Functional Genomics and Bioinformatics

Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution

Hugo Ceulemans,* Willy Stalmans, and Mathieu Bollen
Summary We have used the (nearly) completed eukaryotic genome sequences to trace the evolution of thirteen families of established vertebrate regulators of type-1 protein phosphatases (PP1). Two of these families are present in all lineages of the eukaryotic crown and therefore qualify as candidate primordial regulators that determined the surface of PP1. The set of regulators of PP1 has continued to expand ever since, often in response to functional innovations in different eukaryotic lineages. In particular, the development of metazoan multicellularity was accompanied by an explosive increase in the number of regulators of PP1. The further increase in the functional diversity of PP1 in the vertebrate lineage was mainly achieved by the duplication of genes for regulatory subunits and by the conversion of already existing proteins into regulators of PP1. Unexpectedly, our analysis has also enabled us to classify nine poorly characterized proteins as likely regulators of PP1. BioEssays 24:371381, 2002. 2002 Wiley Periodicals, Inc.
Afdeling Biochemie, Katholieke Universiteit Leuven, Belgium. Funding agency: A Flemish concerted research action. H.C. is a postdoctoral fellow of the National Fund for Scientific Research-Flanders. *Correspondence to: Hugo Ceulemans, Afdeling Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Hugo.Ceulemans@med.kuleuven.ac.be DOI 10.1002/bies.10069 Published online in Wiley InterScience (www.interscience.wiley.com).

Abbreviations: BLAST, basic local alignment search tool; CPI-17, 17kDa calcium-dependent kinase potentiated phosphatase inhibitor; DARPP-32, 32-kDa dopamine- and cAMP-regulated phosphoprotein; FHA-domain, forkhead-associated domain; GADD34, 34-kDa growth arrest and DNA damage inducible protein; G-subunit, glycogentargetting subunit; Mypt, myosine-phosphatase targetting subunit; NIPP1, nuclear inhibitor of PP1; p53BP2, p53-binding protein 2; PDZdomain, PSD-95/DlgA/ZO-1 domain; PHI, phosphatase holoenzyme inhibitor; PHI-BLAST, position hit initiated BLAST; PNUTS, PP1 nuclear targetting subunit; PP1, protein phosphatase-1; PPP, serine/ threonine-specific phosphoprotein phosphatase; PPP1R*, gene encoding regulatory subunit * of PP1; PSI-BLAST, position-specific iterative BLAST; R-subunit, regulatory subunit; SAM-domain, sterile a-domain; SH3, src-homology domain 3; TGFb, transforming growth factor.

Introduction Since up to one third of all proteins in eukaryotes are regulated by reversible phosphorylation, it is not surprising that 23% of their genes encode protein kinases or protein phosphatases.(1) These enzymes are classified according to the conservation of their catalytic domain and their ability to (de)phosphorylate serine/threonine or tyrosine residues. In most eukaryotes studied, the number of protein tyrosine phosphatases approximates that of protein tyrosine kinases. In contrast, the protein serine/threonine kinases tend to outnumber the protein serine/ threonine phosphatases severalfold. The human genome, for instance, is predicted to encode well over 300 protein serine/ threonine kinases, but only about 20 protein serine/threonine phosphatases.(1) This relative under-representation may be accounted for by the alternative diversification strategy of some protein serine/threonine phosphatases, involving the evolution of interacting polypeptides rather than gene duplication and subsequent sequence diversification. This is most evident for the widely expressed and abundant protein serine/ threonine phosphatases of type-1 (PP1), which regulate such diverse processes as intermediate metabolism, mRNA splicing, transcription and apoptosis. PP1 holoenzymes consist of an exceptionally well-conserved catalytic subunit and one or two variable regulatory (R-) subunits that target the phosphatase to a particular cellular compartment and/or act as substrate specifiers (Fig. 1). For example, the glycogen-targetting subunits not only anchor PP1 to glycogen, but also bind the substrate glycogen synthase and furthermore increase the specific activity of the associated PP1 towards glycogen synthase.(2) To date, dozens of different R-subunits of PP1 have already been described and for some of them a function has already been delineated (Fig. 1, Table 1). The large number of PP1-specific families of R-subunits, combined with the availability of six (nearly) complete eukaryotic genomes, provides the molecular resolution required to trace the functional evolution and diversification of PP1. This analysis led us to propose that the acquisition of novel functions through the complexation with R-subunits underlied the emergence of the PP1 subfamily very early in eukaryotic evolution and continued throughout the further evolution of the mammalian lineage. Unexpectedly, our study also identified

BioEssays 24:371381, 2002 Wiley Periodicals, Inc.

