- Email: [email protected]
threonine protein phosphatases in apoptosis
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Serine/threonine protein phosphatases in apoptosis Susanne Klumpp* and Josef Krieglstein† The importance of phosphorylation and dephosphorylation in intracellular signaling pathways has long been recognized, although attention has focused mainly on kinases. Recent studies have highlighted the importance of serine/threonine protein phosphatases in many processes including apoptosis. The phosphorylation state of antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (BAD, Bid, Bik) Bcl-2 proteins regulates their cellular activity and, therefore, cell survival and cell death. For example, dephosphorylation of BAD by the protein phosphatases PP1, PP2A and PP2B allows BAD to interact with Bcl-XL and initiate cell death. Caspases are also important in cell death and phosphorylation/dephosphorylation of caspases themselves, their targets and their regulators modulates apoptotic pathways. The activity of serine/threonine protein phosphatases needs further study, but it is clear that these enzymes are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases and neurodegeneration. Addresses *Westfälische Wilhelms-Universität, Institut für Pharmazeutische und Medizinische Chemie, Hittorfstrasse 58–62, D-48149 Münster, Germany; e-mail: [email protected] † Philipps-Universität, Institut für Pharmakologie und Toxikologie, Ketzerbach 63, D-35032 Marburg, Germany; e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:458–462 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BAD Bcl-2/Bcl-X-associated death promoter MAPK mitogen-activated protein kinase NFAT nuclear factor for activated T cells PKA cAMP-dependent protein kinase PKC Ca2+-dependent protein kinase PP1 type-1 protein phosphatase PP2 type-2 protein phosphatase PPM Mg2+-dependent protein phosphatase PPP phosphoprotein phosphatase PTP protein tyrosine phosphatase
Introduction Apoptosis is mainly regulated by the Bcl-2 family of proteins, by apoptotic protease-activating factor 1 (Apaf-1) and by the caspase family. Neurons share the same basic apoptosis program with all other cell types; however, different types and developmental stages of neurons express a different combination of these protein factors, which is one way of providing specificity and regulation. The other significant mechanism that regulates cell death is the reversible phosphorylation of proteins. The level of phosphorylation dictates the activity of these factors and, consequently, protein kinases and protein phosphatases play a pivotal role in cell viability and cell death. This review focuses on the role of dephosphorylation processes
in apoptosis. Because of space constraints it is not possible to discuss all of the phosphatases involved and we concentrate on the role of serine/threonine protein phosphatases and their regulation of Bcl-2 family proteins.
Reversible phosphorylation of proteins Reversible phosphorylation regulates almost all aspects of cell life, from classical metabolic pathways to memory and even cell death. At least one-third of human proteins contain covalently bound phosphate. The phosphorylation levels can be modulated by changes in the activities of protein kinases (enzymes that add phosphate groups to target proteins) and protein phosphatases (enzymes that hydrolyze phosphate esters and amidates). An estimated 600 protein kinases and 200 protein phosphatases are encoded by the human genome. Because a single phosphatase catalytic moiety often associates with several different regulatory or targeting subunits, the total number of functional phosphatase holoenzymes is expected to be similar to the number of protein kinases. Phosphorylation and dephosphorylation affect the function of proteins in every conceivable way [1••]. This includes increasing or decreasing enzyme activities (phospholipase C [PLC], glycogen synthase kinase 3 [GSK3]), marking a protein for destruction (inhibitor of NFκB [IκB]), allowing a protein to move from one cellular compartment to another (nuclear factor for activated T cells [NFAT]), or enabling a protein to interact with or dissociate from other proteins (Bcl-2/Bcl-X-associated death promoter [BAD]). Kinases and phosphatases may even be colocalized on the same protein but at different docking sites [2]. Yotiao is one of these adapter proteins, which are also known as AKAPs (A-kinase-anchoring proteins). Yotiao associates with the NMDA receptor — one of the ion channel receptors for the neurotransmitter glutamate — and acts as a scaffold protein that physically attaches a phosphatase (type-1 enzyme) and a protein kinase (cAMP-dependent) to the NMDA receptor to regulate the ion flow through the channel. Although protein phosphorylation and dephosphorylation may look simple at first glance — the phosphoryl donors ATP and GTP are readily available — protein phosphorylation and dephosphorylation does have finesse. First, kinases and phosphatases themselves may be subject to multiple and reversible phosphorylation (as in the mitogen-activated protein kinase [MAPK] cascades). Second, most proteins carry multiple phosphorylation sites, with corresponding kinases and phosphatases acting interdependently and hierarchically (e.g. dopamine and cAMP regulated phosphoprotein-32 [DARP-32]).
Serine/threonine protein kinases Traditionally, much attention has been focused on protein kinases. The classification of serine/threonine protein
Serine/threonine protein phosphatases in apoptosis Klumpp and Krieglstein
kinases is straightforward. If the kinase is second-messengerdependent, it is named according to the stimulus involved. For example, cAMP-dependent protein kinase is designated PKA, Ca2+-dependent protein kinase is PKC and calmodulindependent protein kinase is CaM-K. One of the few exceptions is PKB (also known as RAC and Akt), where the acronym refers to the relatedness of this kinase (the cellular homolog of the oncogenic form v-Akt) to both PKA and PKC. The nomenclature of the second-messengerindependent serine/threonine protein kinases mostly refers to specific components of the system that they regulate.
