- Email: [email protected]
threonine protein phosphatases
seminars in
CANCER BIOLOGY, Vol 6, 1995: pp 239–248
Regulation of gene expression by serine/threonine protein phosphatases Axel H. Sch¨onthal
additional mammalian PPases that are sensitive to low concentrations of okadaic acid complicates this analysis. For example, bovine PP-3 has an IC50 of 5 nM,4 the ubiquitously expressed rabbit PP-X (PP-4) is inhibited at 0.2 nM,5 and the predominantly nuclear human PP5 has an IC50 of 1–10 nM.6 Furthermore, PCR analysis indicates that many more phosphatases are yet to be discovered.7 Thus, the resulting effects of okadaic acid treatment, even at low concentrations, cannot easily be ascribed to a single PPase. Despite these limitations, however, the use of okadaic acid has spread tremendously over the last few years and has generated a wealth of useful information.
The activation of signal transduction pathways by extracellular stimuli, such as growth factors or hormones, ultimately results in changes in the expression of specific genes. The altered pattern of expression eventually determines the resulting cellular consequences, e.g. cell growth, division, or differentiation. It has been well established that the reversible phosphorylation of proteins is a major regulatory mechanism in these processes. However, while much has been learned about the role of kinases, the involvement of protein phosphatases is less clear and has only recently begun to be investigated in more detail. This review will present some of the new findings that demonstrate a crucial regulatory function of serine/threonine protein phosphatases (PPases) in gene regulatory processes. Key words: expression
phosphatases
/
okadaic
acid
/
gene
Effects of okadaic acid on kinase activity
©1995 Academic Press Ltd
Although no direct effect of okadaic acid on any kinase has been found, it may exert indirect regulatory effects on various kinases.8 Protein kinases whose catalytic activities are regulated by reversible phosphorylation may be stimulated in the absence of (inhibitory) phosphatase activity. This, in turn, may lead to the accumulation of phosphorylated substrates, which themselves may be kinases and further amplify the signal. Thus, the balance of reversible phosphorylations would be shifted towards the increased net phosphorylation of proteins. This has indeed been observed. When added to intact cells or cell extracts, okadaic acid causes a change in the apparent phosphorylation of many cellular proteins.9,10 Using high definition two-dimensional gel electrophoresis, Guy et al11 identified 74 proteins that exhibited altered levels of phosphorylation in response to okadaic acid treatment of human fibroblasts. Intriguingly, this pattern of phosphorylation was strikingly similar to that induced by tumour necrosis factor alpha (TNFα), but rather different from that induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The mimicry of TNFα by okadaic acid indicates that either agonist may initiate similar signal transduction pathways in these cells, which may affect the expression of the
Inhibition of PPases by okadaic acid A novel drug that has proven to be extremely useful for the study of gene regulation by PPases is the tumour promoter okadaic acid.1 This compound is a complex polyether derivative of a 38-carbon fatty acid that is synthesized by marine dinoflagellates. Importantly, okadaic acid has been found to bind to and inhibit the two major intracellular PPases, type-1 (PP1) and type-2A (PP-2A).2,3 Inhibition of PP-2A is more efficient than inhibition of PP-1, with 50% inhibition (IC50) in vitro occurring at 0.1–1.0 nM and 20–100 nM, respectively. Due to this differential effect, the consequences of treatment with low doses of okadaic acid have generally been attributed to a reduction of PP-2A activity. However, the recent discovery of Department of Molecular Microbiology and Immunology and K. Norris Jr. Comprehensive Cancer Center, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90033-1054, USA ©1995 Academic Press Ltd 1044-579X/95/040239 + 10$12.00/0
239
A. H. Sch¨onthal Table 1. Overview of genes that were activated in response to treatment of cells with okadaic acid Activated genes c-fos fos B fra-1 c-jun jun B jun D egr-1 egr-2, egr-3 krox-24 grp78 collagenase stromelysin-1 TIMP transin u-PA u-PA-R t-PA PAI-2 mal-1, mal-2 TNFα IL-2β IP-10 Id HPV16-LCR HIV-1-LTR progesteroneresponse element
Cell lines
Transcriptional regulation
Post-transcriptional regulation
References
yes
yes
35,38,5,6,7,78,79
yes
78 38,78 38,76,79,80,77,78,79
fibr., Jurkat, HT-1080, A-549, U937, C2C12 myobl., mouse skin C2C12 myobl. Jurkat, C2C12 myobl. fibr., Jurkat, LLC-PK1, U937 mouse skin, C2C12 myobl. Jurkat, HT-1080 Jurkat, HT-1080, U937 fibr., U937 fibr. lymphoc. gliosarc. synoviocytes, A-549, U937 HT-1080 HT-1080 mouse skin LLC-PK1, U937, mouse skin A549, U937, Hela, WI-38 U937 U937 mouse skin macroph. B-cells monoc. macroph. C2C12 myobl. fibr. Jurkat, BJA-B, U251 CV-1
yes
yes
yes
yes yes
yes yes yes yes yes yes yes
yes weak no
38,75 38,75,77 76,81,82 76 83 84 35,70 75 75 79 70,79,80,85 69 70 70 79 86,87,88 87 86 89 90 91 99
Numerous examples that indicate such events have been found. For instance, okadaic acid produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types.17 Several kinases that are important components of gene regulatory signal transduction pathways, such as Raf-1, MEK, and MAP kinases (ERKs) have been found to be hyperphosphorylated in response to okadaic acid treatment of cells.18-20 In not all cases, however, does increased phosphorylation lead to stimulation of the respective kinase activity. For MEK-1 it has been shown that its kinase activity is negatively regulated by threonine phosphorylation.21 The regulation of kinases by PPases has been further indicated by in-vitro experiments where purified phosphatases were shown to use phosphorylated kinases as substrates. (see detailed references in ref 22). Experiments with SV40 small t antigen, a protein that binds to and inactivates PP-2A,23 showed that its overexpression in cells led to the increased activity of ERK-2 and MEK-1,24 and increased transcriptional activity of an AP-1-regulated reporter construct.22 These experiments strongly suggested that some of these kinases may be negatively regulated by PP-2A in vivo.
same set of genes. In a different study using human breast cancer cells,12 it was shown that okadaic acid induced a protein phosphorylation pattern that overlapped that of TPA, and both agents stimulated similar biochemical and cellular events. It had been suggested earlier that some of the effects of TPA and okadaic acid were mediated via the same pathways: TPA-activated protein kinase C (PKC) initiates a phosphorylation cascade that can be reversed by okadaic acid sensitive PPases.13-15 Blockage of these PPases may lead to the sustained phosphorylation of substrates of the PKC pathway and thereby may affect gene expression in a manner similar to TPA. Indeed, many genes that are activated by TPA are also stimulated by okadaic acid, for example, c-fos, c-jun, and collagenase (Table 1; see 16 for TPA-inducible genes). Gene regulatory functions of PPases are likely exerted by a wide variety of mechanisms. At the gene proximal site, PPases may directly regulate the activity of transcription factors. Further upstream, PPases may affect components of signal transduction pathways, such as growth factor receptors or intermediate signal transduction molecules (kinases), that transmit a signal from the cell membrane into the nucleus. 240
Regulation of gene expression these enzymes in cellular growth control. This was somewhat surprising because these PPases had been thought of as negative regulators of cell growth or even as anti-oncogenes.13 Earlier work with the use of okadaic acid had supported this view. Because okadaic acid is a tumour promoter, it was suggested that inhibition of the respective PPases would contribute to the process of cellular transformation.13,33 This view was backed further by the finding that okadaic acid was able to induce the expression of several proto-oncogenes, such as c-fos and c-jun.34-38 The promoter elements mediating this activation were the same as those that confer the transcriptional activation in response to growth stimulatory signals or the tumour promoter TPA, e.g. the serum responsive element (SRE)39 of the c-fos gene and the TPA responsive element (TRE or AP-1 binding site)40 of c-jun and collagenase35,37,38 (see later). Combining the above described studies, it appears that okadaic acid-sensitive PPases have positive as well as negative effects on the expression of different growth-regulatory genes (see Tables 1–3, Figure 1). Immediate early genes, such as c-fos and c-jun, are negatively regulated by these PPases, and G0-arrested cells that are treated with okadaic acid re-enter the cell cycle and progress through early G1. On the other hand, at least two genes that are required later in the cell cycle, cdc2 and cyclinA, are positively regulated by PPases, and therefore cell cycle progression from G0 of okadaic acid-treated cells is blocked in late G1.31,32 It appears that the differential effects of okadaic acid on histone H1 kinase, namely stimulation of its enzymatic activity and inhibition of its gene, may both be due to the inhibition of PP-2A. Biochemical analysis identified PP-2A as the post-translational inhibitor of histone H1 kinase activity.27,28 Inhibition of cdc2 gene expression occurs at okadaic acid concentrations that inhibit PP-2A, but not PP-1, in cell culture31 (also, Sch¨onthal, unpublished). Moreover, co-transfection of an expression vector for PP-2A stimulates transcription of a reporter plasmid under the control of the cdc2 promoter (Sch¨onthal, unpublished). These data point to a positive role of PP-2A in the transcriptional regulation of cdc2. How can one reconcile these opposing effects of PP-2A? The post-translational stimulation of histone H1 kinase activity by okadaic acid is transient, and is restricted to cells in G2 of the cell cycle.27 In contrast, the transcriptional inhibition of cdc2 gene expression by the tumour promoter is permanent and occurs in G0/G1-synchronized cells.31 Although this latter
Other okadaic acid-regulated phosphoproteins that are significant regulators of gene expression are the histone H1 kinases and the tumour suppressor gene products retinoblastoma (Rb) and p53. Both Rb and p53 are hyperphosphorylated in response to shortterm okadaic acid treatment of cells.25,26 In the case of p53, increased phosphorylation appeared to reduce its transcriptional activation function.26 Moreover, p53 was shown to be dephosphorylated by PP-2A in vitro.23 Thus, PPase activity is likely to contribute to the cell cycle-regulatory functions of this tumour suppressor protein. Hyperphosphorylation of Rb protein is thought to inactive its growth-suppressive function and allows cells to proceed through the cell cycle. The principal kinase to phosphorylate Rb is histone H1 kinase, which has been shown to be activated by okadaic acid in a transient fashion; (27; further references in 1). It is conceivable that hyperphosphorylation of Rb in response to okadaic acid is brought about by a twofold mechanism: the activation of histone H1 kinase in combination with the inhibition of PPases that dephosphorylate Rb. This idea has been further substantiated by the finding that histone H1 kinase is negatively regulated by PP-2A,28 and that Rb is a substrate for PP-1 in vitro and in vivo.29,30 However, the regulation of histone H1 kinase by PPases appears to be rather complex. In addition to its negative regulation at the posttranslational level,27,28 histone H1 kinase appears to be positively regulated by PPases at the transcriptional level.31 In serumdeprived fibroblasts, which are growth-arrested in G0 of the cell cycle, several of the genes that code for histone H1 kinase subunits, such as cyclinA or cdc2, are transcriptionally inactive (see detailed references in 31). Upon stimulation of these cells with growth factors or serum, expression of these genes greatly increases and subsequently allows cells to proceed through the cell cycle. However, when growtharrested cells are stimulated with serum in the presence of okadaic acid, the activation of cdc2 and cyclinA gene expression is blocked.31 Consequently, the cells lack histone H1 kinase proteins and do not have the respective catalytic activity. Phosphorylation (inactivation) of Rb protein does not occur, and the cells accumulate in late G1 of the cell cycle.31 Similar inhibitory effects of okadaic acid were observed when murine embryonic fibroblasts were growth-stimulated with TGF-β.32 The finding that okadaic acid-sensitive PPases were required for the expression of at least some of the histone H1 kinase genes indicated a positive role for 241
A. H. Sch¨onthal Table 2. Overview of genes that were repressed in response to treatment of cells with okadaic acid Activated genes c-myc p53 cdc2 cyclinA myoD1 myogenin MLC u-PA t-PA IL-2 IL-2-Rα cyclophilin SV40 early rRNA
Transcriptional regulation
Cell lines U937 Hela, A549, fibrosarc., lymphoc. fibr., U937 fibr. C2C12 myobl. C2C12 myobl. C2C12 myobl. HT-1080 HT-1080 lymphoc. lymphoc. lymphoc. LLC-PK1, T47D Xenopus extr.
