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Threonine Phosphatase Inhibitors and Human Disease
Chapter 89
Protein Serine/Threonine Phosphatase Inhibitors and Human Disease Shirish Shenolikar 1,2 and Matthew H. Brush1 1
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School Singapore.
2
Introduction Coordinating the activity of protein kinases and phosphatases allows cells to amplify and accelerate hormonal signals and elicit a robust and decisive physiological response. Hormonal signals have been shown to modulate the expression or activity of endogenous phosphatase inhibitor proteins (described in Chapter 105) that dampen the functions of specific protein phosphatases and enhance the phosphorylation of their cellular substrates. This paradigm is best exemplified by protein serine/threonine phosphatases, many of which are targeted by an increasing number of endogenous protein inhibitors. By suppressing the functions of the relatively few phosphatase catalytic subunits that catalyze serine/threonine dephosphorylation in eukaryotic cells, these inhibitor proteins have the potential to modulate a broad range of physiological events and orchestrate an integrated physiological response that coordinates such diverse processes as metabolism, migration, gene transcription, and growth. Emerging studies suggest that some phosphatase inhibitors physically associate with cellular phosphatase complexes comprised of a catalytic subunit bound to one or more regulatory proteins that restrict the actions of the phosphatases to control specific cellular events. The functions of these phosphatase inhibitors are in turn controlled by their expression, reversible phosphorylation, and dynamic association with target protein phosphatases. In this manner, endogenous serine/threonine phosphatase inhibitors achieve both spatial and temporal control of phosphoproteins that regulate normal cell physiology, and growing evidence suggests that aberrant expression and/or activity of phosphatase inhibitors may be associated with many human diseases. This chapter focuses on new information on the mode of action of phosphatase inhibitors and discusses their potential contributions to the pathophysiology of human disease. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
Environmental toxins as phosphatase inhibitors Okadaic acid, responsible for human diarrheretic shellfish poisoning, was first identified as a toxin concentrated by marine sponges, and functions as a potent inhibitor of several eukaryotic protein serine/threonine phosphatases. By inhibiting one or more phosphatases, okadaic acid enhances smooth muscle myosin phosphorylation and the contractility of the gut [1]. Subsequent studies identified cyclic peptides like microcystin and nodularin in freshwater ponds and lakes infested with cyanobacteria, which cause severe hepatotoxicity that is often lethal to animals [2]. Like okadaic acid, these compounds also inhibited the major mammalian serine/threonine phosphatases. To date, more than a dozen environmental toxins or natural products have been shown to inhibit protein serine/threonine phosphatases. Almost all of these compounds have the potential to promote cell growth and transformation, and induce a variety of cancers in experimental animals. However, at least one report hinted at other cellular actions of these toxins, specifically that okadaic acid impaired DNA repair mechanisms and elicited DNA damage through an unknown mechanism independent of phosphatase inhibition [3]. In any case, a number of these cell-permeable compounds are now commercially available and, by enhancing cellular protein phosphorylation, have become valuable experimental tools in elucidating the physiological functions of phosphoproteins. Some compounds contain a phosphate moiety and yet others possess an acidic residue, which serves as a phosphomimetic and directly interacts with the metals in the catalytic site of serine/threonine phosphatases [4]. Although compounds like okadaic acid and calyculin-A show differing selectivity for the inhibition of protein serine/ threonine phosphatases (e.g., type 1 versus type 2) at the 699
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exposures that promote cell growth and transformation, they most likely inhibit as many as five of the seven families of mammalian serine/threonine phosphatases to elicit their broad effects on cell physiology. By comparison, the deletion of individual genes encoding protein serine/threonine phosphatases in the model eukaryotes (yeast and flies) suggests that several phosphatases play critical and nonoverlapping roles in eukaryotic biology such that the loss of function of any of these enzymes inhibits cell growth and reduces cell viability [5]. In this regard, the enhanced growth and transformation of mammalian cells elicited by xenobiotics most likely reflects an incomplete or partial inhibition of protein phosphatases that increases or prolongs the phosphorylation of proteins involved in cell division and growth. However, higher concentrations of these compounds associated with greater inhibition of cellular phosphatases are generally cytotoxic, severely impairing cell growth and often inducing programmed cell death. Due to their broad actions, these compounds have not provided a clear-cut link between the inhibition of individual protein phosphatases and the development of human disease. However, it is also worth stressing that compounds like fostriecin, which inhibits several type 2 phosphatases but has no discernable activity against type 1 enzymes, have been extensively analyzed as potential anticancer drugs [6]. Indeed, the two natural products, cyclosporin A and FK506, are known to be potent and selective inhibitors of calcineurin (protein phosphatase-2B), and are among the most effective immunosuppressive drugs that are widely used to prevent rejection of transplanted organs [7]. These drugs are also clinically approved for the treatment of psoriasis, rheumatoid arthritis, aplastic anemia, nephritic syndrome, and atopic dermatitis, and the list may soon be extended to include other autoimmune diseases. However, these drugs are costly and have some serious side effects. For example, the inhibition of calcineurin has also been implicated in their renal toxicity. Thus, the potential of natural products to be either cytotoxic or valuable therapeutic agents may depend on their phosphatase specificity, degree of enzyme inhibition and physiological roles of the targeted enzymes in mammalian tissues. The availability of natural product inhibitors has also provided scientists with valuable tools to affinity-isolate and -analyze a wide variety of cellular phosphatase complexes [8].
