(Received for publication, September 8, 1995; and in revised form, December 18, 1995)
From the
Several lines of evidence indicate that serine/threonine protein phosphatases may act as negative regulators of cellular growth. For example, treatment of cells with the tumor-promoter okadaic acid, an inhibitor of certain types of these phosphatases, resulted in the increased expression of several proto-oncogenes, indicating a negative role of the respective phosphatases in gene regulation. However, it was puzzling to find that okadaic acid-treated cells, even in the presence of highly expressed proto-oncogenes, did not proliferate, but were arrested at certain points of the cell cycle. To further analyze this discrepancy, we investigated the involvement of protein phosphatases in the control of other cell cycle regulatory genes, such as cdc2, which encodes an essential cell cycle regulatory kinase. We found that cdc2 gene expression was blocked by okadaic acid, but stimulated by protein phosphatase 2A. Protein phosphatase 2A is shown to be a positive regulator of cdc2 gene activity and to be required for cdc2 expression. Thus, our findings identify protein phosphatase 2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control.
Reversible protein phosphorylation appears to be one of the most
important reactions contributing to cellular growth control and
carcinogenesis. While the role of protein kinases in these processes
has been well established, the contribution of protein phosphatases
(PPs) ()is much less clear and has only recently gained more
widespread attention.
The majority of intracellular phosphatase activity has been attributed to the serine/threonine protein phosphatases type 1 (PP-1) and type 2A (PP-2A)(1, 2, 3, 4) . These two enzymes have generally been considered as negative regulators of cellular growth. They are assumed to be necessary to revert growth-stimulatory phosphorylation cascades initiated by binding of growth factors to their cognate cell surface receptors. Further support for this view came from the use of okadaic acid, an inhibitor of PP-1 and PP-2A(5) , and of other recently discovered serine/threonine protein phosphatases (PP-3, PP-4, and PP-5) (6, 7, 8) . Since okadaic acid is a tumor promoter(9) , it has been concluded that inhibition of these phosphatases may contribute to the tumorigenic transformation of cells. This idea was supported by the finding that the treatment of cells with okadaic acid led to the increased expression of several proto-oncogenes, such as c-fos and c-jun (see detailed references in the review by Schönthal(10) ). Moreover, when added to cells synchronized in the S phase of the cell cycle, okadaic acid caused an increase in the enzymatic activity of histone H1 kinase, an enzyme that is necessary for cell cycle progression(11) . The drug also stimulated premature mitosis and increased phosphorylation of mitosis-specific proteins(10) .
However, while the above referenced evidence demonstrates that okadaic acid is stimulatory for various aspects of cellular growth control, and thus indicates a negative role of the respective phosphatases in these processes, there is also some evidence to the contrary. In many experiments it has been shown that okadaic acid-treated cells do not proliferate, but are blocked at certain stages of the cell cycle(12, 13, 14, 15) . Therefore, these latter findings actually may indicate a positive function of the respective phosphatases. Moreover, treatment of cells with okadaic acid blocks the proliferation of cells stimulated with platelet-derived growth factor (16) , reverts raf and ret-II oncogene-transformed cells(17) , and induces apoptosis in certain primary as well as transformed cells(18) . At least some of the growth-inhibitory effects of okadaic acid may be mediated via its block of cdc2 and cyclinA gene expression(12) . The gene products of cdc2 (= cyclin-dependent kinase, cdk1) and cyclins constitute the catalytic and regulatory subunits of histone H1 kinase, a group of enzymes necessary for cell cycle progression (19) . Because okadaic acid blocks the synthesis of H1 kinase, there is no H1 kinase activity, and consequently no phosphorylation (inactivation) of the retinoblastoma tumor-suppressor gene product(12, 19, 20) . As a result, the cells are arrested in the cell cycle. However, because okadaic acid affects the activity of several phosphatases, these experiments did not reveal the identity of the phosphatase(s) mediating these effects.
Because the negative effect of okadaic acid on cdc2 gene expression indicated a positive role of the respective okadaic acid-sensitive phosphatase, we used this system to further define the role of protein phosphatases in the regulation of growth-regulatory genes. We describe here that okadaic acid blocks cdc2 promoter activity. This control is exerted via the inhibition of transcription factor binding to a CCAAT box located at -80 with respect to the start site of transcription. Transfected PP-2A is able to stimulate cdc2 promoter activity and to diminish the negative effect of okadaic acid. Thus, our results identify PP-2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control.
