A number of cellular pathways are regulated through the reversible phosphorylation of proteins orchestrated by kinases and phosphatases, a central mechanism in the regulation of signal transduction (1). Protein phosphatases can be classified into four gene families each with distinct functions in cells: (i) Serine/threonine phospho-protein phosphatase (PPP); (ii) Mg2＋-dependent protein phosphatase (PPM/PP2C); (iii) Phospho-tyrosine phosphatase (PTP); (iv) Asp-based protein phosphatase (1-4). PPPs are the most highly conserved phosphatases in eukaryotes, and more than 80% phosphatase activity is contributed to this family. PPPs are further divided into seven subgroups (PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7) based on sequence, structure, and biochemical properties. Approximately 40 catalytic subunits derived from PPPs and PPM/PP2C are responsible for dephosphorylating the majority of Ser/Thr phosphoresidues on proteins, whereas more than 400 kinases are in charge of phosphorylation of Ser/Thr residues. This implies that these phosphatases should be highly regulated, and the majority of catalytic activities is usually attributed to a wide variety of regulatory subunits, forming functionally unique complexes with a limited number of catalytic subunits (Fig. 1) (1-3).
Protein phosphatase 4 (PP4) complex is comprised of one catalytic subunit (PP4C) and five regulatory subunits (PP4R1, PP4R2, PP4R3α, PP4R3β, and PP4R4), typically forming the functional heterodimer (PP4C/PP4R1 or PP4C/PP4R4) or heterotrimer (PP4C/PP4R2/PP4R3α or PP4C/PP4R2/PP4R3β) in cells (Fig. 2) (3-10). PP4C, a catalytic subunit of PP4, was the first identified in 1993 as PPX (designated as PPP4 in human genome nomenclature) and indicated 65% identical to PP2ACα and PP2ACβ. Given the < 70% identity to other phosphatases including PP1, PP2A and PP2B, PP4C cannot be considered as isoforms of these proteins (4). Also, it did not show physical interaction with other phosphatases tested and had the distinct functions (1, 4). The regulatory subunits of PP4 are highly conserved across mammals, yeast and plants. The heterotrimer complex, PP4C-PP4R2-PP4R3 (PPH3-YBL1046W-PSY2), sharing a high homology between human and
In this review, we will summarize the recent understanding and the biological significance of the roles of PP4 in a wide range of cellular physiology including genomic stability, immune response, glucose homeostasis, neuronal development, and plant physiology (Fig. 3).
Genome is consistently exposed to DNA damages caused by endogenous or exogenous events. If genomic aberration occurs, cells will suffer from disasters such as cell death and transformation. Thus, it is necessary to maintain genomic integrity.
Most studies for cell cycle regulation-related PP4 functions have focused on the mitosis and meiosis events. However, considering that PP4 is required for recovery from a checkpoint-induced arrest by dephosphorylating and inactivating p53 in G1 phase (41), it is feasible that PP4 could have essential functions in other cell cycle phases.
Phosphorylated form of histone H2AX, named as γ-H2AX, is induced upon DNA damage and thus, regarded as the DNA damage marker. Aberration of dephosphorylation of γ-H2AX by PP4 on time clearly impairs proper DDR and causes genomic instability (9, 42). PP4 also dephosphorylates RPA2, whose phosphorylation status is essential for DNA synthesis after DNA damage, G2/M phase checkpoint, and HR (5). Also, phosphorylation of 53BP1, one of most critical proteins in genomic stability and tumorigenesis, is mainly regulated by PP4 (8). 53BP1 must be highly phosphorylated at T1609/S1618 during mitosis so as not to be activated and thus, block error-prone NHEJ repair. However, PP4-mediated dephosphorylation of 53BP1 promotes the recruitment of 53BP1 to the DNA double strand break sites at the onset of G1 phase to secure genomic stability, but not in mitosis (8). When phospho-null mutants of 53BP1 (T1609A/S1618A) are recruited to DNA lesions in mitosis, chromosome segregation is largely impaired. Besides, DNA damage sites should be accessible to facilitate recruitment of DNA repair factors. When DNA lesions are generated, KAP-1, a transcriptional corepressor, is rapidly phosphorylated by ATM kinase, inducing relaxation of DNA lesions and thus promoting transcription of stress-induced genes, such as p21 and Gadd45α (growth arrest and DNA damage-inducible alpha) to initiate DNA repair in heterochromatin. Right after DNA repair is completed, PP4 induces KAP-1 dephosphorylation to recover its corepressive function (6). PP4 is also involved in NHEJ pathway, possibly through altering KAP-1 phosphorylation (43). According to phosphoproteomic analysis (6), PP4 could participate in other DNA repair pathways, such as DNA mismatch repair and base excision repair in addition to DNA DSB repairs, implying that PP4 could be universal regulator for DNA repair.