Serine/threonine protein phosphatases The variety and regulation of phosphatases has only been appreciated more recently. The major drawback in phosphatase research has been that phosphatases were originally thought not to be subject to regulation. This turned out to be wrong. Phosphatases were classified according to the phospho-amino acids they act on, simply because stimulating or inhibitory factors were unknown at the beginning. Phosphatases that dephosphorylate serine and threonine residues are encoded by the phosphoprotein phosphatase (PPP) and Mg2+-dependent protein phosphatase (PPM) gene families, which are defined by distinct amino acid sequences and crystal structures [3]. The PPP family includes the most abundant protein phosphatases — PP1, PP2A and PP2B — as well as more recently cloned enzymes such as PP4 (also known as PPX), PP5, PP6 (a functional homolog of budding yeast Sit4) and PP7. The signature phosphatases were initially divided according to biochemical parameters [4]. Type-1 protein phosphatases (PP1) are inhibited by heat-stable inhibitor proteins and preferentially dephosphorylate the β-subunit of phosphorylase kinase. In contrast, type-2 protein phosphatases (PP2) are insensitive to these inhibitors and preferentially dephosphorylate the α subunit of phosphorylase kinase. The type-2 enzymes were further subdivided into spontaneously active protein phosphatase (PP2A), Ca2+dependent protein phosphatase (PP2B, also known as calcineurin) and Mg2+-dependent protein phosphatase (PP2C). Later on, cDNA cloning revealed that PP1, PP2A and PP2B belong to the same gene family, whereas PP2C is structurally different. Five PP2C isoforms, together with the pyruvate dehydrogenase phosphatase, constitute the distinct gene family PPM. A third branch of protein phosphatases, the protein tyrosine phosphatase (PTP) superfamily — including the dual-specificity phosphatases (DSPs), which act on MAPKs — and the protein histidine phosphatases are not covered in this article. Several naturally occurring toxins are potent inhibitors of the serine/threonine protein phosphatases [5]. These include diverse structures such as okadaic acid (a fatty-acid derivative) or microcystin (a cyclic heptapeptide). These inhibitors are often used as tools to determine the involvement of PP1 and PP2A. However, one should be aware
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that some of the less abundant protein phosphatases (at least PP4, PP5 and PP6) are inhibited by these compounds in the same nanomolar concentration range as PP1 and PP2A are inhibited. PP2B is sensitive to higher (micromolar) concentrations of these inhibitors as well. In contrast, members of the PPM and PTP families are not affected by okadaic acid or microcystin.
Phosphorylation and dephosphorylation in cell death The signaling pathways that mediate neuroprotective and neurodegenerative effects are extremely complex and have been intensively investigated for the past decade. As it turns out, almost any protein molecule involved and almost every single step of signaling cascades are associated with phosphorylation and dephosphorylation processes. It starts with receptors spanning the cellular membrane, for example, transforming growth factor β (TGF-β) receptors undergo reversible phosphorylation and they are serine/threonine protein kinases themselves. Phosphorylation continues via intracellular proteins (e.g. proapoptotic and antiapoptotic Bcl-2 family proteins, caspases and numerous other proteins). Finally, phosphorylation goes as far as the nucleus, where transcription factors (e.g. NFκB) that affect the expression of certain genes are sensitive to phosphorylation. In this section we focus on the significance and complexity of phosphorylation and dephosphorylation of the Bcl-2 family proteins Bcl-2, BAD and Bik, and on the caspases. Bcl-2 family proteins
Bcl-2 family proteins play an important role in both induction (BAD, Bid, Bik) and suppression (Bcl-2, Bcl-XL) of apoptosis [6]. External and internal stimuli have been shown to affect Bcl-2 family proteins in at least three different ways. First, slow effects (in the timescale of hours) on the level of transcription, which result in an altered expression rate; second, fast and irreversible proteolytic cleavage of Bcl-2 proteins by caspases and third, fast but reversible post-translational modification by phosphorylation and dephosphorylation. Proapoptotic and antiapoptotic Bcl-2 family members can heterodimerize and can modify one another’s function. However, reversible phosphorylation and dephosphorylation by kinases and phosphatases are the main mechanisms that directly regulate the functional activity of both antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (BAD, Bid, Bik) proteins of the Bcl-2 family. In addition, there are cases in which phosphorylation and dephosphorylation can affect the formation of heterodimers. Bcl-2
The antiapoptotic molecule Bcl-2 can be modulated by dimerization with family members like Bax and by phosphorylation. The dynamic phosphorylation and dephosphorylation of Bcl-2 causes conformational change within the protein and has been suggested to serve as survival sensor during stress stimuli [7]. If favorable conditions are achieved, Bcl-2 is phosphorylated. As
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Figure 1
14-3-3
Bcl-XL
Kinases BAD
BAD-P Phosphatases
Bcl-XL BAD Cell death
BAD-P 14-3-3 Survival
[MAPKAP-K1]) [15], PKC [15], PKA [16], PKB [17] and phosphatidylinositol-3-kinase (PI3K) [18], most of which act on more than one of the phosphorylation sites. The situation for phosphatases is just as complicated: at least PP1 [19], PP2A [20] and PP2B [21] are responsible for the dephosphorylation of BAD. The function of the various phosphorylation sites, independent of their position, is to render the cell unable to induce apoptosis. The mechanisms, however, are not the same for all positions. Phosphorylation of Ser112 and Ser136 creates binding sites for the interaction of BAD with 14-3-3 proteins [11], whereas phosphorylation of Ser155 triggers dissociation of BAD from Bcl-XL [13].
