(Received for publication, December 6, 1996, and in revised form, March 17, 1997)
From the Friedrich Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland
Calyculin-A (CA), okadaic acid (OA), and
tautomycin (TAU) are potent inhibitors of protein phosphatases 1 (PP1)
and 2A (PP2A) and are widely used on cells in culture. Despite their
well characterized selectivity in vitro, their exact
intracellular effects on PP1 and PP2A cannot be directly deduced from
their extracellular concentration because their cell permeation
properties are not known. Here we demonstrate that, due to the tight
binding of the inhibitors to PP1 and/or PP2A, their cell penetration
could be monitored by measuring PP1 and PP2A activities in cell-free
extracts. Treatment of MCF7 cells with 10 nM CA for 2 h simultaneously inhibited PP1 and PP2A activities by more than 50%. A
concentration of 1 µM OA was required to obtain a similar
time course of PP2A inhibition in MCF7 cells to that observed with 10 nM CA, whereas PP1 activity was unaffected. PP1 was
predominantly inhibited in MCF7 cells treated with TAU but even at 10 µM TAU PP1 inhibition was much slower than that observed
with 10 nM CA. Furthermore, binding of inhibitors to PP2Ac
and/or PP1c in MCF7 cells led to differential posttranslational
modifications of the carboxyl termini of the proteins as demonstrated
by Western blotting. OA and CA, in contrast to TAU, induced
demethylation of the carboxyl-terminal Leu309 residue of
PP2Ac. On the other hand, CA and TAU, in contrast to OA, elicited a
marked decrease in immunoreactivity of the carboxyl terminus of the
-isoform of PP1c, probably reflecting proteolysis of the protein.
These results suggest that in MCF7 cells OA selectively inhibits PP2A
and TAU predominantly affects PP1, a conclusion supported by their
differential effects on cytokeratins in this cell line.
The serine/threonine protein phosphatase family was initially restricted to four biochemically distinct entities, protein phosphatases-1 (PP1),1 -2A (PP2A), -2B (also called calcineurin) (PP2B), a Ca2+-dependent enzyme, and -2C (PP2C), a Mg2+-dependent enzyme (1). This class of enzymes has now been considerably extended by molecular approaches (1, 2). Nevertheless, among its numerous members, PP1 and PP2A can still be considered as the two principal enzymes because of their ubiquitous abundance and broad specificity (1).
PP1 consists of a catalytic subunit (PP1c) associated with various
regulatory subunits, forming numerous oligomeric enzymes (1, 3). For
instance, in muscles PP1c interacts with the G and M subunits that
target it to glycogen and myosin, respectively (reviewed in Refs. 1 and
4). PP1c is a monomeric protein of about 37 kDa. It has a compact,
ellipsoidal structure containing in the amino-terminal domain a
-
-
-
-
metal-coordinating unit typical for
metal-dependent hydrolases (5). Four isoforms,
,
(or
),
1, and
2, encoded by three genes (
1 and
2 resulting from alternative splicing) (6) are differently expressed in mammalian
tissues (7). Protein sequence variations among these isoforms are
mainly confined to their carboxyl termini (6), which play a regulatory
role in the catalytic activity, as demonstrated by proteolysis (8) and
phosphorylation studies (9, 10). Cyclin-dependent protein
kinases (cdk) phosphorylate a TPPR consensus sequence present in the
carboxyl terminus of all PP1c isotypes and inhibit their activity (9,
10). PP1c is also specifically inhibited by small acidic thermostable
proteins, such as inhibitor-1, inhibitor-2, NIPPs (1), and IP (11).
In contrast to PP1c, the catalytic subunit of PP2A (PP2Ac) is always
associated with a constant regulatory subunit of 65 kDa (PR65 or A
subunit). To this dimeric core, various third or variable regulatory
subunits can bind and modulate the enzymatic activity of PP2Ac (1,
12-16). PP2A has long been considered as a predominantly cytosolic
enzyme, in contrast to PP1 which is distributed among most cellular
compartments (1). However, recent studies have questioned this dogma by
demonstrating the presence of PP2Ac in fibroblast nuclei (17) and its
association with microtubules (18) and neurofilaments (19). PP2Ac is a
35.6-kDa protein. Two highly homologous isoforms, and
, encoded
by two genes, are expressed in mammals (1). Except for their
extremities PP2Ac and PP1c are 67% homologous and most of the amino
acids crucial for the 3-dimensional structure of PP1c are conserved in
PP2Ac (5). The carboxyl terminus of PP2Ac also plays a regulatory role
in the enzymatic activity. PP2Ac is phosphorylated by receptor and Src
family tyrosine kinases on Tyr307, two residues upstream of
its carboxyl terminus (Leu309), which suppresses its
activity (20). In vitro PP2Ac can also be inhibited by
phosphorylation on Thr residue(s) catalyzed by the
autophosphorylation-activated protein kinase (21). Moreover, PP2Ac is
methylated on Leu309 by a novel type of carboxyl protein
methyltransferase (22-25). Demethylation is catalyzed by a specific
protein carboxylmethylesterase (26). Interestingly, PP2Ac methylation
is regulated during the cell cycle (17). Potent protein inhibitors of
PP2A, different from those of PP1c, have been recently identified
(27-29).