New insights in cellular phosphatase inhibitors As reviewed in Chapter 105, endogenous inhibitor proteins are much more selective than the environmental toxins discussed above, targeting selected members of the protein serine/threonine phosphatase family and, at the highest concentrations found in mammalian tissues, having little or no effect on other phosphatases [9]. The largest group of
PART | II Transmission: Effectors and Cytosolic Events
known mammalian protein inhibitors targets type 1 phosphatases (PP1). An emerging theme from studies of PP1 inhibitors is that they can exist in unique cellular complexes that contain not only the PP1 catalytic subunit but also selected regulatory or targeting subunits that dictate PP1’s localization and substrate specificity. Thus, some PP1 inhibitors appear to target highly specific physiological events. The first suggestion that cells utilized PP1 inhibitors to target certain physiological events came from the finding that inhibitor-1 (I-1), a PKA-activated PP1 inhibitor, bound both PP1 and its regulator, GADD34. Later experiments showed that the I-1 C-terminus, which directs its interaction with GADD34, was essential for the efficient transduction of cyclic AMP signals that inhibit PP1 activity and promote the phosphorylation of the eukaryotic translation initiation factor, eIF2, a substrate of the GADD34/PP1 complex [10]. This raised the possibility that the heterotrimeric complex of PP1/GADD34/I-1 tranduces hormonal signals that inhibit protein translation in some mammalian tissues. Moreover, the region of I-1 mRNA encoding its Cterminus that binds GADD34 is alternately spliced, and is also not conserved in protein products of other predicted human genes encoding I-1 isoforms. This also suggests that mammalian tissues may contain multiple I-1 isoforms, only one of which regulates protein translation, while the other I-1 polypeptides transduce cAMP signals that control other physiological events [11]. It is also noteworthy that I-1 expression and alternate splicing may be developmentally controlled, peaking after birth to reach much lower steadystate levels in many adult tissues. The recent co-crystallization of PP1 with inhibitor-2 (I-2) provided new insights into the mechanism by which this inhibitor protein regulates PP1 activity [12]. Acute modulation of PP1 most likely occurs by I-2’s binding at an allosteric site shared with many PP1 inhibitors, including I1, which lies some distance from the PP1 catalytic site. In addition, I-2 may chronically suppress phosphatase activity by a direct interaction with the PP1 active site, resulting in the displacement of one of the two catalytic metals and leading to a more prolonged inactivation of PP1. These and other studies demonstrated that I-2 displays two modalities of PP1 regulation, one short-term or transient, and the other prolonged or more persistent. Following the identification of several mammalian PP1 complexes containing I-2, it was speculated that I-2 also targets selected cellular PP1 pools. Thus, I-2 may control the duration of protein phosphorylation at the actin cytoskeleton through its binding to the actin- and PP1-binding protein, neurabin [13], or at mitotic spindles through its association with the kinase, Nek2 [14]. It should be noted that the persistent inactivation of the PP1/I-2 complex is reversed in vitro by the phosphorylation of I-2 (threonine-72) by GSK-3, which also requires the prior phosphorylation of I-2 at several serines by casein kinase-II [15]. The precise physiological
Chapter | 89 Protein Serine/Threonine Phosphatase Inhibitors and Human Disease
circumstances under which GSK-3 and casein kinase-II phosphorylate I-2 and reactivate the latent PP1 complexes in mammalian cells are still unclear. Most recently, a novel complex containing the PP1 catalytic subunit, the nuclear regulatory subunit, Sds22, and inhibitor-3 (I-3) was identified in yeast and mammalian cells [16,17]. While the precise function of the PP1/ Sds22/I-3 complex remains to be investigated, the subcellular localization of this complex suggests that this complex controls nucleolar phosphorylation events. Yet other studies have shown that a PKC-activated PP1 inhibitor, CPI-17, and its structural relatives, the PHI proteins, target a PP1 catalytic subunit bound to the regulatory subunit, MYPT1. This in turn targets the heterotrimeric PP1/MYPT1/CPI-17 complex to smooth muscle myosin, and transduces signals from PKC and other kinases to enhance the calcium sensitivity of muscle contraction and elicit rapid and robust contraction of smooth muscle tissue [18]. Together, these studies begin to highlight a critical difference in the actions of endogenous phosphatase inhibitors from those of environmental toxins, distinguishing them on the basis of their selectivity for phosphatase catalytic subunits and their ability to target defined pools of phosphatases within all eukaryotic cells. Recent work shows that cytokines such as IL-6 downregulate CPI-17 expression in smooth muscle cells [19], while the structurally related PKC-activated PP1 inhibitor, KEPI, is upregulated in neurons in response to opioids [20]. Prior studies also show that levels of I-2 mRNA, and protein, as well as the localization of I-2 in the nucleus, are regulated during the cell division cycle [21]. This highlights a critical mode of modulating the signaling capacity of mammalian cells by rapid changes in cellular content and subcellular distribution of phosphatase inhibitors.
Cellular phosphatase inhibitors and human disease Reduced expression of phosphatase inhibitors and human disease A widely-held view in cell signaling is that in some tissues, under unstimulated or basal conditions, cellular protein phosphatase activity effectively antagonizes or clamps the functions of protein kinases, especially those displaying significant basal activity. This eliminates leaky or unwarranted signaling, but it also means that, following cell stimulation, intracellular signals that are transduced by these protein kinases are severely blunted or sluggish. Thus, cells require mechanisms to activate endogenous phosphatase inhibitors to remove the “brake” imposed by their target phosphatases in parallel with the activation of the protein kinases to achieve speedy and effective signal transduction. Such a “necessary” role for phosphatase inhibitors
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in hormone signaling may be limited to some tissues and processes, where speed and accuracy of signaling is vital. Thus far, the best evidence for a necessary or essential role for phosphatase inhibitors in cell signaling was obtained by the genetic deletion of mouse genes encoding I-1 and its structural homologue, DARPP-32. DARPP-32 is primarily expressed in dopaminergic neurons. The remarkable finding in the DARPP-32 null mice was a dramatic diminution or near complete loss in many aspects of dopamine (D1 receptor) signaling [22]. Thus, the mutant mice demonstrate an altered response to neurotransmitters and drugs of abuse as well as a variety of other behavioral defects [23]. This led to the hypothesis that a loss of DARPP-32 function may also contribute to human neurological disease, specifically schizophrenia. Indeed, several studies reported reduced levels of DARPP-32 protein in brain samples from individuals with schizophrenia [24]. However, analysis of the human DARPP-32 gene in the post mortem brain samples provided no insight into the molecular mechanism underlying DARPP-32 reduction in schizophrenia patients, and a role for DARPP-32 deficiency in human neurological disease remains to be established. Interestingly, a search for candidate genes from mouse models of neurological disease identified DARPP32 as a potential disease-causing gene [25], but more work is needed to establish a clear link between DARPP-32 and bipolar disease and other neurological disorders. In contrast to DARPP-32, I-1 is widely expressed in mammalian tissues, with the highest content of I-1 protein found in brain and muscle. While the I-1 null mice showed defects in excitatory neurotransmission in some areas of the brain, no major behavioral abnormalities were noted in the mutant mice [26]. This argued for the presence of redundant mechanisms, possibly DARPP-32 or other PKAregulated PP1-binding proteins, for amplifying cAMP signaling in the mammalian brain. The I-1 null mice did, however, display defects in cardiac contractility, similar to those previously reported in transgenic mice that overexpressed PP1 catalytic subunit in the heart [27]. Indeed, levels of PP1 inhibitors, both I-1 and I-2, were diminished in experimental models of heart failure [28]. While these data pointed to changes in PP1 inhibitors as a contributing factor in heart disease, direct evidence that PP1 inhibitors improved cardiac function and alleviated or delayed heart failure was obtained from the generation of transgenic mice that overexpressed I-1 [29] or I-2 [30] in the heart. These animals showed significantly improved -adrenergic signaling and enhanced calcium cycling which results from the phosphorylation of the sarcoplasmic reticulum protein, phospholamban, a known PP1 substrate. Additional support for a specific role of I-1 in human heart disease came from the observation that I-1 levels were greatly reduced in the myocardium from failing human hearts [31]. Together, the above studies make a compelling case for I-1 as a major contributor in effective hormonal signaling and cardiac
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function in the normal human heart, and suggest that errors in I-1 expression and/or activity play an important role in heart disease.
