Protein phosphatase 2A — a ‘ménage à trois’

The protein phosphatase 2A (PP2A) family of holo- enzymes has been implicated in many different facets of cellular functiont-L The major characteristics that distinguish these enzymes from other protein phos- phatases are summarized in Box 1. The dephos- phorylation of critical Ser-P and Thr-P residues in a wide range of proteins changes their properties, such as enzymatic activity or interaction with other mol- ecules. Besides regulating metabolic enzymes, PP2A appears to modulate man)" proteins involved in sig- nal transduction, including cell surface receptor mol- ecules, cytosolic protein kinases, and transcription factors. The modification of proteins important for cell cycle regulation, cell transformation and cell fate determination affects essential processes of every cell.

Although much is known about the structure of PP2A enzymes, the regulatory features that control their activity remain to be elucidated. Recent work has focused on elucidating how PP2A can perform in vivo the myriad of regulatory functions predicted from in vitro studies, and has provided some new insights into events controlled by the dephosphoryl- ation of target proteins by PP2A. This article con- centrates on some of the advances in understanding the regulatory mechanisms of PP2A. The reader is referred to other reviews on the metabolic function of PP2A ~'2.

Structures of PP2A holoenzymes Many different trlmeric holoenzymes of PP2A have

been purified and evaluated with regard to substrate specificity and subunlt composition t,2. Molecular cloning and the development of specific antisera have now made It possible to present a fairly com- prehensive picture of this group of phosphatases (summarized in Fig. 1). The core structure of PP2A consists of a 36 kDa catalytic subunlt tightly com- plexed with a 65 kDa regulatory subunlt [termec~ PR65 (phosphatase regulatory subunlt of 65 kDa), or A-subuntt]. The core dlmer complexes with a third, variable subunlt which confers d)stinct properties on the holoenzyme. Four types of these variable sub- units (referred to as PR subunlts with their apparent molecular mass, or B-subunlts) are known, ranging from 54 to 74 kDa. Furthermore, recent data suggest that other proteins may associate with the trimeric holoenzymes. The presence of these multiple forms of PP2A holoenzymes suggests that they might have various functions in vivo.

Molecular cloning of the individual PP2A subunits has been an essential step towards the elucidation of the function of the various holoenzymes. This showed that the catalytic subunit is one of the most highly conserved enzymes known and that the regu- latory subunits are also highly conserved from man to yeast to plants. Molecular cloning also revealed that PP2A is more complex than was first thought, since in mammals there are two isoforms of the cata- lytic and PR65 subunit, and three of the PR55 sub- unit; in addition the PR72 and PR130 subunits appear to be derived from the same gene by alternative splicing ~. The sequences of the other variable regu- latory subunits have not yet been reported, but the

m

Protein phosphatase 2 A - a 'm6nage trois'

%!i i!!i!iiiii!i i: ii!! iiiiii!i!!iii!ililil ¸ !i ! ii ! ¸ i iil !iii iili iil

Protein phosphorylation is probably the major regulatory

meclmnism employed by eukaryotic cells. Mudz work has been

devoted to the role of protein kinases and their modulation by

hormones, growth factors and neurotransmitters. It is now

appreciated that protein phosphatases are also key players in

actively regulating many cellular processes. In this article we

discuss recent adwmces in our understanding of the function of

protein phosphatase 2A, one of the major serine/threonine-specific

protein phosphatases.

characteristics of the proteins clearly exclude the possibility that they are further isoforms of the cloned subunits.

The PR65 subunit has a very distinctive structure consisting of 15 leucine.rich Imperfect repeating units of 38-40 amino acids that produce a rod.like structure important for its interaction with other sub- units. The four C.terminal repeats are essential for Interaction with the catalytic subunlt, where is the N.termlnal repeats appear to Interact with the variable subunlts and with viral tumour antigens s (see below). Interestingly, a similar structure was identified In a protein phosphatase 1 regulatory subunit from yeast (the sds22 gene product 6) and a protein kinase (VpslSp) 7, suggesting that this motif may generally play a role in protein-protein interactions.