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Bik Induction of apoptosis by dephosphorylation of BAD. In its phosphorylated state (BAD-P), BAD interacts with 14-3-3 proteins in the cytosol, resulting in cell survival. In contrast, unphosphorylated BAD forms a complex with Bcl-XL at the mitochondrial membrane, preventing the antiapoptotic actions of Bcl-XL and causing cell death.
death-inducing conditions are encountered, Bcl-2 is dephosphorylated and this is associated with cell death. Ser70 was identified as the functional phosphorylation site of Bcl-2, and PKCα is the major Bcl-2 kinase. In addition to phosphorylation at this single site, multisite hyperphosphorylation of Bcl-2 occurs at Thr69 and Ser87 via the kinases Erk1 (p44) and Erk2 (p42). The apoptosis-inducing agent staurosporine was a valuable tool in identifying these additional phosphorylation sites, because it inhibits the activity of classical PKCs but not Erk1 and Erk2. The rapid phosphorylation of Bcl-2 does require phosphatase activities for the reverse reaction. So far, only PP2A has been found to colocalize at the mitochondrial membrane with Bcl-2 and to dephosphorylate Bcl-2, and the proapoptotic sphingolipid ceramide has been shown to activate the PP2A involved [8]. BAD
The proapoptotic molecule BAD was initially isolated as an interacting protein of Bcl-2; later it was found to bind much more strongly to Bcl-XL [9]. BAD modulates the function of Bcl-XL by direct interaction through BH3 homology domains [10]. However, whether or not such heterodimers are formed is clearly determined by, and in this case solely determined by, the phosphorylation status of BAD (Figure 1). In its phosphorylated form, BAD associates in the cytoplasm with 14-3-3 proteins. Free Bcl-XL is then capable of preventing cell death. In its unphosphorylated state, BAD is targeted to the mitochondrial surface where it binds Bcl-XL, preventing the antiapoptotic activity of this protein and causing cell death. So far, a total of four phosphorylation sites have been identified on BAD (Figure 2); Ser112 [11], Ser136 [11], Ser155 [12,13] and Ser170 [14]. The kinases involved are Rsk (also known as MAPK-activated protein kinase 1
BAD is not the only proapoptotic molecule of the Bcl-2 family that undergoes reversible phosphorylation with a fundamental outcome on cell survival. The proapoptotic molecule Bik is also sensitive to phosphorylation, although it is not yet known exactly how phosphorylation of Bik affects its activity [22]. Mutating the phosphorylation sites Thr33 and Ser35 to alanine reduces the apoptotic activity of Bik but, in contrast to BAD, reversible phosphorylation of Bik does not affect heterodimerization. Phosphorylation of Bik is carried by an enzyme related to casein kinase II. The phosphatases involved have not been identified yet. Overall, the study of phosphorylation and dephosphorylation of Bcl-2 family proteins is a rapidly growing field. We are far from knowing all the phosphorylation sites and their functional consequences. Caspases
Caspases, a group of cysteine proteases, cleave a number of different substrates. They are synthesized as minimally active precursors and are important players in cell death processes. Sequential activation of caspases results in cleavage of key target proteins. This finally dismantles a cell. Regulators of the caspases are the inhibitor of apoptosis proteins (IAPs). In addition, phosphorylation and dephosphorylation are crucial in caspase signaling pathways of apoptosis. First, the phosphorylation status of a caspase substrate protein may decide whether the protein gets cleaved or not. For instance, the proapoptotic protein Bid plays an essential role in Fas-mediated apoptosis and, when phosphorylated, Bid is insensitive to cleavage by caspase-8 [23]. Second, caspases directly affect protein phosphatases. Activation of caspase-3 causes cleavage of the regulatory Aα subunit of PP2A [24]. This in turn increases PP2A activity, thus affecting the phosphorylation state of a cell dramatically. Caspase-3 has also been shown to act on PP2B, removing the C-terminal calmodulin-binding site and the autoinhibitory region from the catalytic subunit of PP2B [25]. This results in an increase in PP2B activity, coupled with a loss of calmodulin sensitivity and subsequently loss of Ca2+ sensitivity.
Serine/threonine protein phosphatases in apoptosis Klumpp and Krieglstein
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Figure 2
Protein kinases
Rsk
BAD (amino acid)
Protein phosphatases
PKA
PKC
Ser 112
PP1
PKA
PKB
Rsk
Ser 136
PP2B PP1 PP2B (PP2A also dephosphorylates; location site not identified)
PKA
?
Ser 155
Ser 170
?
?
Binding to 14-3-3
yes
yes
no
?
Dissociation from Bcl-XL
no
no
yes
?
survival
survival
survival
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Phosphorylation results in
Current Opinion in Pharmacology
Summary of the phosphorylation sites identified on BAD so far. The kinases and phosphatases that act at each site are indicated, whether phosphorylation/dephosphorylation at each site affects binding to 14-3-3 or dissociation from Bcl-XL is
shown and the effect of phosphorylation on cell survival is indicated. PKB is also known as Akt; PP2B is also known as calcineurin; Rsk is also known as MAPK-activated protein kinase 1 (MAPKAP-K1).