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Figure 1. Function and subcellular localisation of primary regulators of PP1. Red, yellow, green and blue boxes correspond to ancient, opisthokont, metazoan and vertebrate regulators, respectively.

nine mostly uncharacterised proteins as putative R-subunits of PP1, as judged from extensive sequence homology with known regulators. The catalytic subunit Together with most other protein serine/threonine phosphatases and the structurally related (eu)bacterial diadenosine tetraphosphatases, the type-1 protein phosphatases constitute a superfamily of phosphohydrolases that is represented in all three phylogenetic domains, i.e. (eu)bacteria, archaea and eukaryota.(3) Most prokaryotes manage without or with just a few of these phosphohydrolases,(4) but, in eukaryotes, the phosphohydrolase superfamily and, in particular, the serine/ threonine-specific phosphoprotein-phosphatase (PPP) family have flourished. Indeed, all the hitherto (nearly) completed metazoan, fungal and plant genomes harbour at least ten PPP-encoding genes. Further sequence analysis delineates distinct branches of highly conserved protein phosphatases within the eukaryotic PPP family, one of which unites the protein phosphatases of type-1. The genome of the earlybranching eukaryote Giardia lamblia(5) encodes a protein phosphatase that is still 72% identical to mammalian PP1

isoforms. Because Giardia lamblia and other diplomonads are thought to have separated from the main eukaryotic lineages shortly after the divergence of archaea and eukaryotes (Fig. 2), this suggests an early eukaryotic origin for the PP1 branch. The catalytic domains of eukaryotic members of the PPP family have about 40% of their residues in common, most of which contribute to the structural core and the catalytic center. Accordingly, these domains are expected to adopt a nearly identical three-dimensional structure and catalytic mechanism.(6) However, the residues that are common to all PPPs amount to only about half of the residues that are conserved among PP1 isoforms. The remainder of the (nearly) invariant residues constitutes the greater part of the unusually wellconserved protein surface of PP1, which mediates the interaction with the R-subunits. Accumulating evidence suggests that the catalytic subunit establishes multiple contacts with each of its R-subunits, although some interaction sites are shared by several R-subunits.(2) For example, a hydrophobic channel near the C terminus of PP1 can accommodate the [K/R]-X0-1-[V/I/L]-X-[F/W] motif that is present in most R-subunits and that is generally referred to as the RVXF-motif.

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Table 1. Novel nomenclature for human genes encoding primary PP1 regulators. Only official gene symbols are listed, with the exception of PPP1R13A (bold), which is an alias to TP53BP2. Accession numbers for the gene products and the PPP1R-related pseudogenes can be found on www.gene.ucl.ac.uk/nomenclature/ and in Tables 2 and 3. The products of the genes marked with an asterisk have not been recognized before as regulators of PP1.
Gene symbol PPP1R1A PPP1R1B PPP1R1C* PPP1R2 PPP1R3A PPP1R3B PPP1R3C PPP1R3D PPP1R3E* PPP1R3F* PPP1R3G* PPP1R7 PPP1R8 PPP1R9A PPP1R9B PPP1R10 PPP1R11 PPP1R12A PPP1R12B PPP1R12C PPP1R13A PPP1R13B* PPP1R14A PPP1R14B PPP1R14C* PPP1R14D* PPP1R15A PPP1R15B* PPP1R16A PPP1R16B* Product Inhibitor-1 DARPP-32 Inhibitor-2 RGL, GM GL, FLJ14005 PTG, R5 R6 FLJ00089 H2bE Sds22 NIPP1, Ard-1 Neurabin-I, FLJ20068 Neurabin-II, Spinophilin PNUTS Inhibitor-3 Mypt1, M110 Mypt2, M20 p85, DKFZp434D0412 p53BP2, ASPP2 KIAA0771, ASSP1 CPI-17 PHI NY-BR-81 FLJ20251 GADD34 FLJ14744 MGC14333, Mypt3 TIMAP, KIAA0823 (Putative) function Conditional inhibition of PP1

Folding of PP1 Glycogen metabolism

Mitosis mRNA splicing Morphogenesis and membrane receptor signalling Transcription Nuclear process Actomyosin function