A 126 amino acid region in the PRSSot and PR55[~ subunits shows a limited similarity to subdomains VI to IX of the c-Abl protein tyrosine kinase ~. These sub ................................................................... domains have been implicated in substrate binding, raising the possibility that this region in the PR55

BOX 1 - ENZYMATIC CHARACTERISTICS OF PP2A

ICs0 for okadaic acid 0.05-0.10 nM (phosphatase 1: 10-20 nM)

Stimulated by polycations No requirement for Ca 2÷ or Mg z* Insensitive to heat.stable inhibitors 1 and 2 Preference for the oc subunit of phosphorylase kinase Preference for Thr-P over Ser-P sites in syntheUc peptides

Regina Mayer. laekel is at the Arbeitsgruppe fur Zellbiologie- Tumorbiologie, Universitat Konstanz, 78434 Konstanz, Germany; and Brian Hemmings is at tile Friedrich Miescher-lnstitut, 4002 Basel, Switzerland.

TRENDS IN CELL BIOLOGY VOL. 4 AUGUST 1994 © 1994 Elsevier Science lad 0962.89241941507.00 287

B

core dimer / ",.

FIGURE 1

Structure of PP2A holoenzymes. The core dimer, consisting of a 36 kDa catalytic subunit (C36) and a 65 kDa regulatory subunit (PR6$), can associate with a third, variable regulatory subunit of 54, 55, 72, 74 or 130 kDa (PR$4, PRSS, PR72, PR74

and PR130). Holoenzymes containing the PR$SI3 or PR130 subunits have not been isolated and are only predicted from the cDNA data. The PR130 protein is

identical to PR72 in its C-terminal part but has a different N-terminus. The catalytic subunit isoforms are 98% identical, and the PR65 and PR$$ subunits are -86% identical. An additional regulatory protein (PTPA) has been identified that

stimulates tyrosine dephosphorylation by the PP2A core dimer but does not apparently bind to it.

subunit has a similar function. This prediction is still to be tested.

Whereas the ,'atalytic and PR65 subunits appear to be ublqttltousl~ expressed, some of the variable subunits show tissue- and development.specific expression pat, eros. For Instance, the transcripts encoding the n~,ammallan PR551'i subunit are mainly expressed in neuronal tissues and In testis ~,'~, and the transcripts encoding the PR72 subunlt are detected ~:,nly In muscle 4, in Drosophila, the PR55 subuntt tran- scripts are mainly detected during the syncytlal phase, and during later development In the ner- vous system, the lmaglnal discs and gonads, which are all proliferative organs~L These expression pat- terns suggest tissue-specific assembly of individual holoenzymes with distinct functions. Obviously, Identifying the tissue-specific factors that control PP2A expression will be of crucial importance In developing an overall understanding of PP2A function.

The various possible interactions between subunit isoforms have not yet been fully investigated. However, based on western blot analysis it seems that PR65~t and I~ can both interact with the ~t isoform of the catalytic subunit and with the PR55~t or PR72 subunlts ~ L

Modulation of activity 1"o understand and characterize the role of PP2A

holoenzymes in cellular processes more fully it will be necessary to identify factors and mechanisms that modulate their activity. Given the plethora of

regulatory subunits now identified, the control of PP2A activity is likely to be complex.

The activity of several different trimeric holo- enzymes can be stimulated in vitro by polycations, such as polyamines, but the relevance of this is not yet clear. Recently, control of the trimeric PP2A holoenzymes by ceramide, a lipid second messenger, was reported :z. Ceramide, a product of the sphingo- myelin cycle, specifically activates the PR54- and PR55a-containing PP2A holoenzymes. This suggests that these holoenzymes may be controlled by extra- cellular signals that trigger sphingomyelin hydroly- sis, such as vitamin D:v tumour necrosis factor ct and interleukin 1, and thus be a target of antiproliferative signals 13.

Furthermore, PP2A activity is known to be regu- lated by covalent modifications (summarized in Fig. 2). The catalytic subunit is phosphorylated on a tyro- sine residue close to the C-terminus (Tyr307) con- comitant with an inhibition of its catalytic activity 14. Phosphorylation is enhanced in v-src-transformed cells, or following serum stimulation or EGF treat- ment of norma; cells ~s. Downregulation of PP2A fol- lowing auitogenic activation of cells would have the effect of enhancing the activating protein kinase cas- cades, since several of the protein kinases are dephos- phorylated and inhibited by PP2A. However, the generality of this type of regulation is still to be explored. In addition, threonine phosphorylation of the catalytic subunit by a novel protein kinase also leads to inactivation of PP2A activity ~', suggesting that PP2A may be modulated by multiple signal transductlon pathways. Both types of phosphoryl- atlon can apparently be reversed by autodephos- phorylatlon (of both Thr=P and 'l'yr.-! ~ residues).

