physiological kinase phosphorylating the MPM-2 epitope, Che
et al. (1997) constructed a substrate formed by a fusion protein
between glutathione S-transferase and a 19-residue peptide
containing two representative MPM-2 epitope sequences
(LTPLQ and LSPMK) overlapping with two potential MAPK
phosphorylation sites. They found that the two MPM-2 epitope
sequences could be phosphorylated by MAPK but not by cdc2
nor ME kinase-H, a kinase extracted from Xenopus eggs that
generated MPM-2 reactivity on multiple polypeptides (Kuang
and Ashorn, 1993). These results suggest that the MPM-2
epitope sequence is only part of the phosphorylation consensus
sequence and that additional structural information is required
for recognition and phosphorylation by ME-kinase H or other
mitotic kinases (Che et al., 1997). It cannot be excluded that the
epitope sequences tested in this assay were not the type
phosphorylated by such kinases. This would imply that subtypes
of the MPM-2 epitope exist, that the antibody could not
discriminate. These results also provide an explanation for the
selective phosphorylation of a small subset of MPM-2 antigens
by MAPK and the selective recognition of only some MAPK
substrates by MPM-2 (Kuang and Ashorn, 1993). In line with
this deduction, MAPK might be responsible for the acquisition
of the MPM-2 epitope by RNAP IIo. The consensus sequence
for MAPK phosphorylation, PXS/TP, exists integrally in the
heptapeptide repeats of the CTD (YSPTSPS) and MAPK family
members have been involved in the enhanced phosphorylation
of the CTD of RNAP II upon serum stimulation of quiescent
cells (Dubois et al., 1994b) or in response to heat-shock and
chemical treatments (Trigon and Morange, 1995; Venetianer et
al., 1995). In heat-shocked cells, the CTD kinase associated with
the general transcription factor TFIIH is impaired concomitantly
with the decrease of the CC-3 epitope while the overall
phosphorylation of the RNAP II large subunit is increased by a
MAPK (Dubois et al., 1997). It is tempting to relate the
augmentation of MPM-2 epitope reactivity that we observed
RNAP II large subunit interacts with Pin1 2499
upon a heat treatment with this heat-shock induced CTD-kinase
activity. mAbs MPM-2 and CC-3 may thus discriminate between
two distinct forms of RNAP IIo, phosphorylated by a stressactivated MAPK and the TFIIH-associated kinase, respectively.
These distinct phosphorylation sites may correspond to different
functions of the CTD in transcription or in pre-mRNA
processing.
Yaffe et al. (1997) found that Pin1, a PPIase involved in the
regulation of mitosis, binds and regulates members of a highly
conserved set of mitotic phosphoproteins that overlaps with
antigens recognized by MPM-2. When incubated with a
mitotic extract, agarose beads containing a glutathione Stransferase (GST)-Pin1 fusion protein precipitated a large
number of proteins reacting with MPM-2 in immunoblot (Yaffe
et al., 1997). Pin1 preferentially isomerizes proline residues
preceded by phophorylated serine or threonine in mitotic
phosphoproteins. It has been suggested that phosphorylation at
these specific S/T-P sites creates a binding site for Pin1, which
induces conformational changes by catalyzing prolyl
isomerization (Yaffe et al., 1997). In interphase cells, Pin1
localizes to the splicing factor-enriched nuclear speckle
domain (Lu et al., 1996), which is also labelled by CC-3
(Bisotto et al., 1995) and MPM-2 antibodies (Fig. 1D). When
incubated with interphase extracts, GST-Pin1 only precipitated
a few MPM-2-reactive polypeptides including a major species
around 240 kDa (Yaffe et al., 1997; Shen et al., 1998). From
our observations, we anticipated that this Pin1-binding
interphase protein might be the RNAP II largest subunit. This
prediction was directly verified using a recombinant Histagged Pin1 protein immobilised on a resin. When incubated
with homogenates of HeLa cells, the resin specifically retained
RNAP IIo, indicating that Pin1 interacts with a complex
containing the hyperphosphorylated form of the polymerase.
