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.
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.
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.
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.
- Brock AK, Willmann R, Kolb D, Grefen L, Lajunen HM, Bethke G, et al. The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression. Plant Physiol. 2010;153(3):1098–111. doi: 10.1104/pp.110.156109.PubMedPubMedCentralCrossRefGoogle Scholar
- Schweighofer A, Kazanaviciute V, Scheikl E, Teige M, Doczi R, Hirt H, et al. The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis. Plant Cell. 2007;19(7):2213–24. doi: 10.1105/tpc.106.049585.PubMedPubMedCentralCrossRefGoogle Scholar