Phosphoinositide-Specific Phospholipase C (PI-PLC)
In 1953, Lowell and Mabel Hokin found that stimulation of pancreatic slices by acetylcholine increased the incorporation of 32P into inositol lipids and phosphatidic acid. This phenomenon became known as the “PI effect,” and a comprehensive exploration of the PI effect was later initiated (Hokin and Hokin 1953). It was described that the rapid turnover of membrane phosphoinositides was increased in various tissues and cells in response to a wide range of external stimuli. Richard Rodnight showed the liberation of inositol phosphate from inositol phosphatide in 1956, and it was suggested that stimulated phospholipid metabolism appears to be specific for phosphatidylinositol. In 1959, it was proposed that the hormonal degradation of phosphatidylinositol is mainly hydrolyzed by a PLC-like enzyme into diacylglycerol (DAG) and inositol phosphate. With the finding that polyphosphoinositides are contained in the brain, the release of inositol di- and triphosphates from polyphosphoinositides was also detected (Dawson 1959; Kemp et al. 1959). The precise existence of PLC was identified by separating polyphosphoinositide phosphomonoesterase and PLC (as phosphodiesterase) from dialyzed extracts of ox brain (Thompson and Dawson 1964).
Before the purification of PLCs, the importance of membrane inositol lipid was established in the 1970s/1980s. In 1975, Bob Michell suggested that the hydrolysis of phosphoinositides is responsible for the mobilization of calcium because of the coincidence of phosphoinositide metabolism with changes in calcium homeostasis (Michell 1975). Four years later, this hypothesis of Michell was proven by Mike Berridge and John Fain (Fain and Berridge 1979). They showed that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium in isolated blowfly salivary glands. Furthermore, Robin Irvine found that inositol 1,4,5-trisphosphate (IP3), the product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC, acts as a second messenger and induces Ca2+ release from non-mitochondrial intracellular stores (Streb et al. 1983). Then, the source was discovered as the endoplasmic reticulum (ER), where IP3 binds a ligand-gated Ca2+ channel, IP3R, and triggers channel opening. The other major product of PIP2 hydrolysis is DAG, which was also characterized as a second messenger that activates protein kinase C (PKC) (Nishizuka 1984).
In 1981, the first purification of homogeneous phosphatidylinositol-specific PLC was achieved by two groups. Tadaomi Takenawa and Yoshitaka Nagai purified a 68-kDa enzyme from the cytosolic fraction of rat liver (Takenawa and Nagai 1981). Sandra Hofmann and Phillip Majerus isolated two PLCs (65 kDa and 85 kDa) from sheep seminal vesicles and immunologically identified them using specific antibodies to 65 kDa PLC (Hofmann and Majerus 1982). In fact, it was revealed that the 65–68 kDa enzyme was not an indigenous PLC but rather was a contamination by other enzymes or fragments of other PLCs. Sue Goo Rhee purified and characterized 150 (PLCβ1), 145 (PLCγ1), and 85 (PLCδ1) kDa PLC isozymes from bovine brain by using both polyclonal and a series of monoclonal antibodies against these three enzymes. They also isolated and sequenced the cDNA of PLCs (Ryu et al. 1987; Suh et al. 1988b). Later, other PLC isozymes were identified, and a total of 13 mammalian PLC isozymes (six subtypes) were discovered to date as follows: PLCβ2 (Park et al. 1992), PLCβ3 (Jhon et al. 1993), PLCβ4 (Kim et al. 1993), PLCγ2 (Banno et al. 1990), PLCδ3 (Bristol et al. 1988), PLCδ4 (Lee and Rhee 1996), PLCε (Kelley et al. 2001; Lopez et al. 2001; Song et al. 2001), PLCζ (Cox et al. 2002), PLCη1, and PLCη2 (Hwang et al. 2005; Nakahara et al. 2005; Stewart et al. 2005; Zhou et al. 2005).
Receptor-mediated activation of PLCs was investigated, and receptor specificity was suggested in the late 1980s and early 1990s. Receptor tyrosine kinases (RTKs), including PDGF and the EGF receptor, phosphorylated and activated PLCγ; however, PLCβ and PLCδ did not respond to RTK (Margolis et al. 1989; Meisenhelder et al. 1989; Wahl et al. 1989). Meanwhile, John Exton and Melvin Simon found that all four members of the Gq and G11 subfamily of heterotrimeric G proteins activated PLCβ but did not activate PLCγ or PLCδ (Blank et al. 1991; Taylor et al. 1991). In addition, nuclear PLCs were suggested by Lucio Cocco to be involved in processes such as cell proliferation, differentiation, and survival (Martelli et al. 1992). In 1996, the crystal structure of PLCδ was determined, and the structural mechanism of lipase activity was revealed by Roger Williams (Essen et al. 1996).