Further experiments will be needed to verify whether this
interaction is direct or not. The Far Western analysis performed
by Shen et al. (1998) using GST-Pin1 as a probe indicates that
two major bands appearing on both sides of the 200 kDa
marker are bound by the Pin1 probe on G1-phase cell extracts
(see Fig. 2A in Shen et al., 1998). These bands may correspond
to IIo and p180, shown here to be retained on the His-tagged
Pin1 column.
Another PPIase (human SRcyp/CASP10) has been shown to
interact with the CTD of RNAP II (Bourquin et al., 1997). This
enzyme, and its rat homologue matrin CYP (Mortillaro and
Berezney, 1998), are members of the cyclophilin family and
show 93% identity in amino acid sequence. SRcyp/CASP10
was identified using the two hybrid-system as a protein
interacting with the CTD (Bourquin et al., 1997). In addition
to a prolylisomerase cis-trans domain related to the
immunophilins/cyclophilins PPIases, this protein contains a
serine/arginine-rich (SR) domain, required for interaction with
the CTD, similar to that found in the SR protein family of premRNA splicing factors (Bourquin et al., 1997). The matrin
CYP is also a SR-rich protein, and a fusion protein containing
the cyclophilin domain of matrin CYP exhibits cyclosporin Asensitive peptidyl-prolyl cis-trans isomerase activity typical of
this family of PPIases (Mortillaro and Berezney, 1998). Like
RNAP IIo and Pin1, both enzymes localize to the
nuclear speckles. Although suspected, the interaction of
SRcyp/CASP10 with the phosphorylated form of the CTD has
not been demonstrated formally (Bourquin et al., 1997). Our
results indicate that Pin1 interacts specifically with the
hyperphosphorylated form of RNAP II. This finding is not
surprising given the remakably high content of pS/pT-P
dipeptides in the phosphorylated CTD and the fact that Pin1
preferentially recognizes and isomerises proline residues
preceded by phosphorylated serine or threonine residues in
synthetic peptides when compared to their unphosphorylated
counterparts (Yaffe et al., 1997). Using such synthetic peptides,
Yaffe et al. (1997) demonstrated the failure of members of the
cyclophilin (Cyp18) and FKBP (FKBP12) families to
effectively catalyze the isomerization of the pS/pT-P bond. In
contrast, their catalytic activity was increased towards peptides
containing S/T-P or Y/pY-P bonds, suggesting the existence of
a substrate specificity distinguishing Pin1 from the cyclophilin
and FKBP families (Yaffe et al., 1997). It would thus be
interesting to compare the phosphorylation dependence of
the CTD-interacting PPIases as they could represent
complementary factors involved in the modulation of CTD
conformation and therefore, in the regulation of its interactions
with many such partners. The CTD was indeed demonstrated,
in the recent years, to function as a platform for the assembly
of multiprotein complexes that proceed to the capping, the
splicing, the cleavage and the polyadenylation of pre-mRNA
(Corden and Patturajan, 1997; Neugebauer and Roth, 1997;
Steinmetz, 1997). By catalyzing phosphorylation-dependent
conformation changes of the CTD, Pin1 may be a new
important player in such complexes. Our results further suggest
that the biological function of Pin1 is not restricted to a role in
regulation of mitotic progression, as generally proposed.
The authors gratefully acknowledge Lynn Davis for technical
assistance and for reading the manuscript. This research was
supported by the Natural Sciences and Engineering Research Council
of Canada (grant OGP0122625). A.A. and S.L. hold studentships
from NSERC (Canada) and FCAR-FRSQ (Québec), respectively.
REFERENCES
Archambault, J., Pan, G., Dahmus, G. K., Cartier, M., Marshall, N.,
Zhang, S., Dahmus, M. E. and Greenblatt, J. (1998). FCP1, the RAP74interacting subunit of a human protein phosphatase that dephosphorylates
the carboxyl-terminal domain of RNA polymerase IIO. J. Biol. Chem. 273,
27593-27601.
Bisotto, S., Lauriault, P., Duval, M. and Vincent, M. (1995). Colocalization
of a high molecular mass phosphoprotein of the nuclear matrix (p255) with
spliceosomes. J. Cell Sci. 108, 1873-1882.