Third, some caspases are themselves subject to reversible phosphorylation. For instance, Akt has been demonstrated to promote cell survival by phosphorylating and inactivating caspase-9 [26].
enter the nucleus, production of interleukin-2 is suppressed and T-cell proliferation is reduced [28].
Clinical relevance Of the drugs in development that target protein kinases and protein phosphatases, the development of protein kinase inhibitors is most advanced [27••]. Tyrosine kinase inhibitors are used for the treatment of particular cancers, their prime targets being MAPKs and cyclin-dependent protein kinases. Inhibitors of protein kinases to treat chronic inflammatory diseases just have entered human clinical trials. For instance, pyrimidinyl imidazoles are effective in suppressing lipopolysaccharide-induced production of tumor necrosis factor-α (TNF-α) in animal models of arthritis. These drugs act as selective inhibitors of a cytokine-suppressive binding protein that turned out to be the stress-activated protein kinase-2a (SAPK-2a or p38), an activator of MAPK-activated protein kinase-2 (MAPKAP-K2) [27••]. The immunosuppressant drug cyclosporine, which made organ transplantation possible, was the first drug to exert its effect by inhibiting a protein phosphatase, although it was in use clinically before the mechanism of action was elucidated in 1990. Cyclosporine, in association with its cellular binding protein cyclophilin, is a potent and specific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase PP2B. Inhibition of PP2B prevents dephosphorylation of an isoform of NFAT. As a result, this transcription factor cannot
Studying the pathogenesis of neurodegeneration usually reveals an apoptotic component that contributes to disease progression. As the apoptotic program is fundamentally regulated by phosphorylation of specific regulatory proteins, influencing these phosphorylation processes offers the ability to modulate the life or death of a neuron. In particular, inhibitors of phosphatases could become promising candidates for therapy of neurodegenerative diseases.
Conclusions Half a century after the pioneering work of Fisher and Krebs, which recognized the overwhelming importance of reversible phosphorylation processes, we’re still just at the initial stages of investigation. Kinases and phosphatases were recently found to act as key players in the signal transduction cascades of apoptosis by ‘regulating the regulators’. Serine/threonine protein phosphatases obviously play a distinct role in life-or-death decisions. They are even considered as important as the kinases; therefore, current thinking considers phosphatases as targets for drug therapy. A large scale industrial search for activators and inhibitors of protein phosphatases is currently under way. In contrast, a second level of regulatory interference so far remains untouched. In most cases little, or even nothing, is known about the cross-talk of the multiple phosphorylation sites within a single protein. Studying the hierarchy of kinases
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and phosphatases involved may become increasingly important in the future.
15. Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P: Protein kinase C θ and ε promote T-cell survival by a Rsk-dependent phosphorylation and inactivation of Bad. J Biol Chem 2000, 275:37246-37250.
References and recommended reading
16. Harada H, Becknell B, Wilm M, Mann M, Huang LJ, Taylor SS, Scott JD, Korsmeyer SJ: Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A. Mol Cell 1999, 3:413-422.
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1. Hunter T: Signaling – 2000 and beyond. Cell 2000, •• 100:113-127. A comprehensive review on phosphorylation and dephosphorylation. Topics range from the past to the future with a number of extraordinary chapters embedded in-between (e.g. signaling specificity, spatiotemporal aspects etc.). 2.
Sim ATR, Scott JD: Targeting of PKA, PKC and protein phosphatases to cellular microdomains. Cell Calcium 1999, 26:209-217.
3.
Cohen PTW: Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv Prot Phosphatases 1994, 8:371-376.
4.
Ingebritsen TS, Cohen P: Protein phosphatases: properties and role in cellular regulation. Science 1983, 221:331-338.
5.
Cohen P, Holmes CFB, Tsukitani Y: Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 1990, 15:98-102.
17.
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg MA: Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death-machinery. Cell 1997, 91:231-241.
18. Pastorino JG, Tafani M, Farber JL: Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway. J Biol Chem 1999, 274:19411-19416. 19. Ayllon V, Martinez AC, Garcia A, Cayla X, Rebollo A: Protein α is a Ras-activated Bad phosphatase that phosphatase 1α regulates interleukin-2 deprivation-induced apoptosis. EMBO J 2000, 19:2237-2246. 20. Chiang CW, Harris G, Ellig C, Masters SC, Subramanian R, Shenolikar S, Wadzinski BE, Yang E: Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. Blood 2001, 97:1289-1297.
6.
Adams JM, Cory S: Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 2001, 26:61-66.
21. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC: Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 1999, 284:339-343.
7.
Ruvolo PP, Deng X, May WS: Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia 2001, 15:515-522.
22. Verma S, Zhao LJ, Chinnadurai G: Phosphorylation of the pro-apoptotic protein BIK. J Biol Chem 2001, 276:4671-4676.
8.
Ruvolo PP, Deng X, Ito T, Carr BK, May WS: Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 1999, 274:20296-20300.
23. Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, Hochmann A, Journot L, Antonsson B, Martinou JC: Phosphorylation of Bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 2001, 8:601-611.
9.
Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ: Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995, 80:285-291.
10. Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ: BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem 1997, 272:24101-24104. 11. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ: Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell 1996, 87:619-628. 12. Lizcano JM, Morrice N, Cohen P: Regulation of BAD by cAMPdependent protein kinase is mediated via phosphorylation of a novel site, Ser155. Biochem J 2000, 349:547-557. 13. Tan Y, Demeter MR, Ruan H, Comb MJ: BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 2000, 275:25865-25869. 14. Dramsi S, Scheid MP, Maiti A, Hojabrpour P, Chen X, Schubert K, Goodlett DR, Aebersold R, Duronio V: Identification of a novel phosphorylation site, ser-170, as a regulator of Bad pro-apoptotic activity. J Biol Chem 2002, 277:6399-6405.
24. Santoro MF, Annand RR, Robertson MM, Peng YW, Brady MJ, Mankovich JA, Hackett MC, Ghayur T, Walter G, Wong WW, Giegel DA: Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J Biol Chem 1998, 273:13119-13128. 25. Mukerjee N, McGinnis KM, Gnegy ME, Wang KK: Caspasemediated calcineurin activation contributes to IL-2 release during T cell activation. Biochem Biophys Res Commun 2001, 285:1192-1199. 26. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC: Regulation of cell death protease caspase-9 by phosphorylation. Science 1998, 282:1318-1321. 27. Cohen P: The role of protein phosphorylation in human health and •• disease. Eur J Biochem 2001, 268:5001-5010. The Sir Hans Krebs Lecture Report presents an overview of the progress that is being made in developing specific inhibitors of protein kinases for the treatment of cancer and chronic inflammatory diseases. Recommendations for their effective use in cell-based assays are made. 28. Aramburu J, Rao A, Klee CB: Calcineurin: from structure to function. Curr Top Cell Regul 2000, 36:237-295.
Serine/threonine protein phosphatases in apoptosis Susanne Klumpp* and Josef Krieglstein† The importance of phosphorylation and dephosphorylation in intracellular signaling pathways has long been recognized, although attention has focused mainly on kinases. Recent studies have highlighted the importance of serine/threonine protein phosphatases in many processes including apoptosis. The phosphorylation state of antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (BAD, Bid, Bik) Bcl-2 proteins regulates their cellular activity and, therefore, cell survival and cell death. For example, dephosphorylation of BAD by the protein phosphatases PP1, PP2A and PP2B allows BAD to interact with Bcl-XL and initiate cell death. Caspases are also important in cell death and phosphorylation/dephosphorylation of caspases themselves, their targets and their regulators modulates apoptotic pathways. The activity of serine/threonine protein phosphatases needs further study, but it is clear that these enzymes are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases and neurodegeneration. Addresses *Westfälische Wilhelms-Universität, Institut für Pharmazeutische und Medizinische Chemie, Hittorfstrasse 58–62, D-48149 Münster, Germany; e-mail: [email protected] † Philipps-Universität, Institut für Pharmakologie und Toxikologie, Ketzerbach 63, D-35032 Marburg, Germany; e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:458–462 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BAD Bcl-2/Bcl-X-associated death promoter MAPK mitogen-activated protein kinase NFAT nuclear factor for activated T cells PKA cAMP-dependent protein kinase PKC Ca2+-dependent protein kinase PP1 type-1 protein phosphatase PP2 type-2 protein phosphatase PPM Mg2+-dependent protein phosphatase PPP phosphoprotein phosphatase PTP protein tyrosine phosphatase
Introduction Apoptosis is mainly regulated by the Bcl-2 family of proteins, by apoptotic protease-activating factor 1 (Apaf-1) and by the caspase family. Neurons share the same basic apoptosis program with all other cell types; however, different types and developmental stages of neurons express a different combination of these protein factors, which is one way of providing specificity and regulation. The other significant mechanism that regulates cell death is the reversible phosphorylation of proteins. The level of phosphorylation dictates the activity of these factors and, consequently, protein kinases and protein phosphatases play a pivotal role in cell viability and cell death. This review focuses on the role of dephosphorylation processes
in apoptosis. Because of space constraints it is not possible to discuss all of the phosphatases involved and we concentrate on the role of serine/threonine protein phosphatases and their regulation of Bcl-2 family proteins.
Reversible phosphorylation of proteins Reversible phosphorylation regulates almost all aspects of cell life, from classical metabolic pathways to memory and even cell death. At least one-third of human proteins contain covalently bound phosphate. The phosphorylation levels can be modulated by changes in the activities of protein kinases (enzymes that add phosphate groups to target proteins) and protein phosphatases (enzymes that hydrolyze phosphate esters and amidates). An estimated 600 protein kinases and 200 protein phosphatases are encoded by the human genome. Because a single phosphatase catalytic moiety often associates with several different regulatory or targeting subunits, the total number of functional phosphatase holoenzymes is expected to be similar to the number of protein kinases. Phosphorylation and dephosphorylation affect the function of proteins in every conceivable way [1••]. This includes increasing or decreasing enzyme activities (phospholipase C [PLC], glycogen synthase kinase 3 [GSK3]), marking a protein for destruction (inhibitor of NFκB [IκB]), allowing a protein to move from one cellular compartment to another (nuclear factor for activated T cells [NFAT]), or enabling a protein to interact with or dissociate from other proteins (Bcl-2/Bcl-X-associated death promoter [BAD]). Kinases and phosphatases may even be colocalized on the same protein but at different docking sites [2]. Yotiao is one of these adapter proteins, which are also known as AKAPs (A-kinase-anchoring proteins). Yotiao associates with the NMDA receptor — one of the ion channel receptors for the neurotransmitter glutamate — and acts as a scaffold protein that physically attaches a phosphatase (type-1 enzyme) and a protein kinase (cAMP-dependent) to the NMDA receptor to regulate the ion flow through the channel. Although protein phosphorylation and dephosphorylation may look simple at first glance — the phosphoryl donors ATP and GTP are readily available — protein phosphorylation and dephosphorylation does have finesse. First, kinases and phosphatases themselves may be subject to multiple and reversible phosphorylation (as in the mitogen-activated protein kinase [MAPK] cascades). Second, most proteins carry multiple phosphorylation sites, with corresponding kinases and phosphatases acting interdependently and hierarchically (e.g. dopamine and cAMP regulated phosphoprotein-32 [DARP-32]).
