Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

PP2C

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_249

Synonyms

Historical Background

Protein phosphatase 2C (PP2C) was first defined as magnesium (or manganese)-dependent Ser/Thr-specific dephosphorylation activity in mammalian tissue extract (Cohen 1989). This activity was also found to be resistant to okadaic acid, a potent inhibitor of Ser/Thr phosphatases. Because of its cation dependency, PP2C is sometimes referred as PPM (protein phosphatase, magnesium or manganese dependent). Genes encoding PP2C were subsequently isolated from yeast to humans, revealing a conserved protein phosphatase family with no apparent sequence similarity to the other Ser/Thr phosphatases such as PP1, PP2A, and PP2B. It is also notable that eukaryotic species have more genes encoding for PP2C than those for the other Ser/Thr phosphatase families. For example, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have seven and six PP2C or PP2C-related phosphatase genes, respectively. The human genome contains at least 16 PP2C genes that express at least 22 isoforms by alternative splicing (Lammers and Lavi 2007). Genome projects in different organisms are rapidly identifying more PP2C genes, ten genes in the fruit fly Drosophila melanogaster and eight genes in the nematode Caenorhabditis elegans. A record-breaking number, 80, of PP2C family genes have been identified in the popular plant model system Arabidopsis thaliana (Xue et al. 2008; Fuchs et al. 2013).

Despite the lack of apparent sequence similarity between PP2C and PP1, PP2A, and PP2B families, the determined crystal structures of their catalytic cores share significant resemblance (Barford et al. 1998), suggesting a common catalytic mechanism. On the other hand, most PP2C enzymes are found to be monomeric, while PP1, PP2A, and PP2B usually require regulatory subunits for their activity/function. PP2C and PP2C-like enzymes are involved in many different biological processes in diverse organisms; in addition to eukaryotic PP2Cs, several metal ion-dependent phosphatases in bacteria also exhibit sequence similarities to PP2C enzymes (Hecker et al. 2007; Hilbert and Piggot 2004). Some of the well-characterized PP2C functions in eukaryotic signal transduction systems will be reviewed here.

PP2C Negatively Regulating the Stress-Activated MAPK

Because of the relatively broad substrate specificity of PP2C enzymes, it is not easy to search cellular substrates of PP2C through biochemical approaches. Genetic screens were instrumental to uncover important roles of PP2C in the negative regulation of the stress-activated MAP kinase (MAPK) cascades.

Two PP2C isoforms in budding yeast, Ptc1 and Ptc3, were identified in a screen for genes whose overexpression suppresses the lethality caused by hyperactivation of Hog1, a MAPK responsive to high osmolarity stress (Hohmann 2002). Studies in the fission yeast S. pombe found that loss of Ptc1 and Ptc3 brings about a defective phenotype which can be suppressed by inactivation of a Hog1-like Spc1 MAPK, implying hyperactivation of Spc1 in the absence of PP2C (Hohmann 2002). Indeed, the following biochemical studies in S. pombe showed that Ptc1 and Ptc3 inhibit Spc1 MAPK by dephosphorylating its Thr-171, one of the activating phosphorylation sites conserved in the T-loop of all MAPKs (Fig. 1). Furthermore, activation of Spc1 MAPK induces expression of Ptc1, indicating that Ptc1 is part of the negative feedback loop to suppress Spc1 activity. In budding yeast, Thr-174 of Hog1 MAPK, the equivalent of Spc1 Thr-171, is known to be the target of three PP2C enzymes, Ptc1, Ptc2, and Ptc3; Ptc1 dephosphorylates Thr-174 to maintain the low basal activity of Hog1 as well as to inactivate Hog1 during cellular adaptation to osmostress. Ptc2 and Ptc3 appear to limit the maximum activation level of Hog1 during the stress (Martín et al. 2005).
PP2C, Fig. 1

Negative regulation of the stress MAPK by PP2Cs in fission yeast. In response to environmental stress, Wis1 MAP kinase kinase (MAPKK) phosphorylates Thr-171 and Tyr-173 in the T-loop of Spc1 MAPK. Activated Spc1 in turn phosphorylates the downstream transcription factor to induce stress-resistance genes as well as the PP2C gene encoding Ptc1. Dephosphorylation of Spc1 Thr-171 by Ptc1 or by constitutively expressed Ptc3 inactivates Spc1 MAPK. The other activating phosphorylation of Spc1 at Tyr-171 is removed by the Pyp1 and Pyp2 tyrosine-specific phosphatases