Rather surprisingly, another novel form of covalent modification has been discovered by two groups studying carboxymethylases. They identified the C- terminal leuclne residue of PP2A as the major cellular target for these enzymes ~'~,~. Independent immunological studies of the PP2A catalytic subuW, t showed that the antigenicity of the C-terminus is affected by carboxymethylation ~'~. These studies also revealed that methylation leads to an increase in PP2A activity, suggesting that this modification may play a role in controlling PP2A activity in vivo. It is also possible that this modification plays a role in the assembly of PP2A hoioenzymes.

Thus, the C-terminus of the catalytic subunlt seems to play an important role in the modulation by co- valent modifications. Besides being a target for meth- ylatlon and tyrosine phosphorylatlon, the threonine phosphorylation site may lie within this region. The C-terminal sequence 'DYFL', containing the meth- ylation and tyrosine phosphorylatlon sites, is highly conserved between PP2A catalytic subunits from plants to mammals and is also found in several other PP2A-related catalytic subunits such as PPX, PPV, PPH3, SIT4, PPG and ppe I :'~ Thus, this mechanism of regulation may also be of si~nific,mce for other protein phosphatases.

The availability of cloned subunits makes it poss- ible to reconstitute holoenzymes with known specific

2,. TRENDS IN CELL BIOLOGY VOL. 4 AUGUST 1994

m

gene products and analyse their enzymatic proper- ties. The data available so far indicate that the PR65 subunit suppresses the activity of the catalytic sub- unit and the addition of a third subunit leads to a further reduction in activity, the extent of which is dependent on the substrate usedZ~L A further variable in the regulation of PP2A was introduced by the iso- lation of a protein (PTPA) that activates phospho- tyrosine-specific phosphatase activity of the core dimer2L

These observations all point towards a complex network of signals responsible for the control of PP2A, but which modification is relevant for a par- ticular cell type, and which primary extracellular signal controls particular modifications, remains to be elucidated. The situation may be further compli- cated by covalent modification of the various regu- latory subunits, which has not yet been extensively investigated.

PP2A and cell transformation Several discoveries suggest an important role for

the modulation of PP2A in cell transformation, but the exact mechanisms are still to be delineated 22. One of the first observations was the identification of PP2A as a major target for the tumour protnoter okadaic acid, which was followed by the discovery of many more toxins that inhibit PP2A and pro- tein phosphatase 1 with different potenciesZ:L This implied a role for PP2A in normal cell proliferation and even suggested that it may be an anti-oncogene product.

A role for PP2A in transforming viral infections was evident from four different fronts. First, the PP2A catalytic subunit stimulates in vitro SV40 DNA rcpli- cation dependent o21 large T antigen by dephosphoryl- ating two serine residues on large T antigen, which allows it to hind to the origin of replication more efflclently2'L This functh)n of PP2A appears to be restricted to S phase 2:'. Second, PP2A catalytic and PR65 subunlts associate with polyoma virus middle T antigen, which is an essential step in transform- ation by polyoma viruses. Furthermore, these two PP2A subunlts are the only cellular proteins that as- sociate with either SV40 or polyoma virus small t antigens (summarized in Fig. 3). Small t replaces the third variable subunit in the PP2A holoenzyme, influencing the catalytic activity in a similar but not identical nlanner 2z. The interaction of SV40 small t antigen with PP2A apparently induces cell prolifer- ation by inhibiting PP2A-mediated dephosphoryl- ation of MEK and MAP kinases z~'. Third, a study by Smits et al. ~-7 also suggests a role of PP2A in human papilloma virus (HPV 16) expression. Transactivation of the HPV16 enhancer/promoter can occur via either SV40 small t antigen, an increased expression of the PP2A PR551~ subunit (and to a lesser extent PR55cO or by treatment with okadaic acid. All three events appear to inhibit PP2A activity, and except in the last case this is likely to be substrate specific. Fourth, adenovirus E4orf4 binds to the trimeric PP2A holoenzyme containing the PR55 subunit, leading to the downregulation of cAMP- or E1A-mediated

NH2 Carboxy methyl transferase NH2 ( ~ Tyrosinekinase ~ - _ _

Autoactivating kinase

Autodephosohorvlation T - - P Phosphatase

TRRTPDYFL Demethylase TRRTPDYFL-'CH3

I P

FIGURE 2 Covalent modifications of PP2A. The figure illustrates the reactions known to modify the PP2A catalytic subunit. The relationship between these reactions and whether they take place on the free catalytic subunit alone or when it is complexed with regulatory subunits are not yet known.

transactivation of JunB 2~. These studies show that PP2A can be recruited by different viral proteins to perform or enhance their growth-promoting effects.

