Regulation of protein kinase cascades by protein phosphatase 2A

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Regulation of protein kinase cascades by protein phosphatase 2A Thomas A. Millward, Stanislaw Zolnierowicz and Brian A. Hemmings Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades. PROTEIN PHOSPHATASE 2A (PP2A) is a multimeric serine/threonine phosphatase that has been highly conserved during the evolution of eukaryotes: the catalytic (C) subunits of PP2A from humans and budding yeast, for example, share 86% amino acid sequence similarity. A wealth of data implicate PP2A in the regulation of cellular metabolism, DNA replication, transcription, RNA splicing, translation, cell-cycle progression, morphogenesis, development and transformation1,2, but the mechanisms by which PP2A affects such diverse cellular functions are not well understood. In classical models of regulation by reversible protein phosphorylation, protein phosphatases reverse the effects of protein kinases by dephosphorylating the substrates of these kinases. However, over the past ten years, we have realized gradually that one of the major classes of PP2A substrate is in fact the protein kinases themselves. The realization that PP2A can directly regulate the activities of multiple protein kinase cascades begins to answer the question of how a single enzyme can have such pleiotropic effects. In addition, PP2A is a structurally complex protein in which a single catalytic subunit can associate with a wide array of regulatory and targeting subunits. T. A. Millward and B. A. Hemmings are at the Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; and S. Zolnierowicz is at the Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland.

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Here, we discuss evidence for the role of PP2A as a kinase phosphatase.

The structure of protein phosphatase 2A In the cell, the C subunit of PP2A forms a complex with an array of regulatory subunits that modulate its activity, substrate specificity and subcellular localization. In keeping with the most popular tradition of signal transduction research, multiple nomenclature systems are currently in use. The C subunit and a 65-kDa regulatory subunit called the PR65 or A subunit exist as a constitutive complex (Fig. 1). The PR65 subunit (whose structure has been solved recently3) is a scaffold protein, the primary function of which is to recruit further regulatory subunits; in fact, the PR65–C core dimer can interact with any one of several, variable regulatory subunits. Three gene families that encode such subunits are known, and encode proteins referred to as PR55 (also known as the B subunit), PR61 (also known as the B9 or B56 subunit) and PR72 (also known as the B99 subunit). Multiple isoforms of each of the three types of B subunit exist, and additional, as-yet-unidentified B-type subunits might exist. To date, the PR61 family, in which five genes produce 11 splice variants, seems to be the most elaborate. The many known regulatory subunits (including splice variants) theoretically could produce .50 different trimeric holoenzymes. Different holoenzyme assemblies might dephosphorylate distinct substrates in distinct cellular compartments. Indeed, complementation data

from yeast have confirmed that the different B subunits perform non-redundant functions4. This unparalleled complexity is almost certainly one explanation for the multifunctional nature of PP2A.

Protein phosphatase 2A is a target for viruses and toxins Several types of viral protein form complexes with PP2A and change its activity or substrate specificity. The SV40 small t antigen can displace some (but not all) types of B subunit from the PR65–C core dimer and, in doing so, inhibit PP2A activity5; to date, PP2A is the only known molecular target of SV40 small t antigen. Polyomavirus small t and middle T antigens bind to the PP2A catalytic subunit and, as in the case of the endogenous cellular protein PTPA, impart tyrosine phosphatase activity upon it6. An adenovirus protein, E4orf4, associates with trimeric PP2A through an interaction with the PR55a B subunit; the PP2A– E4orf4 complex is believed to be responsible for the downregulation of AP-1 transcriptional activity that occurs during late stages of viral infection7. A complex formed by two HIV-encoded proteins, NCp7 and Vpr, directly binds to and activates a PR61-containing PP2A trimer8. Why have so many viruses chosen PP2A as one of their cellular targets? In order to reproduce, a virus must subvert the signal transduction machinery of a host cell to promote its survival and replication – and, given the constraints of a small genome, must achieve this in the most economical way possible. As we discuss below, the central role of PP2A as a regulator of protein kinase cascades therefore makes it an ideal target. The importance of PP2A in cellular homeostasis is further underscored by the fact that several organisms produce PP2A-inhibiting toxins, presumably as a self-defence mechanism. Okadaic acid (OA) and calyculin A (both from marine sponges), tautomycin (an antibiotic from Streptomyces spiroverticillatus), microcystin and nodularin (both cyanobacterial hepatotoxins), and cantharidin (from blister beetles) are all potent PP2A inhibitors9. They are also powerful tools for the study of PP2A function. OA is one of the most widely used agents in PP2A research. In vitro, it inhibits PP2A with an IC50 of 0.1 nM. Moreover, conditions for the selective inhibition of PP2A in intact cells, which take into account the kinetics of penetration of cells by OA, have been established10. OA can be applied to cells at levels of up to 1 mM without any detectable inhibitory