Apoptotic signalling Inhibition of PP1 holoenzymes

Apoptotic and stress signalling TGFb signalling

The R-subunits The current analysis is limited to families of vertebrate `primary' regulators of PP1 (Tables 1 and 2). Thus, we have excluded the `secondary' R-subunits, such as the NimArelated kinase 2, AKAP149, Bcl2 and the retinoblastoma protein, that did not originate as regulators of PP1 and only acquired binding sites for the catalytic subunit during their further evolution. R-subunits, like the G-substrate, Hox11, I1PP2A and I2PP2A, that do not specifically interact with PP1 are also excluded. For the comparison of related sequences, we used the BLAST family algorithms, including the PHI-BLAST and the PSI-BLAST programs.(7) Reported structural and functional features were taken into account to obtain an optimal sensitivity/specificity cut-off.

The ancient families of PP1 regulators

Four mammalian R-subunits of PP1 belong to families that originated before or during the divergence of the four supertaxa that constitute the eukaryotic crown (Fig. 2). Indeed, homologues of Sds22 and Inhibitor-3 have been identified in all

four supertaxa, and homologues of Inhibitor-2 and NIPP1 are present in at least three, and two of these supertaxa, respectively. Yeast Sds22 is required for the completion of mitosis, at least in part because of its involvement in the nuclear distribution of PP1.(8,9) Little is known about the function of Inhibitor-3, other than that it is an inhibitor of PP1 in vitro.(10) Considerable evidence suggests that Inhibitor-2 assists in the folding of newly synthesised PP1 but, at higher concentrations, Inhibitor-2 also acts as a negative regulator of the catalytic subunit.(11) NIPP1 targets PP1 to phosphorylated forms of the splicing factors Cdc5L(12) and SAP155 (our unpublished data), and is also a potent inhibitor of PP1.(13) As judged from northern and EST analysis, the genes that encode the ancient mammalian R-subunits, i.e. PPP1R7 (Sds22), PPP1R11 (Inhibitor-3), PPP1R2 (Inhibitor-2) and PPP1R8 (NIPP1), are expressed ubiquitously in humans. The human genome also harbours numerous loci that are related in sequence to PPP1R11, PPP1R2 or PPP1R8 (Table 3). However, these are likely to be processed pseudogenes because, unlike the genuine genes, they contain no introns

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Figure 2. The emergence (arrows) of PP1 and its regulators in eukaryotic evolution. The tree is adapted from Ref. 54. Poorly resolved regions of the tree are drawn as dashed lines. Phylogenetic relationships between metazoans are shown according to Ref. 55. Model species are indicated in italic.

and most of them contain premature stop codons. Moreover, in contrast to the genuine genes, which are all represented by hundreds of ESTs, the putative pseudogenes match either no ESTs or at most a few ESTs that may well be derived from contaminating genomic fragments in cDNA libraries. Our analysis also suggests that a protein recently described as Inhibitor-4(14) is actually derived from a PPP1R2-related pseudogene (PPP1R2P9). As expected, many of the conserved sequence elements in the ancient mammalian R-subunits have been shown to be involved in the interaction with PP1 (Fig. 3A). Thus, all homologues of Inhibitor-3 and of NIPP1 contain an RVXF-motif that mediates their binding to PP1. However, docking of the RVXF-motif onto PP1 is not inhibitory in itself.(2) Consequently, additional interaction sites must be invoked to account for the inhibitory properties of these proteins. For instance, the in vitro inhibition of PP1 by Inhibitor-3 may require a conserved module that lies adjacent to the RVXF-domain (Fig. 3A). Likewise, for full inhibitory potency, NIPP1 depends on a universally conserved [Y/F]-N-T-X2-N-R motif and a polybasic stretch that precede the RVXF-motif (Fig. 3A), as well as a

C-terminal module that is only conserved among metazoans.(13,15) Interestingly, the inhibitory C-terminal module of NIPP1 also functions as an RNA-binding module.(13) In addition to PP1-binding sites, all members of the NIPP1 family contain an N-terminal FHA-domain that mediates the interaction with phosphorylated Cdc5L and SAP155. Remarkably, although both budding and fission yeast encode homologues of Cdc5L and SAP155, they lack a NIPP1 homologue. The absence of NIPP1 in yeast correlates with the loss of most of the TP-dipeptide motifs that are conserved in metazoan and plant homologues of Cdc5L and SAP155 and that are essential for the interaction with NIPP1 (Ref. 12 and our unpublished results). Collectively, these data suggest that fungi have given up on an ancient PP1-dependent mechanism for regulation of mRNA splicing. No less than five PP1 binding sites have been postulated for Inhibitor-2, a largely extended protein that is predicted to wrap around the catalytic subunit.(16) Strikingly, three of the implicated regions roughly coincide with universally conserved motifs (numbers 2, 3 and 4 in Fig. 3A) and a fourth binding site corresponds to a conserved stretch of acidic residues. The fifth