References 77 83,92 31,77 31 78,89 89 78 70 70 83 83 83 80,93 72
yes yes yes
yes
H1 kinase. In longer term assays, Rb phosphorylation is reduced or completely inhibited because of a lack of histone H1 kinase expression and activity. Since Rb has been shown to be involved in the regulation of several genes,41 it can be expected that the modification of its phosphorylation status in response to okadaic acid treatment may have differential effects on the expression of its target genes.
effect has been observed in log phase cells, it cannot be ruled out that only a subpopulation of cells at a certain cell cycle stage is affected. Moreover, it is likely that PP-2A activity is regulated in a fashion that is dependent on the cell cycle, its (micro-)compartmental localization, and its interaction with various regulatory subunits. Thus, it is conceivable, that PP-2A may be required to ensure expression of sufficient amounts of histone H1 kinase subunits during G1, before its inactivation may contribute to the rapid activation of kinase activity at later stages of the cell cycle. Similarly, the seemingly contradictory effects of okadaic acid on Rb protein phosphorylation could be explained by the different experimental approaches. In short-term assays, okadaic acid treatment of cells increases Rb phosphorylation which is likely due to the transient post-translational activation of histone
Regulation of transcription factor activity by PPases In order to delineate the precise role of PPases in gene expression, it is important to analyse how the activity of defined promoter elements and their respective transcription factors is regulated by these enzymes. Recent work yielded exciting results for
Table 3. Overview of genes whose expression was not affected by okadaic acid treatment alone, but where the presence of okadaic acid inhibited or enhanced the gene-regulatory effects of other stimuli
Regulated genes HSP70 c-myb IL-1α UL-6 IP-10 CRP; SAA fibrinogen PEPCK MMTV-LTR MMTV-LTR thyroid hormone response element
Cell lines
Stimulus
Okadaic acid effects inhibitory (I) stimulatory (S)
Hela, Hep-2, IMR-90 neuroblastoma erythroid monoc. monoc. monoc. Hep3B Hep3B, NPLC hepatoc. T47D CV-1, COS COS-1, CV-1
heat shock
S
84
erythropoietin LPS LPS IFNγ IL-6 + IL-1 IL-6 cAMP progestin dexamethasone T3
S S S I I I I S S S
95 87 87 96 97 97 98 93 100 101,102
242
References
Regulation of gene expression tion of TCF, and that TCF was an in-vitro substrate for PP-2A.42 Using microneedle injections of purified PPases, it has been demonstrated that serum-stimulated induction of SRE activity was inhibited in the presence of exogenous PP-2A.43 Moreover, elevated expression of small t contributed to increased c-fos promoter activity.22 Together, these data indicate that PPases, most likely PP-2A, confer a negative regulation of c-fos promoter activity via the SRE. The transcription factor AP-1 is positively regulated in response to many cell growth-stimulatory signals, and recognizes primarily the TRE.40 It is a heterodimer consisting of polypeptides of the Fos and Jun family, that are regulated by reversible phosphorylation reactions (see further references in 19,44). A detailed analysis of the post-translational modification of c-Jun protein revealed two domains that are modified by phosphorylation. Phosphorylation on two N-terminal sites in combination with the dephosphorylation of two to three C-terminal sites is required for its binding to the TRE and full transcriptional activity.45,46 In a reticulocyte extract, okadaic acid enhanced phosphorylation of in-vitro synthesized c-Jun protein, and immunoprecipitated c-Jun was dephosphorylated in the presence of purified PP2A.47 In other studies it was shown that the purified catalytic subunit of PP-2A (PP-2AC) efficiently dephosphorylated the inhibitory C-terminal phosphorylation sites of c-Jun in vitro,22,48 indicating that this PPase may contribute to the activation of transcription factor AP-1. However, because PP-2AC is normally complexed with two other regulatory subunits, it is not clear whether this activity of PP-2AC truly reflects what occurs in vivo. Nevertheless, these data indicate a role for PP-2A in the transcriptional regulation by AP1 proteins. Maybe the best characterized, and at the same time most controversial, example of gene regulation by PPases is the transcriptional control of the CRE. The CRE has been found in several cAMP-responsive genes and is the binding site for transcription factor CREB.49 CREB activity is regulated by the cAMPdependent protein kinase A (PKA), which phosphorylates CREB at a single serine and thereby stimulates its transcriptional activity.49 Using completely different approaches, two groups have studied the regulation of phospho-CREB (P-CREB) dephosphorylation and transcriptional inactivation by PPases. The first group used microneedle injections and plasmid transfections to introduce either purified phosphatases or the respective expression vectors into
several of these elements, and three of them, the SRE, the TRE, and the cyclic AMP responsive element (CRE), will be discussed below. Several proteins that bind to the SRE have been shown to be phosphorylated and thus are potential targets for modification and regulation by PPases. These proteins form a multiprotein complex consisting of serum response factor (SRF) and ternary complex factor (TCF), a family of proteins which include p62TCF/Elk-1 and SAP-1.39,42 Recent experiments showed that increased c-fos expression in response to okadaic acid correlated with phosphoryla-
Figure 1. Differential regulation of gene expression by okadaic acid. 3C10T1/2 murine fibroblast were treated with the indicated concentrations of okadaic acid for 24 h. PolyA + RNA was isolated and analysed by Northern transfer and subsequent hybridization to specific probes for the immediate early gene c-fos, the histone H1 kinase regulatory subunit gene cyclin A (cycA), and, as a control for equal amounts of RNA loaded in each lane, choA, an abundant RNA species that was originally isolated from Chinese hamster ovary (CHO) cells.74 For experimental details see 31.
243
A. H. Sch¨onthal a number of different cell lines.50,51 They demonstrated that microinjected PP-1, but not PP-2A, inhibited transcription of a CRE-regulated reporter plasmid. Similarly, transfected PP-1, but not PP-2A, was able to inhibit CRE activity. Microinjection of an expression plasmid for inhibitor-1 (I-1), a PP-1 specific inhibitor, resulted in increased phosphorylation and activity of CREB. In contrast, microinjection of SV40 small t had no effect. Thus, these data provided evidence for a primary role of PP-1 in the regulation of CREB function. The second group used biochemical fractionation and immunological analysis to characterize CREB regulation.52 They demonstrated that P-CREB phosphatase activity in nuclear extracts coeluted with PP2A from various columns, and was chromatographically resolved from nuclear PP-1. Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by inhibitor-2 (I-2), a PP-1 specific inhibitor. Moreover, P-CREB treated with PP-2A lost its transcriptional activity in-vitro, whereas P-CREB treated with PP-1 remained transcriptionally active. Further experiments demonstrated that SV40 small t inhibited the dephosphorylation of P-CREB and enhanced transcription from a CRE-containing promoter.53 Taken together, these data provided evidence for a primary role of PP-2A in the regulation of CREB function. It is not clear, why the two different approaches described above provided contradictory results (see discussion in 50,52). They indicate however, that it may be important to study the native forms of the PPases, in which the catalytic subunits are complexed with the appropriate regulatory subunits. The latter appear to play important roles in determining substrate specificity, catalytic activity, and cellular localization14,54 (see further references in 50).