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PII: S0968-0004(99)01375-4

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TIBS 24 – MAY 1999 effect on protein phosphatase 1 or on the other major serine/threonine-phosphatase activities [i.e. protein phosphatase 2B and protein phosphatase 2C (also known as calcineurin)]. Where we cite studies of the effects of OA in intact cells, we cite only studies in which concentrations of 1 mM or less were used. Certain caveats apply to the use of OA, however. Recently, several groups have discovered additional PP2A-like phosphatases that, although they represent only a small fraction of the total cellular serine/threonine phosphatase activity, also show sensitivity to OA. OA inhibits protein phosphatase 4 in vitro with an IC50 comparable to that for PP2A (Ref. 11), whereas protein phosphatase 5 is inhibited with an IC50 of 1–10 nM (Ref. 12). Thus some of the functions currently ascribed to PP2A are probably, in reality, functions of PP4 or PP5. In studies of intact cells, distinguishing these enzymes from PP2A by the use of OA alone will be difficult, although the use of a panel of the toxins listed above might help. Transient or inducible expression of PP2Ainhibitor proteins, such as I1PP2A or I2PP2A (Refs 13 and 14), might also be a useful strategy in the future. Finally, genetic approaches have proven to be very effective in unravelling PP2A function in yeasts and Drosophila melanogaster.

Protein phosphatase 2A as a kinase phosphatase As Table 1 shows, .30 protein kinase activities are known to be modulated by PP2A in vitro, and several kinases form stable complexes with PP2A (Table 2). Even if only a subset of these turn out to be physiological substrates of PP2A, PP2A must still play a major role in kinase regulation. In fact, extensive biochemical, pharmacological and genetic evidence suggests that PP2A controls the activities of several major protein-kinase families in the cell – in particular, those that belong to the AGC subgroup [which includes kinases such as protein kinase B (PKB; also known as Akt), protein kinase C (PKC) and p70 S6 kinase], the calmodulin-dependent kinases, ERK MAP kinases, cyclin-dependent kinases and the IkB kinases. Protein kinase B and p70 S6 kinase. Binding of agonists to cell-surface receptors leads to increases in the intracellular concentrations of several second messengers, which can then trigger activation of protein kinase cascades. The activation of such kinases (although initiated by the second messenger) often depends upon changes in their phosphorylation state.

PR65 (a or b) PPP2R1

PR55 (a, b or g ) PPP2R2

B

A

C

B¢

PR61 (a, b, g, d, or e) (B56) PPP2R5

B¢¢

PR72, PR130 or PR59 PPP2R3

PP2AC (a or b) PPP2C

Figure 1 Mammalian protein phosphatase 2A (PP2A) holoenzymes. The catalytic subunit (C) is bound constitutively to a scaffold subunit (A or PR65). This heterodimer can further complex with any one of an array of B regulatory subunits, whose binding to the core dimer appears to be mutually exclusive. Two isoforms of the C subunit (a and b) and two isoforms of the PR65 (a and b) exist. Three isoforms of PR55 have been identified (a, and the brain-specific b and g isoforms). Five genes (a, b, g, d and e) encode members of the PR61 (also known as the B9 or B56) family, and some transcripts undergo alternative splicing to generate up to ~11 isoforms. The B99 family comprises 72-kDa (PR72) and 130-kDa (PR130) subunits, which are generated by alternative splicing of a single gene, as well as PR59, the product of a closely related gene. The genes that encode the human PP2A subunits are shown in red.

