(Received for publication, October 1, 1996, and in revised form, November 18, 1996)
From the pRb controls cell proliferation by restricting
inappropriate entry of cells into the cell division cycle. As
dephosphorylation of pRb during mitotic exit activates its growth
suppressive function, identification of the protein phosphatase that
dephosphorylates pRb, and characterization of the mechanism of its
regulation, are essential to elucidating the mechanisms of cell growth
control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing
analyses indicate that the catalytic enzyme (PP1c) is present in a high
molecular weight complex, with a predicted molecular mass of 166 kDa.
PP1-interacting proteins in the mitotic cell extracts are identified.
Two PP1-interacting proteins (41 and 110 kDa) are shown to form
distinct complexes with PP1c from fractions of separated mitotic cell
extracts containing phosphorylase phosphatase activity. However, only
the 110-kDa PP1-interacting protein is present in fractions containing
pRb-directed phosphatase activity, identifying this protein as a
putative activator of PP1 function toward pRb during mitosis.
Reversible protein phosphorylation controls many of the events
that coordinate cell division. The cyclin-dependent kinases are recognized as the major protein kinases required for all aspects of
cell division (1-4). One identified substrate for the
cyclin-dependent kinases is the protein product of the
retinoblastoma susceptibility gene (pRb), a prototype for tumor
suppressor genes (5-13). pRb functions in the control of cell
proliferation and differentiation (reviewed in Refs. 13-16). In
resting cells, hypophosphorylated pRb prevents inappropriate entry of
cells into the cell division cycle. Phosphorylation of pRb by the
cyclin-dependent kinases relieves pRb-mediated growth
suppression and allows for cell proliferation (5, 17-25). Conversely,
dephosphorylation of pRb during G1 progression induces
growth arrest or cell differentiation (23, 24, 26, 27). In dividing
cells, pRb is dephosphorylated during mitotic exit and G1
entry (28, 29). This dephosphorylation activates pRb for the ensuing
G1 phase of the cell cycle, during which pRb exerts its
growth suppressive effects.
Protein phosphatase type 1 (PP1)1 has been
implicated in the mitotic dephosphorylation of pRb (28, 30). PP1 is a
major cellular phosphoprotein phosphatase that is active on
phosphoserine and phosphothreonine residues (31). Genetic evidence has
indicated that PP1 is required for successful mitotic progression (1, 32-37). Inhibitor studies have confirmed the requirement for PP1 activity during mitosis in mammalian cells, where successful gene knockout experiments have yet to be reported (36, 38). PP1 has also
been implicated to play a role in other phases of the cell cycle,
although the requirements during G1, S, and G2
are less well defined (reviewed in Ref. 36). Identification of pRb as a
mitotic target for PP1 (28) suggests that at least one important aspect
of PP1-mediated mitotic exit may involve the activation of
G1-specific regulators, such as pRb, concomitant with
G1 entry. Hence, characterization of the cell
cycle-dependent regulation of PP1 activity toward pRb
remains an important part of elucidating the mechanism of pRb-mediated
growth control.
PP1 exists in holoenzyme complexes with noncatalytic, or regulatory,
components that modulate catalytic activity and restrict the
subcellular localization of the catalytic subunit (PP1c). Multiple such
regulatory proteins have been described, and more are anticipated
(39-41). Identifying regulatory components of protein phosphatase
complexes, and elucidating their role in controlling catalytic
phosphatase activity during cell cycle progression, are essential
aspects of characterizing cell growth control.
In this report, we confirm the identity of the mitotic pRb-directed
protein phosphatase as PP1 and present characterization of the
phosphatase designed to elucidate the mechanism of its regulation.
Molecular sizing analyses reveal that the catalytic phosphatase (PP1c)
is present in a high molecular weight protein complex, suggesting
association of PP1c with regulatory components during mitosis. Several
PP1-interacting proteins in the mitotic cell extracts are also
identified. One PP1-associated protein, identified as an approximate
110-kDa polypeptide, may function to activate PP1 toward pRb during
mitosis.
CV-1P cells, a continuous
line of monkey kidney cells, were grown in Dulbecco's modified
Eagle's medium containing 10% newborn calf serum in a 5%
CO2-containing atmosphere at 37 °C. M-phase cells were
isolated by mitotic shake-off after 18 h in Dulbecco's modified
Eagle's medium containing 0.4 µg/ml nocodazole supplemented with
10% newborn calf serum.
M-phase cells were
washed three times with ice cold wash buffer (20 mM
imidazole, pH 7.0, 150 mM NaCl) and lysed by 15 times passage through a 28-gauge syringe needle in the same buffer
supplemented with 14 mM 2-mercaptoethanol and the following
concentrations of protease inhibitors: 1 mM benzamidine, 50 µM
N 32P-pRb
substrate was prepared by immunoprecipitating lysates from
32P-orthophosphate-labeled cells with monoclonal antibody
RB-PMG3-245 (PharMingen) as described previously (28).