Bourquin, J.-P., Stagljar, I., Meier, P., Moosmann, P., Silke, J., Baechi, T.,
Georgiev, O. and Schaffner, W. (1997). A serine/arginine-rich nuclear
matrix cyclophilin interacts with the C-terminal domain of RNA polymerase
II. Nucl. Acids Res. 25, 2055-2061.
Butschak, G., Harborth, J., Osborn, M. and Karsten, U. (1995). New
monoclonal antibodies recognizing phosphorylated proteins in mitotic cells.
Acta Histochem. 97, 19-31.
Capony, J. P., Picard, A., Peaucellier, G., Labbé, J. C. and Dorée, M.
(1986). Changes in the activity of the maturation-promoting factor during
meiotic maturation and following activation of amphibian and starfish
oocytes: their correlations with protein phosphorylation. Dev. Biol. 117, 112.
Chabot, B., Bisotto, S. and Vincent, M. (1995). The nuclear matrix
phosphoprotein p255 associates with splicing complexes as part of the
U4/U6.U5 tri-snRNP particle. Nucl. Acids Res. 23, 3206-3213.
Che, S., Weil, M. M., Nelman-Gonzalez, M., Ashorn, C. L. and Kuang, J.
(1997). MPM-2 epitope sequence is not sufficient for recognition and
phosphorylation by ME kinase-H. FEBS Lett. 413, 417-423.
Cho, E.-J., Takagi, T., Moore, C. R. and Buratowski, S. (1997). mRNA
2500 A. Albert, S. Lavoie and M. Vincent
capping enzyme is recruited to the transcription complex by phosphorylation
of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11, 33193325.
Corden, J. L. (1990). Tails of RNA polymerase II. Trends Biochem. Sci. 15,
383-387.
Corden, J. L. (1993). RNA polymerase II transcription cycles. Curr. Opin.
Gen. Dev. 3, 213-218.
Corden, J. L. and Patturajan, M. (1997). A CTD function linking
transcription to splicing. Trends Biochem. Sci. 22, 413-416.
Crenshaw, D. G., Yang, J., Means, A. R. and Kornbluth, S. (1998). The
mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.
EMBO J. 17, 1315-1327.
Dahmus, M. E. (1994). The role of multisite phosphorylation in the regulation
of RNA polymerase II activity. Prog. Nucl. Acids Res. Mol. Biol. 48, 143179.
Dahmus, M. E. (1996). Reversible phosphorylation of the C-terminal domain
of RNA polymerase II. J. Biol. Chem. 271, 19009-19012.
Davis, F. M., Tsao, T. Y., Fowler, S. K. and Rao, P. (1983). Monoclonal
antibodies to mitotic cells. Proc. Nat. Acad. Sci. USA 80, 2926-2930.
Davis, F. M. and Rao. P. N. (1987). Antibodies to mitosis-specific
phosphoproteins. In Molecular Regulation of Nuclear Events in Mitosis and
Meiosis (ed. R. A. Schlegel, M. S. Halleck and P. N. Rao), pp. 259-294.
Academic Press, New York.
Ding, M., Feng, Y. and Vandré, D. D. (1997). Partial characterization of the
MPM-2 phosphoepitope. Exp. Cell. Res. 231, 3-13.
Dolinski, K. and Heitman, J. (1997). Peptidyl-prolyl isomerases – an
overview of the cyclophilin, FKBP and parvulin families. In Molecular
Chaperones and Protein-folding Catalysts (ed. M.-J. Gething), pp. 359-369.
Oxford University Press.
Dubois, M.-F., Nguyen, V. T., Bellier, S. and Bensaude, O. (1994a).
Inhibitors
of
transcription
such
as
5,6-dichloro-1-β-Dribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8
and H-7) promote dephosphorylation of the carboxyl-terminal domain of
RNA polymerase II largest subunit. J. Biol. Chem. 269, 13331-13336.
Dubois, M.-F., Nguyen, V. T., Dahmus, M. E., Pagès, G., Pouysségur, J.
and Bensaude O. (1994b). Enhanced phosphorylation of the C-terminal
domain of RNA polymerase II upon serum stimulation of quiescent cells:
possible involvement of MAP kinases. EMBO J. 13, 4787-4797.