Phosphoinositide 3-kinase (PI 3-kinase) is important for transmission of mitogenic and anti-apoptotic signals generated by insulin and various other growth factors; in its activated state, it produces the lipid second messengers phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Both of these bind to the PH domain of PKB. This has two consequences: firstly, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 cause recruitment of PKB to the plasma membrane and co-localize it with a constitutively active kinase kinase, PDK1; secondly, they induce a conformational change in PKB that allows it to be phosphorylated by PDK1 (Ref. 15). PtdIns(3,4)P2 and PtdIns (3,4,5)P3 thus facilitate phosphorylation of PKB by PDK1 in a substrate-directed manner (Fig. 2). PKB is also phosphorylated by a second, as-yet-unidentified kinase. The major opponent of PDK1 in the regulation of PKB appears to be PP2A: PKB is inactivated by PP2A in vitro and is stimulated in cells upon treatment with OA (Ref. 16). In addition, PI 3-kinase itself associates with an as-yet-unidentified, wortmannin-insensitive serine-kinase activity that is stimulated by okadaic acid17. The p70 S6 kinase, which is important in control of translation, is also activated by PDK118,19. In this case, however,

phosphorylation by PDK1 is independent of PtdIns(3,4)P2/PtdIns(3,4,5)P3 because the conformational change in p70 S6 kinase necessary for recognition by PDK1 is triggered by prior phosphorylation events rather than by lipid binding. The major p70 S6 kinase inactivator in cell extracts appears to be a type 2A phosphatase (on the basis of chromatographic properties and inhibitor sensitivity). The fact that p70 S6 kinase is inactivated by purified PP2A in vitro is consistent with this20. Moreover, p70 S6 kinase associates stably with PP2A in vivo21. Protein kinase C. The PKC isozymes (which represent a third group of PDK1 targets22,23) are regulated by phosphorylation in a complex fashion. PKCbII contains three phosphorylation sites, and at least one of these is required for its activity; the other two play a more subtle role in maintaining the stability of the enzyme. The conservation of these sites in most other PKC isoforms suggests that they are probably generally important. However, the phosphorylation sites in PKC are not regulated as acutely as they are in most other kinases. Rather, phosphorylation of conventional and novel PKC isoforms is part of a maturation process that allows them to respond to acute changes in the levels of diacylglycerol. One major characteristic of PKC is its downregulation in response

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Table 1. In vitro protein kinase substrates of protein phosphatase 2A (PP2A) Protein kinase Kinases inactivated by PP2A cAMP-dependent kinase cGMP-dependent kinasea PKB (Rac, Akt) PKC PKCµ (PKD) p70 S6 kinase CaM kinase I CaM kinase II CaM kinase IV AMP-activated kinase RAF-1 MEK Ste7 ERK MAP kinase Fus3p SEK1b p38/RK JNK1/SAPKc MAPKAP kinase 2 p90 RSK1, p90 RSK3 CDC2 (CDK1) CDK2 Polo-like kinase (Plk) IkB kinase (IKK) 31-kDa S6 kinase p21-activated kinase (PAK1c) Mck1 Casein kinase IId pp60 SRCe Kinases activated by PP2A Casein kinase I GSK-3, GSK-3 MST1 WEE1

Comments

Ref.

The major PKA phosphatase activity in cell extracts is PP2A-like

57

PKB is activated in vivo by OA PKC is dephosphorylated by membrane-associated PP2A

16 25 58 20 59 60 61 62 63 33 64 34 65

p70 S6 kinase forms a stable complex with PP2A Downregulation of CaM-KII is prevented by OA in vivo CaMKIV forms a stable complex with PP2A in vivo The physiological AMPK phosphatase is probably PP2C RAF-1 forms a stable complex with PP2A MEK is activated in vivo by OA and by expression of small t antigen ERK is activated in vivo by OA and by expression of small t antigen

66

CyclinB-Cdc2 is activated in vivo by OA

IKK is activated in vivo by OA

67 68 69 70 71 56 72

PAK1 forms a stable complex with PP2A 73

WEE1 from mitotic extracts is phosphorylated on inhibitory sites

74 75 76 77

a F. Hofmann, pers. commun. bJ. R. Woodgett, pers. commun. cD. L. Brautigan, pers. commun. dO. G. Issinger, pers. commun. eJ. L. Maller, pers. commun. Abbreviations used: cAMP, cyclic AMP; CaM, calmodulin; GSK-3, glycogen-synthase kinase 3; OA, okadaic acid; PKA, cyclic-AMP-dependent protein kinase; PKB, protein kinase B; PKC, protein kinase C.