The identification of serine/threonine protein phosphatases as the
target of okadaic acid (OA), a polyketal fatty acid, provided novel
insights into their essential functions (30). OA is a potent tumor
promoter (31) that binds to and inhibits PP1c and PP2Ac with
dissociation constants (Ki) of 147 and 0.032 nM, respectively (32). Other polycarbon inhibitors of
serine/threonine protein phosphatases include calyculin-A (CA) and
tautomycin (TAU) (31). CA has a higher affinity for PP2A than PP1,
Ki 0.12 and 1 nM, respectively,
whereas TAU has the opposite preference, Ki
30
and 0.5 nM, respectively (32). OA, CA, and TAU appear to
bind to the same site on PP1c or PP2Ac (32). The two other major
protein Ser/Thr phosphatases, PP2B and PP2C, are either weakly
sensitive (in the µM range) or completely insensitive, respectively, to these inhibitors. Other classes of Ser/Thr phosphatase inhibitors have been identified (31, 33-37).
CA and OA are often used to demonstrate the involvement of PP1 or PP2A in a biological process. These conclusions are usually based on the in vitro selectivity of CA and OA and extracellular concentration of the inhibitors required to observe an effect on cells. However, such conclusions can be biased by differential cell penetration rates of CA and OA. Here we show that CA, OA, and TAU have very distinct cell permeation properties and induce specific posttranslational modifications of PP1c and/or PP2Ac in accordance to their in vitro selectivity, suggesting that the systematic use of these three inhibitors should facilitate the assignment of the cellular functions of PP1 and PP2A.
MCF7 cells were cultured as described previously (24). Where stated, the medium from a 6-cm dish containing approximately 80% confluent cells (time "0") was replaced by 5 ml of fresh medium supplemented with protein phosphatase inhibitor or the same volume of solvent, N,N-dimethylformamide for OA (Diagnostic Chemicals Limited) and CA (LC Services Corp.), and dimethyl sulfoxide for TAU (Drs. Kiyoshi Isono and Hiroyuki Nagata, RIKEN, Japan). Protein synthesis inhibitors, cycloheximide and puromycin (Sigma), were added similarly. For 32P labeling MCF7 cells growing on a 6-cm dish were incubated with 5 ml of medium containing 37 MBq of 32Pi (Amersham Corp.). After 4 h an appropriate volume of protein phosphatase inhibitor or solvent was added to the medium, and cells were further incubated for 2 h before being collected. All experiments were performed at least twice.
Preparation of Cell-free ExtractsCells were collected, frozen, and homogenized as described previously (24). Cell extracts were centrifuged at 10,000 × g for 10 min. Protein concentration in the supernatant, henceforth termed the soluble fraction, was determined with the Bradford's reagent from Bio-Rad, using bovine serum albumin (BSA) as standard. The pellet obtained from the 10,000 × g centrifugation (henceforth termed the particulate fraction) was washed with 1.5 ml of extraction buffer and dissolved in SDS sample buffer containing 4% (w/v) SDS (150 µl per 6-cm dish). To prepare whole cell extracts, frozen cells were directly lysed in SDS sample buffer containing 4% (w/v) SDS (300 µl per 6-cm dish).
Precipitation of Cell Extracts with EthanolSoluble fractions from MCF7 cells were mixed with 4 volumes of absolute ethanol, left on ice for 15 min, and then centrifuged at 10,000 × g for 5 min. Supernatants were discarded, and the pellets were resuspended in 300 µl of 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl (TBS) containing 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 50 mM 2-mercaptoethanol. After 15 min on ice with mixing, samples were centrifuged at 10,000 × g for 10 min. Protein concentration in the supernatants was measured, and both protein amount and volume were equalized between different samples (the variation of protein concentration between the samples did not exceed 15%) before the next round of ethanol precipitation/solubilization. An aliquot of the protein fraction obtained after each ethanol precipitation and solubilization was used for phosphatase activity determination, SDS-polyacrylamide gel electrophoresis (PAGE), and Western blotting.