Dubois, M.-F., Vincent, M., Vigneron, M., Adamczewski, J., Egly, J.-M.
and Bensaude, O. (1997). Heat-shock inactivation of the TFIIH-associated
kinase and change in the phosphorylation sites on the C-terminal domain of
RNA polymerase II. Nucl. Acids Res. 25, 694-700.
Karsenti, E., Bravo, R. and Kirschner, M. (1987). Phosphorylation changes
associated with the early cell cycle in Xenopus eggs. Dev. Biol. 119, 442453.
Kim, W. Y. and Dahmus, M. E. (1986). Immunochemical analysis of
mammalian RNA polymerase II subspecies: Stability and relative in vivo
concentration. J. Biol. Chem. 261, 14219-14225.
Krämer, A., Haars, R., Kabish, R., Will, H., Bautz, F. A. and Bautz, E. K.
F. (1980). Monoclonal antibody directed against RNA polymerase II of
Drosophila melanogaster. Mol. Gen. Genet. 180, 193-199.
Kuang, J., Zhao, J., Wright, D. A., Saunders, G. F. and Rao, P. (1989).
Mitosis-specific monoclonal antibody inhibits Xenopus oocyte maturation
and depletes maturation-promoting activity. Proc. Nat. Acad. Sci. USA 86,
4982-4986.
Kuang, J. and Ashorn, C. L. (1993). At least two kinases phosphorylate the
MPM-2 epitope during Xenopus oocyte maturation. J. Cell Biol. 123, 859868.
Kuriyama, R. (1989). 225-kilodalton phosphoprotein associated with mitotic
centrosomes in sea urchin eggs. Cell Motil. Cytoskel. 12, 90-103.
Lohka, M. J., Kyes, J. L. and Maller, J. L. (1987). Metaphase protein
phosphorylation in Xenopus laevis. Mol. Cell. Biol. 7, 760-768.
Lu, K. P., Hanes, S. D. and Hunter, T. (1996). A human peptidyl-prolyl
isomerase essential for regulation of mitosis. Nature 380, 544-547.
McCracken S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G.,
Siderovski, D., Hessel, A., Foster, S., Amgen EST Program, Shuman, S.
and Bentley, D. L. (1997). 5′-capping enzymes are targeted to the premRNA by binding to the phosphorylated carboxy-terminal domain of RNA
polymerase II. Genes Dev. 11, 3306-3318.
Mortillaro, M. J. and Berezney, R. (1998). Matrin CYP, an SR-rich
cyclophillin that associates with the nuclear matrix and splicing factors. J.
Biol. Chem. 273: 8183-8192.
Neugebauer, K. M. and Roth, M. B. (1997). Transcription units as RNA
processing units. Genes Dev. 11, 3279-3285.
Nigg, E. A., Blangy, A. and Lane, H. A. (1996). Dynamic changes in nuclear
architecture during mitosis: on the role of protein phosphorylation in spindle
assembly and chromosome segregation. Exp. Cell Res. 229, 174-180.
Nurse, P. (1990). Universal control mechanism regulating onset of M-phase.
Nature 344, 503-508.
O’Brien, T. O., Hardin, S., Greenleaf, A. and Lis, J. T. (1994).
Phosphorylation of RNA polymerase II C-terminal domain and
transcriptional elongation. Nature 370, 75-77.
Patturajan, M., Schulte, R. J., Sefton, B. M., Berezney, R., Vincent, M.,
Bensaude, O., Warren, S. L and Corden, J. L. (1998). Growth-related
changes in phosphorylation of yeast RNA polymerase II. J. Biol. Chem. 273,
4689-4694.
Payne, J. M., Laybourn, P. J. and Dahmus, M. E. (1989). The transition of
RNA polymerase II from initiation to elongation is associated with
phosphorylation of the carboxy-terminal domain of subunit IIa. J. Biol.
Chem. 264, 19621-19629.
Schmid, F. X. (1995). Prolyl isomerases join the fold. Curr. Biol. 5, 993-994.
Steinmetz, E. J. (1997). Pre-mRNA processing and the CTD of RNA
polymerase II: the tail that wags the dog? Cell 89, 491-494.