The full-length cDNAs encoding the catalytic subunits of PP-1 (25) and PP-2A (26) were cloned into the multiple cloning site of pCMV to generate pCMV-PP-1 and pCMV-PP-2A. Plasmid pCMV was generated by Sheay et al.(27) (there it is called pWS-4) and contains the tripartite leader sequence of an adenovirus in order to enhance translation efficiency.
We have shown earlier that the treatment of cells with
okadaic acid resulted in the inhibition of cdc2 mRNA
accumulation(12) . To analyze whether this effect could be
mediated via the promoter of the gene, we transiently transfected the cdc2 promoter, linked to the chloramphenicol acetyltransferase
(CAT) reporter gene, into NIH3T3 cells. Fig. 1shows that the
CAT activity from cells that were treated with okadaic acid was greatly
reduced as compared to untreated control cells. In contrast, okadaic
acid did not reduce CAT activity when the promoters of Rous sarcoma
virus (RSV), cytomegalovirus (CMV), c-fos,
or the human -actin gene were analyzed (Fig. 1).
Figure 1:
Activity of various promoters in the
presence of okadaic acid (OA). Cells were transiently
transfected with various CAT-reporter plasmids harboring the cdc2, -actin, c-fos, RSV, or CMV promoter. Then
cells were treated with 50 nM okadaic acid for 24 h, and CAT
activity was determined as described under ``Experimental
Procedures.'' Shown is the average and standard deviation of
several repetitions of this experiment (n > 5). The cdc2 promoter used here span sequences from -3200 to
+75(23) .
Several regulatory elements in the cdc2 promoter have been identified(23, 28, 29) , such as a high affinity binding site for transcription factor E2F and a consensus CAAT box. Because the E2F binding site appears to be regulated by the retinoblastoma tumor suppressor protein (Rb), and because the activity of Rb is strictly controlled by its phosphorylation status, we speculated that inhibition of cdc2 transcription by okadaic acid might be mediated via E2F. To analyze this in more detail, we generated various deletion mutants of the cdc2 promoter that were linked to a luciferase reporter construct and analyzed their response to okadaic acid treatment.
As summarized in Fig. 2, the CAAT box at -80 is the major element that mediates the negative effects of okadaic acid. Construct -245, which contained cdc2 promoter sequences from -245 to +75, was as strongly down-regulated by okadaic acid as the full-length construct -3200, which is shown in Fig. 1. Deletion of two putative transcription factor binding sites, an E-box and a SP-1 site, did not affect the okadaic acid response (construct -129). Further deletion of the E2F site (construct -92), or mutation of the E2F site (construct -245 mE2F), likewise had only a weak effect. However, when the CAAT box was deleted (construct -74), or mutated (construct -245 mCAAT), the down-regulation of cdc2 promoter activity by okadaic acid was lost (Fig. 2). A double mutant, where both the E2F site and the CAAT box were mutated (construct -245 mEmC), also was not affected by okadaic acid.
Figure 2: Repression of various cdc2 deletion mutants by okadaic acid (OA). Cells were transiently transfected with luciferase-reporter constructs harboring various portions of the cdc2 promoter. Then cells were treated with 50 nM okadaic acid and harvested 24 h later. Luciferase activity was analyzed as described under ``Experimental Procedures.'' Repetitions of this experiment yielded similar results. The luciferase activity of construct -245 in the absence of okadaic acid was arbitrarily set to 100. Numbers to the right indicate fold reduction of luciferase activity in the presence of okadaic acid. The boxes in the promoter region indicate transcription factor binding sites. Crossed boxes indicate that the respective site has been inactivated by mutation. In the bottom three constructs the sites for CAAT, mutated CAAT, and AP-1 were generated with synthetic oligonucleotides (see ``Experimental Procedures'').
To confirm the importance of the CAAT box in mediating the negative effects of okadaic acid, we generated synthetic oligonucleotides representing either the CAAT box, an AP-1 binding site, or a mutated form of the CAAT box. Each oligo was inserted into the construct -74 in place of the original CAAT box. As shown in Fig. 2, insertion of the synthesized CAAT box (construct -74/CAAT) restored the negative effect of okadaic acid, whereas the mutated form (construct -74/mCAAT) did not. In contrast, insertion of the AP-1 binding site was not able to restore the negative effects of okadaic acid (Fig. 2, construct -74/AP-1).
