Phosphatase Family PPM

Phosphatase Classification: Fold PPM: Superfamily PPM: Family PPM

PPM (a.k.a. PP2C) is serine/threonine phosphatase found in all eukaryotes, and related to bacterial sporulation protein SpoIIE.

Human PPMs exclusively dephoshorylate pSer/pThr. All PPMs are active, except that TAB1 has been reported as pseudophosphatase (Conner et al. 2006). Unlike the other major Ser/Thr phosphatase family, PPP, PPM does not generally rely on targeting subunits.

Most PPM require two even three metal ions, either Mg²⁺ or Mn²⁺ to activate its phosphatase activity [1].

The PPM subfamilies have different insertions to the core of phosphatase domain, some quite conserved and long, suggesting their importance to the specific functions of individual subfamilies. For instance, the PPM1G subfamily has an inserted acidic region of 54 aa long; PPM1H has an inserted region of ~60 aa; PPM1A has a ~80 aa residues insert to the C-terminal, which consists of three antiparallel alpha helices and form a cleft between it and the catalytic domain.

Subfamilies

PPM1A

The subfamily is named after one of the three human copies, PPM1A (PP2Cα), PPM1B (PP2Cβ) and PPM1N. It is involved in different pathways, such as MAPK, SAPK/JNK, TGF-beta, NF-kappaB signaling. PPM1As were found across holozoa.

PPM1G (PP2Cγ): mRNA splicing and histone regulation

PPM1G is found across metazoa. PPM1G has a predicted N-terminal myristoylation site, C-terminal nuclear localization signaling, and a characteristic phosphatase domain inserted by an acidic domain. It is involved in pre-mRNA splicing, histone regulation, and cell cycle.

PPM1D (WIP1): oncogene in different cancer types

The PPM1D subfamily is an oncogene conserved from Monosiga to human. It regulates cell homeostasis in response to DNA damage. It dephosphorylates the DNA damage response kinases ATM and ATR as well as their phosphorylation targets, p53, Mdm2, Chk2 and gH2AX. It also dephosphorylates p38/MAPK, the tumor suppressors INK4A and ARF, and the RelA subunit of NF-kappaB.

PPM1E (POXP): the phosphatases of CAMKs and PAK

The PPM1E subfamily is named after two human PPMs, PPM1E (also known as POXP1, PP2CH, caMKN, CaMKP-N) and PPM1F (also known as POXP2, CAMKP, FEM-2, hFEM-2, CaMKPase). The subfamily has a single copy in most non-vertebrates from Monosiga to ciona, and duplicated when vertebrates emerged. Both PPM1E and PPM1F dephosphorylate kinases CaMK2g and PAK, and PPM1E can also dephosphorylate CaMK4 (of different families from CaMK2g).

PPM1H

The PPM1H subfamily is named after one of the three copies in human, PPM1H (URCC2, ARHCL1, NERPP-2C), PPM1J (PP2Cζ) and PPM1M (PP2Cη), which are expressed in distinct tissues. The PPM1H subfamily are conserved in animals from sponge to human. Usually, it is single-copy in invertebrates from sponge to ciona. The three copies are found in mammals probably arose by two independent duplication events (not whole-genome duplication).

PPM1K (PP2Cκ, PP2Cm, BDP): mitochondrial phosphatase lost in ecdysozoa

The PPM1K subfamily is a mitochondrial phosphatase that regulates mitochondrial permeability transition pore (MPTP). It also dephosphorylates branched-chain alpha-ketoacid dehydrogenase complex. The PPM1K subfamily emerged in holozoan and lost in ecdysozoa.

PPM1L (PP2Cε, PP2Ce)

Human PPM1L is an ER-anchored phosphatase, where it dephoshorylates ceramide transport protein (CERT). It also dephosphorylates two kinases TAK1 and ASK1. While PPM1L emerged in bilateria, all its known substrates emerged in holozoa or earlier.

PTC7: activating Q6 biosynthesis

The PTC7 subfamily is conserved through eukaryotes, dephosphorylates the mitochondrial hydroxylase COQ7 and activates coenzyme Q biosynthesis.

PDPc: the catalytic subunit of pyruvate dehyrogenase phosphatase

The PDPc subfamily is the catalytic subunit of Pyruvate Dehyrogenase Phosphatase (PDP). It is found throughout eukaryotes, so are Pyruvate Dehyrogenase Kinase (PDK) and its substrate Pyruvate Dehyrogenase Complex (PDC).

ILKAP (PP2Cδ): a subunit of TAK1-TAB1 complex

integrin-linked kinase (ILK) associated phosphatase binds to ILK and specifically regulates one of its two substrates, Ser-9 on glycogen synthase kinase 3 β (GSK3β). It also dephosphorylates p90 ribosomal S6 kinase 2 (RSK2) at multiple serine or threonine sites in the nuclear. All the three, ILKAP, ILK and RSK2, emerged in holozoa, but ILKAP was lost in arthropods, while ILK and RSK2 were not.

PHLPP: AGC kinase phosphatase

The subfamily is characterized by PH domain and Leucine rich repeats. It dephosphorylates AKT/PKB, PKC and S6 kinase families of AGC kinase group at serines in hydrophobic motif site. The subfamily is found across bilateria.

TAB1: binding to TAK1 and p38 and inducing their autophosphorylation

The TAB1 subfamily is most well known for its binding to MAPKs TAK1 and p38. It does not dephosphorylate them. Instead, it binds to them and induces their autophosphorylation. It is found in metazoa except Drosophila. It is found in most other arthropods, which indicates a lineage-specific gene loss in Drosophila. Interestingly, TAK1 and p38 are found in Drosophila and both of them emerged earlier, in holozoa and opisthokont, respectively.