to prolonged activation, a process that involves both dephosphorylation to yield an inactive form, as well as proteolytic degradation24. A PP2A trimer that contains the 55-kDa B subunit (PR55) inactivates PKCa in vitro25, and experiments aimed at characterizing the PKC-phosphatase activity in cell extracts also identified a membrane-bound, PR55-containing PP2A trimer as the phosphatase responsible for dephosphorylation of PKCa (Ref. 26). Importantly, in this study, treatment of intact cells with OA prevented dephosphorylation of PKCa after prolonged agonist stimulation. Vitamin E (a-tocopherol) can bind directly to and activate PP2A in vitro and, when applied to smooth muscle cells, causes isoform-specific dephosphorylation and inactivation of PKCa (Ref. 27). Co-treatment of these cells with calyculin A prevents the inhibitory effect of a-tocopherol on PKCa. Ca21–calmodulin-dependent kinases. Ca21 activates kinase cascades primarily by binding to its intracellular receptor, calmodulin (CaM), and to the regulatory

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domains of various PKC isoforms. The Ca21– CaM-dependent kinases (CaM kinases) are activated by binding to Ca21–CaM but also depend upon phosphorylation. These stimulatory phosphorylation events seem to be the combined result of phosphorylation by upstream CaM-kinase kinases, and Ca21–CaM-induced autophosphorylation. PP2A can dephosphorylate activated preparations of CaM kinase I, CaM kinase II and CaM kinase IV in vitro. In the cell, a subpopulation of trimeric PP2A is found in a constitutive complex with CaM kinase IV – an arrangement that allows rapid downregulation of CaM kinase IV activity after activation, even in the presence of sustained high levels of calcium28. Similarly, CaM kinase II in hippocampal synaptosomes is activated in response to membrane depolarization, but this activation lasts only for ~30 s, at which point a rapid downregulation occurs. Treatment of synaptosomes even with low levels of OA during depolarization prevents the inactivation of CaM kinase II (Ref. 29). The fact that a phosphatase that has the

chromatographic and enzymatic characteristics of PP2A is responsible for most of the CaM-kinase-II-phosphatase activity in rat brain extracts is consistent with this observation30. PP2A can also dephosphorylate CaM itself, which exists as several differently phosphorylated forms that have different potentials to activate effector proteins31. PP2A therefore might control the activity of CaM kinases indirectly by modulating interactions between CaM and CaM kinases. MEK and ERK. The RAS–RAF–MEK–ERK MAP-kinase cascade plays a central role in mediating cell-cycle re-entry and transcriptional responses downstream of many cell-surface receptors. As in the cases of many other signalling pathways, this pathway is usually only transiently activated, and its constitutive activation is sufficient to cause oncogenic transformation of some cell types32. Numerous observations suggest that PP2A plays a major role in downregulation of the ERK MAP-kinase pathway and probably acts at multiple points in the cascade. In vitro, PP2A can dephosphorylate and inactivate MEK1 and ERK-family kinases33,34, and both kinases are activated after treatment of cells with OA35,36. Transient expression of SV40 small t antigen (which inhibits PP2A) activates the MEK1 and ERK, which might explain how small t antigen promotes transformation37; conversely, overexpression of casein kinase 2a (which activates PP2A) suppresses the activity of MEK1 (Ref. 38). Chromatographic separation and immunodepletion experiments demonstrate that the major ERK-phosphatase activity in extracts from PC12 cells is attributable to PP2A plus an unidentified tyrosine-specific phosphatase39. Finally, genetic evidence also implicates PP2A in positive and negative regulation of the ERK MAP-kinase pathway during Drosophila photoreceptor development40. Note that PP2A is not the only phosphatase that is important for the inactivation of ERKs. As mentioned above, PP2A operates in several cell types in conjunction with an unidentified tyrosinespecific phosphatase to dephosphorylate the regulatory threonine and tyrosine residues in the activation loop of ERK. However, in other cell types, both phosphates are removed by a single dualspecificity phosphatase, MKP-1. MKP-1 is the product of an immediate-early gene that is transcriptionally upregulated following activation of the ERK MAP-kinase cascade, and therefore forms part of a negative feedback loop41. Several MKP-1related phosphatases also exist. The