Protein Phosphatase Activity AssayFor PP2A and PP1 assays, the soluble fraction was diluted to a concentration of 120 and 50 µg/ml, respectively, in extraction buffer (24) containing 1% BSA. PP2A activity was measured, using a heptapeptide substrate (LRRASVA, also termed Kemptide Val6-Ala7) (Bachem), phosphorylated by the catalytic subunit of protein kinase A in a final volume of 30 µl containing 60 µM phosphopeptide, 0.4 µg of MCF7 soluble protein, 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.1 mM EDTA, 30 mM 2-mercaptoethanol, 0.7 mg/ml BSA, and where stated, the appropriate concentration of CA, OA, or TAU (24). Phosphorylase a phosphatase activity was assayed according to Ref. 38 in a final volume of 30 µl consisting of 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.1 mM EDTA, 30 mM 2-mercaptoethanol, 3.3 mM caffeine, 0.7 mg/ml BSA, 0.25 mg/ml phosphorylase a, 0.17 µg of MCF7 soluble protein, and where stated, the appropriate concentrations of OA, CA, TAU, or partially purified inhibitor-2 (39). Samples were incubated for 15 min at 30 °C and processed as described (24, 38). Both phosphatase assays were in the linear range of activities (less than 20% substrate dephosphorylation) and 1 unit corresponds to the release of 1 nmol of phosphate per min at 30 °C. Each determination was performed in duplicate.
Western BlottingProtein samples were fractionated by 12%
SDS-PAGE and either stained with Coomassie Blue or transferred onto
Immobilon-P (polyvinylidine difluoride membrane, Millipore). When
required, the soluble fraction was concentrated by precipitating 50 µg of protein with 10% (w/v, final) trichloroacetic acid. After
centrifugation, the pellet was dissolved in 30 µl of SDS sample
buffer and the pH adjusted to 6.8. The amount of proteins contained in
the particulate fraction or whole extracts from cells incubated for
various times was normalized to that from cells collected at time 0 by
scanning Coomassie Blue-stained gels. Blots were incubated with
affinity purified rabbit polyclonal antibodies raised against the
internal peptide Ab169/182 or the carboxyl terminus
Ab299/309 of PP2Ac (24), or the carboxyl terminus of
-PP1 (Ab
317/330) (40), or with affinity purified
sheep polyclonal antibodies generated against the 33,000-35,000
amino-terminal fragment of PP1 from rabbit skeletal muscle
(Ab33k) (8), as described previously (17, 24). Monoclonal
antibodies against cytokeratins 8 (clone M20) and 18 (clone CY-90) or
pan cytokeratin (mixture of clones) (all purchased from Sigma) were diluted 1,000-, 2,000-, and 8,000-fold, respectively, in skimmed milk
blocking buffer (24). Secondary antibodies used were donkey 125I-anti-rabbit immunoglobulins-G (Amersham Corp.), rabbit
horseradish peroxidase-conjugated anti-sheep immunoglobulins-G
(Kirkegaard & Perry, Laboratories) or sheep horseradish
peroxidase-conjugated anti-mouse immunoglobulins-G antibodies (Amersham
Corp.). Membranes decorated with 125I-secondary antibodies
were exposed to preflashed X-OMAT AR films (Kodak) and those with
horseradish peroxidase-conjugated secondary antibodies developed with
enhanced chemiluminescent reagents according to the manufacturer's
protocol (Amersham Corp.). Where indicated, the same membrane was
incubated with a different antibody after having been stripped
according to the enhanced chemiluminescent protocol (Amersham Corp.).
Quantitative estimation of Western blots was done by scanning
preflashed x-ray films exposed in the linear range at 600 nm with a
Shimadzu CS-930 scanner.
Radiolabeled cells were washed consecutively with phosphate-buffered saline and TBS and directly lysed in TBS containing 1 mM EDTA and 0.5% SDS (0.4 ml/6 cm dish). Immunoprecipitations were performed exactly as described (24) with either anti-cytokeratin 8 (50 µg/ml) or anti-cytokeratin 18 (40 µg/ml) monoclonal antibodies purchased from Boehringer Mannheim.
Northern AnalysisNorthern blot analysis was performed
according to Ref. 41 with total RNA isolated from untreated MCF7 cells
(at time 0 or 24 h) or cells treated with 100 nM OA or
10 nM CA, every 6 h until 24 h. The probes used
were human PP2Ac or -PP1c cDNA and, as controls,
oligonucleotides complementary to the human
-actin gene (nucleotides
4-30 of the coding region, accession number X00351[GenBank]) and
-tubulin
gene (nucleotide 1049-1076 of the coding region, accession number
K00558[GenBank]) (41).