When the PLC isozymes were identified in the 1980s, they were purified from various sources, including rat liver, bovine brain, and human platelets. They had diverse molecular masses, and the PLC enzymes were thus unsystematically named according to their molecular mass or their purified order by the discoverer. Thus, the nomenclature of PLC isozymes was in disarray. Sue Goo Rhee analyzed the similarity of PLC enzymes by using three specific antibodies to bovine brain PLC isozymes (150-, 145-, and 85-kD forms) and another antibody to the 62-kD guinea pig uterus enzymes (Bennett and Crooke 1987; Suh et al. 1988a). Then, he suggested using Greek letters to designate the PLC isozymes with different primary structures. The Greek letters were assigned according to the chronological order of their purification, and an Arabic numeral after the letter was used to indicate the proteolysis or alternative splicing variant: α to the 62~68-kDa, β to the 150~154-kDa, γ to the 130~145-kDa, and δ to the 85~88-kDa enzyme (Rhee et al. 1989). From PLCε, the Greek letter was used according to the primary structure and discovery order. Because PLCα was identified as a contaminant from ERP60 or as a fragment of a PLCδ, the name “PLCα” ceased to exist. “PLCδ2” was also removed because PLCδ2 is a species homolog of PLCδ4.
Catalytic Function and Mechanism
A putative pan PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), has generally been used to suppress PLC activity. It is thought that the inhibition of U73122 is achieved by blocking the translocation of PLC to the membrane. However, the nonspecific effects, specifically the inhibition of G protein-activated inwardly rectifying potassium channels, were reported and can even activate some PLCs (Horowitz et al. 2005). Edelfosine (1-octadecyl-2-O-methyl-glycero-3-phosphocholine) has been used as a PLC inhibitor with higher selectivity.
The TIM barrel consists of an X and Y catalytic domain and an X-Y linker. The TIM barrel is highly conserved among the PLCs, with 60–70% sequence identity. The X and Y domain form the Ca2+-binding site and a catalytic active site in which they construct outer α-helices and inner β-strands; the active site is at the C-terminal ends of β-strands. Although the active site residues and structures of the X and Y domains are conserved among all PLC subtypes, the sequence and size of the X-Y linker differ between the subtypes. The X-Y linker is crucial for the regulation of activity and is the target of autoinhibition.
Pleckstrin Homology (PH) Domain
The PH domain is conserved among the PLCs, except PLCζ. It has a sandwich structure of seven β-strands, which are closed off at each end by a C-terminal α-helix and three loops. The length and sequence differ among isozymes. The PH domain serves to anchor to PIP2-rich membranes by binding PIP2. However, the PH domain of PLCβ2 cannot bind to phosphoinositides. The PH domain regulates the activation and translocation of PLCs with other anchoring and activating ligands, for example, PLCδ-IP3, PLCγ-PIP3, PLCβ-Gβγ, or PLCβ-small GTPase Rac. In the case of PLCγ and PLCβ2/PLCβ3, the PH domain is involved in the binding of Gβγ. The PH domain of PLCβ also directly interacts with the catalytic region during activation. PLCγ has an additional split PH domain and the C-terminal half interacts with the TRPC3 Ca2+ channel.
The EF hand is a Ca2+-binding domain composed of two helixes that have a high degree of flexibility. In the case of PLCβ2, PLCβ3, or PLCδ1, Ca2+ is not bound to the EF hand. The EF hand of PLCβ lacks the residues for Ca2+ binding but can still interact with Gαq and accelerate GTP hydrolysis by Gαq.
The C2 domain is tightly packed against the TIM barrel and maintains the structural integrity of the catalytic core. The C2 domain also has a Ca2+-binding site to recruit PLCδ1 to the plasma membrane. The C2 domain of PLCβ does not act as a Ca2+-binding domain. The C-terminal extension of C2 is required for activation by Gαq in PLCβ (Waldo et al. 2010).
Although PLC isozymes have been purified from the cytosolic fraction, PLC activity was initially reported in various membrane preparations. This finding suggested that there are mechanisms responsible for the translocation of PLCs between the cytosol and membrane and that this translocation mechanism might constitute a way of regulating PLC-dependent second messenger generation. Different PLC isozymes have various recruitment mechanisms, including binding to phosphoinositide or the phosphorylation site of tyrosine kinase receptors and interacting with small GTPases or heterotrimeric G protein subunits.
The X-Y linker domain in the TIM barrel is highly mobile or disordered and occludes the active site. Because the active site of the PLCs is superficial and the substrate does not protrude far from the membrane, covering the active site of the X-Y linker effectively inhibits PLC lipase activity and maintains the PLC in an autoinhibited state. For activation of PLC, the X-Y linker recedes from the active site. However, the X-Y linker of PLCζ apparently acts to constitutively activate PLCζ. Contrary to the negative charge of other PLCs, the X-Y linker has a positive charge in PLCζ (Nomikos et al. 2011). When the X-Y linker is deleted, the basal activity of PLC is elevated, and this elevated PLC activity could be further enhanced by activators. This result suggests an additional regulatory mechanism for each PLC isozyme.