Shen, M., Stukenberg, P. T., Kirschner, M. W. and Lu, K. P. (1998). The
essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosisspecific phosphoproteins. Genes Dev. 12, 706-720.
Taagepera, S., Rao, P. R., Drake, F. H. and Gorbsky, G. J. (1993). DNA
topoisomerase IIα is the major chromosome protein recognized by the
mitotic phosphoprotein antibody MPM-2. Proc. Nat. Acad. Sci. USA 90,
8407-8411.
Taagepera, S., Dent, P., Her, J., Sturgill, T. W. and Gorbsky, G. J. (1994).
The MPM-2 antibody inhibits mitogen-activated protein kinase activity by
binding to an epitope containing phosphothreonine-183. Mol. Biol. Cell 5,
1243-1251.
Thibodeau, A., Duchaine, J., Simard, J.-L. and Vincent, M. (1989).
Localization of molecules with restricted patterns of expression in
morphogenesis: an immunohistochemical approach. Histochem, J. 21, 348356.
Thibodeau, A. and Vincent, M. (1991). Monoclonal antibody CC-3
recognizes phosphoproteins in interphase and mitotic cells. Exp. Cell Res.
195, 145-153.
Trigon, S. and Morange, M. (1995). Different carboxyl-terminal domain
kinase activities are induced by heat-shock and arsenite. Characterization of
their substrate specificity, separation by Mono Q chromatography and
comparison with the mitogen-activated protein kinases. J. Biol. Chem. 270,
13091-13098.
Vandré, D. D., Davis, F. M., Rao, P. N. and Borisy, G. (1984).
Phosphoproteins are components of the mitotic microtubule organizing
centers. Proc. Nat. Acad. Sci. USA 81, 4439-4443.
Vandré, D. D., Davis, F. M., Rao, P. N. and Borisy, G. (1986). Distribution
of cytoskeletal proteins sharing a conserved phosphorylated epitope. Eur. J.
Cell Biol. 41, 72-81.
Vandré, D. D., Centonze, V. E., Peloquin, J., Tombes, R. M. and Borisy,
G. G. (1991). Proteins of the mammalian mitotic spindle: phosphorylation/
dephosphorylation of MAP-4 during mitosis. J. Cell Sci. 98, 577-588.
Venetianer, A., Dubois, M.-F., Nguyen, V. T., Bellier, S., Seo, S. J. and
Bensaude, O. (1995). Phosphorylation state of the RNA polymerase II Cterminal domain (CTD) in heat-shocked cells. Possible involvement of the
stress-activated mitogen-activated protein (MAP) kinases. Eur. J. Biochem.
233, 83-92.
Vincent, M., Lauriault, P., Dubois, M.-F., Lavoie, S., Bensaude, O. and
Chabot, B. (1996). The nuclear matrix protein p255 is a highly
phosphorylated form of RNA polymerase II largest subunit which associates
with spliceosomes. Nucl. Acids Res. 24, 4649-4652.
Westendorf, J. M., Rao, P. N. and Gerace, L. (1994). Cloning of cDNAs for
M-phase phosphoproteins recognized by the MPM2 monoclonal antibody
and determination of the phosphorylated epitope. Proc. Nat. Acad. Sci. USA
91, 714-718.
Woychik, N. A. and Young, R. A. (1990). RNA polymerase II: subunit
structure and function. Trends Biochem. Sci. 15, 347-351.
Yaffe, M. B., Schutkowski, M., Shen, M., Zhou, X. Z., Stukenberg, P. T.,
Rahfeld, J.-U., Xu, J., Kuang, J., Kirschner, M. W., Fischer, G., Cantley,
L. C. and Lu, K. P. (1997). Sequence-specific and phosphorylationdependent proline isomerization: a potential mitotic regulatory mechanism.
Science 278, 1957-1960.
Young, R. A. (1991) RNA polymerase II. Annu. Rev. Biochem. 60, 689-715.
Zawel, L. and Reinberg, P. (1992). Advances in RNA polymerase II
transcription. Curr. Opin. Cell Biol. 4, 488-495.