Serine/threonine protein kinases Traditionally, much attention has been focused on protein kinases. The classification of serine/threonine protein
Serine/threonine protein phosphatases in apoptosis Klumpp and Krieglstein
kinases is straightforward. If the kinase is second-messengerdependent, it is named according to the stimulus involved. For example, cAMP-dependent protein kinase is designated PKA, Ca2+-dependent protein kinase is PKC and calmodulindependent protein kinase is CaM-K. One of the few exceptions is PKB (also known as RAC and Akt), where the acronym refers to the relatedness of this kinase (the cellular homolog of the oncogenic form v-Akt) to both PKA and PKC. The nomenclature of the second-messengerindependent serine/threonine protein kinases mostly refers to specific components of the system that they regulate.
Serine/threonine protein phosphatases The variety and regulation of phosphatases has only been appreciated more recently. The major drawback in phosphatase research has been that phosphatases were originally thought not to be subject to regulation. This turned out to be wrong. Phosphatases were classified according to the phospho-amino acids they act on, simply because stimulating or inhibitory factors were unknown at the beginning. Phosphatases that dephosphorylate serine and threonine residues are encoded by the phosphoprotein phosphatase (PPP) and Mg2+-dependent protein phosphatase (PPM) gene families, which are defined by distinct amino acid sequences and crystal structures [3]. The PPP family includes the most abundant protein phosphatases — PP1, PP2A and PP2B — as well as more recently cloned enzymes such as PP4 (also known as PPX), PP5, PP6 (a functional homolog of budding yeast Sit4) and PP7. The signature phosphatases were initially divided according to biochemical parameters [4]. Type-1 protein phosphatases (PP1) are inhibited by heat-stable inhibitor proteins and preferentially dephosphorylate the β-subunit of phosphorylase kinase. In contrast, type-2 protein phosphatases (PP2) are insensitive to these inhibitors and preferentially dephosphorylate the α subunit of phosphorylase kinase. The type-2 enzymes were further subdivided into spontaneously active protein phosphatase (PP2A), Ca2+dependent protein phosphatase (PP2B, also known as calcineurin) and Mg2+-dependent protein phosphatase (PP2C). Later on, cDNA cloning revealed that PP1, PP2A and PP2B belong to the same gene family, whereas PP2C is structurally different. Five PP2C isoforms, together with the pyruvate dehydrogenase phosphatase, constitute the distinct gene family PPM. A third branch of protein phosphatases, the protein tyrosine phosphatase (PTP) superfamily — including the dual-specificity phosphatases (DSPs), which act on MAPKs — and the protein histidine phosphatases are not covered in this article. Several naturally occurring toxins are potent inhibitors of the serine/threonine protein phosphatases [5]. These include diverse structures such as okadaic acid (a fatty-acid derivative) or microcystin (a cyclic heptapeptide). These inhibitors are often used as tools to determine the involvement of PP1 and PP2A. However, one should be aware
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that some of the less abundant protein phosphatases (at least PP4, PP5 and PP6) are inhibited by these compounds in the same nanomolar concentration range as PP1 and PP2A are inhibited. PP2B is sensitive to higher (micromolar) concentrations of these inhibitors as well. In contrast, members of the PPM and PTP families are not affected by okadaic acid or microcystin.