Characteristic of PLCβ
In mammals, there are four genes encoding PLCβs. Individual PLCβ isozymes have different distributions in tissues and cells; PLCβ1 and PLCβ4 are preferentially expressed in the brain. PLCβ1 is particularly enriched in the cerebral cortex and hippocampus, whereas PLCβ4 is highly expressed in the cerebellum and the retina. PLCβ2 is preferentially expressed in hematopoietic cells, and PLCβ3 is widely expressed. PLCβs are mainly stimulated by Gαq subunits, Gβγ subunits, and Ca2+. PLCβs are also regulated by the small GTP-binding protein Rac, phosphatidic acid, and phosphorylation by several kinases. Unlike other PLC isozymes, PLCβs have the distinguished feature of an elongated C-terminus consisting of a coiled-coil domain (CC domain) of approximately 450 residues, which is important for membrane binding, nuclear localization, and activation by Gαq.
Regulation of PLCβs
Gαq stimulates the activation of PLCβ by up to several hundredfold (EC50, 1–2 nM), and the sensitivity for PLCβ1 and PLCβ3 is higher than PLCβ2. Other Gαq family members, Gα11, Gα14, and Gα16, also activate PLCβs. There are four binding sites of Gαq to PLCβ; the EF hand, X-Y linker and CT domains, and a helix-turn-helix motif at the C-terminal of the C2 domain. This interaction was suggested by structural and mutagenic studies of PLCβs (Wang et al. 1999a; Waldo et al. 2010). The interaction between PLCβ and Gαq recruits the PLCβ to the membrane and removes the autoinhibitory X-Y linker from the active site to evoke or contribute to activation. The C-terminal coiled-coil domain is required for stimulation of PLCβs (except PLCβ3) by Gαq and also contributes to Gq GTPase-activating protein activity. The helix-turn-helix motif at the C-terminal of the C2 domain is highly conserved in all PLCβ isozymes but is not found in other PLCs.
PLCβ1, PLCβ2, and PLCβ3, but not PLCβ4, are activated by Gβγ subunits (Smrcka and Sternweis 1993; Philip et al. 2010). Through this Gβγ signaling, PLCβs also mediate Gi-coupled receptor signaling. The order of susceptibility to Gβγ is PLCβ2>>PLCβ3>PLCβ1. Only PLCβ2 has a high affinity to Gβγ. The Ca2+ response of the activated Gi-coupled receptor is regulated mostly by PLCβ2. PLCβ4 is not activated by Gβγ signaling. The putative binding sites of Gβγ are the PH domain and the C-terminal half of the TIM barrel domain (Wang et al. 1999b). The structural mechanism suggests a similarity to that of Gαq, stabilization of the active state. Although Gβγ stimulates PLCβs, PLCβ3 only synergistically responds to Gαq and Gβγ, which are simultaneously activated by the Gq- and Gi-coupled receptor. PLCβ2 responds to both Gαq and Gβγ, but they do not display supra-additive activation.
PLCβ2 is stimulated by the small GTP-binding proteins Rac1, Rac2, and Cdc42, and PLCβ3 is weakly activated by these GTP-binding proteins. However, the binding of these GTP-binding proteins to PLCβ1 or PLCβ4 has not been detected. Rac binds to the PH domain through switch regions and recruits PLCβ to a specific region of the plasma membrane, but it does not induce a conformational change. PLCβ1, PLCβ2, and PLCβ3 are phosphorylated by protein kinase A, C, and G and calmodulin-stimulated kinase II. Phosphorylation inhibits activity and positive effects have not been reported.
Scaffolding of PLCβ Through the PDZ-Binding Motif
All four mammalian PLCβ isozymes have a PDZ-binding motif, which include an –X(S/T)X(V/L)-COOH sequence at the extreme C-terminal end. Scaffold proteins that have PDZ domains keep PLCβs in contact with its upstream receptor because about half of the Gq-coupled receptors have a C-terminal PDZ-binding motif. Each PLCβ isozyme has different sequences of the PDZ-binding motif. This configuration provides diverse involvement for PLCβ in various GPCR-mediated signaling pathways. PAR3 interacts with PLCβ1 and couples it to the bradykinin receptor. PLCβ1 and PLCβ2 interact with NHERF1. NHERF2 binds an LPA2 receptor or muscarinic receptor and PLCβ3. NHERF3 links PLCβ3, and the somatostatin receptor. Shank2 binds PLCβ3 and regulates the Gq GAP activity of PLCβs. WDR36 tethers PLCβ to the β isoform of the thromboxane A(2) receptor and Gαq. Par-3 specifically binds to PLCβ1 and forms a ternary complex with bradykinin receptor B2.