[32P]phosphorylase a was prepared as described
by Cohen et al. (42). Briefly, phosphorylase b
(Sigma) at a concentration of 10 mg/ml was incubated
with 0.2 mg/ml phosphorylase kinase (Sigma) for 1.5 h at 30 °C in kinase reaction buffer (100 mM
Tris-HCl, 100 mM sodium glycerol-1-phosphate, pH 8.2, 0.1 mM CaCl2, 10 mM magnesium acetate,
and 0.2 mM [ pRb-directed phosphatase activity was assayed as
described previously (28). Briefly, equal amounts of
32P-labeled, immunocomplexed pRb (from approximately 1 µg
of antibody/reaction) were aliquoted, mixed with cell extracts, column
fractions, or immunocomplexes of PP1 isoforms and incubated for 40 min
at 30 °C. Reactions were terminated by the addition of 1/2 volume of
3 × SDS-PAGE sample buffer (7% SDS, 33% glycerol, 0.32 M dithiothreitol, and 0.001% bromphenol blue) and boiled
for 10 min. Proteins were separated on 8% SDS-polyacrylamide gels
(43). Gels were then fixed, dried, and exposed to x-ray film for
autoradiography. Images were quantified by densitometry. Total pRb
radioactivity present in the assays was determined from control
aliquots of 32P-pRb substrate, which were not subjected to
incubation at 30 °C in the presence of cell extract, fractions, or
immunocomplexed PP1. Results are presented as percent decrease in pRb
band densities on autoradiographs for experimental lanes relative to
untreated control lanes. For inhibitor studies, okadaic acid
(Sigma), glutathione S-transferase (GST),
and glutathione S-transferase-thiophosphorylated-inhibitor 1 (GST-S-I1) fusion protein were mixed with the protein fractions and
preincubated for 15 min at 30 °C prior to mixing with
32P-pRb. Reactions were then allowed to proceed as
described above. Purified GST and GST-I1 fusions were kindly provided
by Dr. Shirish Shenolikar (Duke University) and were thiophosphorylated
in vitro using ATP Phosphorylase
phosphatase activity was measured as the release of trichloroacetic
acid soluble counts from 32P-phosphorylase a
according to Cohen et al. (42). For inhibitor studies,
okadaic acid, GST, and GST-S-I1 were mixed with the protein fractions
and preincubated for 15 min at 30 °C prior to mixing with
[32P]phosphorylase a.
Mitotic CV-1P cell extracts were diluted to 50 mM NaCl in 20 mM imidazole, pH 7.0, supplemented with 14 mM 2-mercaptoethanol and the following
concentrations of protease inhibitors: 1 mM benzamidine, 20 µM TLCK, 20 µM TPCK, 20 µM
leupeptin, and 1 µM pepstatin. Diluted extracts were then
applied to a 5-ml HiTrap Heparin column (Pharmacia Biotech Inc.)
equilibrated in 20 mM imidazole, pH 7.0, 50 mM
NaCl with the same supplements. The flow-through fractions were
monitored by absorbance at 280 nm (A280).
Protein measurements were made using the Bio-Rad protein assay system. Fractions containing proteins were pooled and brought to 0.2 M NaCl. The column was washed with the same buffer until
the A280 profile returned to base line. Bound
proteins were eluted with 20 mM imidazole, pH 7.0, 0.4 M NaCl with the same supplements as the loading buffer.
Protein fractions were pooled and diluted to 0.2 M NaCl
with 20 mM imidazole, pH 7.0, containing the same supplements as the column buffers.
Mitotic
CV-1P cell extracts were run through a calibrated Superose 6 HR 10/30
column (Pharmacia, exclusion limit approximately 4 × 107 daltons) in 20 mM imidazole, pH 7.0, 150 mM NaCl supplemented with 14 mM
2-mercaptoethanol and the aforementioned protease inhibitor concentrations. Stokes radius was determined as described (44). Void
volume determination was made with blue dextran, and the included
volume determination was made with glycine. Molecular standards used
were: chymotrypsinogen, 20.9 Å; Mitotic CV-1P cell extracts were layered onto a
10-30% glycerol gradient in 20 mM imidazole, pH 7.0, 100 mM NaCl supplemented with 14 mM
2-mercaptoethanol and the aforementioned concentrations of protease
inhibitors. Centrifugation in a Beckman SW65 rotor was for 19 h at
55 krpm and 4 °C. Gradient was fractionated from top to bottom in
0.5-ml aliquots. Sedimentation coefficients were determined by
comparison with standards (45) (cytochrome c, 1.9 s;
bovine serum albumin, 4.3 s; glyceraldahyde-3-phosphate dehydrogenase, 7.01 s; and catalase, 11.3 s) separated in a
parallel gradient.
The molecular mass and
frictional ratio of the pRb-directed phosphatase complex was determined
by the method of Siegel and Monty (44), assuming an average value of
0.7350 for partial specific volume (v, ml/g) (46). Molecular
weight and frictional coefficient were calculated using the equations
M = 6 Protein samples were separated by SDS-PAGE
and transferred to nitrocellulose (47). Antisera used were kindly
provided as follows: PP1 and PP2A-specific antibodies from Dr. Norbert
Berndt (Children's Hospital of Los Angeles), PP1 isoform-specific
antibodies from Dr. Emma Villa-Moruzzi (Pisa, Italy), and NIPP-1B
antibodies from Dr. Mathieu Bollen (Leuven, Belgium). Blots were
developed using horseradish peroxidase-conjugated secondary antibodies
and enhanced chemiluminescence detection (Amersham Corp.) according to
the manufacturer's instructions.
Protein samples were separated on
10% SDS-polyacrylamide gels and transferred to nitrocellulose.
PP1-interacting proteins were detected as described by Jagiello
et al. (48). Digoxygenin-labeled PP1c and detection
materials were kindly provided by Dr. Mathieu Bollen.