Increased Expression of Phosphatase Inhibitors While some tissues express high levels of specific phosphatase inhibitors, no inhibitor proteins are detected in other tissues. For example, compared to muscle and brain, mammalian liver expresses very low levels of I-1. Similarly, no measurable DARRP-32 protein can be seen in most non-neural tissues, and significantly less CPI17 is expressed in tissues other than smooth muscle. The transcriptional mechanisms that dictate the tissue-specific expression of phosphatase inhibitors are just being understood. In addition, there are undoubtedly mechanisms that can also downregulate these proteins. Thus, it comes as no surprise that errors in the regulatory processes that control the phosphatase inhibitors can result in their aberrant expression, and this in turn may contribute to the pathophysiology of many human diseases. Indeed, a growing number of publications have linked an elevated expression of I-1 with human hepatic cancers [32], DARPP-32 overexpression in gastric, esophageal, gastrointestinal, and colorectal cancers [33,34], and I-2 in prostrate cancer [35,36]. As the tumor-promoting activity of environmental toxins that inhibit protein serine/threonine phosphatases is well documented, the development of cancer associated with abnormally elevated expression of phosphatase inhibitors simply points to the ability of protein serine/threonine phosphatases to regulate critical events in the mammalian cell division cycle and phosphorylation of substrates that promote cell proliferation. For example, the PP1/MYPT1 complex, which dephosphorylates smooth muscle myosin, also targets members of the ERM family of actin-binding proteins, including merlin, the product of the human neurofibromatosis NF2 gene. By inhibiting the PP1/MYPT1 complex in mammalian cells, CPI-17 can elevate merlin phosphorylation and lead to activation of the oncogene, Ras, and cell transformation [37]. Elevated expression of the putative PP2A inhibitor, SET, has also been linked with cell growth and transformation, but direct evidence has not been obtained to show that PP2A activity is inhibited in human cancers [38]. However, emerging studies show that a growing number of cell-cycle regulators, such as Cdc25, are subject to regulation by dephosphorylation events catalyzed by both PP1 and PP2A [39,40]. In conclusion, the overall concept that abnormal levels of PP1 inhibitors contribute to rapid growth and metastasis of human cancers is perhaps not surprising. However, the cause-or-effect relationship between cellular changes in phosphatase inhibitors and the disease process needs further clarification.
PART | II Transmission: Effectors and Cytosolic Events
The product of the Down Syndrome Critical Region 1 or DSCR1 gene (also known as Adapt78, a gene induced by oxidative stress) is elevated in Down syndrome patients, who also display neural pathology similar to that of early onset Alzheimer’s disease [41]. Studies of post mortem tissue from Alzheimer’s patients also showed that increased expression of DSCR1 correlated with the severity of disease and the abundance of neurofibrillar tangles. DSCR1 encodes RCAN1 (regulator of calcineurin 1), a protein previously termed calcipressin 1, Rcn1 (the yeast regulator of calcineurin) and MCIP1 (myocyte-enriched calcineurin-interacting protein), and is highly conserved in all eukaryotes. RCAN proteins share a signature sequence also found in NFAT (nuclear factor of activated T cells), the calcineurin substrate that directly binds the phosphatase. Biochemical and genetic studies indicate some similarities between calcineurin regulation by RCAN1 and PP1 regulation by I-2. For example, several factors, including oxidative stress, elevated intracellular calcium, and amyloid (A) peptide induce RCAN1 expression. RCAN1 binds and inhibits calcineurin or PP2B activity, specifically towards the substrate, NFAT. Like I-2, RCAN1 may also function as a possible chaperone to elevate cellular functions of its target phosphatase, and, like I-2, phosphatase inhibition by RCAN1 appears to be subject to regulation by GSK-3-mediated phosphorylation [42]. While RCAN1 and calcineurin are highly expressed in the brain, calcium and calcineurin also play a critical role in cardiac output and promote cardiac hypertrophy. Genetic studies in mice suggest that excessive NFAT dephosphorylation by calcineurin promotes cardiac hypertrophy, and transgenic mice with elevated RCAN1 levels in the heart tissue are protected from hypertrophy [43]. However, these animals show defects in heart valve formation. Thus, either reduced inhibition of calcineurin or excessive levels of its inhibitor, RCAN1, may contribute to different aspects of cardiac disease. Other studies suggest that that aberrant RCAN1 function may also contribute to diabetes, immunological diseases, and skin disorders. However, more work is needed to establish the relevance of these findings to human disease.
Concluding remarks The availability of protein and non-protein inhibitors of protein serine/threonine phosphatases has provided researchers with an extensive toolkit to study the role of phosphatases and phosphatase inhibitors in modulating the mammalian physiology. Rapid progress is also being made in the understanding of cellular phosphatase complexes and their functions. In addition, the mechanisms that regulate the expression and activity of phosphatase inhibitors and their mode of action in controlling specific pools of cellular protein phosphatases are being actively investigated. Many studies have correlated changes in phosphatase inhibitors with human diseases, including cancer,
Chapter | 89 Protein Serine/Threonine Phosphatase Inhibitors and Human Disease
Alzheimer’s, and cardiomyopathy. Thus, it is anticipated that it will not be long before experimental evidence clearly demonstrates the mechanism or mode of action of phosphatase inhibitors in the genesis and progression of human disease.
References 1. Bialojan C, Takai A. Inhibitory effects of marine sponge toxin on protein phosphatases : specificity and kinetics. Biochem J 1988;256:283–90. 2. Herfindal L, Selheim F. Microcystin produces disparate effects on liver cells in a dose dependent manner. Mini Rev Med Chem 2006;6:279–85. 3. Nakagama H, Kaneko S, Shima H, Inamori H, Fukuda H, Kominami R, Sugimura T, Nagao M. Induction of minisatellite mutation in NIH3T3 cells by treatment with the tumor promoter okadaic acid. Proc Natl Acad Sci USA 1997;94:10,813–10,816. 4. Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN. Molecular enzymology underlying regulation of protein phosphatase1 by natural toxins. Curr Med Chem 2002;9:1981–9. 5. Stark MJ, Black S, Sneddon AA, Andrews PD. Genetic analyses of yeast protein serine/threonine phosphatases. FEMS Microbiol Letts 1994;117:121–30. 6. Roberge M, Tudan C, Hung SMF, Harder KW, Jirik FR, Anderson H. Antitumor drug Fostriecin inhibits the mitotic entry checkpoint and protein phosphatases 1 and 2 A. Cancer Res 1994;54:6115–21. 7. Keown PA. Emerging indications for the use of cyclosporin in organ transplantation and autoimmunity. Drugs 1990;40:315–25. 