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Table 2. Accession numbers for the protein sequences of the putative primary regulators of PP1 from Arabidopsis thaliana (At), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm) and Homo sapiens (Hs). Only a single accession number is given for each gene. Where no protein sequence is available, an accession number for a DNA sequence is provided (italic).
At R11-family R7-family AC006593 AF296838 Sc BAA09242 AAB34673 Sp CAB11073 AAA35342 Ce CAA88441 CAB05783 CAA85336 AAA81047 AAA62539 AAF60485 AAC17655 AAB42352 AAF39789 Dm AAF50877 AAF52335 AAF55413 Hs CAC16919 AAD26611

R2-family R8-family R3-family

BAA97463 BAB11326

AAA53673 CAA45371 AAB67365 CAA86906 AAB64590

CAB11509

AAF50198 AAF53602 AAF57920 AAF49732 AAF49172

AAC51206 AAD24669 AAB94596 BAB14811 CAA77082 CAA77081 BAB15779 AAF62521 AL035653 BAA86536 CAC37685 BAA22378 BAA28376 AAG60045 AC025166 2208307A AAG48264 BAA91037 AAC50557 BAA34491 AAC25631 BAA34491 AAHO7854 AAK52796 CAA73697 AAB02402 AAB30129 AC078936

R9-family R12-family

AAB37620 AAB47273

AAF47657 AAF49547

R14-family

Z74036

AAF53006

R13-family R15-family R16-family R10-family R1-family

CAB03115 CAA99881

AAF46699 AAF47124 AAF49247 AAF51438

PP1-binding site corresponds to the [K/R]-[G/S]-I-L-K-motif that is conserved only among opisthokonts and amoebozoa. Remarkably, Inhibitor-2 contains no canonical RVXF-motif but a conserved R-[K/R]-X-H-Y-motif has been reported to interact with the hydrophobic RVXF-binding channel.(16) Sds22 and its homologues consist largely of leucine-rich repeats and a leucine-rich repeat cap that are predicted to fold into a curved superhelix.(17) A handful of conserved residues that are exposed at the concave side of the superhelix appear to be essential for the binding of PP1 (our unpublished results). Members of the Sds22 family lack a canonical RVXF-motif. Furthermore, disruption of the RVXF-binding channel of PP1 does not affect the interaction with Sds22.(18)

The family of glycogen-targetting subunits

The storage of glucose-based polysaccharides as a reserve of energy is widespread but quite distinct enzymes have evolved

for their synthesis. Thus, the closely related UDP-glucosespecific glycogen synthases from opisthokonts and amoebozoa bear no similarity to vegetal starch synthases and bacterial glycogen synthases, which prefer ADP-glucose as a substrate. Before the fungal/metazoan divergence, a mechanism evolved for regulation of UDP-glucose-specific glycogen synthases by reversible phosphorylation. Indeed, both in yeast and in mammals, glycogen synthase is inactivated by phosphorylation and re-activated by PP1. Glycogen-targetting subunits (G-subunits) direct PP1 to glycogen and increase its specific activity towards glycogen synthase. Remarkably, humans can boast no less than seven genes that encode G-subunits (Table 1). Northern(19) and/or EST analysis indicate that the expression of PPP1R3A is restricted to striated muscle, whereas PPP1R3B through F are expressed ubiquitously, albeit at variable levels. These genes are all represented by numerous ESTs, but only a single EST

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bilateria.(22) Accordingly, many of the metazoan R-subunits of PP1 are involved in functions linked to multicellularity, such as programmed cell death and the organisation of the actin cytoskeleton at cellcell and cellmatrix contact sites.