these immunophilin-ligand complexes is PP-2B (see 58,59 for reviews). Most of this work so far has concentrated on T cells, where CsA and FK506 have been shown to inhibit signals arising from the activated T cell receptor. As a consequence of drug treatment, expression of early T cell activation genes, such as interleukin 2 (IL-2), is inhibited, and the cells do not progress through G1.60,61 Two of the promoter elements in the IL-2 promoter that mediate this inhibition are the binding sites for the transcription factors NFAT and NFIL2A.62,63 These sites have also been shown to be synergistically activated by overexpression of a constitutively active form of PP-2B in the presence of an activated Ha-ras oncogene or simultaneous treatment of cells with TPA.60,63 From these studies it has been inferred that PP-2B is a necessary mediator of T cell receptor signalling and IL-2 gene activation.57,58 These studies were extended to show that PP-2B also stimulated the NF-kB-dependent element of the IL-2 promoter through the inactivation of IkB, an inhibitor of NF-kB.64 Gene regulatory effects of PP-2B occur in other cell types as well. For example, in PC12 cells CsA and FK506 exhibited differential effects on the Ca2 + stimulated expression of two immediate early genes: while transcription of the NGFI-A gene was enhanced by these drugs, transcription of NGFI-B was inhibited.65 In human fibroblasts, CsA enhanced cytokine and phorbol ester-induced expression of the collagenase, c-jun, and junB genes.66 Clearly, with the identification of PP-2B as the CsA- and FK506-sensitive enzyme, numerous observations of gene regulation by CsA and FK506 (see detailed references in 64) imply that this PPase may be involved in the regulation of many genes in a variety of cell types.67 Furthermore, the demonstration that Ras protein and PP-2B can cooperate to activate T cells has led to the suggestion that PP-2B could be a protooncogene.63
Gene regulation by PP-2B/calcineurin Conclusions The analysis of gene regulatory pathways that are regulated by phosphatases other than PP-1/PP-2A received a strong impetus through the finding that PP-2B (also called calcineurin or Ca2 + , calmodulindependent PPase) activity could be inhibited by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506).55-57 CsA and FK506 are natural microbial products that form complexes with the immunophilins cyclophilin and FK506-binding protein (FKBP). Importantly, the only characterized target for
Great strides have been made towards a more complete understanding of PPase function in gene regulatory processes. Nearly 50 genes have been found so far that appear to be regulated by these enzymes. However, because in most studies okadaic acid was used to establish the role of PPases, and because okadaic acid affects the activity of more than one PPase, it was often not possible to determine which of the various okadaic acid-sensitive PPases was 244
Regulation of gene expression
References
responsible for the observed effect. Nevertheless, in order to gain preliminary insight into gene regulatory processes, okadaic acid has certainly proven to be extremely useful. In the future, a combination of various approaches will be necessary in order to characterize the precise roles of PPases in gene regulatory processes. For example, biochemical and immunological analysis together with cellular studies may be most useful. However, as presented above for the analysis of the transcription factor CREB, even these types of studies may yield equivocal data. Clearly, the analysis of PPase function is greatly complicated by the presence of several isoforms and subunits for each enzyme, as well as by its varying subcellular localization and by its cell cycle dependence.1,14,54 Moreover, in many cases strong cell cycle specificities have been observed that may be due to a difference in the balance or availability of the regulatory components.22,68-70 In addition to the regulation of specific transcription factors, PPases may also be involved in the control of the basal transcriptional machinery. For instance, since the C-terminal domain (CTD) of RNA polymerase II (Pol II) is phosphorylated at multiple sites in response to serum stimulation of quiescent cells,71 it is conceivable that PPases may also be involved in the regulation of Pol II activity. This control could be exerted directly via the dephosphorylation of the CTD, or indirectly via MAP kinase, which has been implicated in the enhanced phosphorylation of Pol II.71 Moreover, using a Xenopus transcription extract, the activity of a type 1 PPase has been implicated in the transcriptional initiation of ribosomal gene expression by Pol 1.72 Other experiments suggested a role for PP-1 in the regulation of mammalian spliceosome assembly.73 Thus, it appears that PPases play a crucial role at various levels of gene expression: from the signal input site via transcription factor regulation to posttranscriptional and post-translational controls.
1. Sch¨onthal A (1992) Okadaic acid — a valuable new tool for the study of signal transduction and cell cycle regulation? New Biologist 4:16–21 2. Bialojan C, Takai A (1988) Inhibitory effect of a marinesponge toxin okadaic acid, on protein phosphatases. Biochem J 256:283–290 3. Cohen P, Klumpp S, Schelling DL (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett 250:596–600 4. Honkanen RE, Zwiller J, Daily SL, Khatra BS, Dukelow M, Boynton AL (1991) Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain. J Biol Chem 266:6614–6619 5. Brewis ND, Street AJ, Prescott AR, Cohen PTW (1993) PPX, a novel protein serine/threonine phosphatase localized to centrosomes. EMBO J 12:987–996 6. Chen MX, McPartlin AE, Brown L, Chen YH, Barker HM, Cohen PTW (1994) A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J 13:4278–4290 7. Chen MX, Chen YH, Cohen PTW (1992) Polymerase chain reactions using Saccharomyces, Drosophila and human DNA predict a larger family of protein serine/threonine phosphatases. FEBS 306:54–58 8. Sassa T, Richter WW, Uda N, Suganuma M, Suguri H, Yoshizawa S, Hirota M, Fujiki H (1989) Apparent ‘activation’ of protein kinases by okadaic acid class tumor promoters. Biochem Biophys Res Comm 159:939–944 9. Haystead TAJ, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG (1989) Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337:78–81 10. Haystead TAJ, Weiel JE, Litchfield DW, Tsukitani Y, Fischer EH, Krebs EG (1990) Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. J Biol Chem 265:16571–16580 11. Guy GR, Cao X, Chua SP, Tan YH (1992) Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1. J Biol Chem 267:1846–1852 12. Kiguchi K, Giometti C, Chubb CH, Fujiki H, Huberman E (1992) Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A. Biochem Biophys Res Comm 189:1261–1267 13. Cohen P, Cohen PTW (1989) Protein phosphatases come of age. J Biol Chem 264:21435–21438 14. Shenolikar S, Nairn AC (1991) Protein phosphatases: recent progress, in Advances in Second Messenger and Phosphoprotein Research (Greengard P, Robison GA, eds), Vol 23, pp 3-38. Raven Press, New York 15. Clarke PR, Siddhanti SR, Cohen P, Blackshear PJ (1993) Okadaic acid-sensitive protein phosphatases dephosphorylate MARCKS, a major protein kinase C substrate. FEBS Lett 336:37–42 16. Rahmsdorf HJ, Herrlich P (1990) Regulation of gene expression by tumor promoters. Pharmac Ther 48:157–188 17. Hern´andez-Sotomayor SMT, Mumby M, Carpenter G (1991) Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. J Biol Chem 266:21281–21286 18. Kharbanda S, Saleem A, Emoto Y, Stone R, Rapp U, Kufe D (1994) Activation of raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. J Biol Chem 269:872–878 19. Hipskind RA, Baccarini M, Nordheim A (1994) Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with
Acknowledgements I thank Norbert Berndt for critical reading of this manuscript and Laura Steel for secretarial assistance. I am grateful to Tanya Kormeili for her smile and other multifaceted means of sustenance. Work in the author’s lab was supported by grants from the Tobacco Related Disease Research Program (TRDRP) of California and the Margaret E. Early Medical Research Trust.
245
A. H. Sch¨onthal
20.
21.
22.
23.
24.
25. 26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
36.
ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo. Mol Cell Biol 14:6219–6231 Miyasaka T, Miyasaka J, Saltiel AR (1990) Okadaic acid stimulates the activity of microtubule associated protein kinase in PC-12 pheochromocytoma cells. Biochem & Biophys Res Comm 168:1237–1243 Rossomando AJ, Dent P, Sturgill TW, Marshak DR (1994) Mitogen-activated protein kinase 1 (MKK1) is negatively regulated by threonine phosphorylation. Mol Cell Biol 14:1594–1602 Frost JA, Alberts AS, Sontag E, Guan K, Mumby MC, Feramisco JR (1994) Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity. Mol Cell Biol 14:6244–6252 Scheidtmann KH, Mumby MC, Rundell K, Walter G (1991) Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol Cell Biol 11:1996–2003 Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M (1993). The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell 75:887–897 Yatsunami J, Komori A, Ohta T, Suganuma M, Fujiki H (1993) Hyperphosphorylation of retinoblastoma protein and p53 by okadaic acid, a tumor promoter. Cancer Res 53:239–241 Zhang W, McClain C, Gau J, Guo X-YD, Deisseroth AB (1994) Hyperphosphorylation of p53 induced by okadaic acid attenuates its transcriptional activation function. Cancer Res 54:4448–4453 Yamashita K, Yasuda H, Pines J, Yasumoto K, Nishitani H, Ohtsubo M, Hunter T, Sugimura T, Nishimoto T (1990) Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitotis-like state in BHK21 cells. EMBO J 9:4331–4338 Lee TH, Solomon MJ, Mumby MC, Kirschner MW (1991) INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415–423 Alberts AS, Thorburn AM, Shenolikar S, Mumby MC, Feramisco JR (1993) Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc Natl Acad Sci USA 90:388–392 Ludlow JW, Glendening CL, Livingston DM, DeCaprio JA (1993) Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol 13:367–372 Sch¨onthal A, Feramisco JR (1993) Inhibition of histone H1 kinase expression, retinoblastoma protein phosphorylation, and cell proliferation by the phosphatase inhibitor okadaic acid. Oncogene 8:433–441 Kim TA, Velasquez BR, Wenner CE (1993) Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts. Proc Natl Acad Sci USA 90:5460–5463 Suganuma M, Fujiki H, Suguri H, Yoshizawa S, Hirota M, Nakayasu M, Ojika M, Wakamatsu K, Yamada K (1988) Okadaic acid: an additional non-phorbol-12-tetradecanoate13-acetate-type promoter. Proc Natl Acad Sci USA 85:1768–1771 Herschman HR, Lim RW, Brankow DW, Fujiki H (1989) The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression. Carcinogenesis 10:1495–1498 Kim SJ, Lafyatis R, Kim KY, Angel P, Fujiki H, Karin M, Sporn MB, Roberts AB (1990) Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases. Cell Reg 1:269–278 Sch¨onthal A, Tsukitani Y, Feramisco JR (1991) Transcriptional and posttranscriptional regulation of c-fos proto-oncogene
37. 38. 39. 40. 41.
42.
43.