8. Moorhead G, MacKintosh RW, Morrice N, Gallagher T, Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography. FEBS Letts 1994;356:46–50. 9. Oliver CJ, Shenolikar S. Physiological importance of protein phosphatase inhibitors. Front Biosci 1998;3:961–72. 10. Brush MH, Weiser DC, Shenolikar S. The growth arrest and DNA-damage-inducible gene product, GADD34, targets protein phosphatase-1 to endoplasmic reticulum and promotes the dephosphorylation of -subunit of the eukaryotic translation initiation factor 2. Mol Cell Biol 2003;23:1292–303. 11. Weiser DC, Sikes S, Li S, Shenolikar S. Inhibitor-1 C-terminus facilitates hormonal regulation of cellular protein phosphatase1: functional implications for inhibitor-1 isoforms. J Biol Chem 2004;279:48,904–48,914. 12. Hurley TD, Yang J, Zhang L, Goodwin KD, Zou Q, Cortese M, Dunker AK, DePaoli-Roach AA. Structural basis for regulation of protein phosphatase 1 by inhibitor-2. J Biol Chem 2007;282:28,874–28,883. 13. Eto M, Elliott E, Prickett TD, Brautigan DL. Inhibitor-2 regulates protein phosphatase-1 complexed with NimA-related kinase to induce centrosome separation. J Biol Chem 2002;277:44,013–44,020. 14. Terry-Lorenzo RT, Elliot E, Weiser DC, Prickett TD, Brautigan DL, Shenolikar S. Neurabins recruit protein phosphatase-1 and inhibitor-2 to actin cytoskeleton. J Biol Chem 2002;277:46,535–46,543. 15. DePaoli-Roach AA. Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by Fa/GSK-3 and casein kinase II (PC0.7). J Biol Chem 1984;259:12,144–12,152. 16. Pedelini L, Marquina M, Joaquin Ariño J, Casamayor A, Sanz L, Bollen M, Sanz P, Garcia-Gimeno MA. YPI1 and SDS22 proteins
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regulate the nuclear localization and function of yeast type 1 phosphatase Glc7. J Biol Chem 2007;282:3282–92. 17. Lesage B, Beullens M, Pedelini L, Garcia-Gimeno MA, Waelkens E, Sanz P, Bollen M. A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3. Biochemistry 2007;46:8909–19. 18. Ito M, Nakano T, Erdodi F, Hartshorne DJ. Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 2004;259:197–209. 19. Ohama T, Hori M, Sato K, Ozaki H, Karaki H. Chronic treatment with interleukin-1 attenuates contractions by decreasing the activities of CPI-17 and MYPT-1 in intestinal smooth muscle. J Biol Chem 2003;278:48,794–48,804. 20. Liu QR, Zhang PW, Zhen Q, Walther D, Wang XB, Uhl GR. KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine. J Biol Chem 2002;277:13,312–13,320. 21. Brautigan DL, Sunwoo J, Labbe JC, Fernandez A, Lamb NJ. Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction. Nature 1990;344:74–8. 22. Greengard P. The neurobiology of slow synaptic transmission. Science 2001;294:1024–30. 23. N Hiroi H, Fienberg AA, Haile CN, Alburges M, Hanson GR, Greengard P, Nestler EJ. Neuronal and behavioural abnormalities in striatal function in DARPP-32-mutant mice. Eur J Neurosci 1999;11:1114–18. 24. Manji HK, Gottesman II, Gould TD. Signal transduction and genes-to-behaviors pathways in psychiatric diseases. Science STKE 2003;2003:49. 25. Sunkin SM, Hohmann JG. Insights from spatially mapped gene expression in the mouse brain. Hum Mol Genet 2007;16:209–19. 26. Allen PB, Havlby O, Jensen V, Errington ML, Ramsay M, Chaudhry FA, Bliss TV, Storm-Mathisen J, Morris RG, Andersen P, Greengard P. Protein phosphatase-1 regulation in the induction of long-term potentiation: heterogeneous molecular mechanisms. J Neurosci 2000;20:3537–43. 27. Carr AN, Schmidt AG, Suzuki Y, del Monte F, Sato Y, Lanner C, Breeden K, Jing SL, Allen PB, Greengard P, Yatani A, Hoit BD, Grupp IL, Hajjar RJ, DePaoli-Roach AA, Kranias EG. Type-1 phosphatase, a negative regulator of cardiac function. Mol Cell Biol 2002;22:4124–35. 28. Gupta RC, Mishra S, Yang XP, Sabbah HN. Reduced inhibitor-1 and -2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats. Mol Cell Biochem 2005;269:49–57. 29. Pathak A, del Monte F, Zhao W, Schultz JE, Lorenz JN, Bodi I, Weiser D, Hahn H, Carr AN, Syed F, Mavila N, Jha L, Mareez Y, Chen G, McGraw DW, Heist EK, Guerrero L, DePaoli-Roach AA, Hajjar RJ, Kranias EG. Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase1. Circ Res 2005;96:756–66. 30. Kirchhefer U, Baba HA, Boknik P, Breeden KM, Mavila N, Bruchert N, Justus I, Matus M, Schmitz W, DePaoli-Roach AA, Neumann J. Enhanced cardiac function in mice overexpressing protein phosphatase inhibitor-2. Cardiovasc Res 2005;68:98–108. 31. El-Armouche A, Pamminger T, Ditz D, Zolk O, Eschenhagen T. Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts. Cardiovasc Res 2004;61:87–93. 32. Aleem E, Flohr T, Thielman HW, Bannasch P, Mayer D. Protein phosphatase inhibitor-1 mRNA expression correlates with neoplastic transformation in epithelial liver cells and progression of hepatocellular carcinomas. Intl J Oncol 2004;24:869–77.
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33. El-Rafai W, Smith MF, Li G, Beckler A, Carl VS, Montgomery E, Knuutila S, Moskaluk CA, Frierson HF, Powell SM. Gastric cancers overexpress DARPP-32 and a novel isoform, t-DARPP. Cancer Res 2002;62:4061–4. 34. Wang MS, Pan Y, Liu N, Guo C, Hong L, Fan D. Overexpression of DARPP-32 in colorectal adenocarcinoma. Intl J Clin Pract 2005;59:58–61. 35. Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K, Pienta KJ, Rubin MA, Chinnaiyan AM. Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412:822–6. 36. Lapointe J, Li C, Higgins JP, van de Rijn M, Bair E, Montgomery K, Ferrari M, Egevad L, Rayford W, Bergerheim U, Ekman P, DeMarzo AM, Tibshirani R, Botstein D, Brown PO, Brooks JD, Pollack JR. Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci USA 2004;101:811–16. 37. Jin H, Sperka T, Herrlich P, Morrison H. Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase. Nature 2006;442:576–9. 38. Li M, Makkinje A, Damuni Z. The myeloid leukemia-associated protein, SET is a potent inhibitor of protein phosphatase-2A. J Biol Chem 1996;271:11,059–11,062,.
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39. Margolis SS, Walsh S, Weiser DC, Yoshida M, Shenolikar S, Kornbluth S. PP1 control of M-phase entry exerted through 14-3-3regulated Cdc25 dephosphorylation. EMBO J 2003;22:5734–45. 40. Margolis SS, Perry J, Forester C, Nutt LK, Guo X, Jardim MJ, Thomenius MJ, Freel CD, Darbandi R, Ahn JH, Aroyo JD, Wang XF, Shenolikar S, Nairn AC, Dunphy WG, Han WC, Virshup DM, Kornbluth S. Role for the PP2A/B56?phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 2006;127: 759–73. 41. Harris CD, Ermak G, Davies KJA. Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product Calcipressin 1 (or RCAN1) in disease. Cell Mol Life Sci 2005;62:2477–86. 42. Hilioti Z, Gallagher DA, Low-Nam ST, Ramaswamy P, Gajer P, Kingsbury TJ, Birchwood CJ, Levchenko A, Cunningham KW. GSK3 kinases enhance calcineurin signaling by phosphorylation of RCNs. Genes Dev 2004;18:35–47. 43. Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P, Yang J, Antos CL, Shelton JM, Bassel-Duby R, Olson EN, Williams RS. Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci USA 2001;98:3328–33.