Since we were
interested in the selectivity of CA, OA, and TAU in MCF7 cells, we
first analyzed their apparent affinity for PP1 and PP2A in cell
extracts. To assay PP2A activity we used a synthetic phosphorylated
heptapeptide whose sequence, LRRASVA, corresponds to the protein kinase
A phosphorylation site in pyruvate kinase. About 85% of the total
soluble phosphopeptide phosphatase activity from MCF7 cell extracts
(specific activity without inhibitor: 1.77 ± 0.08 units/mg
protein, ± S.E., n = 36) was inhibited by 5 nM OA (Fig. 1A). The 15%
phosphopeptide phosphatase activity insensitive to 5 nM OA
was not suppressed by higher concentrations of OA (up to 1 µM), indicating that this activity could not be attributed to PP1. CA exhibited inhibitory properties similar to OA. In
contrast, TAU was approximately 10-fold less potent than OA and CA
(Fig. 1A). Henceforth PP2A activity is defined as the
phosphopeptide phosphatase activity inhibited by 10 nM OA.
Both PP1 and PP2A are active toward phosphorylase a and
account for 100% of the cellular activity (42). Total phosphorylase a phosphatase activity in the soluble fraction of MCF7 cells
(1.57 ± 0.18 units/mg protein, ± S.E., n = 21)
was very sensitive to CA, as expected from the similar high affinity of
CA for both PP1 and PP2A (32) (Fig. 1B). With OA an
intermediate plateau of inhibition at about 1 nM was
observed, PP2A activity corresponding to the phosphatase activity most
sensitive to OA (1 nM), 0.54 ± 0.06 units/mg
protein (± S.E., n = 21) (32), and PP1 activity to the
least OA-sensitive (
1 nM OA), 1.03 ± 0.12 units/mg
protein (± S.E., n = 21). When an increasing amount of
inhibitor-2 was added, which specifically affects PP1 (1, 39), the
inhibition reached a plateau, corresponding to a suppressed PP1
activity of 0.85 ± 0.07 units/mg protein (± S.E.,
n = 21) (not shown). The exact reason for the
difference consistently observed between OA and inhibitor-2 (around 0.2 units/mg protein, see above) is unclear (longer preincubation times
with inhibitor-2 did not alter the results) but might be related to the
presence of PP1-like activities relatively insensitive to inhibitor-2,
as described previously in other systems (43-45). PP1 activity is
defined in all further experiments as the phosphorylase a
phosphatase activity that was inhibited by a saturating concentration
of inhibitor-2. TAU inhibited phosphorylase a phosphatase
activity with a potency slightly weaker than that of CA. Addition of 1 nM OA to 1 nM TAU almost completely suppressed
the activity (92% in comparison with 66% inhibition observed in the
presence of TAU alone). In contrast, inhibitor-2 negligibly increased
caused (4%) the inhibition promoted by 1 nM TAU,
demonstrating that the activity more sensitive to TAU was catalyzed by
PP1 (32).
In our experimental conditions to assay PP2A activity
from MCF7 cells a concentration of 3 nM OA was required to
completely inhibit PP2A activity (Fig. 1A). This value is
much higher than the dissociation constant of OA for PP2Ac
(Ki 40 pM) (32). Consequently, upon MCF7
cell treatment with OA, the inhibitor bound to PP2Ac should not
dissociate during the preparation of MCF7 cell extracts, since the
concentration of PP2Ac is always higher than its Ki
for OA. Incubation of cells with OA indeed induced a concentration- and
time-dependent inhibition of PP2A (Fig.
2A).
OA Treatment of MCF7 Cells Affects the Immunoreactivity of PP2Ac
The possibility that OA altered the level of PP2Ac in MCF7 cells was investigated by Western blot analysis of the soluble and particulate fractions with Ab169/182. With these antibodies (as well as with Ab299/309) no signal could be detected in the particulate fraction from untreated or OA-treated cells (see Ref. 17 for factors affecting the distribution of PP2A in cell-free extracts), whereas in the soluble fraction from cells treated with 100 nM OA for 24 h the immunoreactivity of PP2Ac was 1.7-fold ± 0.15 (± S.E., n = 7) higher than that from untreated cells (Fig. 2B). This OA-induced increase was due to an up-regulation of the protein since it could be almost prevented by cotreatment with the protein synthesis inhibitor cycloheximide (Fig. 2C, upper panel) or puromycin (data not shown). The precise mechanism underlying this modest increased expression of PP2Ac upon treatment of MCF7 cells with OA is unknown. Northern blot analysis did not reveal any significant effect of OA treatment on the level of mRNA transcripts encoding PP2Ac (see "Experimental Procedures," data not shown).