Although all four PLCβ isozymes are detected in the nucleus, PLCβ1 is apparently the most abundant (PLCβ1>>PLCβ3>PLCβ2>PLCβ4). In particular, among the PLCβ1 splicing variants, the shorter PLCβ1 variant (PLCβ1b) localizes preferentially to the nucleus, and the longer form (PLCβ1a) is expressed in both the cytoplasm and nucleus. Nuclear PLCβ1 has been reported to localize to nuclear speckles. Nuclear PLCβs are associated with cell cycle progression, proliferation, and differentiation. Nuclear PLCβ1 is involved in the G2/M transition through the PKC phosphorylation of lamin B, which leads to nuclear envelope disassembly and mitosis progression. PLCβ1 also regulates G1 progression through cyclin D3/cdk4. However, the detailed mechanisms of the interaction between nuclear PLCβ1 products, DAG and IP3, with the cell cycle machinery are still unclear. Nuclear PLCβ1 is also involved in erythroid differentiation by targeting the transcription factors p45/NF-E2, SRp20, and CD24 and myogenesis through the regulation of cyclin D3 gene expression.
PLCβ1 in Hematological Malignancies
In myelodysplastic syndromes (MDSs), PLCβ1 appears to play a fundamental role. High-risk MDS patients who bear the monoallelic deletion of PLCβ1 had a worse clinical outcome, and they rapidly progressed to acute myeloid leukemia. In addition, MDS cells always show a higher level of PLCβ1b mRNA than PLCβ1a mRNA. MDS patients who are in almost complete remission with azacitidine, a DNA methyltransferase inhibitor, showed a correlation between PLCβ1 expression with drug responsiveness. This finding suggests that there is an association between PLCβ1 and azacitidine effectiveness and that PLCβ1 is potentially a good candidate for both MDS prognosis and as a marker of the epigenetic effects of antileukemic drugs (Follo et al. 2009).
PLCβs as a GAP for Gαq
PLCβs accelerate deactivation of Gαq through GAP activity. This GAP activity not only immediately terminates Gq-PLCβ signaling in response to the deactivation of the receptor, but it also markedly modulates the dynamics of PLCβ-mediated signaling nodes. Therefore, the rate of activation and deactivation is robustly increased, and the signaling acuity is sharpened. The loop between the third and fourth EF hand domain interacts with the switch II region of Gαq and stabilizes the structure for the hydrolysis of GTP. The coiled-coil domain at the C-terminal is also required for GAP activity because it allows high-affinity binding to Gq; this domain also has alternative GAP activity (Ilkaeva et al. 2002). PLCβ also increases the rate of GTP binding by tethering the receptor-PLCβ-Gq complex even after GTP hydrolysis and potentiating the intrinsic nucleotide exchange activity of the receptor (Waldo et al. 2010).
Physiology of PLCβ
The essential function of PLCβ1 in inhibitory neuronal pathways was suggested by studies using PLCβ1 null mice (Kim et al. 1997). PLCβ1 null mice exhibited epileptic seizures and sudden death. PLCβ1 also regulates the plasticity of the M1 muscarinic receptor in the adult neocortex and the expression of RGS4, which is a susceptibility gene for schizophrenia, in the CA1 region of the hippocampus. Deficiency of PLCβ1 results in an imbalance between the muscarinic and dopaminergic system and decreased RGS4 and the PLCβ1 null mice showed schizophrenia phenotypes (McOmish et al. 2008). PLCβ2 negatively regulates chemotaxis in leukocytes. Not surprisingly, neutrophils isolated from PLCβ2-deficient mice exhibited increased rates of chemotaxis (Jiang et al. 1997). PLCβ3 null mice demonstrated higher sensitivity to morphine, implicating PLCβ3 as a negative modulator for μ-opioid receptor signaling. However, PLCβ3-deficient mice did not respond to sensory stimuli and prematurely died owing to the development of lymphomas and carcinomas. Myeloproliferative disease developed in PLCβ3 null mice because of the dysregulation of STAT5. PLCβ4 null mice exhibited ataxia, motor defects, and impaired visual processing (Jiang et al. 1996; Kim et al. 1997)
Characteristic of PLCγ
There are two PLCγ isozymes (PLCγ1 and PLCγ2) in mammals. PLCγ has a large multidomain insert between the X and Y domains that consists of two split PH (sPH) domains, two SH2 domains, and an SH3 domain. The sPH domains locate at either end of the insert and form the tertiary structure of the standard PH domain after folding. When PLCγ is phosphorylated, it is activated by removing autoinhibition of the X-Y linker. PLCγ1 is widely expressed and PLCγ2 is mainly expressed in immune cells. PLCγ1 responds to both receptor tyrosine kinases and soluble tyrosine kinases that are recruited to the plasma membrane. Activation of the T cell receptor (TCR) results in activation of PLCγ1 rather than PLCγ2; however, PLCγ2 functions downstream of the B cell receptor (BCR) rather than PLCγ1. Therefore, PLCγ1 is critical for TCR signaling and T cell selection, whereas PLCγ2 is important for BCR signaling and B cell maturation.