Table 3. Accession numbers of DNA sequences that contain human pseudogenes related to genes encoding primary regulators of PP1. Only one accession number is provided for each pseudogene.
Pseudogene PPP1R1AP1 PPP1R1AP2 PPP1R2P1 PPP1R2P2 PPP1R2P3 PPP1R2P4 PPP1R2P5 PPP1R2P6 PPP1R2P7 PPP1R2P8 PPP1R2P9 PPP1R8P PPP1R10P PPP1R11P1 PPP1R11P2 PPP1R12CP PPP1R13AP PPP1R14BP1 PPP1R14BP2 PPP1R14BP3 PPP1R14BP4 PPP1R14BP5 Chromosome 11 11 6 21 5 13 2 7? 4 5 X 1 11 1 X 2 8 22 2 4 7 6 Accession number AC040946 nt. 108290107706 AC016985 nt. 2364723178 X87344 nt. 7437674427 AP001724 nt. 122914124285 AC008491 nt. 3003331400 AL138686 nt. 9137989989 AC005040 nt. 7726575958 AC091440 nt. 7789276271 AC022463 nt. 86738055 AC026295 nt. 8853186948 Z94277 nt. 113300112217 AL359959 nt. 8636187531 AP000824 nt. 6315061179 AL365182 nt. 121590123156 Z83745 nt. 6735867849 AC079249 nt. 112822113151 AC007868 nt. 3382234708 AC002073 nt. 2975030094 AC009409 nt. 134537134192 AC068096 nt. 109466109122 AC087072 nt. 135808135484 AL162580 nt. 162141161798

matches PPP1R3G. Yet, we believe that PPP1R3G is a genuine gene because, in contrast to the processed pseudogenes described in the previous section, it shows no significant similarity to any other human gene at the nucleotide level and it is conserved in mice. All G-subunits share a canonical RVXF-motif for interaction with PP1 as well as a targetting module with motifs for binding to glycogen and glycogen synthase (Fig. 3B and Ref. 20). Based on a proposed distant relationship with a family of glycoside hydrolases and glycosyl-transferases, this targetting module was predicted to fold into a b-sandwich.(21) Some G-subunits combine the RVXF-motif and the targetting module with specific C-terminal modules, such as the putative sarcoplasmic reticulum-binding module of RGl/GM and the allosteric binding site for phosphorylase-a in GL (reviewed in Ref. 2).

Comparison of the completed genomes of man, Drosophila melanogaster and Caenorhabditis elegans with those of fungi and Arabidopsis thaliana indicates that at least seven new families of PP1 regulators emerge in metazoans before the divergence of the bilateria (Fig. 2). This observation hints at a pronounced functional diversification of PP1 during the transformation of a unicellular proto-metazoon into the complex animal that is believed to be the common ancestor of all

Metazoan families of PP1 regulators

Actin-associated regulators of PP1. These belong to three metazoan families of R-subunits, i.e. the neurabin family, the Mypt family and the PHI family (Table 1; Fig. 3C). The neurabin family unites a number of PP1-binding proteins that link integral membrane proteins to the subcortical actin cytoskeleton at cellcell adhesion sites. They have been implicated in the formation of dendritic spines(23) and in morphogenesis.(24) The myosin-phosphatase targetting (Mypt) subunits come in large and small isoforms.(25) Heterodimers that consist of a large and a small Mypt tether PP1 to class-II myosin and promote the dephosphorylation of the myosin regulatory light chain, thereby causing relaxation of the actomyosin fibers. Although myosin-II based actomyosin is involved in cytokinesis in all opisthokonts, Mypt subunits occur only in metazoans. Thus, their emergence roughly coincides with the acquirement of a novel function for these actomyosin fibers. Indeed, the actin cytoskeleton at cellcell and cell matrix adhesion sites in metazoans is reinforced by tensile and contractile stress fibers that contribute to the shape of cells. Interestingly, two other proteins that are associated with the subcortical actin cytoskeleton at cellcell adhesion sites, adducin and moesin, were also found to be substrates of Mypt-associated PP1.(26,27) It is currently believed that the contractile apparatus of metazoan striated and vertebrate smooth muscles evolved independently from myosin-II based actomyosin fibres of non-muscle cells.(28) Therefore, it comes as no surprise that striated and smooth muscles have inherited the PP1-dependent mechanism for downregulation of myosin contractility from their non-muscle ancestors. The activity of Mypt-PP1 complexes is tightly regulated, not only by reversible phosphorylation of their regulatory module, but also by members of the PHI family of phosphatase holoenzyme inhibitors, such as PHI and the closely related CPI-17.(29) These become potent inhibitors of Mypt-associated PP1 and possibly also of other PP1 holoenzymes after phosphorylation on a conserved threonine. Expression of the neurabin-encoding PPP1R9A and PPP1R9B genes is most pronounced in neural tissues, but occurs also to a limited extent in various other tissues. All Myptencoding genes (PPP1R12A, PPP1R12B and PPP1R12C) are ubiquitously expressed and give rise to large (85 to 130 kDa) Mypt subunits. In addition, small Mypt isoforms (about 20 kDa) are derived from PPP1R12B by alternative splicing. Four genes, PPP1R14A, PPP1R14B, PPP1R14C and PPP1R14D, encode members of the PHI family. PPP1R14A, PPP1R14B(29) and PPP1R14C have a broad expression pattern, but the product of PPP1R14A, CPI-17, is