PP2Ac is reversibly methylated on its carboxyl terminus Leu309 residue (22-26) and phosphorylated on Tyr307 (20). We have demonstrated that carboxyl methylation of PP2Ac negatively affects the affinity of Ab299/309 for the protein (17, 24). It is probable that tyrosine phosphorylation has similar consequences. Upon cell treatment with 100 nM OA, the immunoreactivity of the carboxyl terminus of PP2Ac progressively increased with prolonged incubation time to reach 420% ± 115 (± S.E., n = 14) in comparison with that of untreated cells (Fig. 2B). In contrast to what was previously observed with Ab169/182, cycloheximide (or puromycin) did not affect the large OA-induced increase of the carboxyl terminus immunoreactivity (Fig. 2C, lower panel). Since PP2Ac is predominantly carboxyl-methylated and dephosphorylated in asynchronously growing MCF7 cells (24), results obtained with Ab299/309 indicate that the degree of methylation of PP2Ac was lower in OA-treated cells than in untreated cells. Similar demethylation of PP2Ac in response to OA treatment was observed in COS-1 and BHK-21 cells (data not shown).
OA-mediated Inhibition of PP2Ac Is Due to the Tight Binding of the Inhibitor to PP2AcAssociation of OA with PP2Ac is reversible but
very slow (32). Therefore, we tested whether precipitation of soluble
extracts with 80% ethanol, which releases the regulatory subunits from PP2Ac (38), could also dissociate OA from PP2Ac. PP2A activity in the
soluble fraction from OA-treated cells increased after one ethanol
precipitation but recovery was incomplete. It reached a plateau only
after three to four successive ethanol precipitations (Fig.
3A, lower panel). After these repetitive
ethanol precipitations PP2A activity from OA-treated cells was slightly
higher (1.6-fold ± 0.1, ± S.E., n = 3) than that
from untreated cells, whereas the OA-insensitive phosphopeptide
phosphatase activity was similar in both extracts (Fig. 3A, upper
panel). Addition of 100 nM OA to extracts from
untreated cells, before starting the ethanol precipitations, did not
induce a comparable elevation in PP2A activity ruling out a
stabilization effect of OA during the precipitation-solubilization cycles.
Ethanol precipitation of PP2A in MCF7 cell-free extracts also elicits demethylation of PP2Ac, which should negatively influence PP2Ac activity (24, 25). Western blot analyses with Ab299/309 of the effect of three successive ethanol precipitations indicated that demethylation, monitored by an increase in immunoreactivity of the carboxyl terminus of PP2Ac, mainly occurred during the first ethanol precipitation/solubilization (Fig. 3B). Therefore, the progressive recovery of PP2A activity in OA-treated samples by repetitive ethanol precipitation/solubilization cycles was essentially due to the dissociation of OA from PP2Ac and could not be related to the degree of methylation of PP2Ac.
PP1 Activity and Immunoreactivity Is Unaffected in OA-treated MCF7 CellsTreatment of MCF7 cells with 100 nM OA for
24 h did not affect PP1 activity (Fig.
4A). Western blot analyses with
Ab317/330 did not reveal any significant changes due to
cell treatment with 100 nM OA when compared with untreated
cells (Fig. 4B).
CA Has Comparable Effects to Those Induced by OA on PP2A Activity and Immunoreactivity
If the observed effects of OA on PP2A were a
direct consequence of its binding to the protein, similar results
should be obtained with other PP2A inhibitors. Treatment of MCF7 cells
with 10 nM CA also led to the inhibition of PP2A activity.
CA apparently entered the cells much faster than OA, but a plateau was
reached when about 60-70% of PP2A activity was inhibited (Fig.
5A). The time course of penetration was
dependent on CA concentration as with OA, but even at 100 nM CA inhibition was incomplete. To obtain a similar rate
of PP2A inhibition to that induced by 10 nM CA, a
concentration of 1 µM OA was necessary (Fig.
5A). Upon treatment of MCF7 cells with CA the level of
PP2Ac, monitored with Ab169/182, slightly increased while
the degree of methylation of the protein, estimated with
Ab299/309, diminished (Fig. 5B).
CA Negatively Affects PP1 Activity and Immunoreactivity in MCF7 Cells
PP1 activity in the soluble fraction of cells treated with CA was inhibited in a concentration- and time-dependent manner as for PP2A (Fig. 5C). Similar to the inhibition of PP2A activity by CA, the suppression of PP1 activity in response to cell incubation with CA was also incomplete. A plateau was reached when about 75% of the activity was inhibited (Fig. 5C).
Surprisingly, treatment of cells with CA resulted in a time- and
concentration-dependent decrease in immunoreactivity of the carboxyl terminus of -PP1c both in the soluble and particulate fractions (50% decrease after about 60 and 90 min, respectively, with
10 nM CA and 40 and 60 min, respectively, with 100 nM CA) (Fig. 5D). A similar phenomenon was
observed in the four other cell lines we tested, namely HeLa, COS-1,
BHK-21, and REF-52 (data not shown). Since a concentration of 1 µM OA was required to obtain a similar time course of
PP2A inhibition to that of 10 nM CA, we also tested the
effects of 1 µM OA on PP1c. Neither PP1 activity (Fig.