PP2D1

The function of the PP2D1 subfamily is unknown. It is found across the eumetazoa, but frequently lost, including from C. elegans, Drosophila and zebrafish. It has an N-terminal predicted nuclear localization signaling (NLS).

CG9801

The CG9801 subfamily found in metazoa but lost in deuterostomes. Its function is unknown.

PPM1Z: a phosphatase lost in vertebrates

The function of this subfamily is unclear. It emerged in holozoa or metazoa through the duplication of its common ancestral gene with PPM1A.

LRR-PPM

The LRR-PPM subfamily has a combination of leucine riches repeats (LRRs) and PPM phosphatase domain. It is found in Amoebozoa. It does not have any clear orthology to the other LRR-PPM family, PHLPP

KAPP-like

The subfamily is similar to the plant PPM phosphatase KAPP (see [1]), a regulator of the receptor-like kinase (RLK) signaling pathway. Plant RLKs are counterpart of animal receptor kinases. This subfamily is found in Dictyostelids and lacks the FHA domain found in plant KAPPs.

Unclassified phosphatases

Below are unclassified phosphatases of PPM family, the functions of which have been known.

• Budding yeast PTC1. Its functions have been reviewed in [2, 3]. PTC1 is found in most fungi (see our internal data).

• Budding yeast PTC6 is found in a broad of fungi. In budding yeast, Ptp6p locates both in the intermembrane and mitochondria. Along with Ptc5p, it regulates the phosphorylation state of Pda1p, the E1alpha subunit of the pyruvate dehydrogenase (PDH). PTC6 and PTC5 (of PDPc subfamily share a large overlap of phenotypes, but they may have distinct molecular functions [2].

• Budding yeast CYR1 encodes adenylate cyclase in budding yeast. The protein has a domain combination of 1) Adenylate cyclase G-alpha binding domain, 2) Ubiquitin domain CYR1 adenylate cyclase, 3) Leucine repeats, 4) PPM phosphatase domain, and 5) cyclase homology domain. CYR1 is found in most fungi including both Ascomycota and Basidiomycota (also by BLAST NR database and Newton's review). CYR1 is close to PHLPP in phosphatase domain sequence, but they have distinct domain structure and molecular function.

• Dictyostelium spnA has two domains, Galpha subunit family of GTP binding proteins at N-terminal, and PPM phosphatase domain at C-terminal. It functions cell autonomously for prestalk differentiation, and cell non-autonomously for prespore differentiation (see summary from DictyBase).

Phosphatase domain structure

The PPM phosphatase domain (PD) has a central β sandwich, flanked by helices. The catalytic core locates at the cleft between the two β sheets. The dephosphorylation reaction is mediated by two or three metal ions located at the base of the cleft (reviewed in [4]). Both of the two β sheets are anti-parallel.

The PPM PDs have largely the same secondary structure combination, E1, E2, E3, H1, H2, E4, E5, E6, E7, H3, E8, E9, H4, H5, H6, E10. The E1, E10, E9, E6, E7 forms one of the anti-parallel beta sheet; the E2, E3, E4, E5, E8 forms another anti-parallel beta sheet.

Based upon the structure-based sequence alignment, there are 7 conserved motifs:

• M1: ED at E2
• M2: VxDG at E3
• M3: GxT at E4
• M4: GDS between E5 and E6
• M5: RxxG at eukaryotic flap
• M6: DG at H4
• M7: DN at E10

The motifs carry the residues coordinate the metal ions mediated the reaction, which are:

• R and D at M1
• D and G at M2
• D at M4
• D at M7

The available structures of PPM PDs show diversity in the regions beyond catalytic core. For instance, the region of H1 and H2 has the helices of different numbers and lengths. The so-called flap region is different in eukaryotes and prokaryotes.

Note:

• A few PPMs (e.g. PPM1A) have an additional N-terminal beta strand (E1'). PPM1A has three helices at C termini. Because these SS elements are not conserved across PPM PDs, we do not include them as part of PPM PD.
• The flag is a surface loop close to the active site was shown to play an important role in regulating substrate access to the catalytic center.
• D of M6 coordinate the third metal is required for activity as shown by D to A substitution [5].

References

1. Tanoue K, Miller Jenkins LM, Durell SR, Debnath S, Sakai H, Tagad HD, Ishida K, Appella E, and Mazur SJ. Binding of a third metal ion by the human phosphatases PP2Cα and Wip1 is required for phosphatase activity. Biochemistry. 2013 Aug 27;52(34):5830-43. DOI:10.1021/bi4005649 |
2. Ariño J, Casamayor A, and González A. Type 2C protein phosphatases in fungi. Eukaryot Cell. 2011 Jan;10(1):21-33. DOI:10.1128/EC.00249-10 |
3. Sharmin D, Sasano Y, Sugiyama M, and Harashima S. Effects of deletion of different PP2C protein phosphatase genes on stress responses in Saccharomyces cerevisiae. Yeast. 2014 Oct;31(10):393-409. DOI:10.1002/yea.3032 |
4. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009 Oct 30;139(3):468-84. DOI:10.1016/j.cell.2009.10.006 |
5. Su J, Schlicker C, and Forchhammer K. A third metal is required for catalytic activity of the signal-transducing protein phosphatase M tPphA. J Biol Chem. 2011 Apr 15;286(15):13481-8. DOI:10.1074/jbc.M109.036467 |