Phosphorylation and dephosphorylation in cell death The signaling pathways that mediate neuroprotective and neurodegenerative effects are extremely complex and have been intensively investigated for the past decade. As it turns out, almost any protein molecule involved and almost every single step of signaling cascades are associated with phosphorylation and dephosphorylation processes. It starts with receptors spanning the cellular membrane, for example, transforming growth factor β (TGF-β) receptors undergo reversible phosphorylation and they are serine/threonine protein kinases themselves. Phosphorylation continues via intracellular proteins (e.g. proapoptotic and antiapoptotic Bcl-2 family proteins, caspases and numerous other proteins). Finally, phosphorylation goes as far as the nucleus, where transcription factors (e.g. NFκB) that affect the expression of certain genes are sensitive to phosphorylation. In this section we focus on the significance and complexity of phosphorylation and dephosphorylation of the Bcl-2 family proteins Bcl-2, BAD and Bik, and on the caspases. Bcl-2 family proteins
Bcl-2 family proteins play an important role in both induction (BAD, Bid, Bik) and suppression (Bcl-2, Bcl-XL) of apoptosis [6]. External and internal stimuli have been shown to affect Bcl-2 family proteins in at least three different ways. First, slow effects (in the timescale of hours) on the level of transcription, which result in an altered expression rate; second, fast and irreversible proteolytic cleavage of Bcl-2 proteins by caspases and third, fast but reversible post-translational modification by phosphorylation and dephosphorylation. Proapoptotic and antiapoptotic Bcl-2 family members can heterodimerize and can modify one another’s function. However, reversible phosphorylation and dephosphorylation by kinases and phosphatases are the main mechanisms that directly regulate the functional activity of both antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (BAD, Bid, Bik) proteins of the Bcl-2 family. In addition, there are cases in which phosphorylation and dephosphorylation can affect the formation of heterodimers. Bcl-2
The antiapoptotic molecule Bcl-2 can be modulated by dimerization with family members like Bax and by phosphorylation. The dynamic phosphorylation and dephosphorylation of Bcl-2 causes conformational change within the protein and has been suggested to serve as survival sensor during stress stimuli [7]. If favorable conditions are achieved, Bcl-2 is phosphorylated. As
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Figure 1
14-3-3
Bcl-XL
Kinases BAD
BAD-P Phosphatases
Bcl-XL BAD Cell death
BAD-P 14-3-3 Survival
[MAPKAP-K1]) [15], PKC [15], PKA [16], PKB [17] and phosphatidylinositol-3-kinase (PI3K) [18], most of which act on more than one of the phosphorylation sites. The situation for phosphatases is just as complicated: at least PP1 [19], PP2A [20] and PP2B [21] are responsible for the dephosphorylation of BAD. The function of the various phosphorylation sites, independent of their position, is to render the cell unable to induce apoptosis. The mechanisms, however, are not the same for all positions. Phosphorylation of Ser112 and Ser136 creates binding sites for the interaction of BAD with 14-3-3 proteins [11], whereas phosphorylation of Ser155 triggers dissociation of BAD from Bcl-XL [13].
Current Opinion in Pharmacology
Bik Induction of apoptosis by dephosphorylation of BAD. In its phosphorylated state (BAD-P), BAD interacts with 14-3-3 proteins in the cytosol, resulting in cell survival. In contrast, unphosphorylated BAD forms a complex with Bcl-XL at the mitochondrial membrane, preventing the antiapoptotic actions of Bcl-XL and causing cell death.
death-inducing conditions are encountered, Bcl-2 is dephosphorylated and this is associated with cell death. Ser70 was identified as the functional phosphorylation site of Bcl-2, and PKCα is the major Bcl-2 kinase. In addition to phosphorylation at this single site, multisite hyperphosphorylation of Bcl-2 occurs at Thr69 and Ser87 via the kinases Erk1 (p44) and Erk2 (p42). The apoptosis-inducing agent staurosporine was a valuable tool in identifying these additional phosphorylation sites, because it inhibits the activity of classical PKCs but not Erk1 and Erk2. The rapid phosphorylation of Bcl-2 does require phosphatase activities for the reverse reaction. So far, only PP2A has been found to colocalize at the mitochondrial membrane with Bcl-2 and to dephosphorylate Bcl-2, and the proapoptotic sphingolipid ceramide has been shown to activate the PP2A involved [8]. BAD
The proapoptotic molecule BAD was initially isolated as an interacting protein of Bcl-2; later it was found to bind much more strongly to Bcl-XL [9]. BAD modulates the function of Bcl-XL by direct interaction through BH3 homology domains [10]. However, whether or not such heterodimers are formed is clearly determined by, and in this case solely determined by, the phosphorylation status of BAD (Figure 1). In its phosphorylated form, BAD associates in the cytoplasm with 14-3-3 proteins. Free Bcl-XL is then capable of preventing cell death. In its unphosphorylated state, BAD is targeted to the mitochondrial surface where it binds Bcl-XL, preventing the antiapoptotic activity of this protein and causing cell death. So far, a total of four phosphorylation sites have been identified on BAD (Figure 2); Ser112 [11], Ser136 [11], Ser155 [12,13] and Ser170 [14]. The kinases involved are Rsk (also known as MAPK-activated protein kinase 1
BAD is not the only proapoptotic molecule of the Bcl-2 family that undergoes reversible phosphorylation with a fundamental outcome on cell survival. The proapoptotic molecule Bik is also sensitive to phosphorylation, although it is not yet known exactly how phosphorylation of Bik affects its activity [22]. Mutating the phosphorylation sites Thr33 and Ser35 to alanine reduces the apoptotic activity of Bik but, in contrast to BAD, reversible phosphorylation of Bik does not affect heterodimerization. Phosphorylation of Bik is carried by an enzyme related to casein kinase II. The phosphatases involved have not been identified yet. Overall, the study of phosphorylation and dephosphorylation of Bcl-2 family proteins is a rapidly growing field. We are far from knowing all the phosphorylation sites and their functional consequences. Caspases
Caspases, a group of cysteine proteases, cleave a number of different substrates. They are synthesized as minimally active precursors and are important players in cell death processes. Sequential activation of caspases results in cleavage of key target proteins. This finally dismantles a cell. Regulators of the caspases are the inhibitor of apoptosis proteins (IAPs). In addition, phosphorylation and dephosphorylation are crucial in caspase signaling pathways of apoptosis. First, the phosphorylation status of a caspase substrate protein may decide whether the protein gets cleaved or not. For instance, the proapoptotic protein Bid plays an essential role in Fas-mediated apoptosis and, when phosphorylated, Bid is insensitive to cleavage by caspase-8 [23]. Second, caspases directly affect protein phosphatases. Activation of caspase-3 causes cleavage of the regulatory Aα subunit of PP2A [24]. This in turn increases PP2A activity, thus affecting the phosphorylation state of a cell dramatically. Caspase-3 has also been shown to act on PP2B, removing the C-terminal calmodulin-binding site and the autoinhibitory region from the catalytic subunit of PP2B [25]. This results in an increase in PP2B activity, coupled with a loss of calmodulin sensitivity and subsequently loss of Ca2+ sensitivity.