Mitotic CV-1P cell
extracts or Superose 6 FPLC column fractions were incubated for 1 h at 4 °C with rabbit antibodies specific for PP1c isoforms and
precipitated with protein A-Sepharose. Immunoprecipitates were washed
with the supplemented mitotic cell lysis buffer, diluted in 1 × SDS-PAGE sample buffer (60 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, and 0.01% bromphenol blue), separated
on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose.
Blots were developed by enzyme affinity labeling with
digoxygenin-labeled PP1c.
We demonstrated previously that
in vitro dephosphorylation of pRb by mitotic CV-1P cell
extracts required the activity of the type 1, and possibly the type 2A,
phosphoprotein phosphatases (28). In order to clarify the roles of PP1
and PP2A in the dephosphorylation of pRb in vitro, mitotic
CV-1P cell extracts were fractionated to separate PP1 from PP2A and
tested for the distribution of the pRb-directed protein phosphatase
activity. Since type 1 phosphatases are generally retained by heparin
matrices, whereas the type 2A phosphatases are not (31, 49), we tested
mitotic CV-1P cell extracts for binding of the pRb-directed protein
phosphatase to heparin.
Fig. 1A shows that the pRb-directed protein
phosphatase activity (hatched bars) was retained by heparin
and eluted in a 0.4 M NaCl step. For comparison, the
profile for phosphatase activity toward glycogen phosphorylase was
included (stippled bars), since this is the standard
substrate for measuring PP1 activity in vitro (42).
B shows that the pRb and phosphorylase phosphatase
activities co-segregated in the 0.4 M NaCl heparin step
fraction with the type 1 catalytic subunit (lane 3). In
contrast, the type 2A remained in the flow-through fraction (lane
2). C shows the inhibitor sensitivities of the
phosphatase activities isolated on heparin. As indicated, phosphatase
activity toward both pRb and phosphorylase a was completely inhibited by high concentrations of okadaic acid, which inhibit both
PP1 and PP2A. In contrast, 1 nM okadaic acid, which
selectively inhibits PP2A under the assay conditions employed, did not
inhibit the phosphorylase phosphatase activity of the step fraction.
This confirms that the phosphorylase phosphatase activity of the step is mediated by PP1.
Because in vitro dephosphorylation of pRb requires more cell
extract than in vitro dephosphorylation of phosphorylase
a, okadaic acid cannot be used to selectively inhibit PP2A
relative to PP1 in these assays (50, 51). Hence, we used a PP1-specific
inhibitor fusion protein to further demonstrate that the pRb-directed
phosphatase activity of the heparin step is mediated by PP1 (Fig.
1C). As illustrated, phosphatase activity toward both
substrates was inhibited by the PP1-specific inhibitor fusion protein,
thiophosphorylated GST-inhibitor 1 (GST-S-I1). Thiophosphorylated
fusion protein was used, since thioesters are very poor substrates for
cellular phosphatases (52), thereby preventing dephosphorylation and inactivation of inhibitor 1 (I1) during the assay. Note that
phosphatase activity of the step toward these substrates was unaffected
by GST protein alone, demonstrating the specificity of inhibition for
the I1 portion of the fusion protein.
In summary, these experiments demonstrate that the pRb-directed protein
phosphatase activity cosegregated with the type 1 phosphatase catalytic
subunit on heparin was completely inhibited by okadaic acid and was
markedly inhibited by the type 1-specific protein phosphatase inhibitor
I1. Taken together, these data clearly show that the pRb-directed
protein phosphatase activity in mitotic CV-1P cell extracts is mediated
by a PP1 phosphatase and rule out any additional requirements for
PP2A.
Regulation of PP1 activity in differentiated cell types
is conferred by association of the catalytic subunit (PP1c) with
noncatalytic proteins (reviewed in Refs. 39-41). To elucidate the
mechanism by which PP1 activity toward pRb is regulated during mitosis, we then determined if PP1 in mitotic cell extracts is present in higher
order complexes, suggesting association with regulatory proteins. Fig.
2A shows the distribution of the pRb-directed
phosphatase activity during molecular sizing by gel filtration
chromatography on Superose 6. The phosphorylase phosphatase activity
profile is included for comparison. As illustrated, phosphatase
activity toward pRb eluted in a single peak between approximately 14 and 16 ml. A also shows that the elution position of
phosphorylase phosphatase activity overlapped, but was slightly broader
than, the pRb-directed phosphatase activity. The phosphorylase
phosphatase activity resolved into two overlapping peaks, the first of
which was coincident with the pRb-directed phosphatase peak, the second of which eluted slightly after the pRb-directed phosphatase peak. Furthermore, phosphatase activity toward both substrates eluted much
earlier than expected for the free 38-kDa catalytic subunit of PP1. The
calculated Stokes radius of the pRb-directed phosphatase activity peak
is 54.5 Å. This value is considerably larger than the Stokes radius of
the isolated catalytic subunit, PP1c, with a reported value of 24 Å (53, 54).
In order to obtain a more accurate size estimate for the pRb-directed
protein phosphatase, mitotic CV-1P cell extracts were also analyzed by
density sedimentation using glycerol gradients. As illustrated in Fig.