44. 45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
246
expression by the tumor promoter okadaic acid. Oncogene 6:423–430 Sch¨onthal A, Alberts AS, Frost JA, Feramisco JR (1991) Differential regulation of jun family gene expression by the tumor promoter okadaic acid. New Biol 3:977–986 Th´evenin C, Kim SJ, Kehrl JH (1991) Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. J Biol Chem 266:9363–9366 Treisman R (1990) The SRE: a growth factor responsive transcriptional regulator. Semin Cancer Biol 1:47–58 Angel P, Karin M (1991) The role of Jun, Fos and AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072:129–157 Kim S-J, Lee HD, Robbins PD, Busam K, Sporn MB, Roberts AB (1991) Regulation of transforming growth factor β1 gene expression by the product of the retinoblastoma-susceptibility gene. Proc Natl Acad Sci USA 88:3052–3056 Zinck R, Hipskind RA, Pingoud V, Nordheim A (1993) c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF. EMBO J 12:2377–2387 Sch¨onthal A, Alberts AS, Rahman A, Meinkoth J, Mumby M, Feramisco JR (1992) Involvement of serine/threonine specific protein phosphatases in signal transduction pathways that regulate gene expression and cell growth, in Recent Advances in Cellular and Molecular Biology (Wegmann RJ, Wegmann MA, eds), Vol 2, pp 67–74. Peeters Press, Leuven Hunter T, Karin M (1992) The regulation of transcription by phosphorylation. Cell 70:375–387 Boyle WJ, Smeal T, Defize LHK, Angel P, Woodgett JR, Karin M, Hunter T (1991) Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64:573–584 Lin A, Frost J, Deng T, Smeal T, Al-Alawi N, Kikkawa U, Hunter T, Brenner D, Karin M (1992) Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell 70:777–789 Black EJ, Street AJ, Gillespie DAF (1991) Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun. Oncogene 6:1949–1958 Alberts AS, Deng T, Lin A, Meinkoth JL, Sch¨onthal A, Mumby MC, Karin M, Feramisco JR (1993) Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements. Mol Cell Biol 13:2104–2112 Brindle PK, Montminy MR (1992) The CREB family of transcription activators. Curr Opin Genet Dev 2:199–204 Alberts A, Montminy M, Shenolikar S, Feramisco JR (1994) Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol Cell Biol 14:4398–4407 Hagiwara M, Alberts AS, Brindle P, Meinkoth J, Feramisco J, Tiliang Deng, Karin M, Shenolikar S, Montminy M (1992) Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70:105–113 Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, Klemm DJ (1993) Nuclear protein phosphatase 2A dephosphorylates protein kinase A-Phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 138:222–2834 Wheat WH, Roesler WJ, Klemm DJ (1994) Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 14:5881–5890 Mayer-Jaekel RE, Hemmings BA (1994) Protein phosphatase 2A-a ‘m´enage a` trois’. Trends in Cell Biol 4:287–291
Regulation of gene expression 55. Liu J, Farmer JD, Lane WS, Friedman J, Weissman I, Schreiber SL (1991) Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes. Cell 66:807–815 56. Fruman DA, Klee CB, Bierer BF, Burakoff SJ (1992) Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A. Proc Natl Acad Sci USA 89:3686-3690 57. Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ, Cohen P, MacKintosh C, Klee CB, Schreiber SL (1992) Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochem 31:3896–3901 58. Schreiber SL (1992) Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell 70:365–368 59. McKeon F (1991) When worlds collide: immunosuppressants meet protein phosphatases. Cell 66:823–826 60. O’Keefe SJ, Tamura J, Kincaid RL, Tocci MJ, O’Neill EA (1992) FK-506- and CsA-sensitive activation of the interleukin2 promoter by calcineurin. Nature 357:692–694 61. Clipstone NA, Crabtree GR (1992) Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357:695–697 62. Hain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JH, Verdine GL, Curran T, Rao A (1993) The T-cell transcription factor NFATP is a substrate for calcineurin and interacts with Fos and Jun. Nature 365:352–355 63. Woodrow M, Clipstone NA, Cantrell D (1993) p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells. J Exp Med 178:1517–1522 64. Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, Tocci MJ, O’Keefe SJ, O’Neill EA (1994) Calcineurin acts in synergy with PMA to inactivate IkB/MAD3, an inhibitor of NFkB. EMBO J 13:861–870 65. Enslen H, Soderling TR (1994) Roles of calmodulin-dependent protein kinases and phosphatases in calcium-dependent transcription of immediate early genes. J Biol Chem 269:20872–20877 66. Lohi J, K¨ah¨ari VM, Keski-Oja J (1994) Cyclosporin A enhances cytokine and phorbol ester-induced fibroblast collagenase expression. J Invest Dermatol 102:938–944 67. Tai PK, Albers MW, McDonnell DP, Chang H, Schreiber SL, Faber LE (1994) Potentiation of progesterone receptormediated transcription by the immunosuppressant FK506. Biochem 33:10666–10671 68. Steinmann KE, Belinsky GS, Lee D, Schlegel R (1991) Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation. Proc Natl Acad Sci USA 88:6843–6847 69. Lund LR, Danø K (1992) Okadaic acid strongly increases gene transcription, mRNA and protein level for the urokinase receptor in human A549 cells. FEBS 298:177–181 70. Medcalf RL (1992) Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. J Biol Chem 267:12220–12226 71. Dubois MF, Nguyen VT, Dahmus ME, Pag`es G, Pouyss´egur J, Bensaude O (1994) Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases. EMBO J 13:4787–4797 72. Labhart P (1994) Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation. Mol Cell Biol 14:2011–2020 73. Mermoud JE, Cohen PTW, Lamond AI (1994) Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J 13:5679–5688 74. Harpold MM, Evans RM, Salditt-Georgieff M, Darnell JE (1979) Production of mRNA in Chinese hamster cells: Relationship of the rate of synthesis to the cytoplasmic
75.
76.
77.
78.
79. 80.
81.
82.
83.
84. 85.
86. 87.
88.
89.
90.
91.
247
concentration of nine specific mRNA sequences. Cell 17:1025–1035 Westermarck J, Lohi J, Keski-Oja J, K¨ah¨ari VM (1994) Okadaic acid-elicited transcriptional activation of collagenase gene expression in HT-1080 fibrosarcoma cells is mediated by JunB. Cell Growth & Differ 5:1205–1213 Cao X, Mahendran R, Guy GR, Tan YH (1992) Protein phosphatase inhibitors induce the sustained expression of the Egr-1 gene and the hyperphosphorylation of its gene product. J Biol Chem 267:12991–12997 Adunyah SE, Unlap TM, Franklin CC, Kraft AS (1992) Induction of differentiation and c-jun expression in human leukemic cells by okadaic acid, an inhibitor of protein phosphatases. J Cell Phys 151:415–426 Park K, Chung M, Kim SJ (1992) Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP1. J Biol Chem 267:10810–10815 Holladay K, Fujiki H, Bowden GT (1992) Okadaic acid induces the expression of both early and secondary response genes in mouse keratinocytes. Mol Carcin 5:16–24 Lee JS, Favre B, Hemmings BA, Kiefer B, Nagamine Y (1994) Okadaic acid-dependent induction of the urokinase-type plasminogen activator gene associated with stabilization and autoregulation of c-Jun. J Biol Chem 269:2887–2894 Cao X, Mahendran R, Guy GR, Tan YH (1992) Protein phosphatase inhibitors induce the sustained expression of the Egr-1 gene and the hyperphosphorylation of its gene product. J Biol Chem 267:12991–12997 Kharbanda S, Rubin E, Datta R, Hass R, Sukhatme V, Kufe D (1993) Transcriptional regulation of the early growth response 1 gene in human myeloid leukemia cells by okadaic acid. Cell Growth & Differ 4:17–23 Richards FM, Milner J, Metcalfe S (1992) Inhibition of the serine/threonine protein phosphatases PP1 and PP2A in lymphocytes: effect on mRNA levels for interleukin-2, IL-2Ra, krox-24, p53 hsc70 and cyclophilin. Immunology 76:642–647 Hou MC, Shen CH, Lee WC, Lai YK (1993) Okadaic acid as an inducer of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells. J Cell Biochem 51:91–101 Nagamine Y, Ziegler A (1991) Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition. EMBO J 10:117–122 Tebo JM, Hamilton TA (1994) Okadaic acid stimulates inflammatory cytokine gene transcription in murine peritoneal macrophages. Cell Immun 153:479–491 Sung SSJ, Walters JA, Fu SM (1992) Stimulation of tumor necrosis factor a production in human monocytes by inhibitors of protein phosphatase 1 and 2A. J Exp Med 176:897–901 Rieckmann P, Th´evenin C, Kehrl JH (1992) Okadaic acid is a potent inducer of AP-1, NF-kB, and tumor necrosis factor-a in human B lymphocytes. Biochem Biophys Res Comm 187:51–57 Kim SJ, Kim KY, Tapscott SJ, Winokur TS, Park K, Fujiki H, Weintraub H, Roberts AB (1992) Inhibition of protein phosphatases block myogenesis by first altering MyoD binding activity. J Biol Chem 267:15140–15145 Smits PHM, Smits HL, Minnaar RP, Hemmings BA, MayerJaekel RE, Schuurman R, van der Noordaa J, ter Schegget J (1992) The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11. EMBO J 11:4601–4606 Th¨evenin C, Kim SJ, Rieckmann P, Fujiki H, Norcross MA, Sporn MB, Fauci AS, Kehrl JH (1990) Induction of nuclear
A. H. Sch¨onthal
92.
93.
94. 95. 96.
factor-kB and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A. New Biologist 2:793–800 Skouv J, Jensen PO, Forchhammer J, Larsen JK, Lund LR (1994) Tumor-promoting phorbol ester transiently downmodulate the p53 level and blocks the cell cycle. Cell Growth Diff 5:320–340 Beck CA, Weigel NL, Edwards DP (1992) Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol Endocrin 6:607–620 Chang NT, Huang LE, Liu AYC (1993) Okadaic acid markedly potentiates the heat-induced hsp 70 promoter activity. J Biol Chem 268:1436–1439 Patel HR, Choi HS, Sytkowski AJ (1992) Activation of two discrete signaling pathways by erythropoietin. J Biol Chem 267:21300–21302 Luong H, Winestock KD, Finbloom DS (1994) Inhibitors of serine/threonine phosphatases enhance phosphorylation of the interferon-g receptor while selectively attenuating interferon-g-induced gene expression in human peripheral-blood monocytes. Biochem J 299:799–;803
97. Gapathi MK (1992) Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits induction of acute-phase proteins by interleukin-6 alone or in combination with interleukin-1 in human hepatoma cell lines. Biochem J 284:645–648 98. Morrison MD, Bate AJ, Harding HL, Whatmore AJ, Dickson AJ (1993) Effects of okadaic acid on expression of phospho enol pyruvate carboxykinase in cultured rat hepatocytes. Biochim Biophys Acta 1178:135–140 99. Denner LA, Weigel NL, Maxwell BL, Schrader WT, O’Malley BW (1990) Regulation of progesterone receptor-mediated transcription by phosphorylation. Science 250:1740–1743 100. Somers JP, DeFranco DB (1992) Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptormediated enhancement. Mol Endocrin 6:26–34 101. Lin KH, Ashizawa K, Cheng SY (1992) Phosphorylation stimulates the transcriptional activity of the human B1 thyroid hormone nuclear receptor. Proc Natl Acad Sci USA 89:7737–7741 102. Jones KE, Brubaker JH, Chin WW (1994) Evidence that phosphorylation events participate in thyroid hormone action. Endocrinology 134:543–548
248
CANCER BIOLOGY, Vol 6, 1995: pp 239–248
Regulation of gene expression by serine/threonine protein phosphatases Axel H. Sch¨onthal
additional mammalian PPases that are sensitive to low concentrations of okadaic acid complicates this analysis. For example, bovine PP-3 has an IC50 of 5 nM,4 the ubiquitously expressed rabbit PP-X (PP-4) is inhibited at 0.2 nM,5 and the predominantly nuclear human PP5 has an IC50 of 1–10 nM.6 Furthermore, PCR analysis indicates that many more phosphatases are yet to be discovered.7 Thus, the resulting effects of okadaic acid treatment, even at low concentrations, cannot easily be ascribed to a single PPase. Despite these limitations, however, the use of okadaic acid has spread tremendously over the last few years and has generated a wealth of useful information.