PP2A mutants - a link to the cell cycle and cell fate

Recently, the isolation and characterization of yeast and flies with mutations in the catalytic and regulatory subunits have provided important insights into the roles of PP2A holoenzymes. Distinct PP2A holoenzymes appear to play a crucial role at several points of the cell cycle, and also in cell fate determination.

Dlmer

Variable , \ Transforming s u b u ~ " ~ t l g e n s

~a~lgens

\ " Variable

1 subunits 1

Regulation of metabolism, Growth promotion transcription, signal transduction, ceU cycle, cell fate and cell shape

Redirection of PP2A function by association with transforming antigens of SV40 and polyoma viruses. The PP2A core dimer can associate with either one of the variable subunits, r ," with small t antigen or medium T amig~=n from SV40 and polyoma viruses.

TRENDS IN CELL BIOLOGY VOL. 4 AUGUST 1994 289

(a) Abnormal anaphase

wild type tws P, aar ~, tws 55

(b) Sensory bristle duplication

wild type tws 55, aar l

(c) Pattern duplication in wing imaginal discs

wild type tws P

FIGURE 4

Reduced levels of the PR55 subunit in Drosophila result in mitotic abnormalities and altered cell fate determination. A reduction in the PRS5 protein levels to about

10% of wild type leads to abnormal anaphase figures in larval brain cells (a) and to a duplication of sensory bristles in adult flies (b). A further reduction to about

1% of wild type additionally results in a pattern duplication in wing Imaginal discs from thlrd.lnstar larvae (c). tw~ p mutants die before sensory differentiation. See

Refs 10, 37 and 38 for full descriptions of mutant phenotypes.

Schizosattharomytr~ pombe has two PP2A catalytic suDunlt genes: ppal and ppa2. Disruptions In both genes are lethal, and deletion or inactivation of the major lsoform, ppa2, leads to a premature mitosis characterized by small cell size (the wee pheno- type) 29. A genetic interference between ppa2 and cdc25 (encoding the dual specificity phosphatase regu- lating p34 ,',l~a) suggests that the ppa2 gene product, as well as the weel protein ktnase, are responsible for the negative control of entry into mitosis 3°. A simi- lar conclusion was obtained from biochemical studies wtth Xenopus oocytes, where PP2A was found to promote the dephosphorylation of and thereby inhibit cdc25 (Ref. 31). Furthermore, Xenopus INH, a factor inhibiting hlstone H1 kinase (p34 cd,'z) acti- vation, is a trimeric form of PP2A containing the PRS5~ subunlt 32 thought to function upstream of the kinases and phosphatases that directly modify p34 c,tcz.

The PP2A activity in Saccharomyces cerevisiae is derived mainly from three genes: PPH21, PPH22 and, to a lesser extent, PPH3. Disruption of all three genes is lethal. A muiticopy suppressor of PP2A deficiency, termed PAM-I, can bypass the need for the PP2A cata- lytic subunit 3:~. The protein sequence of PAM-1 does not provide any clues towards its precise function. However, we speculate that this protein should func- tion as an activator subunit of a protein phosphatase that can replace PP2A.

The S. cerevisMe PR65 gene, TPD3, was isolated by complementation of a temperature-sensitive mutant defective in the synthesis of tRNA 34 (the reason for this is not known at the moment). At reduced tempera- tures tpd3 strains have a similar phenotype to cells lacking the PR55 subunit. The PRS5 gene, CDC55, was identified in a screen for cold-sensitive cell cycle mutants 3s. The mutants show defects in morpho- genetic events of the cell cycle, with abnormal elongated buds and a delay or partial block in sep- tation and/or cell separation. It was also suggested that these events correlate with cell fate determi- nation, i.e. the change from normal budding to a pseudohyphal growth form 36. Both the tpd3 and cdc55 mutations presumably disrupt the normal function of the PP2A holoenzyme containing the PR55 subunit, implying that both PR65 and PRSS provide essential regulatory features. The results also clearly underline the fact that specific holoenzymes have specific substrates.