2B, the pRb-directed protein phosphatase sedimented as a
broad activity profile in fractions 5-9. Again, the phosphorylase
phosphatase activity profile overlapped the pRb-directed phosphatase
profile; however the phosphorylase phosphatase activity peak was
slightly smaller in size than the pRb-directed phosphatase activity
peak. The sedimentation coefficient of the pRb-directed phosphatase
activity peak was determined to be 7.1 s. This value is also
considerably larger than the sedimentation coefficients reported for
free PP1c, which range from 2.7 to 4.1 s (53-55).
Fig. 2C presents a summary of the physical characteristics
of the mitotic pRb-directed protein phosphatase. Using the Stokes radius and sedimentation coefficient, the predicted molecular mass of
the pRb-directed phosphatase was calculated to be approximately 166 kDa. This is much larger than the mass of the free catalytic subunit of
PP1, at 38 kDa, suggesting that PP1c is associated with regulatory
components when active on pRb during mitosis. The calculated frictional
ratio (f/f0) of the mitotic
pRb-directed phosphatase is 1.50. This value suggests a rather
asymmetric configuration for the mitotic phosphatase complex, in
contrast to the value of 1.05 reported for isolated PP1c, which is
indicative of a more globular configuration for the catalytic core
enzyme (54). This asymmetry of the pRb-directed phosphatase would lead
to overestimation of the molecular mass based on gel filtration
experiments alone. Hence, it was important to measure both the Stokes
radius and the sedimentation coefficient of the pRb-directed
phosphatase activity in order to estimate the molecular mass of the
active enzyme complex. In summary, the molecular sizing experiments
presented here demonstrate that the mitotic pRb-directed phosphatase
exists as a large protein complex, thus supporting our earlier
suggestion that the catalytic activity of PP1 during mitosis is
modulated by as yet uncharacterized PP1 regulatory proteins (56).
The fractionated mitotic cell extracts were then tested for
the distribution of PP1c. The catalytic subunit of PP1 is expressed as
a family of protein isoforms (36, 40-41, 57). The
Fig. 3B shows the elution positions of the phosphoprotein
phosphatase activities. Comparison of the PP1c immunoreactivity profiles and the phosphatase activity profiles reveals that the peak of
catalytic subunit was associated with only a subset of the phosphatase
activity. As illustrated, the pRb-directed phosphatase activity peaked
around 15 ml. In contrast, the immunoreactivity peaks for all three
isoforms of PP1c examined were present at approximately 16.5 ml within
the included column volume. The distribution of phosphorylase
phosphatase activity was somewhat broader than that of the pRb-directed
phosphatase activity. The first peak was coincident with the
pRb-directed phosphatase activity peak at around 15 ml and eluted
before the peak of PP1c immunoreactivity on the column. The second
phosphorylase phosphatase activity peak, however, which is less active
toward pRb, cosegregated with the immunoreactive peak of PP1 catalytic
subunit on the column at about 16.5 ml. These data suggest that
distinct PP1 enzyme complexes are present in the mitotic cell extracts,
with differential activity toward pRb and phosphorylase.
To identify putative noncatalytic
components of the mitotic pRb-directed phosphatase complex that may
regulate catalytic function, Superose 6 fractions of the mitotic cell
extracts were tested for PP1-interacting proteins by protein affinity
labeling using digoxygenin-labeled PP1c (48, 59). Fig.
4A shows the 4 major bands detected on the
blot after the elution of the column void volumn (which trailed down to
about 10 ml elution volume). The apparent molecular masses of these
four PP1-interacting proteins are approximately 180, 125, 110, and 41 kDa. The enzyme affinity detection of PP1-interacting proteins using
digoxygenin-labeled PP1c in rat liver nuclei (48) revealed two major
regulatory subunits. One of these, R41, was identified as nuclear
inhibitor of protein phosphatase 1 (NIPP-1). To test the possibility
that the 41-kDa protein detected here is NIPP-1, the Superose 6 fractions were immunoblotted using antibodies to NIPP-1 (Fig.
4B). As illustrated, NIPP-1 immunoreactivity was present as
a single peak at approximately 16.5 ml. Note that the NIPP-1 Western
profile overlays the 41-kDa PP1-interacting protein distribution on the
column. These data indicate that the 41-kDa PP1-interacting protein
present in the mitotic cell extracts is likely to be NIPP-1 and suggest
that the NIPP-1 protein is complexed with PP1c during mitosis.
Comparison of the elution positions of NIPP-1 and the phosphatase
activity peaks, however, indicates that complex formation between PP1c and NIPP-1 cannot fully account for either the distribution of the
active pRb-directed protein phosphatase on Superose 6 or the molecular
mass of the active pRb-directed protein phosphatase complex as a
heterodimer (166 kDa, see Fig. 2C). Still, the overlapping distributions of PP1c and NIPP-1 within the phosphorylase phosphatase profile on Superose 6 suggest that NIPP-1 may be associated with, and
regulate the activity of, PP1c during mitosis.
The other PP1 regulatory subunit identified by Jagiello et
al. (48) was designated R111, as its apparent molecular mass by
SDS-PAGE was 111 kDa. Two of the PP1-interacting proteins identified in
the fractionated mitotic CV-1P cell extracts (Fig. 4A,
110-kDa protein and 125-kDa protein) displayed an apparent molecular
mass by SDS-PAGE similar to that of the R111 protein. Either or both of
these PP1-interacting proteins could be identical to the rat R111
protein identified as a subunit of nuclear PP1. The identity of the
approximate 180-kDa PP1-interacting protein is at this point unknown.