The activation of signal transduction pathways by extracellular stimuli, such as growth factors or hormones, ultimately results in changes in the expression of specific genes. The altered pattern of expression eventually determines the resulting cellular consequences, e.g. cell growth, division, or differentiation. It has been well established that the reversible phosphorylation of proteins is a major regulatory mechanism in these processes. However, while much has been learned about the role of kinases, the involvement of protein phosphatases is less clear and has only recently begun to be investigated in more detail. This review will present some of the new findings that demonstrate a crucial regulatory function of serine/threonine protein phosphatases (PPases) in gene regulatory processes. Key words: expression
phosphatases
/
okadaic
acid
/
gene
Effects of okadaic acid on kinase activity
©1995 Academic Press Ltd
Although no direct effect of okadaic acid on any kinase has been found, it may exert indirect regulatory effects on various kinases.8 Protein kinases whose catalytic activities are regulated by reversible phosphorylation may be stimulated in the absence of (inhibitory) phosphatase activity. This, in turn, may lead to the accumulation of phosphorylated substrates, which themselves may be kinases and further amplify the signal. Thus, the balance of reversible phosphorylations would be shifted towards the increased net phosphorylation of proteins. This has indeed been observed. When added to intact cells or cell extracts, okadaic acid causes a change in the apparent phosphorylation of many cellular proteins.9,10 Using high definition two-dimensional gel electrophoresis, Guy et al11 identified 74 proteins that exhibited altered levels of phosphorylation in response to okadaic acid treatment of human fibroblasts. Intriguingly, this pattern of phosphorylation was strikingly similar to that induced by tumour necrosis factor alpha (TNFα), but rather different from that induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The mimicry of TNFα by okadaic acid indicates that either agonist may initiate similar signal transduction pathways in these cells, which may affect the expression of the
Inhibition of PPases by okadaic acid A novel drug that has proven to be extremely useful for the study of gene regulation by PPases is the tumour promoter okadaic acid.1 This compound is a complex polyether derivative of a 38-carbon fatty acid that is synthesized by marine dinoflagellates. Importantly, okadaic acid has been found to bind to and inhibit the two major intracellular PPases, type-1 (PP1) and type-2A (PP-2A).2,3 Inhibition of PP-2A is more efficient than inhibition of PP-1, with 50% inhibition (IC50) in vitro occurring at 0.1–1.0 nM and 20–100 nM, respectively. Due to this differential effect, the consequences of treatment with low doses of okadaic acid have generally been attributed to a reduction of PP-2A activity. However, the recent discovery of Department of Molecular Microbiology and Immunology and K. Norris Jr. Comprehensive Cancer Center, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90033-1054, USA ©1995 Academic Press Ltd 1044-579X/95/040239 + 10$12.00/0
239
A. H. Sch¨onthal Table 1. Overview of genes that were activated in response to treatment of cells with okadaic acid Activated genes c-fos fos B fra-1 c-jun jun B jun D egr-1 egr-2, egr-3 krox-24 grp78 collagenase stromelysin-1 TIMP transin u-PA u-PA-R t-PA PAI-2 mal-1, mal-2 TNFα IL-2β IP-10 Id HPV16-LCR HIV-1-LTR progesteroneresponse element
Cell lines
Transcriptional regulation
Post-transcriptional regulation
References
yes
yes
35,38,5,6,7,78,79
yes
78 38,78 38,76,79,80,77,78,79
fibr., Jurkat, HT-1080, A-549, U937, C2C12 myobl., mouse skin C2C12 myobl. Jurkat, C2C12 myobl. fibr., Jurkat, LLC-PK1, U937 mouse skin, C2C12 myobl. Jurkat, HT-1080 Jurkat, HT-1080, U937 fibr., U937 fibr. lymphoc. gliosarc. synoviocytes, A-549, U937 HT-1080 HT-1080 mouse skin LLC-PK1, U937, mouse skin A549, U937, Hela, WI-38 U937 U937 mouse skin macroph. B-cells monoc. macroph. C2C12 myobl. fibr. Jurkat, BJA-B, U251 CV-1
yes
yes
yes
yes yes
yes yes yes yes yes yes yes
yes weak no
38,75 38,75,77 76,81,82 76 83 84 35,70 75 75 79 70,79,80,85 69 70 70 79 86,87,88 87 86 89 90 91 99
Numerous examples that indicate such events have been found. For instance, okadaic acid produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types.17 Several kinases that are important components of gene regulatory signal transduction pathways, such as Raf-1, MEK, and MAP kinases (ERKs) have been found to be hyperphosphorylated in response to okadaic acid treatment of cells.18-20 In not all cases, however, does increased phosphorylation lead to stimulation of the respective kinase activity. For MEK-1 it has been shown that its kinase activity is negatively regulated by threonine phosphorylation.21 The regulation of kinases by PPases has been further indicated by in-vitro experiments where purified phosphatases were shown to use phosphorylated kinases as substrates. (see detailed references in ref 22). Experiments with SV40 small t antigen, a protein that binds to and inactivates PP-2A,23 showed that its overexpression in cells led to the increased activity of ERK-2 and MEK-1,24 and increased transcriptional activity of an AP-1-regulated reporter construct.22 These experiments strongly suggested that some of these kinases may be negatively regulated by PP-2A in vivo.
same set of genes. In a different study using human breast cancer cells,12 it was shown that okadaic acid induced a protein phosphorylation pattern that overlapped that of TPA, and both agents stimulated similar biochemical and cellular events. It had been suggested earlier that some of the effects of TPA and okadaic acid were mediated via the same pathways: TPA-activated protein kinase C (PKC) initiates a phosphorylation cascade that can be reversed by okadaic acid sensitive PPases.13-15 Blockage of these PPases may lead to the sustained phosphorylation of substrates of the PKC pathway and thereby may affect gene expression in a manner similar to TPA. Indeed, many genes that are activated by TPA are also stimulated by okadaic acid, for example, c-fos, c-jun, and collagenase (Table 1; see 16 for TPA-inducible genes). Gene regulatory functions of PPases are likely exerted by a wide variety of mechanisms. At the gene proximal site, PPases may directly regulate the activity of transcription factors. Further upstream, PPases may affect components of signal transduction pathways, such as growth factor receptors or intermediate signal transduction molecules (kinases), that transmit a signal from the cell membrane into the nucleus. 240
Regulation of gene expression these enzymes in cellular growth control. This was somewhat surprising because these PPases had been thought of as negative regulators of cell growth or even as anti-oncogenes.13 Earlier work with the use of okadaic acid had supported this view. Because okadaic acid is a tumour promoter, it was suggested that inhibition of the respective PPases would contribute to the process of cellular transformation.13,33 This view was backed further by the finding that okadaic acid was able to induce the expression of several proto-oncogenes, such as c-fos and c-jun.34-38 The promoter elements mediating this activation were the same as those that confer the transcriptional activation in response to growth stimulatory signals or the tumour promoter TPA, e.g. the serum responsive element (SRE)39 of the c-fos gene and the TPA responsive element (TRE or AP-1 binding site)40 of c-jun and collagenase35,37,38 (see later). Combining the above described studies, it appears that okadaic acid-sensitive PPases have positive as well as negative effects on the expression of different growth-regulatory genes (see Tables 1–3, Figure 1). Immediate early genes, such as c-fos and c-jun, are negatively regulated by these PPases, and G0-arrested cells that are treated with okadaic acid re-enter the cell cycle and progress through early G1. On the other hand, at least two genes that are required later in the cell cycle, cdc2 and cyclinA, are positively regulated by PPases, and therefore cell cycle progression from G0 of okadaic acid-treated cells is blocked in late G1.31,32 It appears that the differential effects of okadaic acid on histone H1 kinase, namely stimulation of its enzymatic activity and inhibition of its gene, may both be due to the inhibition of PP-2A. Biochemical analysis identified PP-2A as the post-translational inhibitor of histone H1 kinase activity.27,28 Inhibition of cdc2 gene expression occurs at okadaic acid concentrations that inhibit PP-2A, but not PP-1, in cell culture31 (also, Sch¨onthal, unpublished). Moreover, co-transfection of an expression vector for PP-2A stimulates transcription of a reporter plasmid under the control of the cdc2 promoter (Sch¨onthal, unpublished). These data point to a positive role of PP-2A in the transcriptional regulation of cdc2. How can one reconcile these opposing effects of PP-2A? The post-translational stimulation of histone H1 kinase activity by okadaic acid is transient, and is restricted to cells in G2 of the cell cycle.27 In contrast, the transcriptional inhibition of cdc2 gene expression by the tumour promoter is permanent and occurs in G0/G1-synchronized cells.31 Although this latter
Other okadaic acid-regulated phosphoproteins that are significant regulators of gene expression are the histone H1 kinases and the tumour suppressor gene products retinoblastoma (Rb) and p53. Both Rb and p53 are hyperphosphorylated in response to shortterm okadaic acid treatment of cells.25,26 In the case of p53, increased phosphorylation appeared to reduce its transcriptional activation function.26 Moreover, p53 was shown to be dephosphorylated by PP-2A in vitro.23 Thus, PPase activity is likely to contribute to the cell cycle-regulatory functions of this tumour suppressor protein. Hyperphosphorylation of Rb protein is thought to inactive its growth-suppressive function and allows cells to proceed through the cell cycle. The principal kinase to phosphorylate Rb is histone H1 kinase, which has been shown to be activated by okadaic acid in a transient fashion; (27; further references in 1). It is conceivable that hyperphosphorylation of Rb in response to okadaic acid is brought about by a twofold mechanism: the activation of histone H1 kinase in combination with the inhibition of PPases that dephosphorylate Rb. This idea has been further substantiated by the finding that histone H1 kinase is negatively regulated by PP-2A,28 and that Rb is a substrate for PP-1 in vitro and in vivo.29,30 However, the regulation of histone H1 kinase by PPases appears to be rather complex. In addition to its negative regulation at the posttranslational level,27,28 histone H1 kinase appears to be positively regulated by PPases at the transcriptional level.31 In serumdeprived fibroblasts, which are growth-arrested in G0 of the cell cycle, several of the genes that code for histone H1 kinase subunits, such as cyclinA or cdc2, are transcriptionally inactive (see detailed references in 31). Upon stimulation of these cells with growth factors or serum, expression of these genes greatly increases and subsequently allows cells to proceed through the cell cycle. However, when growtharrested cells are stimulated with serum in the presence of okadaic acid, the activation of cdc2 and cyclinA gene expression is blocked.31 Consequently, the cells lack histone H1 kinase proteins and do not have the respective catalytic activity. Phosphorylation (inactivation) of Rb protein does not occur, and the cells accumulate in late G1 of the cell cycle.31 Similar inhibitory effects of okadaic acid were observed when murine embryonic fibroblasts were growth-stimulated with TGF-β.32 The finding that okadaic acid-sensitive PPases were required for the expression of at least some of the histone H1 kinase genes indicated a positive role for 241
A. H. Sch¨onthal Table 2. Overview of genes that were repressed in response to treatment of cells with okadaic acid Activated genes c-myc p53 cdc2 cyclinA myoD1 myogenin MLC u-PA t-PA IL-2 IL-2-Rα cyclophilin SV40 early rRNA
Transcriptional regulation
Cell lines U937 Hela, A549, fibrosarc., lymphoc. fibr., U937 fibr. C2C12 myobl. C2C12 myobl. C2C12 myobl. HT-1080 HT-1080 lymphoc. lymphoc. lymphoc. LLC-PK1, T47D Xenopus extr.