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Figure 3. Conserved modules of thirteen families of vertebrate regulators of PP1. The figure lists the gene families and a prototype protein of each family. The prototype proteins are drawn to scale, with canonical RVXF-motifs depicted as red boxes. Features conserved in all family branches are coloured yellow, whereas lineage-specific conserved features are shown in violet. In addition to earlier defined abbreviations, the following abbreviations were used: LRRs, leucine-rich repeats; TM, targetting module; ABM, actin-binding module; ANK-Rs, ankyrin repeats (dark yellow); RM, regulatory module; MBM, myosin-binding module; TFIIS, domain I of transcription factor IIS; ZF, zinc finger.

enriched in smooth muscle. The available data did not allow us to appreciate the expression of PPP1R14D. The neurabins are characterised by an N-terminal F-actinbinding module, a PDZ-domain fused to an RVXF-motif, a coiled-coil domain and a SAM motif (Fig. 3C). PDZ-domains are protein interaction modules that often bind to the cytosolic C terminus of transmembrane proteins,(30) whereas SAMmotifs and coiled-coil domains act as homo- or heterooligomerising modules and may, consequently, account for the reported oligomerisation of neurabin-I.(31) In addition to an RVXF-motif that is essential for the interaction with PP1,(32) the large Mypt subunits contain a second PP1 interaction site, possibly corresponding to the conserved N-terminal [E/D]-QL-[K/R]-X-W-motif (Fig. 3C). The RVXF-motif is flanked Cterminally by an array of eight, often degenerate ankyrin repeats, which are thought to contain a third interaction site for PP1. Large Mypt subunits also contain a central regulatory

module that inhibits the associated PP1 when phosphorylated.(25) The C terminus of both the large and small Mypt subunits encompasses a myosin-binding module and a module for dimerisation of large and small Mypt subunits. After complexation with a large Mypt, the hydrophobic RVXFbinding channel of PP1 is already occupied. Hence, additional binding of one of the members of the PHI family cannot be mediated by an RVXF-motif. Accordingly, the PHI proteins lack an RVXF-motif. Instead, they largely consist of a single conserved module that includes the essential phosphorylatable threonine.

PP1 regulators modulating apoptotic signalling.

Even though endogenous oxygen radicals produced in response to stress apparently can trigger a primitive type of apoptosis in all supertaxa of the eukaryotic crown,(33) canonical apoptotic regulators, such as p53, caspases and