Importantly, however, the 110-kDa PP1-interacting protein detected here
cosegregated with the first peak of phosphorylase phosphatase activity
and was coincident with the pRb-directed phosphatase activity peak
(Fig. 3). This suggests that the 110-kDa protein could be associated
with PP1c in the active mitotic pRb-directed phosphatase complex. Note
that the 110-kDa protein in combination with PP1c could also very
closely account for the molecular mass of the mitotic pRb-directed
phosphatase complex as a heterodimer (166 kDa; Fig. 2C).
In summary, two peaks of phosphorylase phosphatase activity were
identified in mitotic CV-1P cell extracts separated by gel filtration
chromatography. The first peak was coincident with the mitotic
pRb-directed protein phosphatase activity peak, eluting with a Stokes
radius of approximately 54 Å. This phosphatase activity peak was
coincident with only a subset of the total PP1c during gel filtration.
A 110-kDa PP1-interacting protein was also detected in this fraction by
enzyme affinity labeling, suggesting that the 110-kDa protein may be a
regulatory component of the active pRb-directed protein phosphatase
complex. The second phosphorylase phosphatase peak was inactive toward
pRb. This activity comigrated with the major immunoreactivity peak for
PP1c on Superose 6. Furthermore, NIPP-1 was also found to be present in
this fraction of the column, suggesting that NIPP-1 may also complex
with, and regulate the activity of, PP1c during mitosis.
To determine if the PP1-interacting proteins identified
by enzyme affinity labeling form a complex with PP1c, mitotic CV-1P cell extracts were subjected to immunoprecipitation with PP1c isoform-specific antibodies and the immunoprecipitates tested for
PP1-interacting proteins by enzyme affinity labeling. As illustrated in
Fig. 5A, precipitations of mitotic cell
extracts with each of the isoform-specific antibodies contained
PP1-interacting proteins relative to the control reaction, performed in
parallel with the immunoprecipitations but without PP1-specific
antibodies. All three isoform-specific antibodies (Fig. 5A, lanes
2-4) coprecipitated the 110-kDa PP1-interacting protein relative
to the control reaction (Fig. 5A, lane 1). The 125- and
41-kDa (NIPP-1) proteins were coprecipitated with varying efficiencies
by the three PP1c isoform-specific antibodies. The 125-kDa protein was
coprecipitated by all three PP1c antibodies; however, considerably more
125-kDa protein was detected in the precipitation with the
In summary, the four major PP1c-interacting proteins identified above
were all coprecipitated by the PP1c isoform-specific antibodies
relative to control precipitations from mitotic CV-1P cell extracts.
However, differences in the coprecipitating protein species and their
relative coprecipitation efficiencies were observed with each
individual isoform-specific antisera. Additionally, the PP1 To determine if the
PP1-interacting proteins that coprecipitated with PP1c in whole cell
extracts also coprecipitate with PP1c after separation by gel
filtration, Superose 6 fractions containing known distributions of the
PP1c isoforms (see Fig. 3) and the PP1-interacting proteins (see Fig.
4) were then subjected to PP1c immunoprecipitation and enzyme affinity
labeling. As a control, the column fractions were also subjected to
precipitations using normal rabbit serum. In this case, none of the
precipitating proteins interacted with PP1c by enzyme affinity labeling
(data not shown). As illustrated in Fig. 6, all four
PP1-interacting proteins coprecipitated with PP1c from the gel
filtration column fractions as detected by enzyme affinity labeling.
The 180-kDa protein was coprecipitated by all three PP1c isoform
antibodies from fractions eluting just after the column void volume,
peaking at approximately 11.5 ml. The Coprecipitation of PP1-interacting proteins
with PP1c-specific antibodies from mitotic CV-1P cell extracts after
separation by gel filtration. Mitotic CV-1P cell extracts were
separated on Superose 6. Equal volumes of the resulting fractions were
then subjected to immunoprecipitation antibodies to PP1c
All three antisera also coprecipitated the 41-kDa PP1-interacting
protein (NIPP-1) from the column fractions, peaking at about 16-17 ml
elution volume. This is consistent with the position of this protein in
previous analyses of the Superose 6 fractions (see Fig. 4) and confirms
that the 41-kDa NIPP-1 protein forms a complex with PP1c in the
separated mitotic cell extracts. This complex was active toward
phosphorylase a, but inactive toward pRb.
The 110-kDa PP1-interacting protein also coprecipitated with PP1c from
the separated mitotic cell extracts. The 110-kDa protein was
precipitated by all three antisera from fractions eluting just after
the column void volume, peaking at about 10 ml, and by the PP1c In summary, the PP1c-specific antibodies coprecipitated PP1-interacting
proteins from the expected Superose 6 fractions of the mitotic CV-1P
cell extracts. The pattern of coprecipitating proteins detected by this
method using the three different antibodies were similar, but not
identical. Significantly, however, the 41-kDa (NIPP-1) and 110-kDa
proteins coprecipitated with PP1c from Superose 6 fractions, which
contain active phosphorylase phosphatase, but only the 110-kDa protein
coprecipitated with PP1c from fraction containing pRb-directed protein
phosphatase activity. This suggests that the active mitotic
pRb-directed protein phosphatase is likely to be a PP1c-p110
heterodimer and indicates that p110 may activate PP1, perhaps the
PP1 PP1 is identified as the protein phosphatase which
dephosphorylates and activates the growth suppressive function of pRb
in mitotic CV-1P cell extracts. As PP1 activity is required for mitotic exit, this suggests that one important aspect of PP1-mediated mitotic
exit involves the activation of G1-specific regulators such
as pRb concomitant with G1 entry. This activation of pRb would ensure its growth suppressive function during re-establishment of
cellular interphase. Active pRb would then prevent inappropriate entry
of cells into the cell division cycle, which can result in tumor
formation.