We then used EMSAs to study whether okadaic acid would affect the binding of transcription factors to the CAAT box. For that purpose, cells were treated with okadaic acid for various times, and nuclear extracts were analyzed for their binding activity toward oligonucleotides representing the CAAT box of the cdc2 promoter. A representative result of several repetitions of this experiment is shown in Fig. 3. Okadaic acid caused a time-dependent reduction of binding activity. After 12 h, binding activity was reduced to less than half, whereas after 24 h a reduction to 1-10% was typically observed. In contrast, the use of an AP-1 oligonucleotide in the EMSA resulted in a transient stimulation of binding activity. Taken together, this indicated that okadaic acid reduced cdc2 promoter activity by preventing the binding of a transcription factor(s) to the CAAT box.
Figure 3: Band shift analysis (EMSA) of transcription factor binding during okadaic acid treatment. Cells were treated with okadaic acid for the indicated times, then nuclear extracts were prepared. The extracts were incubated with an oligonucleotide representing either the CAAT box of the cdc2 promoter or a consensus AP-1 binding site, and separated by PAGE. Shown is only the part of the gel that contained protein-DNA complexes. The weak band below the major band with the CAAT oligo was not consistently observed. In some repetitions of the experiment the reduction in binding activity to the CAAT oligo at 24 h was even stronger or lost completely.
Because okadaic acid is able to affect the activity of several phosphatases, we next set out to determine which one might be involved in the regulation of the cdc2 promoter. First, we used increasing concentrations of okadaic acid to compare the kinetics of phosphatase inhibition with the down-regulation of cdc2-promoter activity. Cells were treated with okadaic acid and the activity of PP-1 and PP-2A was measured in parallel to cdc2-luciferase activity. As shown in Fig. 4A, okadaic acid concentrations of up to 50 nM did not affect PP-1 activity. However, the activity of PP-2A was gradually reduced and paralleled the down-regulation of cdc2-luciferase activity that is shown in Fig. 4B.
Figure 4: Effects of various concentrations of okadaic acid on phosphatase and luciferase activity. Cells were treated with increasing concentrations of okadaic acid for 24 h and analyzed for A, PP-1 and PP-2A phosphatase activity, and B, luciferase activity of a transiently transfected cdc2-luciferase construct(-245). See ``Experimental Procedures'' for details.
The results above indicated that PP-2A might be involved in positive cdc2 promoter regulation. To confirm this, we co-transfected cdc2-luciferase together with an expression vector for the catalytic subunit of PP-2A. Because okadaic acid inhibits phosphatase activity and down-regulates cdc2 promoter activity, we expected to see increased cdc2 promoter activity in the presence of elevated PP-2A levels. Indeed, co-transfected PP-2A stimulated cdc2-luciferase activity (Fig. 5A). This effect was specific for this type of phosphatase, as co-transfected PP-1 catalytic subunit did not stimulate, but rather weakly reduced, cdc2 promoter activity (Fig. 5A).
Figure 5: Effects of co-transfected phosphatases on cdc2-luciferase activity. A, plasmid -245 (cdc2-luciferase, 3 µg) was co-transfected with 10 µg of an expression vector containing the cDNA of the catalytic subunit of either PP-2A or PP-1 (CMV PP-2A and CMV PP-1, respectively). As a control, the expression vector without the insert (CMV control), or small denatured salmon sperm DNA (s.d. DNA), was co-transfected. In B, the indicated cdc2-luciferase constructs were co-transfected with either an expression vector for PP-2A (+PP2A) or with the vector without the PP-2A insert (Control). Numbers reflect the fold induction of luciferase activity in the presence of co-transfected PP-2A. In C, construct -245 was co-transfected with PP-2A (+PP2A) or vector without PP-2A insert (-PP2A), and then cells were treated with increasing concentrations of okadaic acid. The luciferase activity of construct -245 without okadaic acid treatment was arbitrarily set to 100.
We next sought to define the promoter elements that mediated the stimulatory effect of PP-2A. Various mutants of the cdc2 promoter linked to the luciferase reporter gene were co-transfected with the catalytic subunit of PP-2A. As shown in Fig. 5B, the stimulatory effect was somewhat reduced when the E2F binding site was mutated (construct -245 mE2F). However, it was lost completely when the CAAT box was inactivated (construct -245 mCAAT), indicating that the positive regulation by PP-2A was mainly mediated via the CAAT box element.