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Bcl2-related polypeptides appear to be unique to metazoans. These regulators confer a more nuanced response to stressful conditions, but also harness apoptosis as a tool for developmental control of differentiated cell populations in multicellular organisms. Two primary R-subunits of PP1, p53BP2 and GADD34, have been linked to apoptotic signalling (Table 1). p53BP2 binds not only to PP1,(34) but also to the classic apoptotic regulators p53 and Bcl2, to the p65 subunit of the antiapoptotic transcription factor NF-kB(35) and to a number of other proteins. Notably, all these interactions are mutually exclusive. Although the precise function of p53BP2 is ill understood, multiple lines of evidence suggest a role in the regulation of apoptosis.(35) GADD34, on the contrary, belongs to a group of proteins that are produced in reaction to severe stress conditions, which result in the downregulation of translation in general. This so-called integral-stress-response is induced by phosphorylation of the eukaryotic translationinitiation factor eIF2a by stress-activated kinases. GADD34associated PP1 is an eIF2a -phosphatase, and consequently a negative regulator of the integral-stress-response.(36) Remarkably, the eIF2a -mediated attenuating effect of PP1 on the integral-stress-response is conserved in budding yeast,(37) which, however, lacks an appreciable homologue of GADD34. Furthermore, GADD34 has also been described as a proapoptotic protein and production of GADD34 appears strictly correlated with an apoptotic outcome after ionising radiation.(38) The genes for p53BP2 and GADD34, termed PPP1R13A and PPP1R15A, respectively, are widely expressed, and so are PPP1R13B and PPP1R15B, which are predicted to encode homologues of p53BP2 and GADD34, respectively. p53BP2, the product of PPP1R13B and their unique homologues in Drosophila and Caenorhabditis, all contain an RVXFmotif fused to four (degenerate) ankyrin repeats and an SH3domain (Fig. 3C and Ref. 39). A third human member of the p53BP2 family, termed RelA-associated inhibitor (RAI),(40) lacks an RVXF-motif and is therefore unlikely to be a PP1 regulator. However, the presence of the RVXF-motif in all other known relatives of p53BP2 suggests that RAI has lost the common PP1-binding RVXF-motif. The GADD34 family is characterised by a relatively small C-terminal module that is also headed by an RVXF-motif (Fig. 3C). This conserved region is proposed to encompass the PP1-regulating features of GADD34. of Mypt family described above, Mypt3 is an inhibitor of the myosin phosphatase activity of PP1. Furthermore, Mypt3, TIMAP and their invertebrate homologues all lack the myosin-binding site and other features of the Mypt family, but instead they contain a prenylatable CaaX box. Finally, comparison of the intronexon conservation of the ubiquitously expressed Mypt3- and TIMAP-encoding genes, PPP1R16A and PPP1R16B, with that of the PPP1R12 genes validates a distinct classification. The involvement of TIMAP in TGFb signalling [B.J. Ballermann, personal communication] may point to yet another function of PP1 in development and apoptosis of metazoans. Mammalian PNUTS has been described as a nuclear regulator of PP1 that colocalises with chromatin during telophase, but not during other mitotic phases.(42) PNUTS and its homologues (Fig. 3C) harbour an N-terminal module that may account for the localisation to chromatin, as the second half of this module appears distantly related to domain I of the transcription-elongation factor TFIIS.(43) Similar modules are also present in other transcription factors, such as Elongin-A and CRSP70. A second conserved domain of unknown function is headed by an RVXF-motif. The extreme C terminus of the PNUTS family members harbours the classical zinc finger motif, which occurs in many transcription factors. Collectively, these observations may hint at a function as a subunit that targets PP1 to a transcription complex. The PNUTS-encoding PPP1R10 gene is expressed ubiquitously in mammals.

Other metazoan regulators of PP1. Little is known about the function of two additional families of R-subunits that appear at this stage of evolution, the TIMAP and the PNUTS families. Like the Mypt subunits, the members of the TIMAP family share an array of eight (degenerate) ankyrin repeats adjacent to an RVXF-motif. On the basis of this similarity, one of the two human representatives of the TIMAP family has been described as Mypt3.(41) However, unlike the members 378 BioEssays 24.4

The explosive increase during pre-bilaterian metazoan evolution in the number of protein families that regulate PP1 apparently slowed down in the lineage that gave rise to the vertebrates (Fig. 2). The only known family of primary R-subunits that is unique to this lineage includes inhibitor-1 and its homologues (Table 1). Inhibitor-1 and DARPP-32 function as switch proteins that can turn off the activity of PP1 in response to phosphorylation on a conserved threonine by cAMP-dependent protein kinase (PKA). Conversely, their dephosphorylation by the Ca2/calmodulin-dependent protein phosphatase PP2B re-activates the associated PP1. Thus, cAMP and Ca2-agonists regulate a pool of PP1 that is associated with Inhibitor-1 or DARPP-32, which in turn modulates transmembrane ion currents by controlling the dephosphorylation state of ion pumps and channels.(44,45) The appearance of the Inhibitor-1 family fits in nicely with the development of multiple regulatory interactions between PKA and PP1 in the vertebrate lineage. Indeed, several vertebrate PKA-anchoring proteins, such as AKAP149, AKAP220 and Yotiao also bind PP1. Furthermore, a number of the more ancient primary R-subunits of PP1 (NIPP1, the muscle-type G-subunit and Neurabin-I) have acquired regulatory phosphorylation sites for PKA after the divergence of the bilateria.(2)