References 77 83,92 31,77 31 78,89 89 78 70 70 83 83 83 80,93 72
yes yes yes
yes
H1 kinase. In longer term assays, Rb phosphorylation is reduced or completely inhibited because of a lack of histone H1 kinase expression and activity. Since Rb has been shown to be involved in the regulation of several genes,41 it can be expected that the modification of its phosphorylation status in response to okadaic acid treatment may have differential effects on the expression of its target genes.
effect has been observed in log phase cells, it cannot be ruled out that only a subpopulation of cells at a certain cell cycle stage is affected. Moreover, it is likely that PP-2A activity is regulated in a fashion that is dependent on the cell cycle, its (micro-)compartmental localization, and its interaction with various regulatory subunits. Thus, it is conceivable, that PP-2A may be required to ensure expression of sufficient amounts of histone H1 kinase subunits during G1, before its inactivation may contribute to the rapid activation of kinase activity at later stages of the cell cycle. Similarly, the seemingly contradictory effects of okadaic acid on Rb protein phosphorylation could be explained by the different experimental approaches. In short-term assays, okadaic acid treatment of cells increases Rb phosphorylation which is likely due to the transient post-translational activation of histone
Regulation of transcription factor activity by PPases In order to delineate the precise role of PPases in gene expression, it is important to analyse how the activity of defined promoter elements and their respective transcription factors is regulated by these enzymes. Recent work yielded exciting results for
Table 3. Overview of genes whose expression was not affected by okadaic acid treatment alone, but where the presence of okadaic acid inhibited or enhanced the gene-regulatory effects of other stimuli
Regulated genes HSP70 c-myb IL-1α UL-6 IP-10 CRP; SAA fibrinogen PEPCK MMTV-LTR MMTV-LTR thyroid hormone response element
Cell lines
Stimulus
Okadaic acid effects inhibitory (I) stimulatory (S)
Hela, Hep-2, IMR-90 neuroblastoma erythroid monoc. monoc. monoc. Hep3B Hep3B, NPLC hepatoc. T47D CV-1, COS COS-1, CV-1
heat shock
S
84
erythropoietin LPS LPS IFNγ IL-6 + IL-1 IL-6 cAMP progestin dexamethasone T3
S S S I I I I S S S
95 87 87 96 97 97 98 93 100 101,102
242
References
Regulation of gene expression tion of TCF, and that TCF was an in-vitro substrate for PP-2A.42 Using microneedle injections of purified PPases, it has been demonstrated that serum-stimulated induction of SRE activity was inhibited in the presence of exogenous PP-2A.43 Moreover, elevated expression of small t contributed to increased c-fos promoter activity.22 Together, these data indicate that PPases, most likely PP-2A, confer a negative regulation of c-fos promoter activity via the SRE. The transcription factor AP-1 is positively regulated in response to many cell growth-stimulatory signals, and recognizes primarily the TRE.40 It is a heterodimer consisting of polypeptides of the Fos and Jun family, that are regulated by reversible phosphorylation reactions (see further references in 19,44). A detailed analysis of the post-translational modification of c-Jun protein revealed two domains that are modified by phosphorylation. Phosphorylation on two N-terminal sites in combination with the dephosphorylation of two to three C-terminal sites is required for its binding to the TRE and full transcriptional activity.45,46 In a reticulocyte extract, okadaic acid enhanced phosphorylation of in-vitro synthesized c-Jun protein, and immunoprecipitated c-Jun was dephosphorylated in the presence of purified PP2A.47 In other studies it was shown that the purified catalytic subunit of PP-2A (PP-2AC) efficiently dephosphorylated the inhibitory C-terminal phosphorylation sites of c-Jun in vitro,22,48 indicating that this PPase may contribute to the activation of transcription factor AP-1. However, because PP-2AC is normally complexed with two other regulatory subunits, it is not clear whether this activity of PP-2AC truly reflects what occurs in vivo. Nevertheless, these data indicate a role for PP-2A in the transcriptional regulation by AP1 proteins. Maybe the best characterized, and at the same time most controversial, example of gene regulation by PPases is the transcriptional control of the CRE. The CRE has been found in several cAMP-responsive genes and is the binding site for transcription factor CREB.49 CREB activity is regulated by the cAMPdependent protein kinase A (PKA), which phosphorylates CREB at a single serine and thereby stimulates its transcriptional activity.49 Using completely different approaches, two groups have studied the regulation of phospho-CREB (P-CREB) dephosphorylation and transcriptional inactivation by PPases. The first group used microneedle injections and plasmid transfections to introduce either purified phosphatases or the respective expression vectors into
several of these elements, and three of them, the SRE, the TRE, and the cyclic AMP responsive element (CRE), will be discussed below. Several proteins that bind to the SRE have been shown to be phosphorylated and thus are potential targets for modification and regulation by PPases. These proteins form a multiprotein complex consisting of serum response factor (SRF) and ternary complex factor (TCF), a family of proteins which include p62TCF/Elk-1 and SAP-1.39,42 Recent experiments showed that increased c-fos expression in response to okadaic acid correlated with phosphoryla-
Figure 1. Differential regulation of gene expression by okadaic acid. 3C10T1/2 murine fibroblast were treated with the indicated concentrations of okadaic acid for 24 h. PolyA + RNA was isolated and analysed by Northern transfer and subsequent hybridization to specific probes for the immediate early gene c-fos, the histone H1 kinase regulatory subunit gene cyclin A (cycA), and, as a control for equal amounts of RNA loaded in each lane, choA, an abundant RNA species that was originally isolated from Chinese hamster ovary (CHO) cells.74 For experimental details see 31.
243
A. H. Sch¨onthal a number of different cell lines.50,51 They demonstrated that microinjected PP-1, but not PP-2A, inhibited transcription of a CRE-regulated reporter plasmid. Similarly, transfected PP-1, but not PP-2A, was able to inhibit CRE activity. Microinjection of an expression plasmid for inhibitor-1 (I-1), a PP-1 specific inhibitor, resulted in increased phosphorylation and activity of CREB. In contrast, microinjection of SV40 small t had no effect. Thus, these data provided evidence for a primary role of PP-1 in the regulation of CREB function. The second group used biochemical fractionation and immunological analysis to characterize CREB regulation.52 They demonstrated that P-CREB phosphatase activity in nuclear extracts coeluted with PP2A from various columns, and was chromatographically resolved from nuclear PP-1. Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by inhibitor-2 (I-2), a PP-1 specific inhibitor. Moreover, P-CREB treated with PP-2A lost its transcriptional activity in-vitro, whereas P-CREB treated with PP-1 remained transcriptionally active. Further experiments demonstrated that SV40 small t inhibited the dephosphorylation of P-CREB and enhanced transcription from a CRE-containing promoter.53 Taken together, these data provided evidence for a primary role of PP-2A in the regulation of CREB function. It is not clear, why the two different approaches described above provided contradictory results (see discussion in 50,52). They indicate however, that it may be important to study the native forms of the PPases, in which the catalytic subunits are complexed with the appropriate regulatory subunits. The latter appear to play important roles in determining substrate specificity, catalytic activity, and cellular localization14,54 (see further references in 50).