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TIBS 24 – MAY 1999 relative contributions of PP2A and MKP1-like phosphatases to ERK inactivation vary, depending on the cell type and the agonist. In serum- or EGF-treated fibroblasts, for example, MKP-1 provides the major phosphatase activity for ERK inactivation. By contrast, in EGF-treated adipocytes or PC12 cells, MKP-1 is not necessary for ERK inactivation, and PP2A (plus the as-yet-unidentified tyrosine phosphatase) is responsible for ERK inactivation39. An important objective will be to understand why different cell types employ different phosphatases to downregulate MAP kinases. Is the form of PP2A that acts on MAP kinases a special holoenzyme that contains a variable subunit expressed only in certain cell types? Or is PP2A MAP-kinase-phosphatase activity stimulated by a cell-type-specific signal-transduction pathway? The role of PP2A in the regulation of RAF-1 activity is also unclear. PP2A associates stably with RAF-1 (M. Baccarini, pers. commun.), but expression of small t antigen in cultured cells has no effect on RAF-1 activity37. Although PP2A can dephosphorylate the stress-activated MAP kinases p38 and JNK1 in vitro (see Table 1), protein phosphatase 2C is probably more important for negative regulation of these pathways in the cell: overexpression of PP2C suppresses basal and stress-stimulated activity of SEK1, MKK3, MKK6 and MKK7 in transfected cells, but does not affect the basal or growth-factorstimulated activity of MEK142,43. Cyclin-dependent kinases. Genetic evidence points to a role for PP2A in the regulation of cell-cycle progression, especially during mitosis. Strains of budding yeast that carry a mutation in PR55 lack a functional spindle-assembly checkpoint44, and mutations of the corresponding gene in Drosophila cause abnormal sister-chromatid separation45. One explanation for this is undoubtedly the fact that PP2A is required for dephosphorylation of substrates of cyclindependent kinases (CDKs): the PP2A trimer that contains PR55 is the major proline-directed serine/threonine-phosphatase activity in extracts from several different tissues and cell types46. However, addition of OA to intact mammalian cells synchronized in S phase also leads to activation of pre-existing, inactive cyclin-B–CDC2 complexes, and concomitant premature chromosome condensation and breakdown of the nuclear lamina47. Similarly, addition of OA to extracts of interphase Xenopus laevis oocytes activates cyclin-B–CDC2, whereas addition of inhibitor 2 (to inhibit protein

Table 2. Proteins that form stable complexes with protein phosphatase 2A (PP2A) Protein

Comments

Refs

Protein kinases p70 S6 kinase CaM kinase IV Casein kinase IIa

p70 S6 kinase is a PP2A substrate Binds to PR55-containing ABC complex; substrate for C subunit Binds to AC dimer in quiescent cells; stimulates activity of C subunit RAF-1a RAF-1 can be dephosphorylated by PP2A p21-activated kinase (PAK1) PAK1 is a PP2A substrate JAK2 Transient association upon interleukin-11 stimulation of adipocytes

21 78

Other cellular proteins I1PP2A (PHAPI, mapmodulin) I2PP2A (SET) Tap42/a4

13 14 79, 80

Cyclin G1 p107 (pRb-related) HOX11 HRX Caspase-3 PTPA (PPP2R4) TAU Neurofilament proteins eRF1 Viral proteins SV40 small t Polyomavirus small t Polyomavirus middle T Adenovirus E4orf4 HIV NCp7:Vpr