Protein Serine/Threonine Phosphatase Inhibitors and Human Disease Shirish Shenolikar 1,2 and Matthew H. Brush1 1
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School Singapore.
2
Introduction Coordinating the activity of protein kinases and phosphatases allows cells to amplify and accelerate hormonal signals and elicit a robust and decisive physiological response. Hormonal signals have been shown to modulate the expression or activity of endogenous phosphatase inhibitor proteins (described in Chapter 105) that dampen the functions of specific protein phosphatases and enhance the phosphorylation of their cellular substrates. This paradigm is best exemplified by protein serine/threonine phosphatases, many of which are targeted by an increasing number of endogenous protein inhibitors. By suppressing the functions of the relatively few phosphatase catalytic subunits that catalyze serine/threonine dephosphorylation in eukaryotic cells, these inhibitor proteins have the potential to modulate a broad range of physiological events and orchestrate an integrated physiological response that coordinates such diverse processes as metabolism, migration, gene transcription, and growth. Emerging studies suggest that some phosphatase inhibitors physically associate with cellular phosphatase complexes comprised of a catalytic subunit bound to one or more regulatory proteins that restrict the actions of the phosphatases to control specific cellular events. The functions of these phosphatase inhibitors are in turn controlled by their expression, reversible phosphorylation, and dynamic association with target protein phosphatases. In this manner, endogenous serine/threonine phosphatase inhibitors achieve both spatial and temporal control of phosphoproteins that regulate normal cell physiology, and growing evidence suggests that aberrant expression and/or activity of phosphatase inhibitors may be associated with many human diseases. This chapter focuses on new information on the mode of action of phosphatase inhibitors and discusses their potential contributions to the pathophysiology of human disease. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
Environmental toxins as phosphatase inhibitors Okadaic acid, responsible for human diarrheretic shellfish poisoning, was first identified as a toxin concentrated by marine sponges, and functions as a potent inhibitor of several eukaryotic protein serine/threonine phosphatases. By inhibiting one or more phosphatases, okadaic acid enhances smooth muscle myosin phosphorylation and the contractility of the gut [1]. Subsequent studies identified cyclic peptides like microcystin and nodularin in freshwater ponds and lakes infested with cyanobacteria, which cause severe hepatotoxicity that is often lethal to animals [2]. Like okadaic acid, these compounds also inhibited the major mammalian serine/threonine phosphatases. To date, more than a dozen environmental toxins or natural products have been shown to inhibit protein serine/threonine phosphatases. Almost all of these compounds have the potential to promote cell growth and transformation, and induce a variety of cancers in experimental animals. However, at least one report hinted at other cellular actions of these toxins, specifically that okadaic acid impaired DNA repair mechanisms and elicited DNA damage through an unknown mechanism independent of phosphatase inhibition [3]. In any case, a number of these cell-permeable compounds are now commercially available and, by enhancing cellular protein phosphorylation, have become valuable experimental tools in elucidating the physiological functions of phosphoproteins. Some compounds contain a phosphate moiety and yet others possess an acidic residue, which serves as a phosphomimetic and directly interacts with the metals in the catalytic site of serine/threonine phosphatases [4]. Although compounds like okadaic acid and calyculin-A show differing selectivity for the inhibition of protein serine/ threonine phosphatases (e.g., type 1 versus type 2) at the 699
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exposures that promote cell growth and transformation, they most likely inhibit as many as five of the seven families of mammalian serine/threonine phosphatases to elicit their broad effects on cell physiology. By comparison, the deletion of individual genes encoding protein serine/threonine phosphatases in the model eukaryotes (yeast and flies) suggests that several phosphatases play critical and nonoverlapping roles in eukaryotic biology such that the loss of function of any of these enzymes inhibits cell growth and reduces cell viability [5]. In this regard, the enhanced growth and transformation of mammalian cells elicited by xenobiotics most likely reflects an incomplete or partial inhibition of protein phosphatases that increases or prolongs the phosphorylation of proteins involved in cell division and growth. However, higher concentrations of these compounds associated with greater inhibition of cellular phosphatases are generally cytotoxic, severely impairing cell growth and often inducing programmed cell death. Due to their broad actions, these compounds have not provided a clear-cut link between the inhibition of individual protein phosphatases and the development of human disease. However, it is also worth stressing that compounds like fostriecin, which inhibits several type 2 phosphatases but has no discernable activity against type 1 enzymes, have been extensively analyzed as potential anticancer drugs [6]. Indeed, the two natural products, cyclosporin A and FK506, are known to be potent and selective inhibitors of calcineurin (protein phosphatase-2B), and are among the most effective immunosuppressive drugs that are widely used to prevent rejection of transplanted organs [7]. These drugs are also clinically approved for the treatment of psoriasis, rheumatoid arthritis, aplastic anemia, nephritic syndrome, and atopic dermatitis, and the list may soon be extended to include other autoimmune diseases. However, these drugs are costly and have some serious side effects. For example, the inhibition of calcineurin has also been implicated in their renal toxicity. Thus, the potential of natural products to be either cytotoxic or valuable therapeutic agents may depend on their phosphatase specificity, degree of enzyme inhibition and physiological roles of the targeted enzymes in mammalian tissues. The availability of natural product inhibitors has also provided scientists with valuable tools to affinity-isolate and -analyze a wide variety of cellular phosphatase complexes [8].