Considering the critical roles of PP4 as a phosphatase, PP4 should be tightly regulated for efficient DDR. In fact, the post-translational modifications of PP4C and its regulatory subunits have significant impact on PP4 activity. PP4C methylation on C-terminal leucine site (Leu307) by LCMT-1 (leucine carboxyl methyltransferase 1) is required for stable PP4 complex, enzymatic activity to its targets, such as KAP-1 and 53BP1, and thus, DNA DSB repairs (HR and NHEJ) (44, 45). Also, PP4 regulatory subunits, like PP4R2, PP4R3α, and PP4R3β, are phosphorylated to be inactivated in mitosis (30, 46). In particular, PP4R3β phosphorylation is required for maintaining 53BP1 phosphorylation and blocking premature 53BP1 foci formation in mitosis (45).
Recently, some endogenous PP4 inhibitors, such as DHX38 (DEAH box polypeptide 38), TIPRL (Tip41-like protein), and PP4IP (protein phosphatase 4 inhibitory protein), have been reported (47-49). DHX38 specifically interacts with PP4C in constitutive and damage-independent manner, and inhibits PP4 activity
Similar to mammalian cells, PP4 is also regarded as an essential element in yeast DDR. PPH3 prevents hyperphosphorylation of Rad53 (checkpoint kinase 2, CHK2, yeast homolog), thus affecting DNA end resection at DSBs (14), and also promotes telomere healing at accidental breaks by opposing Cdc13 phosphorylation, even though it is unknown whether PPH3 directly dephosphorylates Cdc13 (50). Besides, PPH3 depletion alleviates DNA damage accumulation and rescues the short lifespan in
Not always, but in many cases, protein functions in yeast homolog are reflected in the human system. Thus, it is quite convincing that human PP4 regulates CHK2-mediated activity and telomere-related events. Actually, the phosphorylation of KAP-1 by CHK2 is regulated by PP4 and hyperphosphorylation of 53BP1 in mitosis induced by PP4 inactivation prevents telomere fusion (6, 53).
Immune cell development and activation are regulated by extremely sophisticated pathways, and dysfunction of these processes leads to many hazardous immune disorders. To prevent such catastrophic incidents, PP4 participates in various immune-related processes, such as immune cell lineage development, cytokine secretion, and immune cell receptor signaling.
PP4 is equally essential for B-cell lineage development. Ablation of PP4 in B-cell lineage leads to reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells (59). In the PP4-knockout B cells, immunoglobulin (Ig) class switch recombination is impaired and the basal levels of serum immunoglobulins of all isotypes are reduced (59-61). However, beyond the cell proliferation phase, the conditional deletion of PP4 completely restores normal IgG1 production in B cells of immunized mice (61). The
In addition to roles in T and B cells, PP4 is an essential component in other immune cells including macrophage. Type I IFN production is indispensable for antiviral innate immune response, and TBK1 (TANK-binding kinase 1) plays crucial roles in type I IFN production. PP4 suppresses production of type I IFN and IFN-stimulated genes by dephosphorylating and inhibiting TBK1 (62).
Similar to the conflicting role in genomic stability, the overexpression and depletion of PP4 cause apoptosis in T cells, meaning that PP4 can be proapoptotic or antiapoptotic gene (54, 63, 64). Interestingly, the knockout of PP4 in rodents causes embryonic lethality (54), suggesting that tight regulation or adequate expression of PP4 is pivotal in immune system development, at least in T cell lineage.
The dysfunction of glucose homeostasis leads to critical metabolic disorders, such as diabetes and obesity. Insulin resistance is one of the main causes contributing to impaired glucose dysregulation (65). Recently, accumulating data indicate that PP4 is related to insulin resistance and glucose metabolism.
In type 2 diabetic db/db mice or insulin-resistant mice treated with TNF-α (tumor necrosis factor α), the expression of PP4C and PP4R1 in protein level and PP4R3α/β in mRNA level is increased, and downregulation of PP4 alleviates the insulin resistance (66-69), though the alteration of PP4R2 expression level remains elusive. It was reported that TNF-α induces the phosphorylation and activation of PP4C, subsequently leading to the activation of JNK (70). However, it seems that PP4 may regulate JNK function in an indirect manner, since PP4 does not physically interact with JNK (70). Also, upon the activation of JNK, the interaction of IRS-1 (insulin receptor substrate 1) with PP4 causes the decreased expression of IRS-1 and increased phosphorylation of IRS-1 (68). Additionally, TNF-α downregulates IRS-4 expression, which depends on the phosphatase activity of PP4. But, it is unknown whether PP4 dephosphorylates IRS-4 directly (71).