Mutant alleles of the Drosophila PRS5 gene were found by two independent approaches (the pheno- types are summarized in Fig. 4). The mutant fly strain termed aar I (abnormal anaphase resolution) was in- itially isolated in a screen for mitotic abnormalities in larval brain cells and subsequently found to be due to a P-element insertion in the PRS5 gene 1°. The mutants have various defects such as overcondensed chromosomes and abnormal anaphases, with chromo- somes not segregating to the poles ~r sister chroma- rids bridging between the two poles. These defects can be rescued by reintroducing the PRS5 gene. The PR55 gene was Independently isolated In a search for the gene responsible for wing imaglnal disc abnor- realities :~7. The larvae of the mutant flies (termed tws~) show a partial pattern duplication in wing im. ~ginal discs, indicating a defect in cell fate determi. raatlon. This allele arises from the integration of a dif- ferent P-element in the PRSS gene, at exactly the same position as in aaP flies. Studies of RNA and pro- tein levels indicate that tws ~' Is a stronger allele than aar ~. The generation of a series of weaker mutations of tws allowed the characterization of other effects. These include a bristle duplication, which also appears to be due to a change In cell fate determi- nation of a sensory neuron 38,

Studies of the protein phosphatase activity in these mutants showed that the aar ~ and tws ~' mutant lar- vae have a reduced protein phosphatase activity towards the p34cdcZ-phosphorylated substrate pro- teins caldesmon and histone H 1, whereas the activity towards other substrates is not altered ag, Thus, loss of the PR5$ subunit seems to lead to a dramatic drop in the dephosphorylatlon of rather specific substrates. Furthermore, the results suggest that PP2A can exert its regulatory effect on the cell cycle through effects on both the kinase(s) and their substrates.

Where ,Io we go from here? The PP2A family of holoenzymes clearly can influ-

ence many different cellular events, apparently through the assembly of several different trimeric holoenzymes. Biochemical and genetic data show

290 TRENDS IN CELL BIOLOGY VOL. 4 AUGUST 1994

that the variable components of PP2A play a crucial role in regulating cellular processes. How the variable subunits modulate activity and specificity is an important question for future studies. Several possi- bilities exist:

(1) The variable subunits could perform a substrate- recognition function, thereby giving the different holoenzymes specific functions, as predicted from the Drosophila studies. ,

(2) Alternatively, the variable subunits could be responsible for targeting of the holoenzyme to the appropriate subcellular compartment. The subcellular localization of the various regulatory subunits has not yet been extensively investi- gated.

(3) The variable subunits could perform a transducer function, analogous to that performed by G pro- teins. In this model, a pool of dimeric PP2A (con- sisting of C36 and PR65) would be available to complex with the different 'activated' variable subunits, and hence become able to perform specific dephosphorylation reactions.

(4) The variable subunits might be targets for protein- kinase-mediated activation. This would have the advantage, and elegance, of closing the circle in dny signal transduction pathway, i.e. activated protein kinase activates phosphatase, which inac- tivates the activated protein kinase. In this model, a signal transduction pathway would be auto- matically downregulated and reset for the next round of stimulation.

(5) Finally, the variable subunits could be the targets to whtch putative second messengers bind and subsequently activate the target holoenzyme via a conformational change and/or release of the catalytic subunlt. The activation of two trlmeric holoenzymes by ceramlde as a second messenger Is the first example of such a regulation. Together with the known modifications of the cata- lytic subunlt this could provide a very subtle mechanism of controlling Individual PP2A holoenzymes.

All these possibilities are eminently testable and their assessment will probably provide answers, or at least open alternative avenues of future research. We predict that isolation of yeast or fly mutants in other regulatory subunits could provide important insights into the modulation and regulation of this family of enzymes and ultimately into the numerous cellu- lar processes that are predicte~ to be regulated by PP2A.

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Acknowledgements

We thank our collaborators, especially Hiroyuki Ohkura, David M. Glover, Tadashi Uernura and Iozef Goris, who have contributed to our studies on PP2A, and Torn Millward and Nata~a Andielcovi~ for critical review of U~is manuscnpt

TRENDS IN CELL BIOLOGY VOL. 4 AUGUST 1994 291