Serine/threonine protein phosphatases in apoptosis Klumpp and Krieglstein
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Figure 2
Protein kinases
Rsk
BAD (amino acid)
Protein phosphatases
PKA
PKC
Ser 112
PP1
PKA
PKB
Rsk
Ser 136
PP2B PP1 PP2B (PP2A also dephosphorylates; location site not identified)
PKA
?
Ser 155
Ser 170
?
?
Binding to 14-3-3
yes
yes
no
?
Dissociation from Bcl-XL
no
no
yes
?
survival
survival
survival
survival
Phosphorylation results in
Current Opinion in Pharmacology
Summary of the phosphorylation sites identified on BAD so far. The kinases and phosphatases that act at each site are indicated, whether phosphorylation/dephosphorylation at each site affects binding to 14-3-3 or dissociation from Bcl-XL is
shown and the effect of phosphorylation on cell survival is indicated. PKB is also known as Akt; PP2B is also known as calcineurin; Rsk is also known as MAPK-activated protein kinase 1 (MAPKAP-K1).
Third, some caspases are themselves subject to reversible phosphorylation. For instance, Akt has been demonstrated to promote cell survival by phosphorylating and inactivating caspase-9 [26].
enter the nucleus, production of interleukin-2 is suppressed and T-cell proliferation is reduced [28].
Clinical relevance Of the drugs in development that target protein kinases and protein phosphatases, the development of protein kinase inhibitors is most advanced [27••]. Tyrosine kinase inhibitors are used for the treatment of particular cancers, their prime targets being MAPKs and cyclin-dependent protein kinases. Inhibitors of protein kinases to treat chronic inflammatory diseases just have entered human clinical trials. For instance, pyrimidinyl imidazoles are effective in suppressing lipopolysaccharide-induced production of tumor necrosis factor-α (TNF-α) in animal models of arthritis. These drugs act as selective inhibitors of a cytokine-suppressive binding protein that turned out to be the stress-activated protein kinase-2a (SAPK-2a or p38), an activator of MAPK-activated protein kinase-2 (MAPKAP-K2) [27••]. The immunosuppressant drug cyclosporine, which made organ transplantation possible, was the first drug to exert its effect by inhibiting a protein phosphatase, although it was in use clinically before the mechanism of action was elucidated in 1990. Cyclosporine, in association with its cellular binding protein cyclophilin, is a potent and specific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase PP2B. Inhibition of PP2B prevents dephosphorylation of an isoform of NFAT. As a result, this transcription factor cannot
Studying the pathogenesis of neurodegeneration usually reveals an apoptotic component that contributes to disease progression. As the apoptotic program is fundamentally regulated by phosphorylation of specific regulatory proteins, influencing these phosphorylation processes offers the ability to modulate the life or death of a neuron. In particular, inhibitors of phosphatases could become promising candidates for therapy of neurodegenerative diseases.
Conclusions Half a century after the pioneering work of Fisher and Krebs, which recognized the overwhelming importance of reversible phosphorylation processes, we’re still just at the initial stages of investigation. Kinases and phosphatases were recently found to act as key players in the signal transduction cascades of apoptosis by ‘regulating the regulators’. Serine/threonine protein phosphatases obviously play a distinct role in life-or-death decisions. They are even considered as important as the kinases; therefore, current thinking considers phosphatases as targets for drug therapy. A large scale industrial search for activators and inhibitors of protein phosphatases is currently under way. In contrast, a second level of regulatory interference so far remains untouched. In most cases little, or even nothing, is known about the cross-talk of the multiple phosphorylation sites within a single protein. Studying the hierarchy of kinases
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and phosphatases involved may become increasingly important in the future.
15. Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P: Protein kinase C θ and ε promote T-cell survival by a Rsk-dependent phosphorylation and inactivation of Bad. J Biol Chem 2000, 275:37246-37250.
References and recommended reading
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24. Santoro MF, Annand RR, Robertson MM, Peng YW, Brady MJ, Mankovich JA, Hackett MC, Ghayur T, Walter G, Wong WW, Giegel DA: Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J Biol Chem 1998, 273:13119-13128. 25. Mukerjee N, McGinnis KM, Gnegy ME, Wang KK: Caspasemediated calcineurin activation contributes to IL-2 release during T cell activation. Biochem Biophys Res Commun 2001, 285:1192-1199. 26. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC: Regulation of cell death protease caspase-9 by phosphorylation. Science 1998, 282:1318-1321. 27. Cohen P: The role of protein phosphorylation in human health and •• disease. Eur J Biochem 2001, 268:5001-5010. The Sir Hans Krebs Lecture Report presents an overview of the progress that is being made in developing specific inhibitors of protein kinases for the treatment of cancer and chronic inflammatory diseases. Recommendations for their effective use in cell-based assays are made. 28. Aramburu J, Rao A, Klee CB: Calcineurin: from structure to function. Curr Top Cell Regul 2000, 36:237-295.