Regulation of PLCγs
PLCγs have multiple Tyr residues that are phosphorylated. Among them, Tyr783 in PLCγ1 (Tyr 759 in PLCγ2) is a conserved residue that is necessary and sufficient to induce lipase activity. The phosphorylation is catalyzed by the receptor for EGF, PDGF, NGF, FGF, and multiple non-receptor soluble tyrosine protein kinases, including Src, Syk, Btk, and Tec. These soluble kinases are recruited by TCR, BCR, FcεR1, GPCR, integrins, and other tyrosine kinase receptors. PLCγs are recruited to receptors through the interaction between the nSH2 domain of PLCγs and the phosphotyrosine docking site of the receptor. For example, autophosphorylation of Tyr766 in FGFR1 and Tyr992 in EGFR, Tyr1021 in PDGFR is the docking site for PLCγ1. These interactions induce the phosphorylation (Tyr783, Tyr775, Tyr472, and Tyr 1254) and activation of PLCγ1. Tyr783 lies between the cSH2 and SH3 domain of PLCγ, and the binding of pTyr783 to cSH2 is necessary for activation. This interaction induces a conformational change, reorienting the X-Y linker to allow the substrate to access an active site. Although phosphorylation of Tyr775 is necessary to increase lipase activity, it could not induce the activation of PLCγ1 alone. These phosphorylation sites are conserved in PLCγ2; the equivalent of Tyr775 and Tyr783 in PLCγ1 is Tyr753 and Tyr759 in PLCγ2. Tyr771 has a negative role in the activation of PLCγ1, whereas a mutation at Tyr771 significantly enhances PLCγ1 activity. Phosphorylation of Ser1248 also inactivates PLCγ1 through dephosphorylation and structural changes.
The SH3 domain, which binds a proline-rich motif, is used to form multi-protein complexes, including both regulators and effectors. The SH3 domain binds several scaffolding proteins (Cbl, SWIP, Lcp2), cytoskeleton components (dynamin, cortical actin complexes, microtubule-associated proteins), and signaling proteins (Akt, TrpC3 channels, SHIP1, PIKE). In the case of Grb2, it negatively regulates PLCγ1 by competing with PLCγ1 for binding to FGFR2 via the SH3 domain and direct interaction with PLCγ1 (Choi et al. 2005; Timsah et al. 2014). The SH3 domain of PLCγ1 also can act as a GEF for PIKE and dynamin-1.
PLCγ2 is stimulated by the small GTPases Rac1 and Rac2. Rac2 binds the sPH domain of PLCγ2 and induces a conformational change to move the X-Y linker away from an autoinhibitory position. The activation by Rac is independent of phosphorylation and is also associated with translocation of PLCγ2 to the plasma membrane. PLCγ1 is not activated by Rac 1 and Rac2; however, the requirement of PLCγ1 in Rac1-mediated NFAT5 activation was suggested.
PLCγs are also stimulated by PIP3; however, relatively high concentrations (40–100 μM) of PIP3 are needed to activate PLCγ. This activation of PLCγ by PIP3 is mediated by both the N-terminal PH domain and the cSH2 domains of PLCγ.
Physiology of PLCγ
Homozygous deletion of PLCγ1 results in embryonic death with failure of both vasculogenesis and erythropoiesis at E 8.5–9 (Ji et al. 1997). In addition, PLCγ1 mutant zebra fish displayed defects in the formation of arteries and cardiac contractility, which are regulated by VEGF-PLCγ1 signaling (Rottbauer et al. 2005). In the immune system, PLCγ1 regulates T cell development. PLC-γ1 also participates in a neurotrophin signaling pathway and various neuronal events, such as neurite outgrowth, neuronal cell migration, and synaptic plasticity. Consistent with these functions, abnormal activity, and expression levels, polymorphisms of PLCγ1 have been detected in brain disorders, such as epilepsy, bipolar disorder, depression, and degenerative diseases, such as Huntington’s disease and Alzheimer’s disease (Jang et al. 2013).
Although PLCγ1 is activated by growth factors, its role in the regulation of cell proliferation is controversial. Inhibition of PLCγ1 through neutralizing antibodies and a dominant negative PLCγ1 fragment showed the mitogenic role of PLCγ1. However, growth factor-stimulated proliferation was not defective in PLCγ1 null fibroblasts. The DNA synthesis and mitogenic properties of PLCγ1 were attributed to protein interactions through the SH3 domain rather than lipase activity.
In mammary and prostate cancer, PLCγ1 has crucial roles in metastasis. Recurrent activating PLCγ1 mutation (R707Q) was discovered in cardiac angiosarcomas and hepatic angiosarcomas, and this mutation is associated with cellular proliferation, migration, and invasiveness (Prenen et al. 2015). The roles of PLCγ1 have been established in the survival in oxidative stress and cell differentiation, including keratinocyte differentiation. PLCγ1 also regulates the directionality of cell movement through multiple means, including the regulation of cofilin and Rac/Cdc42 GTPase.