these immunophilin-ligand complexes is PP-2B (see 58,59 for reviews). Most of this work so far has concentrated on T cells, where CsA and FK506 have been shown to inhibit signals arising from the activated T cell receptor. As a consequence of drug treatment, expression of early T cell activation genes, such as interleukin 2 (IL-2), is inhibited, and the cells do not progress through G1.60,61 Two of the promoter elements in the IL-2 promoter that mediate this inhibition are the binding sites for the transcription factors NFAT and NFIL2A.62,63 These sites have also been shown to be synergistically activated by overexpression of a constitutively active form of PP-2B in the presence of an activated Ha-ras oncogene or simultaneous treatment of cells with TPA.60,63 From these studies it has been inferred that PP-2B is a necessary mediator of T cell receptor signalling and IL-2 gene activation.57,58 These studies were extended to show that PP-2B also stimulated the NF-kB-dependent element of the IL-2 promoter through the inactivation of IkB, an inhibitor of NF-kB.64 Gene regulatory effects of PP-2B occur in other cell types as well. For example, in PC12 cells CsA and FK506 exhibited differential effects on the Ca2 + stimulated expression of two immediate early genes: while transcription of the NGFI-A gene was enhanced by these drugs, transcription of NGFI-B was inhibited.65 In human fibroblasts, CsA enhanced cytokine and phorbol ester-induced expression of the collagenase, c-jun, and junB genes.66 Clearly, with the identification of PP-2B as the CsA- and FK506-sensitive enzyme, numerous observations of gene regulation by CsA and FK506 (see detailed references in 64) imply that this PPase may be involved in the regulation of many genes in a variety of cell types.67 Furthermore, the demonstration that Ras protein and PP-2B can cooperate to activate T cells has led to the suggestion that PP-2B could be a protooncogene.63
Gene regulation by PP-2B/calcineurin Conclusions The analysis of gene regulatory pathways that are regulated by phosphatases other than PP-1/PP-2A received a strong impetus through the finding that PP-2B (also called calcineurin or Ca2 + , calmodulindependent PPase) activity could be inhibited by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506).55-57 CsA and FK506 are natural microbial products that form complexes with the immunophilins cyclophilin and FK506-binding protein (FKBP). Importantly, the only characterized target for
Great strides have been made towards a more complete understanding of PPase function in gene regulatory processes. Nearly 50 genes have been found so far that appear to be regulated by these enzymes. However, because in most studies okadaic acid was used to establish the role of PPases, and because okadaic acid affects the activity of more than one PPase, it was often not possible to determine which of the various okadaic acid-sensitive PPases was 244
Regulation of gene expression
References
responsible for the observed effect. Nevertheless, in order to gain preliminary insight into gene regulatory processes, okadaic acid has certainly proven to be extremely useful. In the future, a combination of various approaches will be necessary in order to characterize the precise roles of PPases in gene regulatory processes. For example, biochemical and immunological analysis together with cellular studies may be most useful. However, as presented above for the analysis of the transcription factor CREB, even these types of studies may yield equivocal data. Clearly, the analysis of PPase function is greatly complicated by the presence of several isoforms and subunits for each enzyme, as well as by its varying subcellular localization and by its cell cycle dependence.1,14,54 Moreover, in many cases strong cell cycle specificities have been observed that may be due to a difference in the balance or availability of the regulatory components.22,68-70 In addition to the regulation of specific transcription factors, PPases may also be involved in the control of the basal transcriptional machinery. For instance, since the C-terminal domain (CTD) of RNA polymerase II (Pol II) is phosphorylated at multiple sites in response to serum stimulation of quiescent cells,71 it is conceivable that PPases may also be involved in the regulation of Pol II activity. This control could be exerted directly via the dephosphorylation of the CTD, or indirectly via MAP kinase, which has been implicated in the enhanced phosphorylation of Pol II.71 Moreover, using a Xenopus transcription extract, the activity of a type 1 PPase has been implicated in the transcriptional initiation of ribosomal gene expression by Pol 1.72 Other experiments suggested a role for PP-1 in the regulation of mammalian spliceosome assembly.73 Thus, it appears that PPases play a crucial role at various levels of gene expression: from the signal input site via transcription factor regulation to posttranscriptional and post-translational controls.
1. Sch¨onthal A (1992) Okadaic acid — a valuable new tool for the study of signal transduction and cell cycle regulation? New Biologist 4:16–21 2. Bialojan C, Takai A (1988) Inhibitory effect of a marinesponge toxin okadaic acid, on protein phosphatases. Biochem J 256:283–290 3. Cohen P, Klumpp S, Schelling DL (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett 250:596–600 4. Honkanen RE, Zwiller J, Daily SL, Khatra BS, Dukelow M, Boynton AL (1991) Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain. J Biol Chem 266:6614–6619 5. Brewis ND, Street AJ, Prescott AR, Cohen PTW (1993) PPX, a novel protein serine/threonine phosphatase localized to centrosomes. EMBO J 12:987–996 6. Chen MX, McPartlin AE, Brown L, Chen YH, Barker HM, Cohen PTW (1994) A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J 13:4278–4290 7. Chen MX, Chen YH, Cohen PTW (1992) Polymerase chain reactions using Saccharomyces, Drosophila and human DNA predict a larger family of protein serine/threonine phosphatases. FEBS 306:54–58 8. Sassa T, Richter WW, Uda N, Suganuma M, Suguri H, Yoshizawa S, Hirota M, Fujiki H (1989) Apparent ‘activation’ of protein kinases by okadaic acid class tumor promoters. Biochem Biophys Res Comm 159:939–944 9. Haystead TAJ, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG (1989) Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337:78–81 10. Haystead TAJ, Weiel JE, Litchfield DW, Tsukitani Y, Fischer EH, Krebs EG (1990) Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. J Biol Chem 265:16571–16580 11. Guy GR, Cao X, Chua SP, Tan YH (1992) Okadaic acid mimics multiple changes in early protein phosphorylation and gene expression induced by tumor necrosis factor or interleukin-1. J Biol Chem 267:1846–1852 12. Kiguchi K, Giometti C, Chubb CH, Fujiki H, Huberman E (1992) Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A. Biochem Biophys Res Comm 189:1261–1267 13. Cohen P, Cohen PTW (1989) Protein phosphatases come of age. J Biol Chem 264:21435–21438 14. Shenolikar S, Nairn AC (1991) Protein phosphatases: recent progress, in Advances in Second Messenger and Phosphoprotein Research (Greengard P, Robison GA, eds), Vol 23, pp 3-38. Raven Press, New York 15. Clarke PR, Siddhanti SR, Cohen P, Blackshear PJ (1993) Okadaic acid-sensitive protein phosphatases dephosphorylate MARCKS, a major protein kinase C substrate. FEBS Lett 336:37–42 16. Rahmsdorf HJ, Herrlich P (1990) Regulation of gene expression by tumor promoters. Pharmac Ther 48:157–188 17. Hern´andez-Sotomayor SMT, Mumby M, Carpenter G (1991) Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. J Biol Chem 266:21281–21286 18. Kharbanda S, Saleem A, Emoto Y, Stone R, Rapp U, Kufe D (1994) Activation of raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. J Biol Chem 269:872–878 19. Hipskind RA, Baccarini M, Nordheim A (1994) Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with
Acknowledgements I thank Norbert Berndt for critical reading of this manuscript and Laura Steel for secretarial assistance. I am grateful to Tanya Kormeili for her smile and other multifaceted means of sustenance. Work in the author’s lab was supported by grants from the Tobacco Related Disease Research Program (TRDRP) of California and the Margaret E. Early Medical Research Trust.
245
A. H. Sch¨onthal
20.
21.
22.
23.
24.
25. 26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
36.
ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo. Mol Cell Biol 14:6219–6231 Miyasaka T, Miyasaka J, Saltiel AR (1990) Okadaic acid stimulates the activity of microtubule associated protein kinase in PC-12 pheochromocytoma cells. Biochem & Biophys Res Comm 168:1237–1243 Rossomando AJ, Dent P, Sturgill TW, Marshak DR (1994) Mitogen-activated protein kinase 1 (MKK1) is negatively regulated by threonine phosphorylation. Mol Cell Biol 14:1594–1602 Frost JA, Alberts AS, Sontag E, Guan K, Mumby MC, Feramisco JR (1994) Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity. Mol Cell Biol 14:6244–6252 Scheidtmann KH, Mumby MC, Rundell K, Walter G (1991) Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol Cell Biol 11:1996–2003 Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M (1993). The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell 75:887–897 Yatsunami J, Komori A, Ohta T, Suganuma M, Fujiki H (1993) Hyperphosphorylation of retinoblastoma protein and p53 by okadaic acid, a tumor promoter. Cancer Res 53:239–241 Zhang W, McClain C, Gau J, Guo X-YD, Deisseroth AB (1994) Hyperphosphorylation of p53 induced by okadaic acid attenuates its transcriptional activation function. Cancer Res 54:4448–4453 Yamashita K, Yasuda H, Pines J, Yasumoto K, Nishitani H, Ohtsubo M, Hunter T, Sugimura T, Nishimoto T (1990) Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitotis-like state in BHK21 cells. EMBO J 9:4331–4338 Lee TH, Solomon MJ, Mumby MC, Kirschner MW (1991) INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415–423 Alberts AS, Thorburn AM, Shenolikar S, Mumby MC, Feramisco JR (1993) Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc Natl Acad Sci USA 90:388–392 Ludlow JW, Glendening CL, Livingston DM, DeCaprio JA (1993) Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol 13:367–372 Sch¨onthal A, Feramisco JR (1993) Inhibition of histone H1 kinase expression, retinoblastoma protein phosphorylation, and cell proliferation by the phosphatase inhibitor okadaic acid. Oncogene 8:433–441 Kim TA, Velasquez BR, Wenner CE (1993) Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts. Proc Natl Acad Sci USA 90:5460–5463 Suganuma M, Fujiki H, Suguri H, Yoshizawa S, Hirota M, Nakayasu M, Ojika M, Wakamatsu K, Yamada K (1988) Okadaic acid: an additional non-phorbol-12-tetradecanoate13-acetate-type promoter. Proc Natl Acad Sci USA 85:1768–1771 Herschman HR, Lim RW, Brankow DW, Fujiki H (1989) The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression. Carcinogenesis 10:1495–1498 Kim SJ, Lafyatis R, Kim KY, Angel P, Fujiki H, Karin M, Sporn MB, Roberts AB (1990) Regulation of collagenase gene expression by okadaic acid, an inhibitor of protein phosphatases. Cell Reg 1:269–278 Sch¨onthal A, Tsukitani Y, Feramisco JR (1991) Transcriptional and posttranscriptional regulation of c-fos proto-oncogene
37. 38. 39. 40. 41.
42.
43.