Identification of the mitotic pRb-directed phosphatase as a high
molecular weight species of PP1 suggested that its regulation toward
pRb during mitosis may be conferred by associated regulatory proteins.
This observation conforms to the paradigm for PP1 regulation in
differentiated cell types, where the catalytic portion associates with
a variety of noncatalytic components. These regulatory proteins function to differentially regulate the activity of the PP1 holoenzyme complexes toward the large number of substrates on which PP1 is active
in cells (reviewed in Refs. 39-41).
We further observed that the active pRb-directed protein phosphatase
cofractionated with only a subset of the total PP1c and active
phosphorylase phosphatase complexes in mitotic cell extracts. The
cosegregation of a 110-kDa PP1-interacting protein with the pRb-directed phosphatase suggested that the 110-kDa protein may complex
with, and activate, only a subset of the total cellular PP1 toward pRb.
Coprecipitation of the 110-kDa protein with PP1c confirmed that it
forms a complex with PP1c, thus supporting this suggestion. An
interesting possibility is that the 110-kDa PP1-interacting protein
identified here may represent the R111 protein that was also found to
complex with PP1c in cell nuclei (48). In contrast, the NIPP-1 protein,
which did not cofractionate with the pRb-directed phosphatase activity,
cofractionated with the majority of the PP1c in the separated mitotic
cell extracts. NIPP-1 protein coprecipitation with PP1c confirmed that
it also forms a complex with PP1c in the mitotic cell extracts and gel
filtration column fractions. Recall that the NIPP-1 protein was shown
previously to form a complex with PP1c in differentiated cells, where
it is restricted to the cell nucleus (48). Hence, as many proteins are
dephosphorylated during mitotic exit, future studies will need to
address the role of differential complex formation between PP1 and
these noncatalytic regulatory proteins in dictating substrate
specificity of PP1 catalytic activity during mitotic exit and
G1 entry.
The paradigms for regulation of PP1 activity include reversible
inhibition of catalytic activity and subcellular targeting of PP1
enzyme complexes. The cofractionation of pRb-directed protein phosphatase activity with only a subset of active PP1 enzyme complexes observed in these studies suggests that another mechanism for the
regulation of PP1 activity in cells by noncatalytic components may
involve the activation of PP1 catalytic activity toward only a subset
of potential substrates. In this case, the 110-kDa protein may activate
PP1 toward pRb. Additionally, the observed differences in phosphatase
activity profiles toward pRb and phosphorylase a highlight
the importance of using a variety of protein substrates when evaluating
the role of noncatalytic regulatory proteins in controlling the cell
cycle-dependent regulation of phosphatase activity. Future
studies need to address the role of the 110-kDa protein in controlling
PP1 catalytic activity toward pRb throughout the cell cycle and the
role of noncatalytic components in conferring substrate specificity to
individual PP1 complexes during cell division.
We acknowledge Drs. Norbert Berndt, Mathieu
Bollen, Shirish Shenolikar, and Emma Villa-Moruzzi for their kind gifts
of biological and biochemical reagents.
Department of Biochemistry and Biophysics,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
Cell Culture and Synchronization
-p-tosyl-L-lysine chloromethyl
ketone (TLCK), 50 µM
N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK), 50 µM leupeptin, and 1 µM
pepstatin. Insoluble material was separated from the soluble extracts
by brief centrifugation at 10,000 × g. Protein was
measured using the Bio-Rad protein assay system according to the
manufacturer's instructions.
-32P]ATP (106
cpm/nmol)).
S and the cyclic
AMP-dependent protein kinase.
-lactoglobulin, 29.2 Å; bovine
serum albumin, with two published values of 34.9 and 36.4 Å; catalase,
52.3 Å; apoferritin, 59.5 Å;
-galactosidase, 68.8 Å; and
thyroglobulin, with two published values of 81 and 82.2 Å. Values for
Stokes radii of the standards were obtained from the Handbook of
Biochemistry (45) using the relationship Stokes radius = kT/6
D, where k is the
Boltzman constant (1.3805 × 10
16 erg/K),
T = temperature (K),
= viscosity (1.002), and
D = diffusion coefficient (cm2
s
1 × 10
7).
Nas/(1
v
) and
f/f0 = a/(3vM/4
N)1/3, where M = molecular weight (daltons), f/f0 = frictional ratio, N = Avagadro's number,
a = Stokes radius, s = sedimentation
coefficient, and
= density of the medium (1 g/ml).
The Mitotic pRb-directed Phosphatase Is a Type 1 Serine/Threonine
Phosphoprotein Phosphatase
Fig. 1.
The pRb-directed protein phosphatase is a
type 1 serine/threonine phosphoprotein phosphatase. A,
isolation of phosphoprotein phosphatase activity on heparin-agarose.
Mitotic CV-1P cell extracts were separated on heparin-Sepharose.