PLCγ2 null mice are viable after birth. However, these mice display strong deficiencies in the function of B cells and platelets, mast cells, and natural killer cells. In these mice, pro-B cell differentiation was decreased, and mature B cell and IgM, IgG2a, and IgG3 levels were reduced. In addition, T cell-independent antibody production and the response of B cells to IgM were defective. FcR signaling is also defective in platelets, mast cells, and NK cells (Wang et al. 2000). PLCγ2 deleted mice also showed an osteopetrotic phenotype. PLCγ2 was needed for RANKL-induced osteoclastogenesis where PLCγ2 upregulated NFATc1, AP1, and NF-κB via lipase activity and interaction with ITAMs and GAB2 (Mao et al. 2006). Two gain-of-function PLCγ2 mutant (ALI5 or ALI14) mice exhibit autoimmunity and severe inflammatory arthritis and dermatitis (Yu et al. 2005; Abe et al. 2011). PLCγ2 Ali5 (Ali5, abnormal limb 5) is a gain-of-function mutation in the PLCg2 gene, which enhances membrane adherence and leads to autoimmune diseases depending on the genetic background. In line with these mouse models, PLCG2-associated antibody deficiency and immune dysregulation (PLAID) was discovered in human patients. All patients with PLAID showed an urticarial reaction to cold with other variable manifestations (atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency) and had a mutation in an autoinhibitory cSH2 domain of PLCγ2 (Ombrello et al. 2012).
Characteristic of PLCδ
The three PLCδ isozymes (PLCδ1, PLCδ3, and PLCδ4) consist of only core domains (PH domain, EF hand motif, TIM barrels, and C2 domain) and are expressed in almost all cell types at a low level. The PLCδ isozymes have a different cellular distribution. PLCδ1 is mainly expressed in the cytoplasm. However, PLCδ1 accumulates in the nucleus at the G1-S transition, during which PIP2 increases in the nuclear membrane and recruits PLCδ1. Subsequently, PLCδ1 returns to the plasma membrane via a constitutive nuclear export signal. In the nucleus, PLCδ1 is involved in S phase progression and cell proliferation by reducing cyclin E levels (Stallings et al. 2008). PLCδ4 is principally located in the nucleus and the expression is altered according to the cell cycle. In the G1-S interface, the levels of PLCδ4 is increased in the nucleus and degraded thereafter.
PLCδs tightly bind PIP2 through their PH domain, which has a positive charge and serves as a plasma membrane anchor. Binding is the main mechanism for activity regulation and translocation of PLCδ. During a single binding event of a PH domain to PIP2, PLCδ hydrolyzes multiple PIP2. This recruitment of PLCδ by its substrate allows feedback regulation through a decreased local PIP2 concentration. In addition, the PH domain of PLCδ also binds IP3 with high affinity (Kd = 210 nM) (Ferguson et al. 1995). The interaction with IP3 competes with the binding of PIP2 and inhibits PLCδ.
For the activation of PLCδ, an additional activator is required beyond the recruitment by PIP2. Ca2+ is the significant activator of PLCδ, and PLCδ is the most sensitive to Ca2+ among PLC isozymes. The functional concentration of Ca2+ is 10–100 nM, and Ca2+ alone is sufficient to enhance the lipase activity of PLCδ. In addition to the active site, three or four Ca2+ ions bind to the C2 domains. Binding of Ca2+ to the C2 domain alters the electrostatic potential and recruits PLCδ1 from the cytosol to the plasma membrane and increases lipase activity. The C2 domains of PLCδ1 and PLCδ3, but not PLCδ4, also form a complex with Ca2+ and phosphatidylserine (PS) and then translocate PLCδ1 to PS-enriched regions.
PLCδ is involved in G protein-mediated signaling. The α1-adrenergic receptor activates PLCδ1 through atypical GαH, G-protein transglutaminase II. Moreover, the angiotensin II signal, which is mediated by Gαq/11-PLCβ, is potentiated by PLCδ1. Through these pathways, PLCδ amplifies and sustains the G-protein signal or Ca2+ signals mediated by other PLCs as secondary PLC. PLCδ1 is also regulated by other binding proteins. p122 RhoGAP directly binds and activates PLCδ1. The association of GAP43 with PLCδ1 increased Ca2+ and IP3 formation in response to hypotonicity.
Physiology of PLCδ
PLCδ1 null mice showed a hairless phenotype associated with abnormal hair follicle structures, bent hair shafts, and failure of epidermal penetration (Nakamura et al. 2003). This phenotype is very similar to nude mice, which has a mutation in the transcription factor Foxn1 and a decreased level of PLCδ1. The expression of PLCδ1 is regulated by Foxn1 and PLCδ1 is involved in the expression of hair keratin mHa3 to control hair follicle differentiation. PLCδ1 null mice also display skin inflammation and epidermal hyperplasia in interfollicle epidermis. The skin of PLCδ1 knockout mice showed increased inflammatory cytokines, including IL-1β and IL-6, and in turn promoted keratinocyte proliferation.
PLCδ1 was identified as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). PLCδ1 is located on chromosome 3p22 and is frequently deleted in ESCC. In addition to allelic loss, PLCδ1 is frequently reduced with promoter hypermethylation in ESCC. Consistent with reduced expression, administration of PLCδ1 into ESCC cells suppressed their tumorigenic ability. Downregulation of PLCδ1 correlates with metastasis. In the case of gastric cancer, this effect also suggested the epigenetic regulation and antitumor function of PLCδ1.