5C) nor
-PP1c immunoreactivity was affected (data not
shown).
The decrease in
immunoreactivity detected by Ab317/330 could reflect
changes in either the level of the whole protein or a posttranslational modification occurring at the carboxyl terminus. To discriminate between these two hypotheses we used Ab33k, which are
polyclonal antibodies raised against PP1c whose carboxyl terminus
(about 30 residues) was cleaved off due to partial proteolysis during
its purification (Mr 33,000-35,000) (8). Such
analysis revealed that PP1c was down-regulated following 10 nM CA treatment (Fig. 6A). The
disappearance rate of PP1c (50% decrease after 5 and 3 h in the
soluble and particulate fraction, respectively) was slower than that
observed with Ab
317/330. Further analysis of the mode of
action of CA on PP1c indicated that the level of PP1c transcripts in
MCF7 cells was unaltered by 10 nM CA treatment for 24 h (see "Experimental Procedures," data not shown). Moreover,
protein synthesis inhibitors (cycloheximide or puromycin) did not mimic
the negative effect of 10 nM CA on the carboxyl
immunoreactivity of
-PP1c (Fig. 6B). These results suggest that CA treatment promoted the degradation of PP1c.
TAU Treatment of MCF7 Cells Strongly Affects PP1 but Not PP2A
TAU significantly inhibited cellular PP1 activity at
concentrations 3 µM. The inhibition was both
concentration- and time-dependent (Fig.
7A). Treatment of cells with 10 µM TAU resulted in complete inhibition of PP1 activity.
TAU promoted a decrease in immunoreactivity of the carboxyl terminus of
-PP1c both in the soluble and particulate fractions (50% decrease
in 10 and 7 h, respectively) (Fig. 7B). However, the
effect of TAU was not as great as that induced by CA, despite the
complete inhibition of PP1. The level of PP1c, tested with
Ab33k, was not significantly altered upon treatment with
TAU (data not shown). In contrast to OA and CA, TAU had only a weak
effect toward PP2A in MCF7 cells. PP2A was inhibited by approximately 20% after a 24 h treatment of cells with 10 µM TAU
(Fig. 7A). Neither the methylation state of PP2Ac (Fig.
7C) nor the level of the protein (data not shown) was
altered in MCF7 cells incubated with 10 µM TAU for
24 h.
Hyperphosphorylation of Intermediate Filaments in MCF7 Cells upon Treatment with CA and OA but Not TAU
To confirm the apparent
selectivity of OA for PP2A and TAU for PP1 in MCF7 cells deduced from
our previous experiments, we analyzed their effects, as well as those
of CA, on potential substrates for PP1 and/or PP2A, the intermediate
filaments. Intermediate filaments are abundant proteins that become
rapidly hyperphosphorylated and disorganized following the addition of
protein phosphatase inhibitors to various cells (46-50). This
hyperphosphorylation results in migration shifts on SDS-PAGE that can
be easily detected. MCF7 cells express three intermediate filaments,
cytokeratin 8, 18, and 19, with Mr of 52,500, 45,000, and 40,000, respectively (51). SDS-PAGE analysis of whole
extracts from MCF7 cells treated with 10 nM CA, 100 nM OA, or 10 µM TAU for 24 h revealed
that the migration of two abundant proteins, Mr
52,000 and 44,000, was altered by treatment with either CA or OA (Fig.
8A). Their identity with cytokeratin 8 and 18 was indicated by Western blotting with various monoclonal
anti-cytokeratin antibodies (Fig. 8B). The correlation
between SDS-PAGE migration shift and hyperphosphorylation of
cytokeratins induced by CA and OA in MCF7 cells was confirmed by
labeling the cells with 32Pi and
immunoprecipitating the intermediate filament proteins with either
anti-cytokeratin 8 or anti-cytokeratin 18 antibodies (see
"Experimental Procedures") (data not shown). The mobility of the
third cytokeratin expressed in MCF7 cells, cytokeratin 19, Mr 40,000, which migrated just beneath an
abundant protein identified by Western blotting as actin, was
apparently not affected by cell treatment with any protein phosphatase
inhibitor (Fig. 8, A and B). TAU did not
significantly alter the migration of any cytokeratins (Fig.
8A), indicating that hyperphosphorylation of cytokeratins
was not a consequence of PP1 inhibition and supporting the previously
drawn conclusions that PP1 is selectively inhibited by TAU and PP2A by
OA in MCF7 cells.