Flow-through and step fractions were tested for phosphatase activity
toward pRb (hatched bars) and phosphorylase a
(stippled bars). pRb-directed phosphatase assays each
contained 30 µg of protein for both the flow-through and the step
fractions. Phosphorylase phosphatase assays were done with variable
protein amounts to obtain percent release values for each fraction
within the linear range of the assay. FT, flow-through; Step, 0.4 M NaCl step fractions. B,
Western blot profile of the heparin fractions using PP1- and
PP2A-specific antibodies. Mitotic cell extracts (lane 1, 3 µg for PP1 and 30 µg for PP2A), heparin flow-through (lane
2, 6 µg), and heparin step (lane 3, 4 µg) were immunoblotted using PP1- and PP2A-specific antibodies. Catalytic subunit positions are indicated by arrows. C, sensitivity to
a type 1 phosphatase-specific inhibitor. The 0.4 M NaCl
heparin step was tested for phosphatase activity toward pRb and
phosphorylase a as described in A. Assays were
conducted with okadaic acid (OA), with a GST protein
(GST), or with a thiophosphorylated GST-inhibitor 1 fusion
protein (GST-S-I1). Results are presented as the percent inhibition of phosphatase activity in each condition relative to assays
conducted in the absence of inhibitor. ND indicates not
determined.
[View Larger Version of this Image (28K GIF file)]
Fig. 2.
Molecular size of the mitotic pRb-directed
protein phosphatase activity. A, phosphatase activity
profiles on Superose 6. Mitotic CV-1P cell extracts were separated on
Superose 6, and equal volumes of the resulting fractions were tested
for phosphatase activity toward pRb (solid squares) and
phosphorylase a (open squares). Results are
expressed as the percent of total radioactivity released by the assays
as a function of elution volume. The overall protein profile is
included for reference (open circles). Molecular size
standards are as follows: t, thyroglobulin; g,
-galactosidase; a, apoferritin; c, catalase;
b, bovine serum albumin; l,
-lactoglobulin; and y, chymotrypsinogen. B, phosphatase activity
profiles in the glycerol gradient. Mitotic CV-1P cell extracts were
sedimented through a 10-30% glycerol gradient and fractionated from
the top to the bottom (left to right in figure).
Equal volumes of the resulting fractions, indicated numerically, were
tested for phosphatase activity toward pRb (solid squares)
and phosphorylase a (open squares). Results are
expressed as the percent of total radioactivity released. Distribution
of total protein is included for reference (open circles).
Positions of molecular size standards run on a parallel gradient and
indicated by arrows are as follows: c, cytochrome c; b, bovine serum albumin; g,
glyceraldahyde-3-phosphate dehydrogenase; t, catalase.
C, physical parameters of the mitotic pRb-directed protein phosphatase. Determined as described under "Experimental Procedures."
[View Larger Version of this Image (19K GIF file)]
,
, and
-1
isoforms are expressed in many cell types (40, 58). Fig.
3A shows the distributions of these PP1c
isoforms in mitotic CV-1P cell extracts separated by gel filtration. As
illustrated, the three PP1c isoforms had similar distributions. The
first peak eluted with the column void volume, trailing down through
approximately 9-10 ml. The second peak eluted within the included
column volume, peaking at about 16.5 ml. The
isoform, however,
contained a greater proportion of the total PP1c immunoreactivity in
the second peak, eluting within the included column volume.
Fig. 3.
Distribution of PP1c isoforms after gel
filtration separation of mitotic CV-1P cell extracts. A,
Western profiles for PP1c isoforms. Mitotic CV-1P cell extracts were
separated on Superose 6. Equal volumes of the resulting fractions were
immunoblotted using antibodies to PP1c-, PP1c-
, and PP1c-
-1.
Results are shown as a function of elution volume. The position of PP1c
is indicated by an arrow. Elution positions of molecular
standards are indicated at the top as follows: thy,
thyroglobulin; gal,
-galactosidase; apo,
apoferritin; cat, catalase; bsa, bovine serum
albumin; lac,
-lactoglobulin; and chy,
chymotrypsinogen. B, phosphatase activity profiles.
Phosphatase activity profiles toward pRb (filled squares)
and toward phosphorylase a (open squares) on
Superose 6.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Identification of PP1-interacting proteins in
mitotic CV-1P cell extracts after separation by gel filtration.
A, enzyme affinity labeling of Superose 6 fractions. Mitotic
CV-1P cell extracts were separated on Superose 6. Equal volumes of the
resulting fractions were then subjected to SDS-PAGE and transferred to
nitrocellulose. The blot was developed using digoxygenin-labeled PP1c
to detect the PP1c-interacting proteins present in each fraction.
Results are shown as a function of elution volume. The positions of the major PP1-interacting proteins identified are indicated on the right side of the blot by arrows. Positions of
molecular weight markers included on the gel are indicated on the
left side of the blot. Elution positions of molecular
standards on the Superose 6 column are indicated at the top
of the figure as follows: thy, thyroglobulin;
gal, -galactosidase; apo, apoferritin;
cat, catalase; bsa, bovine serum albumin;
lac,
-lactoglobulin; and chy,
chymotrypsinogen. B, distribution of NIPP-1 on Superose 6. Mitotic CV-1P cell extracts were separated on Superose 6. Equal volumes
of the resulting fractions were then subjected to immunoblotting using
antibodies to NIPP-1. The position of NIPP-1 is indicated by an
arrow.