PLCδ3 regulates microvilli formation in enterocytes and the radial migration of neurons in the cerebral cortex of during development. Disruption of both PLCδ1 and PLCδ3 resulted in embryonic lethality at E11.5–E13.5 because of defective placental development. The number of vessels in the labyrinth layer of PLCδ1 and PLCδ3 double null mice was severely reduced, suggesting that PLCδ1 and PLCδ3 are crucial in trophoblasts for placental development (Nakamura et al. 2005). The significance of PLCδ1 and PLCδ3 in cardiomyocyte survival and normal cardiac function was suggested by studying conditional PLCδ1 and PLCδ3 double null mice, which exhibited cardiac abnormalities with impaired activation of Akt and PKC θ.
PLCδ4 null mice exhibit male infertility although female mice remained fertile. PLCδ4 is concentrated in the anterior acrosomal region of sperm and is involved in the initiation of the acrosome reaction, which is an exocytotic event required for fertilization. Sperm interacts with the zona pellucida, the extracellular glycoprotein layer of egg, and it increases Ca2+ influx, which is critical for the execution of the acrosome reaction. The sperm from PLCδ4 null mice was unable to initiate the acrosome reaction due to a minor increase in Ca2+ influx (Fukami et al. 2003).
Characteristic of PLCε
PLCε was first discovered as a Let-60 Ras-binding protein in Caenorhabditis elegans (Shibatohge et al. 1998). In 2001, PLCε was identified as the largest mammalian PLC isozyme (Lopez et al. 2001). PLCε has the following two unique domains, which are not found in other PLC isozymes: two Ras-/Rap-associating (RA) domains at the C-terminus and a CDC25 homology domain (Ras GEF) at the N-terminus. PLCε acts downstream of almost every signaling pathway. PLCε responds to GPCR signaling (including Gq, Gs, G12/13, and Gi) and RTK signaling mainly through Rho, Ras, and Rap. Ras and Rap localize and activate PLCε by binding RA2 domains. Ras recruits PLCε to the plasma membrane, and Rap moves it to the perinuclear region and Golgi. The binding affinity of Ras to the RA2 domain is eightfold higher than that of Rap. Although PLCε is downstream of Rap/Ras, it can also activate Rap/Ras through the CDC25 homology domain. This relationship serves as a feed-forward loop and produces long-lasting activation. The CDC25 homology domain is also related to dwelling time in the region near the nucleus and the activation of lipase activity with continued activation of Rap1. RhoA activates PLCε directly but requires a 65 amino acid residue insert, which is unique to PLCε, within the Y domain instead of RA domains. Several GPCR ligands, such as endothelin-1, LPA, and thrombin, activate both PLCβ and PLCε. However, activation is achieved in a temporally distinct manner in which PLCβ is acutely activated, and the activation of PLCε is sustained (Kelley et al. 2006). PLCε is also activated by Gβγ; however, this activation is independent of PI3K or Ras signaling and the PH domain. There is controversy about the relation between PLCγ and PLCε; however, activated PLCγ1 triggered by the EGF receptor could stimulate PLCε via Rap2B. However, PLCε is ubiquitinated by the E3 ligase Siah leading to degradation of PLCε upon growth factor stimulation (Yun et al. 2008).
Physiology of PLCε
PLCε null mice showed developmental defects of the aortic and pulmonary cardiac valves (Tadano et al. 2005). PLCε is important for growth factor-mediated heart development. PLCε-deficient mice exhibit a decreased cardiac contractile response to activation of β-adrenergic receptors and a reduced response to isoproterenol, indicating the higher susceptibility of PLCε null mice to hypertrophy in response to chronic cardiac stress (Wang et al. 2005). The decreased cardiac contractility is due to decreased Ca2+- induced Ca2+ release (CICR) in response to β-adrenergic stimulation. CICR is regulated by the Epac/Rap1/PLCε/PKCε pathway.
In contrast to the conventional knockout of PLCε, siRNA-based depletion and conditional knockout of PLCε in mouse cardiac myocytes lead to a protective effect against stress-induced pathological hypertrophy. Pathological heart hypertrophy requires lipase activity of perinuclear PLCε in which phosphatidylinositol 4-phosphate of the Golgi apparatus is used as a substrate and DAG is mainly generated instead of IP3. The lipase activity and scaffolding of PLCε to the nuclear envelope occur by muscle-specific A kinase-anchoring protein (mAKAPβ). mAKAPβ is important in the regulation of nuclear PKD activation and in the expression of hypertrophic genes (Zhang et al. 2013).
PLCε is also important for renal development and is involved in nephritic syndrome. A mutation in the catalytic region of PLCε was reported in patients with severe kidney diseases such as diffuse mesangial sclerosis or focal segmental glomerulosclerosis. Deficiency of PLCε may block normal renal development at the capillary loop stage. The defect of PLCε results in abnormal podocyte development of zebra fish and epidermal morphogenetic failure of C. elegans (Hinkes et al. 2006; Vazquez-Manrique et al. 2008).