Our data show that PP2A activity is inhibited in cell-free extracts upon treatment of MCF7 cells with CA or OA, whereas PP1 inhibition is observed after cell incubation with CA or TAU. A similar decrease in PP2A activity in cell-free extracts upon treatment of GH4 rat pituitary cells and human fibroblasts with OA has also been observed (52, 53). As demonstrated for cell treatment with OA, PP2A inhibition is due to the tight binding of the inhibitor to PP2Ac. Upon cell incubation with CA, binding of the inhibitor to both PP1 and PP2A could also be released by repetitive ethanol precipitations (data not shown). Based on theoretical considerations (32), similar conclusions can be drawn to explain the inhibition of PP1 after cell treatment with TAU. It is, therefore, possible to take advantage of the tight binding of CA and TAU to PP1, as well as CA and OA to PP2A, to estimate their rate of cellular penetration. It cannot, however, be excluded that part of the inhibitors becomes trapped in membranes or compartments separated from protein phosphatases and gains access to the enzymes only after cell homogenization (measurement of the cellular uptake of radioactively labeled inhibitor could not distinguish between bound and unbound inhibitor). Nevertheless, OA, despite its hydrophobicity, does not significantly distribute to artificial membranes (54). Our results indicate that OA must be 100-fold more concentrated than CA and TAU 100-fold more concentrated than OA to cross the membrane at a similar rate.
The structural characteristics underlying the distinct permeation properties of CA, OA, and TAU is unknown. OA permeates through dipalmitoylphosphatidylcholine membranes only when in a liquid crystalline state (54). It has been recently reported that various factors such as pH and temperature affect the penetration rate of OA through rabbit erythrocyte membranes and that CA passes across these membranes much faster than OA (55). No similar studies with artificial or biological membranes have been performed with TAU. TAU is a mixture of two tautomers in solution. It is unclear whether both tautomers are inhibitory to PP1c and PP2Ac. Moreover, because of their distinct electrostatic charges, their membrane permeability is probably dissimilar. In contrast to OA and TAU, CA contains a phosphate group in its chemical structure. This peculiarity might be a determinant for the unique properties of CA, very rapid cell penetration but incomplete protein phosphatase inhibition.
The absence of inhibition in vitro of either PP1 or PP2A
after cell treatment with OA or TAU, respectively, does not necessarily mean that the enzymes were not inhibited within the cell. Because of
the higher Ki of these complexes, preparation and dilution of cell extracts could have led to their dissociation. However, since OA and TAU are 10-fold more selective for PP2A and
PP1, respectively, than the other type of protein phosphatase in MCF7
cell extracts, one can assume that as long as a significant fraction of
the more sensitive phosphatase is not inhibited, the less sensitive
enzyme will be negligibly affected. This conclusion seems to be
confirmed by the effects of CA, OA, and TAU on the carboxyl termini of
PP1c and PP2Ac. Our results suggest that binding of protein phosphatase
inhibitors to PP1c and PP2Ac induces a conformational change in each
protein which results in distinct posttranslational modification of
their carboxyl terminus, since it has been established that inhibitors
do not directly bind to the carboxyl terminus of
-PP1c (5, 8,
56).
Previous studies have demonstrated that an increase in immunoreactivity of PP2Ac from MCF7 cells, detected by Ab299/309, corresponded to demethylation of PP2Ac (17, 24). We found no evidence for a steady, significant phosphorylation level of PP2Ac in asynchronously growing MCF7 cells (24). The question concerning the mechanism of OA-mediated demethylation of PP2Ac in MCF7 cells (in the absence or presence of protein synthesis inhibitors) remains to be clarified. The action of both the carboxylmethyltransferase and -esterase, which are specific for PP2Ac, is inhibited by the binding of OA to PP2Ac (25, 26, 57). Xie and Clarke (58) have identified a phenylmethylsulfonyl fluoride-sensitive carboxylmethylesterase active toward PP2Ac that is different from the PP2A-specific, phenylmethylsulfonyl fluoride-insensitive carboxylmethylesterase (26). We have observed that a high concentration of a mixture of protease inhibitors could prevent the dramatic increase in PP2Ac immunoreactivity induced by ethanol precipitation of MCF7 cell extracts (data not shown). Since one ethanol precipitation completely dissociates the regulatory subunits from PP2Ac (38) but not OA from the protein (Fig. 3A), it is plausible that the nonspecific esterases, which are apparently inhibited by the association of regulatory subunits with PP2Ac, are responsible for the slow demethylation of PP2Ac observed in OA-treated cells. The apparent correlation between inhibition and demethylation of PP2Ac strongly suggests that TAU, in contrast to OA and CA, was not bound to PP2Ac in MCF7-treated cells.