New insights in cellular phosphatase inhibitors As reviewed in Chapter 105, endogenous inhibitor proteins are much more selective than the environmental toxins discussed above, targeting selected members of the protein serine/threonine phosphatase family and, at the highest concentrations found in mammalian tissues, having little or no effect on other phosphatases [9]. The largest group of
PART | II Transmission: Effectors and Cytosolic Events
known mammalian protein inhibitors targets type 1 phosphatases (PP1). An emerging theme from studies of PP1 inhibitors is that they can exist in unique cellular complexes that contain not only the PP1 catalytic subunit but also selected regulatory or targeting subunits that dictate PP1’s localization and substrate specificity. Thus, some PP1 inhibitors appear to target highly specific physiological events. The first suggestion that cells utilized PP1 inhibitors to target certain physiological events came from the finding that inhibitor-1 (I-1), a PKA-activated PP1 inhibitor, bound both PP1 and its regulator, GADD34. Later experiments showed that the I-1 C-terminus, which directs its interaction with GADD34, was essential for the efficient transduction of cyclic AMP signals that inhibit PP1 activity and promote the phosphorylation of the eukaryotic translation initiation factor, eIF2, a substrate of the GADD34/PP1 complex [10]. This raised the possibility that the heterotrimeric complex of PP1/GADD34/I-1 tranduces hormonal signals that inhibit protein translation in some mammalian tissues. Moreover, the region of I-1 mRNA encoding its Cterminus that binds GADD34 is alternately spliced, and is also not conserved in protein products of other predicted human genes encoding I-1 isoforms. This also suggests that mammalian tissues may contain multiple I-1 isoforms, only one of which regulates protein translation, while the other I-1 polypeptides transduce cAMP signals that control other physiological events [11]. It is also noteworthy that I-1 expression and alternate splicing may be developmentally controlled, peaking after birth to reach much lower steadystate levels in many adult tissues. The recent co-crystallization of PP1 with inhibitor-2 (I-2) provided new insights into the mechanism by which this inhibitor protein regulates PP1 activity [12]. Acute modulation of PP1 most likely occurs by I-2’s binding at an allosteric site shared with many PP1 inhibitors, including I1, which lies some distance from the PP1 catalytic site. In addition, I-2 may chronically suppress phosphatase activity by a direct interaction with the PP1 active site, resulting in the displacement of one of the two catalytic metals and leading to a more prolonged inactivation of PP1. These and other studies demonstrated that I-2 displays two modalities of PP1 regulation, one short-term or transient, and the other prolonged or more persistent. Following the identification of several mammalian PP1 complexes containing I-2, it was speculated that I-2 also targets selected cellular PP1 pools. Thus, I-2 may control the duration of protein phosphorylation at the actin cytoskeleton through its binding to the actin- and PP1-binding protein, neurabin [13], or at mitotic spindles through its association with the kinase, Nek2 [14]. It should be noted that the persistent inactivation of the PP1/I-2 complex is reversed in vitro by the phosphorylation of I-2 (threonine-72) by GSK-3, which also requires the prior phosphorylation of I-2 at several serines by casein kinase-II [15]. The precise physiological
Chapter | 89 Protein Serine/Threonine Phosphatase Inhibitors and Human Disease
circumstances under which GSK-3 and casein kinase-II phosphorylate I-2 and reactivate the latent PP1 complexes in mammalian cells are still unclear. Most recently, a novel complex containing the PP1 catalytic subunit, the nuclear regulatory subunit, Sds22, and inhibitor-3 (I-3) was identified in yeast and mammalian cells [16,17]. While the precise function of the PP1/ Sds22/I-3 complex remains to be investigated, the subcellular localization of this complex suggests that this complex controls nucleolar phosphorylation events. Yet other studies have shown that a PKC-activated PP1 inhibitor, CPI-17, and its structural relatives, the PHI proteins, target a PP1 catalytic subunit bound to the regulatory subunit, MYPT1. This in turn targets the heterotrimeric PP1/MYPT1/CPI-17 complex to smooth muscle myosin, and transduces signals from PKC and other kinases to enhance the calcium sensitivity of muscle contraction and elicit rapid and robust contraction of smooth muscle tissue [18]. Together, these studies begin to highlight a critical difference in the actions of endogenous phosphatase inhibitors from those of environmental toxins, distinguishing them on the basis of their selectivity for phosphatase catalytic subunits and their ability to target defined pools of phosphatases within all eukaryotic cells. Recent work shows that cytokines such as IL-6 downregulate CPI-17 expression in smooth muscle cells [19], while the structurally related PKC-activated PP1 inhibitor, KEPI, is upregulated in neurons in response to opioids [20]. Prior studies also show that levels of I-2 mRNA, and protein, as well as the localization of I-2 in the nucleus, are regulated during the cell division cycle [21]. This highlights a critical mode of modulating the signaling capacity of mammalian cells by rapid changes in cellular content and subcellular distribution of phosphatase inhibitors.
Cellular phosphatase inhibitors and human disease Reduced expression of phosphatase inhibitors and human disease A widely-held view in cell signaling is that in some tissues, under unstimulated or basal conditions, cellular protein phosphatase activity effectively antagonizes or clamps the functions of protein kinases, especially those displaying significant basal activity. This eliminates leaky or unwarranted signaling, but it also means that, following cell stimulation, intracellular signals that are transduced by these protein kinases are severely blunted or sluggish. Thus, cells require mechanisms to activate endogenous phosphatase inhibitors to remove the “brake” imposed by their target phosphatases in parallel with the activation of the protein kinases to achieve speedy and effective signal transduction. Such a “necessary” role for phosphatase inhibitors
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in hormone signaling may be limited to some tissues and processes, where speed and accuracy of signaling is vital. Thus far, the best evidence for a necessary or essential role for phosphatase inhibitors in cell signaling was obtained by the genetic deletion of mouse genes encoding I-1 and its structural homologue, DARPP-32. DARPP-32 is primarily expressed in dopaminergic neurons. The remarkable finding in the DARPP-32 null mice was a dramatic diminution or near complete loss in many aspects of dopamine (D1 receptor) signaling [22]. Thus, the mutant mice demonstrate an altered response to neurotransmitters and drugs of abuse as well as a variety of other behavioral defects [23]. This led to the hypothesis that a loss of DARPP-32 function may also contribute to human neurological disease, specifically schizophrenia. Indeed, several studies reported reduced levels of DARPP-32 protein in brain samples from individuals with schizophrenia [24]. However, analysis of the human DARPP-32 gene in the post mortem brain samples provided no insight into the molecular mechanism underlying DARPP-32 reduction in schizophrenia patients, and a role for DARPP-32 deficiency in human neurological disease remains to be established. Interestingly, a search for candidate genes from mouse models of neurological disease identified DARPP32 as a potential disease-causing gene [25], but more work is needed to establish a clear link between DARPP-32 and bipolar disease and other neurological disorders. In contrast to DARPP-32, I-1 is widely expressed in mammalian tissues, with the highest content of I-1 protein found in brain and muscle. While the I-1 null mice showed defects in excitatory neurotransmission in some areas of the brain, no major behavioral abnormalities were noted in the mutant mice [26]. This argued for the presence of redundant mechanisms, possibly DARPP-32 or other PKAregulated PP1-binding proteins, for amplifying cAMP signaling in the mammalian brain. The I-1 null mice did, however, display defects in cardiac contractility, similar to those previously reported in transgenic mice that overexpressed PP1 catalytic subunit in the heart [27]. Indeed, levels of PP1 inhibitors, both I-1 and I-2, were diminished in experimental models of heart failure [28]. While these data pointed to changes in PP1 inhibitors as a contributing factor in heart disease, direct evidence that PP1 inhibitors improved cardiac function and alleviated or delayed heart failure was obtained from the generation of transgenic mice that overexpressed I-1 [29] or I-2 [30] in the heart. These animals showed significantly improved -adrenergic signaling and enhanced calcium cycling which results from the phosphorylation of the sarcoplasmic reticulum protein, phospholamban, a known PP1 substrate. Additional support for a specific role of I-1 in human heart disease came from the observation that I-1 levels were greatly reduced in the myocardium from failing human hearts [31]. Together, the above studies make a compelling case for I-1 as a major contributor in effective hormonal signaling and cardiac
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function in the normal human heart, and suggest that errors in I-1 expression and/or activity play an important role in heart disease.