The Inhibitor-1 family

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In humans, three members of the Inhibitor-1 family are generated from the genes PPP1R1A, PPP1R1B and PPP1R1C (Table 1). All three genes are expressed in a wide variety of tissues. All members of the Inhibitor-1 family share an RVXF-motif that is C-terminally coupled to an inhibitory module (Fig. 3D). The latter includes the pivotal threonine that has been proposed to occupy the catalytic site of PP1 in the phosphorylated state. The conserved N terminus is followed by isoform-specific features, such as the module for phosphorylation-dependent inhibition of PKA in DARPP-32.(46) Conclusion Not only the structurally and catalytically important residues, but also the exposed residues of PP1 are exceptionally well conserved in all eukaryotic lineages. Thus, the origin of the PP1 subfamily may well have been the acquirement by an ancestral PPP of a number of interaction sites for one or more primordial R-subunits. An essential function of PP1 conferred by the binding of the primordial subunit(s) would then account for the conservation of its surface. One of the most ancient and essential functions of PP1 is to antagonise the mitotic Aurorarelated protein kinases by the dephosphorylation of histone H3 and other substrates.(47) It is tempting to speculate that this function corresponds to a primordial function of PP1. Two of the ancient PP1 regulators, Sds22 and Inhibitor-3, are conserved in all completed genomes and are essential, and are consequently excellent candidate primordial R-subunits. Notably, Sds22 has also been implicated in mitosis. An essential primordial function of PP1 would steer the evolution of novel R-subunits, in that the latter would have to be compatible with the interaction sites for the primordial R-subunit(s). Such a scenario may explain the success of common PP1 interaction motifs, such as the RVXF-motif.(2) The typical RVXF-motif resides in a flexible and exposed loop. Such loops are prone to mutation, unless they are of functional importance. Consequently, random mutation can account for the acquirement of an RVXF-motif that will be conserved if binding of PP1 provides a functional advantage. Interestingly, all the PP1 regulators that evolved after the divergence of the eukaryotic crown (Fig. 2) contain an RVXF-motif, with the exception of the members of the PHI-family, which have no use for an RVXF-motif, as they inhibit PP1 that is bound via the RVXF-binding channel to other regulators. The expansion of the set of PP1 regulators is correlated with functional innovations in eukaryotic evolution. Thus, the development of multicellularity in metazoans went hand in hand with the appearance of numerous families of primary R-subunits. This marked increase in the number of regulators of PP1 is parallelled by the metazoan bifurcation of the PP1 subfamily into the a and b subtypes.(48) Even though these two subtypes are biochemically indistinguishable, increasing evidence indicates at least partially distinct functions. Only a single family of primary R-subunits of PP1 (Inhibitor1) appeared after the divergence of the bilateria. Nevertheless, the functional diversification of PP1 has continued in the lineage that gave rise to vertebrates, mainly by the expansion of existing families of primary R-subunits. Indeed, six out of the seven families of R-subunits that first appear in metazoans have only a single representative in Drosophila and in Caenorhabditis, but at least two in mammals. A second factor that has contributed to the functional diversification of PP1 in vertebrates is the acquisition of PP1-binding properties by proteins with a different original function. For example, the secondary R-subunits NimA-related kinase 2 and retinoblastoma protein are the only members of their respective families that contain an RVXF-motif, and our analysis suggests that they gained this motif fairly recently, i.e. after the divergence of the bilateria. Other secondary PP1 regulators, such as BH-protocadherin-c,(49) are the only of a number of isoforms derived from the same gene that are endowed with an RVXFmotif. The latter regulators presumably represent an early stage in the evolution of R-subunits, which suggests that the functional diversification of PP1 is a still ongoing process. It has to be noted that, while we focussed in this article on the evolution of vertebrate PP1 regulators, novel R-subunits have evolved in other eukaryotic lineages as well. For instance, the Drosophila R-subunits Bif(50) and Klp38B,(51) and the fungal PP1 regulators related to Reg1(52) and Gip1(53) have no obvious vertebrate counterparts. Our analysis has uncovered two novel mammalian genes (PPP1R1C and PPP1R3G). Based on the conservation of hallmark motifs and modules, we have also been able to propose a function as a primary regulator of PP1 for nine mostly uncharacterised proteins (Table 1). In vertebrates, eight of the thirteen families of primary regulators of PP1 have at least two members, which has prompted us to propose a streamlined nomenclature for their genes (Table 1). Acknowledgments We greatly appreciate the help from Dr. Hester Wain of the Human Gene Nomenclature Committee in updating the nomenclature of the primary R-subunit genes. References
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