Also in mammals, PP2C enzymes are implicated in the negative regulation of the stress-activated MAPKs called p38 and JNK, through dephosphorylation of their T-loop (Lu and Wang 2008). For example, Thr-180 in the T-loop of p38 is dephosphorylated by PP2Cα and PP2Cδ/Wip1 isoforms. Interestingly, p38 MAPK activated by UV stress phosphorylates the tumor suppressor p53, which induces transcription of Wip1. Thus, as seen with the regulation of the Ptc1 phosphatase in fission yeast, the p38-regulated transcription of Wip1 forms a negative feedback loop (Fig. 2). Moreover, not only MAPKs but also MAPKKs (MKK3, 4, 6, 7) and MAPKKKs (ASK1, TAK1) that function upstream of p38 and JNK MAPKs are under the negative regulation by PP2Cα, β, and ε isoforms.
PP2C, Fig. 2

Negative regulation of the p53 pathway by Wip1 PP2C. Wip1 negatively regulates the p53 tumor suppressor pathway by dephosphorylating p53 and the protein kinases (red) that phosphorylate p53 in response to DNA-damaging stress. In addition, Wip1-dependent dephosphorylation of MDM2 promotes degradation of p53. Expression of Wip1 is induced by active p53, thus forming a negative feedback loop

In Arabidopsis, two PP2C enzymes, AP2C1 and PP2C5 (AP2C3), have been identified as negative regulators of the stress-activated MAPKs (Brock et al. 2010; Schweighofer et al. 2007; Fuchs et al. 2013). In addition, two closely related PP2Cs, AP2C2 and PP2C4, also possess a MAPK-interacting motif at their N-terminus. Environmental stress stimuli activate multiple Arabidopsis MAPKs such as MPK3, MPK4, and MPK6, to which AP2C1 and PP2C5 physically bind. Deletion of the AP2C1 gene in the genome leads to higher activation of MPK4 and MPK6 upon wound stress. Simultaneous deletion of both AP2C1 and PP2C5 phosphatase genes induces extremely high activity of MPK3, 4, and 6 in response to the plant hormone abscisic acid (ABA). On the other hand, overexpression of either AP2C1 or PP2C5 represses the stress-activated MAPKs. Expression of AP2C and PP2C5 is significantly induced by wounding and ABA, respectively, implying a negative feedback mechanism. It has not been determined, however, whether the induction is dependent on the stress-activated MAPKs.

Wip1 (PP2Cδ) Negatively Regulating the p53 Tumor Suppressor

Amplification of the PP2Cδ/Wip1 gene is frequently detected in human breast and ovarian cancers. In addition, PP2Cδ/Wip1 knockout mice are less prone to tumor formation, and fibroblasts derived from the mice are resistant to the transformation activity of oncogenes. Consistent with these observations, recent studies are unveiling the oncogenic activity of the PP2Cδ/Wip1 phosphatase, through inhibition of the p53 tumor suppressor by multiple means (Lu et al. 2008; Goloudina et al. 2016, Fig. 2). As discussed in the last section, Wip1 dephosphorylates and inactivates p38 MAPK, an activator of p53. p53 is also a direct substrate of the Wip1 phosphatase; ionizing radiation and UV stress activate the ATM and ATR kinases that phosphorylate Ser-15 of p53 to induce apoptosis, while dephosphorylation of this residue by Wip1 suppresses apoptosis. The Ser-15 phosphorylation is also inhibitory to the interaction of p53 with MDM2, the E3 ubiquitin ligase involved in p53 degradation, and therefore, dephosphorylation of Ser-15 by Wip1 can destabilize p53. Moreover, Wip1 indirectly affects the stability and activity of p53 through dephosphorylation of MDM2 as well as inhibition of the ATM kinase that phosphorylates MDM2 (Fig. 2). The ATM-dependent phosphorylation of MDM2 at Ser-395 is removed by Wip1 to stabilize MDM2 and promote its interaction with p53 for increased ubiquitination and degradation of p53.

In the regulation of the p53 pathway discussed above, the Wip1 phosphatase counteracts the ATM and ATR kinases on two different substrates, p53 and MDM2 (Fig. 2). It has also been reported that Wip1 dephosphorylates the protein kinases Chk1 and Chk2 that are phosphorylated by ATR (Fig. 2), as well as the histone variant H2AX that is mainly phosphorylated by ATM. Consistent with these observations, Wip1 preferentially dephosphorylates Ser and Thr residues followed by Gln, the consensus sequence motif phosphorylated by the ATM and ATR kinases. Extensive effort to develop Wip1-specific inhibitors has been made, which has delivered peptide and chemical inhibitors that may serve as seeds for future pharmaceutical development (Goloudina et al. 2016).