ACC1 (acetyl-CoA carboxylase 1) is associated with hepatic lipogenesis, and its phosphorylation by AMPK blocks lipid synthesis and is reversed by PP4 (66). Consistently, PP4 dephosphorylates AMPK in Ca2＋ dependent manner, thus blocking lipid consumption (72). Furthermore, PP4 is involved in gluconeogenesis. Overexpression of PP4R3α/β induces dephosphorylation of CRTC2 (cAMP-response element binding protein-regulated transcriptional coactivator 2) and promotes transcription of gluconeogenesis-related genes (69). It remains to be seen whether PP4 directly dephosphorylates CRTC2. Also, as another role in glucose signaling, PPH3 and PSY2 (PP4C and PP4R3 yeast homolog, respectively) dephosphorylate Mth1 (MutT homolog 1) in glucose withdrawal condition, causing the binding of Rgt1 (restores glucose transport protein 1) to the promoters of glucose transporter (HXT) genes and represses their expression (73).
SMA (Spinal muscular atrophy) is an autosomal recessive neurodegenerative disease characterized by progressive loss of motor neurons from the anterior horn of the spinal cord, resulting in paralysis and severe muscular atrophy (74). SMA mainly arises from deletions or mutations in
Hedgehog signaling is related to the development in vertebrates and invertebrates, and Smo (the seven-transmembrane protein smoothened) is an essential factor in Hedgehog signaling (76). In
PP4 functions also have been elucidated in other various biological fields, though requiring more information in future. First,
PP4 can activate or inhibit NF-κB signaling. Signaling to NF-κB is crucial for T cell activation, differentiation, and proliferation as it regulates a wide variety of target genes such as different cytokines (e.g., IL-2, IFN-γ, and TNF-α), chemokines (e.g., IL-8), and anti-apoptotic molecules (e.g., Bcl-2 and c-IAPs; cellular inhibitor of apoptosis protein 1) (93). PP4C-PP4R1 complex functions as the negative regulator between IκB and IKK (IκB kinase) complex. Inactivation of IKK by PP4C/PP4R1-mediated dephosphorylation keeps IκB dephosphorylated and thereby, prevents the activation of NF-κB (93). PP4 also negatively regulates LPS-induced and TRAF6 (TNF receptor-associated factor 6)-mediated NF-κB activation by inhibiting the ubiquitination of TRAF6 (94). Although PP4 physically interacts with TRAF6 and is recruited to TLR4 (toll-like receptor 4) complex upon LPS (lipopolysaccharide) stimulation, whether PP4 regulates TRAF6 by direct dephosphorylation remains to be elucidated.
In non-immune cells, PP4 stimulates c-Rel-mediated DNA binding and NF-κB-mediated transcription, and decreases threonine phosphorylation on p65 subunit of NF-κB for its activation (95, 96). Conversely, PP4 can also inactivate NF-κB activity by dephosphorylation of TRAF2, inhibition of TRAF6 polyubiquitination, and interaction with tAg (small T antigen) of MCPyV (merkel cell polyomavirus) and NEMO (NF-κB essential modulator) adaptor protein (97, 98).
In Neurospora, PP4 dephosphorylates and activates WHITE COLLAR complex (WCC), phosphorylated by FREQUENCY-mediated manner to close circadian negative feedback loop (99). Additionally, PP4 inhibits thiazide-sensitive Na＋-Cl− cotransporter (NCC) in
Emerging evidence clearly suggests that PP4 plays multifaceted roles which control a variety of cellular phenomena. In the light of the significant impact of PP4 in cells, it is obvious that a more intensive study should be pursued to achieve better understanding of the working mechanisms in organism, as well as to clarify conflicting results observed in the different experimental systems. In that scenario, PP4 could be harnessed for the development of therapeutic materials. For example, since PP4C is considered an oncoprotein frequently overexpressed or irrelevantly regulated in a number of tumor types, discovering molecules as an inhibitor suppressing or blocking the gene expression or inhibiting directly enzymatic functions of PP4 is sufficiently persuasive and promising. From another perspective, the synthetic inhibitor, specific to PP4C, can be developed applying an entire or functional region of endogenous protein inhibitors identified. Inhibitors has been used in the studies on PP4 also inhibits other phosphatases meaning that there is no PP4-specific inhibitor developed. The PP4 inhibitor to be produced should be valuable for investigating PP4-specific functions and targeting tumors particularly with the high level of PP4C expression in the long term.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant number: NRF-2017R1A2B4007852).
The authors have no conflicting interests.