[View Larger Version of this Image (44K GIF file)]
isoform
antibodies (Fig. 5A, lane 3). In contrast, the 41-kDa
PP1-interacting protein (NIPP-1) was precipitated much more effectively
by the
and
-1 isoform antibodies than with the
isoform
antibody, which gave almost no enzyme affinity interaction signal in
this experiment. The 180-kDa protein showed only a very faint PP1c
affinity interaction signal in precipitations with the
and
-1
isoform antibodies and no signal in precipitations with the
isoform
antibody. When performing pRb-directed phosphatase activity assays
using immunocomplexed PP1c isoforms as the enzyme source (Fig.
5B), it appears that PP1
dephosphorylates pRb more
effectively that PP1
or PP1
-1.
Fig. 5.
PP1 isoform-interacting proteins and
isoform-specific pRb dephosphorylation. A, coprecipitation
of PP1c isoform-interacting proteins in mitotic CV-1P cell extracts.
Mitotic CV-1P cell extracts (500 µg) were subjected to
immunoprecipitation using PP1c isoform-specific antibodies. Proteins in
the immunoprecipitations were then separated by SDS-PAGE and
transferred to nitrocellulose. The blot was developed using
digoxygenin-labeled PP1c to detect PP1c-interacting proteins present in
the immunoprecipitates. Lane 1, no antibody control; lane 2, PP1c immunoprecipitation; lane 3,
PP1c
immunoprecipitation; lane 4, PP1c
-1
immunoprecipitation. Positions of the major PP1-interacting proteins
identified in the immunoprecipitations are indicated on the right
side by arrows. Positions of molecular weight markers included on the gel are indicated on the left side of the
blot. B, immunoprecipitation of PP1c isoforms and activity
toward pRB. Mitotic cell extracts (500 µg) were individually
immunoprecipitated with approximately 3 µg of rabbit antisera to each
of the PP1 isoforms (PP1
, PP1
-1, and PP1
), mixed with
radiolabeled pRb, and phosphatase activity measurements made as
described under "Experimental Procedures." An immunoprecipitation
with 3 µg of normal rabbit serum (NRS) served as a
negative control. Mitotic cell extracts alone (10 µg) served as a
positive control. Percent activity was calculated by subtracting the
32P-labeled pRb band density for each treated
immunoprecipitation from the band density for an untreated control
immunoprecipitation, then dividing by the untreated control band
intensity and multiplying this number by 100.
[View Larger Version of this Image (28K GIF file)]
isoform
demonstrated the greatest ability to dephosphorylate pRb in
vitro.
isoform antibody
(B), but not the
and
-1 isoform antibodies
(A and C, respectively), coprecipitated the
125-kDa PP1-interacting protein, peaking at about 13 ml elution volume,
and in a pattern consistent with, but slightly broader than, the
distribution seen for this protein by previous enzyme affinity labeling
of the column fractions (see Fig. 4).
Fig. 6.
(A), PP1c
(B), and PP1c
-1 (C). Immunoprecipitated proteins were then
separated by SDS-PAGE, transferred to nitrocellulose, and developed by
enzyme affinity labeling. The positions of the major PP1-interacting proteins identified in the immunoprecipitations are indicated on the
right side of the blot by arrows. Positions of
molecular weight markers included on the gel are indicated on the
left side of the blot. Elution positions of molecular
standards on the Superose 6 column are indicated at the top
of the figure as follows: thy, thyroglobulin;
gal,
-galactosidase; apo, apoferritin;
cat, catalase; bsa, bovine serum albumin;
lac,
-lactoglobulin; and chy,
chymotrypsinogen.
[View Larger Version of this Image (23K GIF file)]
antibodies in a second peak at approximately 14-15 ml elution volume.
This again is similar to the elution position for this protein observed
in the previous enzyme affinity labeling of the Superose 6 fractions
(see Fig. 4). Importantly, the 110-kDa protein coprecipitated with PP1c
from fractions containing the pRb-directed protein phosphatase
activity peak (approximately 15 ml elution volume, see also Fig. 4).
This is also the same isoform that was most effective at
dephosphorylating pRb in vitro (see Fig. 5B).
This demonstrates that p110 forms a complex with PP1c in fractions
containing the active pRb-directed phosphatase complex and supports the
suggestion that the 110-kDa protein may regulate PP1 activity toward
pRb. Furthermore, as noted previously, p110 in combination with PP1c
could account for the mass of the pRb-directed protein phosphatase
complex determined above.
isoform predominantly, toward pRb during mitotic exit.
*
This work was supported in part by National Institutes of
Health Grant CA56940 (to J. W. L.) and Cancer Center Core Grant CA11198
awarded to the late Robert A. Cooper, Jr. 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.
§
Recipient of a predoctoral fellowship from the Oral and Cellular
Molecular Biology Training Grant DE07202.
Supported by National Institutes of Health Cancer Research
Training Grant T32 CAO9363.
**
To whom correspondence should be addressed: University of
Rochester Cancer Center, Division of Developmental Therapeutics, Box 704, 601 Elmwood Ave., Rochester NY 14642. Tel.: 716-275-6325; Fax:
716-273-1042; E-mail: jludlow{at}cc.urmc.rochester.edu.
1
The abbreviations used are: PP1, protein
phosphatase type 1; TLCK,
N-p-tosyl-L-lysine chloromethyl
ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; GST,
glutathione S-transferase; ATP
S, adenosine
5
-O(thiotriphosphate); GST-S-I1, glutathione
S-transferase-thiophosphorylated-inhibitor 1; NIPP-1,
nuclear inhibitor of protein phosphatase 1.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.