Phospholipase C

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Discovery

In 1953, a study reported that adding acetylcholine or carbamoylcholine to pancreatic cells would produce phospholipids. In these studies, ³²P was used to detect a 7-fold increase in phospholipid levels in drug-treated samples compared to control slices, which is the first evidence of the presence of phospholipase C (PLC) function in cells. In 1975, studies showed that PLC preparations can be used to cleave phosphatidylinositol. In 1981, the first purified preparation of PLC was obtained. A few years later, it was discovered that inositol 1,4,5 trisphosphate (IP₃) produced by the cleavage of phosphatidyl inositol 4,5 bisphosphate (also known as PI (4,5) P₂ or PIP₂) can induce the release of Ca²⁺ from intracellular storage. This important observation has promoted people's insights into the function of PLC in living organisms. Eventually, the cDNAs of PLCβ, PLCγ, PLCδ, PLCε, and PLCηPLCζ were all cloned. PIP₂ plays a central role in regulating many cellular processes. PLC is activated following stimulation of cells by either tyrosine kinase receptors, B-cell receptors, T-cell receptors, Fc receptors, G protein-coupled receptors or integrin adhesion proteins via cognate ligands including neurotransmitters, growth factors, histamine, and hormones.

Figure 1. Three major pathways for activating phospholipase C (PLC) (Bill, C.A.; Vines, C.M. 2021)

Cleavage of PIP₂ and signaling

PLC is a cytoplasmic protein that responds to cellular stimuli by locating lipid rafts in the plasma membrane and catalyzing the hydrolysis of phosphorylated form of phosphatidylinositol, ultimately controlling the level of PIP 2 in the cell. It is reported that these enzymes can increase the rate of lysis of phosphatidyl inositol >1000s⁻¹ at 30 °C and low substrate concentration, but it is likely to reach a rate of >5000s^-1. As the preferred substrate for PLC, PIP₂ is a phospholipid that is not commonly found in the plasma membrane, followed by phosphatidyl inositol phosphate (PIP), and then phosphatidyl inositol (PI). The cleavage of PIP₂ leads to the production of two products. One of them is diacylglycerol (DAG), which is responsible for activating calcium-dependent protein kinase C (PKC), which phosphorylates downstream effectors, and activating a series of subsequent cell functions, including regulation of cell proliferation, cell polarity, learning, memory, and spatial distribution of signals, etc. DAG bound to the membrane can be cleaved to produce another signal molecule, arachidonic acid. 

Another product is IP₃, which is a small water-soluble molecule. It diffuses away from the membrane and binds to the IP₃ receptor on the endoplasmic reticulum through the cytosol, thereby inducing the release of Ca²⁺. Cytoplasmic calcium levels rise rapidly and lead to characteristic calcium spikes that signal cell activation. Ca²⁺ in turn activates downstream transcription factors, leading to a plethora of gene activation pathways. In this way, PLC signal transduction plays an essential role in regulating proliferation, differentiation, fertilization, cell division, growth, modification of gene expression, degranulation, secretion, and motility.

Figure 2. Structures of the 6 different identified members of the PLC family (Bill, C.A.; Vines, C.M. 2021)

Methods to inhibit PLC

There are many chemical inhibitors that can be used to block PLC function. A commonly used pan-inhibitor of PLC, 1-[6-((17β-3-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione, (U73122), works by preventing the translocation of enzymes to the membrane. Recent studies have shown that when the phospholipase is associated with mixed micelles, U73122 forms covalent associations with human PLCβ3.

However, Klein et al. proposed that although U73122 has been used as a pan-inhibitor of PLC in many studies, U73122 does not inhibit PLC, but activates human PLCγ1, human PLCβ2, and human PLCβ3, which have been incorporated into micelles to differing magnitudes. In these studies, the purified form of PLC is used, so it is not clear how U73122 functions to regulate the extent of PLC activation. In addition, there are at least three other known PLC inhibitors and two activators, but they are not specific. However, the deletion of heterozygosity of specific PLC family members through siRNA can yield targeted results. In the future, the use of siRNA to identify specific PLC family members involved in a cellular response is a powerful method.

Recently, the use of CRISPR Cas9 technology will likely be used to target specific PLC isoforms. The highly specific 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) can also block PLCγ1 phosphorylation and other functions induced by EGFR in PLCγ1-dependent cells. This interaction occurs through the PH domain of PDK1. The loss of phosphorylation prevents the activity of PLCγ1 and downstream cell migration and invasion, and has been considered as a lead compound of anti-metastatic drugs.