Endogenous, heat-stable inhibitor of PP2A Endogenous, heat-stable inhibitor of PP2A Binds to C subunit; interaction dependent upon TOR1 (FRAP/RAFT) Binds to B subunits of the B¢ (PR61) family Binds PR59-containing AB¢¢C complex Binds to C subunit; inhibits phosphatase activity Binds PP2A through I2PP2A; commonly mutated in acute leukaemias Activates PP2A during apoptosis by proteolysis of the PR65 subunit Binds to AC dimer through A subunit; confers tyrosine phosphatase activity PR55-containing trimer dephosphorylates TAU; promotes microtubule binding AC dimer binds and dephosphorylates NF proteins, promotes assembly Binds to AC dimer through C subunit; might target AC dimer to ribosomes Binds to and inhibits AC dimer; displaces PR55-type B subunits Confers tyrosine phosphatase activity Confers tyrosine phosphatase activity Binds to PR55-containing ABC complex; causes downregulation of AP-1 Binds to PR61-containing AB¢C complex; activates C subunit

21 28 38

81 82 83 84 85 86 87 88 89

5 6 6 7 8

a M. Baccarini, pers. commun. Abbreviations used: CaM, calmodulin; PTPA, phosphotyrosyl-phosphatase activator.

phosphatase 1) has no effect48. The mitotic effects of PP2A therefore might be mediated at least in part through negative regulation of CDK complexes themselves. PP2A could regulate the activity of CDC2 and related kinases by at least three conceivable mechanisms (Fig. 3). (1) The kinase activity of CDC2 depends on phosphorylation on Thr161, which lies in the activation loop. PP2A (either the purified catalytic subunit or an endogenous PP2A holoenzyme from oocyte extracts that is termed INH) can dephosphorylate this site in vitro49. However, a distinct phosphatase, KAP, can also dephosphorylate this site (as well as the equivalent site in CDK2). Dephosphorylation of CDC2 (or CDK2) by KAP is prevented by the binding of cyclin to the kinase, whereas PP2A recognizes both monomeric and cyclinbound CDC2/CDK2 (Ref. 50). In vivo, dephosphorylation of Thr161 of CDC2 seems to be triggered by cyclin B destruction, which suggests that KAP is probably the major physiological phosphatase for this site. (2) PP2A dephosphorylates and activates the kinase WEE1, which is held in

an inactive state during interphase by phosphorylation of inhibitory sites51. Given that WEE1 inhibits CDC2, PP2A could inactivate CDC2 indirectly through activation of WEE1. (3) PP2A is a negative regulator of CDC25, the dual-specificity phosphatase that removes inhibitory phosphate groups from CDC2 and thereby activates it. Two observations suggest that activation of CDC25 is one mechanism by which PP2A activates CDC2: first, OA (but not inhibitor 2) causes activation of CDC25 in oocyte extracts48; second, activation of cyclin-B–CDC2 by OA in intact mammalian cells is reduced by co-treatment with vanadate; this implies that PP2A acts, at least in part, through a vanadatesensitive phosphatase47. The idea that PP2A acts as a positive regulator of WEE1 and as a negative regulator of CDC25 is further supported by data from fission yeast52. Loss of one of the two genes that encode PP2A catalytic subunits causes a semi-Wee phenotype and is lethal when combined with mutations in the wee11 gene; in contrast, the same deletion partially suppresses the growth defect of cdc25 mutants.

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Insulin receptor

P

PtdIns(3,4)P2 or PtdIns(3,4,5)P3

p85

PH

ATP

PI3-K

Inactive PKB

PH

Active PKB kinase

kinase

Inactive PKB

P

IRS 1/2

Ser473 kinase

PDK1

p110

ADP

P P

Active PKB

Pi PH

PH kinase

kinase

PP2A

P P

Figure 2 Regulation of protein kinase B (PKB) by PDK1 and protein phosphatase 2A (PP2A). Binding of insulin to its receptor causes activation of phosphoinositide 3-kinase (PI3-K), which generates phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in the plasma membrane. These phospholipids are ligands for the pleckstrin homology (PH) domain of PKB, and bring about recruitment of PKB to the membrane, as well as a conformational change in PKB. Binding of PtdIns(3,4)P2 or PtdIns(3,4,5)P3 converts PKB into a substrate for two distinct kinase kinases: PDK1 and Ser473 kinase. Each of these phosphorylates PKB on a different site; together they cause its activation. Thr308 of PKB is phosphorylated by PDK1, which is itself membrane localized through a PH domain (not shown); the Ser473 kinase has not yet been identified. Activated PKB detaches from the membrane to phosphorylate various cytosolic and nuclear substrates. The phosphatase responsible for returning PKB to its inactive state is PP2A.