Increased Expression of Phosphatase Inhibitors While some tissues express high levels of specific phosphatase inhibitors, no inhibitor proteins are detected in other tissues. For example, compared to muscle and brain, mammalian liver expresses very low levels of I-1. Similarly, no measurable DARRP-32 protein can be seen in most non-neural tissues, and significantly less CPI17 is expressed in tissues other than smooth muscle. The transcriptional mechanisms that dictate the tissue-specific expression of phosphatase inhibitors are just being understood. In addition, there are undoubtedly mechanisms that can also downregulate these proteins. Thus, it comes as no surprise that errors in the regulatory processes that control the phosphatase inhibitors can result in their aberrant expression, and this in turn may contribute to the pathophysiology of many human diseases. Indeed, a growing number of publications have linked an elevated expression of I-1 with human hepatic cancers [32], DARPP-32 overexpression in gastric, esophageal, gastrointestinal, and colorectal cancers [33,34], and I-2 in prostrate cancer [35,36]. As the tumor-promoting activity of environmental toxins that inhibit protein serine/threonine phosphatases is well documented, the development of cancer associated with abnormally elevated expression of phosphatase inhibitors simply points to the ability of protein serine/threonine phosphatases to regulate critical events in the mammalian cell division cycle and phosphorylation of substrates that promote cell proliferation. For example, the PP1/MYPT1 complex, which dephosphorylates smooth muscle myosin, also targets members of the ERM family of actin-binding proteins, including merlin, the product of the human neurofibromatosis NF2 gene. By inhibiting the PP1/MYPT1 complex in mammalian cells, CPI-17 can elevate merlin phosphorylation and lead to activation of the oncogene, Ras, and cell transformation [37]. Elevated expression of the putative PP2A inhibitor, SET, has also been linked with cell growth and transformation, but direct evidence has not been obtained to show that PP2A activity is inhibited in human cancers [38]. However, emerging studies show that a growing number of cell-cycle regulators, such as Cdc25, are subject to regulation by dephosphorylation events catalyzed by both PP1 and PP2A [39,40]. In conclusion, the overall concept that abnormal levels of PP1 inhibitors contribute to rapid growth and metastasis of human cancers is perhaps not surprising. However, the cause-or-effect relationship between cellular changes in phosphatase inhibitors and the disease process needs further clarification.
PART | II Transmission: Effectors and Cytosolic Events
The product of the Down Syndrome Critical Region 1 or DSCR1 gene (also known as Adapt78, a gene induced by oxidative stress) is elevated in Down syndrome patients, who also display neural pathology similar to that of early onset Alzheimer’s disease [41]. Studies of post mortem tissue from Alzheimer’s patients also showed that increased expression of DSCR1 correlated with the severity of disease and the abundance of neurofibrillar tangles. DSCR1 encodes RCAN1 (regulator of calcineurin 1), a protein previously termed calcipressin 1, Rcn1 (the yeast regulator of calcineurin) and MCIP1 (myocyte-enriched calcineurin-interacting protein), and is highly conserved in all eukaryotes. RCAN proteins share a signature sequence also found in NFAT (nuclear factor of activated T cells), the calcineurin substrate that directly binds the phosphatase. Biochemical and genetic studies indicate some similarities between calcineurin regulation by RCAN1 and PP1 regulation by I-2. For example, several factors, including oxidative stress, elevated intracellular calcium, and amyloid (A) peptide induce RCAN1 expression. RCAN1 binds and inhibits calcineurin or PP2B activity, specifically towards the substrate, NFAT. Like I-2, RCAN1 may also function as a possible chaperone to elevate cellular functions of its target phosphatase, and, like I-2, phosphatase inhibition by RCAN1 appears to be subject to regulation by GSK-3-mediated phosphorylation [42]. While RCAN1 and calcineurin are highly expressed in the brain, calcium and calcineurin also play a critical role in cardiac output and promote cardiac hypertrophy. Genetic studies in mice suggest that excessive NFAT dephosphorylation by calcineurin promotes cardiac hypertrophy, and transgenic mice with elevated RCAN1 levels in the heart tissue are protected from hypertrophy [43]. However, these animals show defects in heart valve formation. Thus, either reduced inhibition of calcineurin or excessive levels of its inhibitor, RCAN1, may contribute to different aspects of cardiac disease. Other studies suggest that that aberrant RCAN1 function may also contribute to diabetes, immunological diseases, and skin disorders. However, more work is needed to establish the relevance of these findings to human disease.
Concluding remarks The availability of protein and non-protein inhibitors of protein serine/threonine phosphatases has provided researchers with an extensive toolkit to study the role of phosphatases and phosphatase inhibitors in modulating the mammalian physiology. Rapid progress is also being made in the understanding of cellular phosphatase complexes and their functions. In addition, the mechanisms that regulate the expression and activity of phosphatase inhibitors and their mode of action in controlling specific pools of cellular protein phosphatases are being actively investigated. Many studies have correlated changes in phosphatase inhibitors with human diseases, including cancer,
Chapter | 89 Protein Serine/Threonine Phosphatase Inhibitors and Human Disease
Alzheimer’s, and cardiomyopathy. Thus, it is anticipated that it will not be long before experimental evidence clearly demonstrates the mechanism or mode of action of phosphatase inhibitors in the genesis and progression of human disease.
References 1. Bialojan C, Takai A. Inhibitory effects of marine sponge toxin on protein phosphatases : specificity and kinetics. Biochem J 1988;256:283–90. 2. Herfindal L, Selheim F. Microcystin produces disparate effects on liver cells in a dose dependent manner. Mini Rev Med Chem 2006;6:279–85. 3. Nakagama H, Kaneko S, Shima H, Inamori H, Fukuda H, Kominami R, Sugimura T, Nagao M. Induction of minisatellite mutation in NIH3T3 cells by treatment with the tumor promoter okadaic acid. Proc Natl Acad Sci USA 1997;94:10,813–10,816. 4. Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN. Molecular enzymology underlying regulation of protein phosphatase1 by natural toxins. Curr Med Chem 2002;9:1981–9. 5. Stark MJ, Black S, Sneddon AA, Andrews PD. Genetic analyses of yeast protein serine/threonine phosphatases. FEMS Microbiol Letts 1994;117:121–30. 6. Roberge M, Tudan C, Hung SMF, Harder KW, Jirik FR, Anderson H. Antitumor drug Fostriecin inhibits the mitotic entry checkpoint and protein phosphatases 1 and 2 A. Cancer Res 1994;54:6115–21. 