To further confirm the stimulatory effect of PP-2A on cdc2 promoter activity, we investigated whether elevated levels of PP-2A could counteract the negative effects of okadaic acid. We therefore co-transfected cdc2-luciferase with PP-2A in the presence of various concentrations of okadaic acid. As shown in Fig. 5C, co-transfected PP-2A significantly reduced the negative effects of okadaic acid. At 50 nM okadaic acid treatment, cdc2-luciferase activity was 2.5-fold higher when PP-2A was co-transfected. Taken together, these results identify PP-2A as a positive regulator of cdc2 promoter activity.
The tumor promoter okadaic acid has proven very useful to study various aspects of serine/threonine protein phosphatase function. However, even at low concentrations this drug inhibits several of these enzymes simultaneously, making it difficult to ascribe a certain effect to a single phosphatase(6, 7, 8) . Moreover, even though several experiments demonstrated stimulatory effects of okadaic acid on certain aspects of cell growth, there are also findings to the contrary. For example, okadaic acid treatment of cells differentially affects the expression of two genes that are required for cellular growth: it induces expression of c-fos(10) , but inhibits expression of cdc2(12) (this study). The latter finding is particularly intriguing because it suggests a positive role of the respective phosphatase in growth-regulatory gene expression.
In this
study, we demonstrate that inhibition of cdc2 expression by
okadaic acid occurs at the promoter level, and that the phosphatase
involved in this regulation is PP-2A. These results indicate that PP-2A
acts as a positive regulator of cdc2 expression. This finding
is somewhat puzzling, because it has been shown earlier that PP-2A is
identical to INH, an inhibitor of histone H1 (cdc2/cyclin)
kinase activity(30) . However, one needs to keep in mind that
these two antagonistic effects of PP-2A occur at different levels and
at different stages of the cell cycle. First, inhibition of the
enzymatic activity of histone H1 kinase by INH is posttranslational,
whereas stimulation of the cdc2 promoter by PP-2A is
transcriptional. Second, INH activity is present in
G(30) , whereas transcriptional regulation occurs
throughout the cell cycle, but mainly in late G
and S (12) . Therefore, it is possible that PP-2A activity, on one
side, contributes to the elevated expression of cdc2 and, on
the other side, participates in keeping the kinase activity in check
until sufficient amounts have accumulated.
Several consensus
transcription factor binding sites have been identified in the cdc2 promoter, such as E2F, CAAT, and SP-1
sites(23, 28, 29) . We show that the effects
of okadaic acid and PP-2A on cdc2 promoter activity are
mediated mainly via the inverted CAAT box element located at -80
(with respect to the start of transcription). The E2F binding site,
which is the main element to confer induction of cdc2 transcription during G to S phase
progression(23, 28) , appears to play only a minor
role in this regulation by PP-2A.
The mechanisms by which the CAAT box serves as a target for negative regulation in response to okadaic acid remain to be determined. The observation that treatment of cells with okadaic acid results in decreased levels of CAAT box binding activity suggests the possibility that inhibition simply reflects the loss of a critical activator. While this mechanism may contribute to negative regulation, additional mechanisms are likely to be involved, because mutation of the CAAT box in the context of the -245-base pair cdc2 promoter (construct -245 mCAAT) does not result in decreased promoter activity. Thus, it remains possible that an active repressive function is mediated through a CAAT box binding protein via interactions with a co-repressor molecule. This scheme would be analogous to the mechanisms by which the retinoblastoma protein actively represses transcription through the E2F binding site(31) . Testing this hypothesis will require the identification of the relevant CAAT box binding proteins that mediate negative regulation in response to okadaic acid.
The finding that PP-2A participates in the control of cdc2 gene expression further supports the idea that this enzyme plays an important role in cellular growth regulation and potentially carcinogenesis. However, it also underscores the notion that protein phosphatases cannot be readily classified as negative regulators of growth. It appears that these types of enzymes play positive as well as negative roles in these processes. The respective function may depend on many variables, such as associated subunits, intracellular distribution, posttranslational modifications, and cell cycle stages (32, 33, 34, 35) .