*Research School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, England; and
Laboratoire de Biophysique Moléculaire et Cellulaire, Département de Biologie Moléculaire et Structurale, Commissariat à l'Energie Atomique-Grenoble, Grenoble, France
All known protein phosphatases specific for phosphorylated Ser and Thr are encoded by two unrelated gene families, termed PPM and PPP (Cohen 1997
; Barford, Das, and Egloff 1998
). The PPM family comprises Mg2+-dependent phosphatases structurally related to PP2C, while the PPP family has traditionally been subdivided into three subfamilies, related to PP1, PP2A, and PP2B (calcineurin). Investigation of these phosphatases has revealed their roles in such processes as glycogen metabolism, regulation of the cell cycle and RNA splicing, regulation of numerous protein kinases, T-cell activation, and memory formation (Cohen 1997
; Klee, Ren, and Wang 1998
; Millward, Zolnierowicz, and Hemmings 1999
; Price and Mumby 1999
; Aggen, Nairn, and Chamberlin 2000
). These three extensively characterized "classical" PPP subfamilies have recently been supplemented by novel members from various species (Cohen 1997
). In addition, a novel divergent group of PPP phosphatases called the PP5/rdgC subfamily has been recognized. It comprises enzymes related to mammalian and fungal PP5/PPT, protein phosphatases with EF-hand domains (PPEF, termed rdgC in Drosophila), and plant PP7 (Becker et al. 1994
; Cohen 1997
; Andreeva and Kutuzov 1999
). Not only do phosphatases of the PP5/rdgC subfamily share relatively low sequence identity with the classical subfamilies, they are also more divergent when presumed orthologs from different species are compared. For example, catalytic domains of mammalian PP1 are 76%88% and
90% identical to those from plants and fungi, respectively, while those of mammalian PP5 are only
65% and 50%58% identical to their presumed counterparts from plants and fungi, respectively. If Trypanosoma species are compared with mammals, the catalytic domains of their PPEF phosphatases are only 39%42% identical, while PP1 and PP2A enzymes are 71%72% and 67% identical, respectively. This situation makes it difficult to ascribe novel divergent PPP phosphatases to the known PPP subfamilies and to anticipate their possible functions, especially when only relatively short incomplete sequences are available or there are no recognizable regulatory domains attached to the catalytic domain. For example, an expressed sequence tag (EST) from watermelon (accession number AA660123) has been annotated as similar to PPX, a PP2A-related phosphatase, while it in fact encodes a homolog of Arabidopsis thaliana PP7. The latest illustration of this complexity is the submission to the sequence databases of the primary structure of a novel protein phosphatase from Plasmodium falciparum (Q9U493), which shares, within its catalytic domain, 35%39%, 37%40%, 30%38%, 38%42%, 38%, and 36%43% identity with PP1, PP2A, PP2B, PP5, PP7, and PPEF, respectively. This enzyme was classified by the authors of Q9U493 as "PP1-like." However, phylogenetic analysis places this phosphatase (PP1Pf in fig. 1
) in the PP5/rdgC subfamily.
|
|
These sequence patterns are confined to the regions adjacent to the residues which coordinate metal ions in the active center (fig. 2 ). They may therefore reflect some differences in the active center structure between PP1/PP2A/PP2B and PP5/PPEF/PP7 phosphatases. Although the nature and significance of these differences will probably not become clear until the three-dimensional structure of a phosphatase of the latter group is solved, the above structural features can be used to tentatively assign new divergent sequences to one of these branches when complete structures are not available.
The observations described in this report suggest that PP1/PP2A/PP2B and PP5/PPEF/PP7 groups are likely to represent two distinct branches which have diverged early in eukaryotic evolution, probably before the divergence of the PP1, PP2A, and PP2B subfamilies and before the acquisition of specific regulatory domains by different catalytic subunits (for diagrams of domain organization of different PPP phosphatases, see fig. 4B in Becker et al. [1994
], fig. 1C
in Li and Baker [1998
], and fig. 2A
in Andreeva and Kutuzov [1999
]). The latter notion is supported by the presence in both branches of phosphatases which have only catalytic but no regulatory domains and are probably regulated by separate regulatory proteins. In the PP1 and PP2A subfamilies, such phosphatases (e.g., PP1, PP2A, and PPX) have been known for a long time. In the PP5/PPEF/PP7 group, PP1Pf appears to have a similar "mono-domain" organization (which possibly led to its assignment to PP1), but a quite distinct primary structure of its catalytic domain, being most closely related to PPEF from T. cruzi (fig. 1
).
There have been at least two different routes to structural diversification of the PPP phosphatases in both branches. Many of them have acquired specific regulatory domains, probably by fusion of their genes with the genes of their cognate regulatory proteins. These fusion events should have occurred in phosphatases of different subfamilies after their structural divergence and functional specialization, since in all known cases regulatory domains in different subfamilies are unrelated. Detailed comparison of the putative regulatory domains in PPEF phosphatases suggests that the fusion of the catalytic domain of the ancestral form with an EF-hand Ca2+-binding protein occurred prior to acquisition of the N-terminal domains. Indeed, the N-terminal domains of PPEF from T. cruzi and those from animals are unrelated, while their C-terminal domains are more similar to each other than to any EF-hand proteins or EF-hand domains in any other enzymes (Andreeva and Kutuzov 1999
).
In addition to the acquisition of and/or C-terminal regulatory domains, there has been another trend in the evolution of PPP phosphatases of both branches: insertion of sequences of various lengths within the catalytic domains. Such inserts are found in some calcineurin isoforms (Barton, Cohen, and Barford 1994
), in PP
from P. falciparum (O96914; Li and Baker 1998
) and its uncharacterized plant homologs distantly related to the PP1 subfamily (represented by F21B7.27At in fig. 1
), in PPEF phosphatases from animals (Montini et al. 1997
; Sherman et al. 1997
), and in PP7 from plants (Andreeva et al. 1998
). To our knowledge, experimental evidence for a functional role of the inserts in the catalytic domains has only been reported for PP7 in A. thaliana. In this phosphatase, proteolytic cleavage of one of the inserts leads to its activation, suggesting that the insert may be an autoinhibitory region (Kutuzov, Evans, and Andreeva 1998
).
The evidence discussed above suggests that the protein phosphatase group called the PP5/rdgC subfamily is as heterogeneous as the combined PP1/PP2A/PP2B group and should rather be considered as consisting of at least three distinct subfamilies: PP5/PPT, probably ubiquitous in eukaryotes; rdgC/PPEF, not found so far in plants and fungi; and PP7, identified so far only in plants. Completion of genome sequencing projects should answer the question of whether two latter subfamilies are indeed kingdom-specific and clarify their origin.
Footnotes
Claudia Kappen, Reviewing Editor
1 Abbreviations: EST, expressed sequence tag; PP, protein phosphatase.
2 Keywords: protein phosphorylation
protein Ser/Thr phosphatases
rdgC/PPEF, PP5/PPT, PP7
3 Address for correspondence and reprints: Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP, United Kingdom. E-mail: p0071233{at}brookes.ac.uk
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