7. Keown PA. Emerging indications for the use of cyclosporin in organ transplantation and autoimmunity. Drugs 1990;40:315–25. 8. Moorhead G, MacKintosh RW, Morrice N, Gallagher T, Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography. FEBS Letts 1994;356:46–50. 9. Oliver CJ, Shenolikar S. Physiological importance of protein phosphatase inhibitors. Front Biosci 1998;3:961–72. 10. Brush MH, Weiser DC, Shenolikar S. The growth arrest and DNA-damage-inducible gene product, GADD34, targets protein phosphatase-1 to endoplasmic reticulum and promotes the dephosphorylation of -subunit of the eukaryotic translation initiation factor 2. Mol Cell Biol 2003;23:1292–303. 11. Weiser DC, Sikes S, Li S, Shenolikar S. Inhibitor-1 C-terminus facilitates hormonal regulation of cellular protein phosphatase1: functional implications for inhibitor-1 isoforms. J Biol Chem 2004;279:48,904–48,914. 12. Hurley TD, Yang J, Zhang L, Goodwin KD, Zou Q, Cortese M, Dunker AK, DePaoli-Roach AA. Structural basis for regulation of protein phosphatase 1 by inhibitor-2. J Biol Chem 2007;282:28,874–28,883. 13. Eto M, Elliott E, Prickett TD, Brautigan DL. Inhibitor-2 regulates protein phosphatase-1 complexed with NimA-related kinase to induce centrosome separation. J Biol Chem 2002;277:44,013–44,020. 14. Terry-Lorenzo RT, Elliot E, Weiser DC, Prickett TD, Brautigan DL, Shenolikar S. Neurabins recruit protein phosphatase-1 and inhibitor-2 to actin cytoskeleton. J Biol Chem 2002;277:46,535–46,543. 15. DePaoli-Roach AA. Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by Fa/GSK-3 and casein kinase II (PC0.7). J Biol Chem 1984;259:12,144–12,152. 16. Pedelini L, Marquina M, Joaquin Ariño J, Casamayor A, Sanz L, Bollen M, Sanz P, Garcia-Gimeno MA. YPI1 and SDS22 proteins
703
regulate the nuclear localization and function of yeast type 1 phosphatase Glc7. J Biol Chem 2007;282:3282–92. 17. Lesage B, Beullens M, Pedelini L, Garcia-Gimeno MA, Waelkens E, Sanz P, Bollen M. A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3. Biochemistry 2007;46:8909–19. 18. Ito M, Nakano T, Erdodi F, Hartshorne DJ. Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 2004;259:197–209. 19. Ohama T, Hori M, Sato K, Ozaki H, Karaki H. Chronic treatment with interleukin-1 attenuates contractions by decreasing the activities of CPI-17 and MYPT-1 in intestinal smooth muscle. J Biol Chem 2003;278:48,794–48,804. 20. Liu QR, Zhang PW, Zhen Q, Walther D, Wang XB, Uhl GR. KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine. J Biol Chem 2002;277:13,312–13,320. 21. Brautigan DL, Sunwoo J, Labbe JC, Fernandez A, Lamb NJ. Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction. Nature 1990;344:74–8. 22. Greengard P. The neurobiology of slow synaptic transmission. Science 2001;294:1024–30. 23. N Hiroi H, Fienberg AA, Haile CN, Alburges M, Hanson GR, Greengard P, Nestler EJ. Neuronal and behavioural abnormalities in striatal function in DARPP-32-mutant mice. Eur J Neurosci 1999;11:1114–18. 24. Manji HK, Gottesman II, Gould TD. Signal transduction and genes-to-behaviors pathways in psychiatric diseases. Science STKE 2003;2003:49. 25. Sunkin SM, Hohmann JG. Insights from spatially mapped gene expression in the mouse brain. Hum Mol Genet 2007;16:209–19. 26. Allen PB, Havlby O, Jensen V, Errington ML, Ramsay M, Chaudhry FA, Bliss TV, Storm-Mathisen J, Morris RG, Andersen P, Greengard P. Protein phosphatase-1 regulation in the induction of long-term potentiation: heterogeneous molecular mechanisms. J Neurosci 2000;20:3537–43. 27. Carr AN, Schmidt AG, Suzuki Y, del Monte F, Sato Y, Lanner C, Breeden K, Jing SL, Allen PB, Greengard P, Yatani A, Hoit BD, Grupp IL, Hajjar RJ, DePaoli-Roach AA, Kranias EG. Type-1 phosphatase, a negative regulator of cardiac function. Mol Cell Biol 2002;22:4124–35. 28. Gupta RC, Mishra S, Yang XP, Sabbah HN. Reduced inhibitor-1 and -2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats. Mol Cell Biochem 2005;269:49–57. 29. Pathak A, del Monte F, Zhao W, Schultz JE, Lorenz JN, Bodi I, Weiser D, Hahn H, Carr AN, Syed F, Mavila N, Jha L, Mareez Y, Chen G, McGraw DW, Heist EK, Guerrero L, DePaoli-Roach AA, Hajjar RJ, Kranias EG. Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase1. Circ Res 2005;96:756–66. 30. Kirchhefer U, Baba HA, Boknik P, Breeden KM, Mavila N, Bruchert N, Justus I, Matus M, Schmitz W, DePaoli-Roach AA, Neumann J. Enhanced cardiac function in mice overexpressing protein phosphatase inhibitor-2. Cardiovasc Res 2005;68:98–108. 31. El-Armouche A, Pamminger T, Ditz D, Zolk O, Eschenhagen T. Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts. Cardiovasc Res 2004;61:87–93. 32. Aleem E, Flohr T, Thielman HW, Bannasch P, Mayer D. Protein phosphatase inhibitor-1 mRNA expression correlates with neoplastic transformation in epithelial liver cells and progression of hepatocellular carcinomas. Intl J Oncol 2004;24:869–77.
704
33. El-Rafai W, Smith MF, Li G, Beckler A, Carl VS, Montgomery E, Knuutila S, Moskaluk CA, Frierson HF, Powell SM. Gastric cancers overexpress DARPP-32 and a novel isoform, t-DARPP. Cancer Res 2002;62:4061–4. 34. Wang MS, Pan Y, Liu N, Guo C, Hong L, Fan D. Overexpression of DARPP-32 in colorectal adenocarcinoma. Intl J Clin Pract 2005;59:58–61. 35. Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K, Pienta KJ, Rubin MA, Chinnaiyan AM. Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412:822–6. 36. Lapointe J, Li C, Higgins JP, van de Rijn M, Bair E, Montgomery K, Ferrari M, Egevad L, Rayford W, Bergerheim U, Ekman P, DeMarzo AM, Tibshirani R, Botstein D, Brown PO, Brooks JD, Pollack JR. Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci USA 2004;101:811–16. 37. Jin H, Sperka T, Herrlich P, Morrison H. Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase. Nature 2006;442:576–9. 38. Li M, Makkinje A, Damuni Z. The myeloid leukemia-associated protein, SET is a potent inhibitor of protein phosphatase-2A. J Biol Chem 1996;271:11,059–11,062,.
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39. Margolis SS, Walsh S, Weiser DC, Yoshida M, Shenolikar S, Kornbluth S. PP1 control of M-phase entry exerted through 14-3-3regulated Cdc25 dephosphorylation. EMBO J 2003;22:5734–45. 40. Margolis SS, Perry J, Forester C, Nutt LK, Guo X, Jardim MJ, Thomenius MJ, Freel CD, Darbandi R, Ahn JH, Aroyo JD, Wang XF, Shenolikar S, Nairn AC, Dunphy WG, Han WC, Virshup DM, Kornbluth S. Role for the PP2A/B56?phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 2006;127: 759–73. 41. Harris CD, Ermak G, Davies KJA. Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product Calcipressin 1 (or RCAN1) in disease. Cell Mol Life Sci 2005;62:2477–86. 42. Hilioti Z, Gallagher DA, Low-Nam ST, Ramaswamy P, Gajer P, Kingsbury TJ, Birchwood CJ, Levchenko A, Cunningham KW. GSK3 kinases enhance calcineurin signaling by phosphorylation of RCNs. Genes Dev 2004;18:35–47. 43. Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P, Yang J, Antos CL, Shelton JM, Bassel-Duby R, Olson EN, Williams RS. Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci USA 2001;98:3328–33.