Abi1/2 PP2Cs in Abscisic Acid Signaling in Plants

The Arabidopsis thaliana genome contains 80 PP2C genes, which can be classified into 13 subgroups based on the encoded amino acid sequences (Xue et al. 2008; Fuchs et al. 2013). The Abi1/2 subgroup that functions in the abscisic acid (ABA) signaling (Cutler et al. 2010; Hubbard et al. 2010; Miyakawa et al. 2013; Ng et al. 2014) has been most extensively characterized. ABA is a plant hormone that has multiple roles in the regulation of plant physiology, including seed dormancy, growth inhibition, and stomatal closure. It has been reported that eight out of the nine Abi1/2 family PP2Cs in Arabidopsis function as negative regulators in the ABA signaling. While mutational inactivation of each Abi1/2-family PP2C has little effect, simultaneous knockout of the multiple phosphatases has profoundly affect seed germination, indicating that the Abi1/2-family PP2Cs possess overlapping functions in the ABA signaling.

Recent attempts to discover the ABA hormone receptor successfully identified PYR/PYL/RCAR (pyrabactin resistance 1/pyrabactin resistance 1-like/regulatory component of ABA receptor) family proteins that directly recognize and bind ABA. The PYR/PYL/RCAR proteins belong to the START-domain superfamily, members of which are soluble proteins with a hydrophobic ligand-binding pocket flanked by two loop structures called the “gate” and the “latch.” ABA binding to the open pocket closes the gate and locks the latch, creating a new interaction surface for the Abi1/2 family PP2C (Fig. 3). When the PP2C binds to the ABA-PYR/PYL/RCAR complex, a Trp residue conserved among the Abi1/2 family PP2Cs is pulled to form a contact with ABA via a water molecule, stabilizing the ternary complex of ABA, the ABA receptor, and the Abi1/2 PP2C. The phosphatase activity of Abi1/2 is inhibited in this ternary complex. In the absence of ABA, however, the Abi1/2 PP2C released from the complex can dephosphorylate and inactivate the SNF1-related protein kinases SnRK2s (Fig. 3). Activated SnRK2s in turn phosphorylate and activate downstream transcription factors to induce ABA-regulated genes.
PP2C, Fig. 3

Abscisic acid (ABA) signaling through the dimeric ABA receptor and the Abi1/2 PP2C. In the absence of ABA (−ABA), the Abi1/2 PP2C represses SnRK2 by removing the activating phosphorylation on SnRK2. Once ABA binds (+ABA), the receptor dimer dissociates to form a complex with the Abi1/2 PP2C and repress its phosphatase activity, resulting in phosphorylation and activation of SnRK2. Active SnRK2 then induces gene expression through activation of the downstream transcription factors

Based on the oligomeric state in the absence of ABA, the ABA receptor proteins are classified into two groups. Four of the receptors (PYR1/RCAR11, PYL1/RCAR12, PYL2/RCAR13, PYL3/RCAR14) form stable homodimers in the absence of ABA and show relatively low affinity to ABA (Kd ≥ 50 μM), as the homo-dimerization interface largely overlaps with the ABA-binding site. Binding of ABA to the dimeric receptors induces conformational changes, which allows dissociation of the receptor homodimers and exposure of the interaction site for the Abi1/2-family PP2C (Fig. 3). Therefore, the dimeric receptors never interact with the Abi1/2-family PP2C in the absence of ABA; thus, complex formation of those receptors with the phosphatases is dependent on ABA. In contrast, the monomeric receptors can interact with and repress the Abi1/2-family PP2C even in the absence of ABA, although the interaction is greatly enhanced by low concentrations (approx. 1 μM) of ABA. Multiple receptors with different affinities to ABA may allow fine-tuning of ABA signaling in response to both biotic and abiotic stresses.

Summary

In eukaryotic species, protein kinases outnumber protein phosphatases. Therefore, PP2C and other protein phosphatases are likely to catalyze dephosphorylation of multiple protein substrates, but our knowledge is still very limited as to the in vivo substrates for each PP2C enzyme. Identification of a complete set of substrates for each PP2C is necessary for a comprehensive understanding of the contribution of the PP2C family enzymes to the cellular signaling network.

Most PP2C enzymes appear to be constitutively active and function as monomer. This is a stark difference from other protein phosphatase families such as PP1 and PP2A, which form multiple different complexes with non-catalytic subunits that determine their substrate specificities (Shi 2009). The ABA receptors in plants are the first example of regulator proteins for PP2C and may represent a breakthrough in the PP2C field. It remains to be studied whether other PP2Cs cooperate with such regulatory proteins.

See Also

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyNaraJapan
  2. 2.Department of Microbiology and Molecular GeneticsUniversity of California, DavisDavisUSA