CA and TAU, in contrast to OA, induced a decrease in the
immunoreactivity of the carboxyl terminus of -PP1c. The extent of this effect does not perfectly match the level of PP1 inhibition measured in vitro. It is higher with CA than TAU, indicating
that the disappearance of the immunoreactivity of the carboxyl terminus is not the primary cause for the inhibition of PP1 activity. A similar
conclusion can be drawn from results obtained with Ab33k.
Disappearance of PP1c upon cell treatment with CA (and TAU) was slower
than both PP1 inhibition and the decrease in carboxyl terminus
immunoreactivity of
-PP1c. A possible explanation for the
discrepancy observed with the two anti-PP1c antibodies is their
distinct specificities. Ab
317/330 can exclusively
recognize the
-isoform of PP1c (40). In contrast, based on the high
homology of the different PP1c isoforms, it is probable that
Ab33k recognizes all isoforms (6, 8). Consequently, the
-isoform of PP1c might be more (or exclusively) susceptible to a
posttranslational modification than other isotypes, if expressed in
MCF7 cells. For instance, only
-PP1c is phosphorylated by tyrosine
protein kinases (59). The use of specific antibodies toward the other PP1c isoforms should answer this question.
A plausible mechanism for the disappearance of the carboxyl-terminal
immunoreactivity of -PP1c is partial proteolysis. The carboxyl
terminus of PP1c is very sensitive to proteolysis in vitro
after dissociation from regulatory subunits (8, 38). However, we could
not detect the presence of a partially proteolyzed form of PP1c, and
incubation of cell extracts (prepared without protease inhibitors) in
the presence of CA at 37 °C did not affect the integrity of PP1c.
Therefore, it cannot be excluded that the decrease in the
immunoreactivity of the carboxyl terminus of
-PP1c is due to a
posttranslational modification different from proteolysis, such as
phosphorylation (9, 10), preventing the binding of the antibodies, with
proteolysis of the complete protein occurring at a later step. However,
the decrease in immunoreactivity of
-PP1c induced by a 2 h
treatment with 10 nM CA could not be reversed by incubating
MCF7 cells with fresh medium for more than 24 h (data not shown).
These results argue against reversible posttranslational modification
of the protein.
The apparent lack of effect of TAU on PP2Ac, and OA on PP1c, in MCF7 cells is in agreement with their in vitro selectivity for both protein phosphatases and strongly suggests that they can be used to investigate the physiological roles for each type of protein phosphatase. Studies on smooth muscle contraction have illustrated their selectivity in vivo. OA up to 1 µM inhibits the Ca2+-dependent contraction of the actomyosin fibers in contrast to CA and TAU, which induce a Ca2+-independent contraction of smooth muscles (60). At concentrations higher than 1 µM, OA apparently mimics the action of CA and TAU, suggesting that PP1 is involved in the dephosphorylation of myosin light chains, a conclusion also supported by biochemical studies (61).
The apparent selectivity of OA and TAU for PP2A and PP1, respectively, is confirmed by their differential effects on intermediate filaments in MCF7 cells. Our results suggest that inhibition of PP2A, induced by cell treatment with OA or CA, is responsible for the hyperphosphorylation of these cytoskeletal proteins. This conclusion is supported by the demonstration of the association of PP2A with neurofilaments that belong to the family of intermediate filaments (19). Based on comparison between in vitro affinities of OA, dinophysistoxin (35-methyl-OA), and CA for PP1 and PP2A and the extracellular concentrations required to induce the hyperphosphorylation of the intermediate filament protein vimentin in BHK-21 cells, Eriksson et al. (46) came to the reverse conclusion, PP1 being probably responsible for the steady dephosphorylation of vimentin. However, the time course of cell permeation by these protein phosphatase inhibitors was not taken into account.
In summary, in MCF7 cells, depending on their concentrations, (i) TAU initially inhibits PP1 and weakly promotes the down-regulation of PP1, (ii) OA specifically inhibits PP2A and induces the demethylation of PP2A, and (iii) CA strongly affects both PP1 and PP2A simultaneously. We suggest that the systematic use of these three inhibitors, while taking into account their cell membrane permeation properties by measuring PP1 and PP2A activities, might help greatly in the assignment of the various functions of PP1 and PP2A in cell regulation. However, it must be kept in mind that protein phosphatases other than PP1 and PP2A are sensitive to inhibitors (35, 62, 63) and cross-talk between PP2A and PP1 is possible (1).
We thank George Thomas and David Brautigan for providing anti-PP1c antibodies, and we are grateful to Kiyoshi Isono and Hiroyuki Nagata from RIKEN for their kind gift of tautomycin. The help of Anne Fernandez and Ned Lamb for the analysis of intermediate filaments and the contribution of Elisabeth MacNulty during the preparation of the manuscript are greatly appreciated.