PLCε is also associated with cell proliferation and carcinogenesis, but the effect of PLCε is controversial. PLCε mediates proliferation induced by agonists, such as PDGF, EGF, and thrombin. It has been reported that PLCε has an oncogenic function in bladder cancers, esophageal cancers, gastric cancers, and squamous cell carcinoma. In addition, Kataoka’s PLCε null mice showed a low incidence of skin tumors induced by UVB or two-stage chemical carcinogenesis (Bai et al. 2004) and a high resistance to spontaneous intestinal tumorigenesis. However, PLCε also acts as a tumor suppressor. Katan’s group showed that PLCε knockout mice had increased tumor number in a two-stage skin chemical carcinogenesis model and that PLCε inhibited cell growth.
Characteristic of PLCζ
PLCζ was first isolated and cloned as a sperm-specific PLC from human and mouse testis (Saunders et al. 2002). PLCζ is the smallest (70 kDa) of the mammalian PLC isozymes and accumulates in the pronucleus. PLCζ consists of a TIM barrel, an EF hand domain, and a C2 domain. Unlike other PLCs, PLCζ lacks a PH domain and is constitutively activated with a positively charged X-Y linker. After sperm-egg fusion, PLCζ is necessary and sufficient to trigger the cytoplasmic Ca2+ oscillations essential for the maturation and development of the fertilization. Moreover, the C2 domain of PLCζ is essential for Ca2+ oscillations in mouse egg by PLCζ. The C2 domain-deleted PLCζ could not induce Ca2+ oscillations. PLCζ is more sensitive to Ca2+ than other PLCs and can respond to low concentrations with an EC50 of 30 nM (Nomikos et al. 2005). In other PLCs, the X-Y linker has a negative charge and deletion of these generally induce activation. However, the X-Y linker of PLCζ is positively charged and removal of this moiety decreases lipase activity. This linker also has a class I nuclear localization signal (NLS), and an additional NLS is located at the C-terminus of the X domain. In the mitotic phase, the translocation of PLCζ to the cytoplasm initiates Ca2+ oscillations, and the return into the nucleus by NLS gradually terminates the Ca2+ oscillations.
Characteristic of PLCη
Two PLCη genes (PLCη1 and PLCη2) were identified in 2005 (Hwang et al. 2005; Nakahara et al. 2005; Stewart et al. 2005; Zhou et al. 2005). Except for a long C-terminal region, PLCη is very similar to PLCδ. This extended C-terminal includes a putative PDZ domain-interacting sequence. PLCη is expressed in the brain, spinal cord, and retina. Interestingly, PLCη2 is predominantly located in the plasma membrane without extracellular stimuli, whereas other PLC isozymes predominantly exist in the cytosol. The N-terminal PH domain acts as a localization signal for the plasma membrane. PLCη promotes an additional PLC signal as a signal amplifier in response to Ca2+ from the ER or outside the cell (Kim et al. 2011; Popovics et al. 2011). PLCη2 is also activated by Gβγ and mediates Gi-coupled receptor signaling. In contrast to PLCη2, the response of PLCη1 to Gβγ is minimal.
PLC is a key enzyme in phospholipid signaling. It generates two second messengers, IP3 and DAG, from PIP2. To date, 13 mammalian PLC isozymes have been identified, and all of these PLC isozymes have a conserved X and Y lipase domain and catalytic activity. However, each PLC isozyme has a distinct regulatory mechanism, different distributions in cells and tissues, and unique function. In particular, PLC isozymes have specific protein interactions mediated by isozyme-specific domains in addition to PLC lipase activity. This characteristic seems to contribute to functional diversity and may explain why many isozymes exist in the same cells or tissues and how PLC has roles in the regulation of diverse cellular events.
Among the PLC isozymes, PLCβ and PLCγ function as the primary PLCs, which are directly stimulated by activation of membrane receptors and acutely regulate signaling. Whereas PLCɛ is activated by the Rho and Ras family as a secondary PLC, PLCɛ amplifies and sustains signaling. Other PLCs, including PLCδ, PLCζ, and PLCη, are also regulated by intracellular Ca2+ mobilization. In addition, PLCδ is competitively inhibited by IP3, the product of lipase activity. They amplify and sustain signals, such as the Ca2+ signal evoked by primary PLC. This functional hierarchy serves as a positive feedback loop within PLC signaling.
Many studies have revealed the regulatory mechanism and function of PLCs in various tissues and cells. Through genetic studies, their physiological roles have been suggested. Few downstream signaling proteins, including IP3R, TRPC, PKC, and NFAT, have been suggested; however, the detailed signaling mechanisms that elucidate the link between PLC with its physiological function remain to be investigated. It will be important in future research to define how different PLC isozymes are activated by various receptors in response to multiple stimuli.
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