44. 45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
246
expression by the tumor promoter okadaic acid. Oncogene 6:423–430 Sch¨onthal A, Alberts AS, Frost JA, Feramisco JR (1991) Differential regulation of jun family gene expression by the tumor promoter okadaic acid. New Biol 3:977–986 Th´evenin C, Kim SJ, Kehrl JH (1991) Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. J Biol Chem 266:9363–9366 Treisman R (1990) The SRE: a growth factor responsive transcriptional regulator. Semin Cancer Biol 1:47–58 Angel P, Karin M (1991) The role of Jun, Fos and AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072:129–157 Kim S-J, Lee HD, Robbins PD, Busam K, Sporn MB, Roberts AB (1991) Regulation of transforming growth factor β1 gene expression by the product of the retinoblastoma-susceptibility gene. Proc Natl Acad Sci USA 88:3052–3056 Zinck R, Hipskind RA, Pingoud V, Nordheim A (1993) c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF. EMBO J 12:2377–2387 Sch¨onthal A, Alberts AS, Rahman A, Meinkoth J, Mumby M, Feramisco JR (1992) Involvement of serine/threonine specific protein phosphatases in signal transduction pathways that regulate gene expression and cell growth, in Recent Advances in Cellular and Molecular Biology (Wegmann RJ, Wegmann MA, eds), Vol 2, pp 67–74. Peeters Press, Leuven Hunter T, Karin M (1992) The regulation of transcription by phosphorylation. Cell 70:375–387 Boyle WJ, Smeal T, Defize LHK, Angel P, Woodgett JR, Karin M, Hunter T (1991) Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64:573–584 Lin A, Frost J, Deng T, Smeal T, Al-Alawi N, Kikkawa U, Hunter T, Brenner D, Karin M (1992) Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell 70:777–789 Black EJ, Street AJ, Gillespie DAF (1991) Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun. Oncogene 6:1949–1958 Alberts AS, Deng T, Lin A, Meinkoth JL, Sch¨onthal A, Mumby MC, Karin M, Feramisco JR (1993) Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements. Mol Cell Biol 13:2104–2112 Brindle PK, Montminy MR (1992) The CREB family of transcription activators. Curr Opin Genet Dev 2:199–204 Alberts A, Montminy M, Shenolikar S, Feramisco JR (1994) Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts. Mol Cell Biol 14:4398–4407 Hagiwara M, Alberts AS, Brindle P, Meinkoth J, Feramisco J, Tiliang Deng, Karin M, Shenolikar S, Montminy M (1992) Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70:105–113 Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, Klemm DJ (1993) Nuclear protein phosphatase 2A dephosphorylates protein kinase A-Phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 138:222–2834 Wheat WH, Roesler WJ, Klemm DJ (1994) Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 14:5881–5890 Mayer-Jaekel RE, Hemmings BA (1994) Protein phosphatase 2A-a ‘m´enage a` trois’. Trends in Cell Biol 4:287–291
Regulation of gene expression 55. Liu J, Farmer JD, Lane WS, Friedman J, Weissman I, Schreiber SL (1991) Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes. Cell 66:807–815 56. Fruman DA, Klee CB, Bierer BF, Burakoff SJ (1992) Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A. Proc Natl Acad Sci USA 89:3686-3690 57. Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ, Cohen P, MacKintosh C, Klee CB, Schreiber SL (1992) Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochem 31:3896–3901 58. Schreiber SL (1992) Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell 70:365–368 59. McKeon F (1991) When worlds collide: immunosuppressants meet protein phosphatases. Cell 66:823–826 60. O’Keefe SJ, Tamura J, Kincaid RL, Tocci MJ, O’Neill EA (1992) FK-506- and CsA-sensitive activation of the interleukin2 promoter by calcineurin. Nature 357:692–694 61. Clipstone NA, Crabtree GR (1992) Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357:695–697 62. Hain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JH, Verdine GL, Curran T, Rao A (1993) The T-cell transcription factor NFATP is a substrate for calcineurin and interacts with Fos and Jun. Nature 365:352–355 63. Woodrow M, Clipstone NA, Cantrell D (1993) p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells. J Exp Med 178:1517–1522 64. Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, Tocci MJ, O’Keefe SJ, O’Neill EA (1994) Calcineurin acts in synergy with PMA to inactivate IkB/MAD3, an inhibitor of NFkB. EMBO J 13:861–870 65. Enslen H, Soderling TR (1994) Roles of calmodulin-dependent protein kinases and phosphatases in calcium-dependent transcription of immediate early genes. J Biol Chem 269:20872–20877 66. Lohi J, K¨ah¨ari VM, Keski-Oja J (1994) Cyclosporin A enhances cytokine and phorbol ester-induced fibroblast collagenase expression. J Invest Dermatol 102:938–944 67. Tai PK, Albers MW, McDonnell DP, Chang H, Schreiber SL, Faber LE (1994) Potentiation of progesterone receptormediated transcription by the immunosuppressant FK506. Biochem 33:10666–10671 68. Steinmann KE, Belinsky GS, Lee D, Schlegel R (1991) Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation. Proc Natl Acad Sci USA 88:6843–6847 69. Lund LR, Danø K (1992) Okadaic acid strongly increases gene transcription, mRNA and protein level for the urokinase receptor in human A549 cells. FEBS 298:177–181 70. Medcalf RL (1992) Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. J Biol Chem 267:12220–12226 71. Dubois MF, Nguyen VT, Dahmus ME, Pag`es G, Pouyss´egur J, Bensaude O (1994) Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases. EMBO J 13:4787–4797 72. Labhart P (1994) Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation. Mol Cell Biol 14:2011–2020 73. Mermoud JE, Cohen PTW, Lamond AI (1994) Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism. EMBO J 13:5679–5688 74. Harpold MM, Evans RM, Salditt-Georgieff M, Darnell JE (1979) Production of mRNA in Chinese hamster cells: Relationship of the rate of synthesis to the cytoplasmic
75.
76.
77.
78.
79. 80.
81.
82.
83.
84. 85.
86. 87.
88.
89.
90.
91.
247
concentration of nine specific mRNA sequences. Cell 17:1025–1035 Westermarck J, Lohi J, Keski-Oja J, K¨ah¨ari VM (1994) Okadaic acid-elicited transcriptional activation of collagenase gene expression in HT-1080 fibrosarcoma cells is mediated by JunB. Cell Growth & Differ 5:1205–1213 Cao X, Mahendran R, Guy GR, Tan YH (1992) Protein phosphatase inhibitors induce the sustained expression of the Egr-1 gene and the hyperphosphorylation of its gene product. J Biol Chem 267:12991–12997 Adunyah SE, Unlap TM, Franklin CC, Kraft AS (1992) Induction of differentiation and c-jun expression in human leukemic cells by okadaic acid, an inhibitor of protein phosphatases. J Cell Phys 151:415–426 Park K, Chung M, Kim SJ (1992) Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP1. J Biol Chem 267:10810–10815 Holladay K, Fujiki H, Bowden GT (1992) Okadaic acid induces the expression of both early and secondary response genes in mouse keratinocytes. Mol Carcin 5:16–24 Lee JS, Favre B, Hemmings BA, Kiefer B, Nagamine Y (1994) Okadaic acid-dependent induction of the urokinase-type plasminogen activator gene associated with stabilization and autoregulation of c-Jun. J Biol Chem 269:2887–2894 Cao X, Mahendran R, Guy GR, Tan YH (1992) Protein phosphatase inhibitors induce the sustained expression of the Egr-1 gene and the hyperphosphorylation of its gene product. J Biol Chem 267:12991–12997 Kharbanda S, Rubin E, Datta R, Hass R, Sukhatme V, Kufe D (1993) Transcriptional regulation of the early growth response 1 gene in human myeloid leukemia cells by okadaic acid. Cell Growth & Differ 4:17–23 Richards FM, Milner J, Metcalfe S (1992) Inhibition of the serine/threonine protein phosphatases PP1 and PP2A in lymphocytes: effect on mRNA levels for interleukin-2, IL-2Ra, krox-24, p53 hsc70 and cyclophilin. Immunology 76:642–647 Hou MC, Shen CH, Lee WC, Lai YK (1993) Okadaic acid as an inducer of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells. J Cell Biochem 51:91–101 Nagamine Y, Ziegler A (1991) Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition. EMBO J 10:117–122 Tebo JM, Hamilton TA (1994) Okadaic acid stimulates inflammatory cytokine gene transcription in murine peritoneal macrophages. Cell Immun 153:479–491 Sung SSJ, Walters JA, Fu SM (1992) Stimulation of tumor necrosis factor a production in human monocytes by inhibitors of protein phosphatase 1 and 2A. J Exp Med 176:897–901 Rieckmann P, Th´evenin C, Kehrl JH (1992) Okadaic acid is a potent inducer of AP-1, NF-kB, and tumor necrosis factor-a in human B lymphocytes. Biochem Biophys Res Comm 187:51–57 Kim SJ, Kim KY, Tapscott SJ, Winokur TS, Park K, Fujiki H, Weintraub H, Roberts AB (1992) Inhibition of protein phosphatases block myogenesis by first altering MyoD binding activity. J Biol Chem 267:15140–15145 Smits PHM, Smits HL, Minnaar RP, Hemmings BA, MayerJaekel RE, Schuurman R, van der Noordaa J, ter Schegget J (1992) The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11. EMBO J 11:4601–4606 Th¨evenin C, Kim SJ, Rieckmann P, Fujiki H, Norcross MA, Sporn MB, Fauci AS, Kehrl JH (1990) Induction of nuclear
A. H. Sch¨onthal
92.
93.
94. 95. 96.
factor-kB and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A. New Biologist 2:793–800 Skouv J, Jensen PO, Forchhammer J, Larsen JK, Lund LR (1994) Tumor-promoting phorbol ester transiently downmodulate the p53 level and blocks the cell cycle. Cell Growth Diff 5:320–340 Beck CA, Weigel NL, Edwards DP (1992) Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol Endocrin 6:607–620 Chang NT, Huang LE, Liu AYC (1993) Okadaic acid markedly potentiates the heat-induced hsp 70 promoter activity. J Biol Chem 268:1436–1439 Patel HR, Choi HS, Sytkowski AJ (1992) Activation of two discrete signaling pathways by erythropoietin. J Biol Chem 267:21300–21302 Luong H, Winestock KD, Finbloom DS (1994) Inhibitors of serine/threonine phosphatases enhance phosphorylation of the interferon-g receptor while selectively attenuating interferon-g-induced gene expression in human peripheral-blood monocytes. Biochem J 299:799–;803
97. Gapathi MK (1992) Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits induction of acute-phase proteins by interleukin-6 alone or in combination with interleukin-1 in human hepatoma cell lines. Biochem J 284:645–648 98. Morrison MD, Bate AJ, Harding HL, Whatmore AJ, Dickson AJ (1993) Effects of okadaic acid on expression of phospho enol pyruvate carboxykinase in cultured rat hepatocytes. Biochim Biophys Acta 1178:135–140 99. Denner LA, Weigel NL, Maxwell BL, Schrader WT, O’Malley BW (1990) Regulation of progesterone receptor-mediated transcription by phosphorylation. Science 250:1740–1743 100. Somers JP, DeFranco DB (1992) Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptormediated enhancement. Mol Endocrin 6:26–34 101. Lin KH, Ashizawa K, Cheng SY (1992) Phosphorylation stimulates the transcriptional activity of the human B1 thyroid hormone nuclear receptor. Proc Natl Acad Sci USA 89:7737–7741 102. Jones KE, Brubaker JH, Chin WW (1994) Evidence that phosphorylation events participate in thyroid hormone action. Endocrinology 134:543–548
248