Cyclin B

Cyclin B

P

WEE1 Y T

CDC2

P

T

P

Y T

CDC2

PP2A

Inactive

T

P

Inactive WEE1 P

CDC25

PP2A

PP2A

An additional possibility for the regulation of CDC25 by PP2A exists. Qian et al.53 recently have identified an OA-stimulated kinase that activates Polo-like kinase (Plk), the upstream activator of CDC25. IkB kinase. Pro-inflammatory cytokines, such as tumor necrosis factor a (TNFa) and interleukin 1 (IL-1), activate signal transduction cascades that promote the induction of genes that encode various proteins involved in the inflammatory response, including other cytokines. Several of the effects of these agonists are mediated by the transcription factor NF-kB. Several years ago, Guy et al.54 noted that OA mimics the effects of TNFa and IL-1 with remarkable accuracy. Following treatment of fibroblasts with either TNFa or IL-1, changes in the phosphorylation states of .100 proteins can be detected by two-dimensional gel electrophoresis of extracts from 32P-labelled cells, and ~95% of these changes can be reproduced by treatment with OA at a concentration that would inhibit PP2A but not PP1. One of the proteins that becomes phosphorylated in response to OA treatment (or TNFa or IL-1) is IkB, an inhibitory subunit of NF-kB that is proteolytically degraded upon phosphorylation. Many of the transcriptional responses to TNFa and IL-1 are therefore faithfully reproduced by OA (Ref. 55). Recently, several labs identified a cytokine-regulated IkB-kinase complex that contains two kinase polypeptides, IKKa and IKKb. IKKa is activated upon exposure of cells to OA and is inactivated by PP2A in vitro56. These results partially explain the fact that OA mimics TNFa and IL-1, and suggest that PP2A plays a central role in negative regulation of cytokine signalling. Clearly, IKK activity is determined by the relative activities of its upstream-activating kinase and PP2A, and inhibition of PP2A is sufficient to activate IKK even in the absence of cytokine stimulation.

Cyclin B

Conclusion Y T

CDC2

Y T

T

CDC25 CDC2

T

P

KAP

Inactive

Active

Figure 3 Possible mechanisms for negative regulation of cyclin-B–CDC2 by protein phosphatase 2A (PP2A). Most genetic data indicate that PP2A negatively regulates progression through mitosis by acting as an inhibitor of cyclin-B-CDC2. On the basis of the available data, we suggest that there are three points at which PP2A might act. The major effect of PP2A is to promote inhibitory phosphorylation of CDC2 on Thr14/Tyr15 by simultaneously activating WEE1 and inactivating CDC25. PP2A might also dephosphorylate Thr161 in the CDC2 T-loop, although KAP is probably the major physiological phosphatase for this site. In addition, PP2A1 (the PP2A holoenzyme that contains the PR55 B subunit) dephosphorylates CDC2 substrates.

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PP2A is an important negative regulator of many protein kinase cascades, but many questions remain open. Genetic and biochemical evidence strongly suggests that B subunits play an important role in conferring specific functions on PP2A, but little is known about how formation of complexes between the PR65–C core dimer and B subunits is regulated or exactly how the B subunits target the core dimer to different substrates. Are different holoenzymes formed purely as a function of subunit abundance in a given subcellular compartment (i.e. is

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TIBS 24 – MAY 1999 holoenzyme structure controlled primarily at the level of gene transcription)? Or is the association of the core dimer with different subunits controlled by additional mechanisms; for example, might post-translational modifications rapidly alter the affinity of the C subunit for a given B subunit? In fact, increasing evidence supports the idea that modifications of the C subunit (phosphorylation and methylation) are acutely regulated upon cell stimulation or during the cell cycle; this might provide a means for signal-dependent regulation of PP2A activity. Answering these questions is a major challenge for the future.

Acknowledgements We thank the Krebsforschung Schweiz (grant number KFS 269-1-1996 to B. A. H.) and the Howard Hughes Medical Institute (grant number 75195-544001 to S. Z. and B. A. H.) for support, and David Evans and Heidi Lane for comments on the manuscript.

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