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Physiological Reviews

December, 2001

Printed in Canada

Phospholipase C: Structure, Mechanisms of Function, Regulation, Homology, and Role in the Cell

JASON J. MCEWAN

Department of Biochemistry, Carleton University, Ottawa, Ontario, Canada

I.                     Introduction

II.                  Enzymology/Structure

III.                Mechanisms of Catalytic Function

IV.               Regulators

V.                  Homology

VI.               References

McEwan, Jason J.  Phospholipase C:  Structure, Mechanisms of Catalytic Function, Regulation, Homology, and Role in the Cell.

I.         INTRODUCTION

The three classes of phospholipase C ß, ?, and d, are related phosphoinositide-specific enzymes that cleave the O-P bond between the lipid and the sugar head in order to generate two very potent intracellular messengers 1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃).

The classes are comprised of a series of core domains each contributing a specific function to phospholipase C.  The common domains include a pleckstrin homology (PH) domain, EF-hands, a TIM a/ß barrel active site, and a C2 domain.  The core residues within these core domains are highly conserved throughout the four isozymes of phospholipase C-ß, the two isozymes of ?, and the four isozymes of d.  The three classes of isozymes are present within the eukaryotic cell and act in unison.  Each of the isozymes contributes a specific aspect to the cellular response.  They also tend to be regulated and affected by slightly different factors.  Phospholipase C-ß, ?, and d, are regulated by the subunits of heterotrimeric G protein, tyrosine or serine/threonine phosphorylation, or by the GTP-binding G_h protein.  Other factors that regulate activity include the concentration of calcium, the concentration of substrate, pH, temperature, activators, inhibitors, cofactor concentrations, and membrane packing density (Rebecchi, 2000). 

The role of phospholipase C in the cell is an important one.  Phospholipase C is at the center of the core machinery for the phosphoinositide signal tranduction pathway (Voet, 1998).  Sequentially, the phosphoinositide signaling system is the triggering of a specific receptor, the activation of phospholipase C, the production of second messengers DAG and IP3, the release of calcium, the activation of protein kinase C, and the recycling of PIP2 (Voet, 1998).  The biological result of this signalling system is vast and wide-reaching.  Specifically, the internal release calcium within the cell increase the concentration of calcium from as little as 0.1µM to as much as 10µM.  This has many fundamental and diverse cellular responses.   Just a few examples of the fundamental cellular processes that are under the control of the phospholinositol signaling include muscle contraction, the prevention of polyspermy, the stimulation of calmodulin, the regulation of exocytosis, cell growth, cell differentiation, neuronal activity, apoptosis, etc (Voet, 1998;Rebecchi, 2000;Becker, 1998)  The signaling system is so diverse in its effects on the cell and the organism as a whole that a recent study has even linked its malfunction to teen suicides and mental disorders (Pandey, 1999).

Phospholipase C activity is regulated ultimately by the binding of an external ligand to seven-membered transmembrane rhodopsin-like receptors (Rebecchi, 2000).  The various classes of phospholipase C have various regulator proteins or kinases that are associated with these receptors.  They are activated by some form of GTP or ATP hydrolysis and dissociate from the receptor.  They migrate through the plane of the inner membrane and activate phospholipase C by association.   The active form of phospholipase moves through the membrane like a combine through a cornfield until the PI(4,5)P2 substrate enters the TIM barrel through its unridged spout.  After the hydrolysis of  PI(4,5)P2, the water soluble IP3 is released into the cytosol and the hydrophobic DAG is released into the membrane.  The IP3 stimulates the release of Ca²⁺ by binding to a IP3 gated membrane port on the membrane of the ER.  The function of DAG is to activate protein kinase C.  Interestingly, phospholipase C is not normally a free protein.  PI(4,5)P2 anchors phospholipase C to the membrane at the PH domain, and catalysis takes place at a different site using a different PI(4,5)P2.  PI(4,5)P2 is a minor component of the membrane’s inner leaflet and serve both as an allosteric regulator and substrate.

The isozymes of phospholipase C are water soluble, multidomained, and range in size from 85kDa to 150kDa (Rebecchi, 2000).  Phospholipase C-d first appeared in ancient single-cell eukaryotes with the ß and ? classes arising between the development of animals  and the parazoaneumetazoan split approximately 940 million years ago (Koyanagi, 1998).   The increase in complexity of the classes coincides with the development of their regulators and signaling components.

Phospholipase C has been studied since the 1950’s but it is only within the last 5 to 10 years that clues to its direct biological function have been elucidated.  What has been learned at this point is that many of the fundamental processes of the cellular are under its control.     

II.                  ENZYMOLOGY/STRUCTURE

The 3 classes of phospholipase C ß, ?, and d, have a series of common domains that surround its catalytic a/ß barrel active site.  These domains include a peckstrin homology (PH) domain, EF-hands, a TIM domain made up of X-Y box regions that are separated by a substrate recognition center Z, and a C2 site.  See Figures 1, 2, and 3 for schematics of the domains.    The ß and ? isozymes have minor additions to these domains of which are not all known (Rebecchi, 2000).  These additional domains account for the increase in their molecular size in comparison with the isozymes of ? (145-150kDa to 85kDa) (Essen, 1997).  But the main domains, throughout the classes and isozymes tend to remain constant.

Figure 1 -   Crystal structure of phospholipase C.  The PH Domain, the EF hands, the catalytic TIM Barrel, and the C2 Domain can all be seen.  3 Calcium ions can bind to the C2 Domain and 1 can bind to the TIM Barrel Domain.  Also shown is the binding of the PH and TIM Domain to PIP2 (Rebecchi, 2000).

Pleckstrin Homology (PH) Domain

The peckstrin homology domain is seen schematically in Figures 1 and 2.  It consists of approximately 100-130 amino acid residues that tend to show very poor sequence but high structure homology within the isozymes of phospholipase C and over 100 other unrelated proteins (Rebecchi, 1998).  See Figure 5 and VI. Homology.  Basically the suggested function of the PH region (if present in the isozyme) is to provide a targetting/linking function by Ser/Thr protein kinases, Tyr kinases, or G-protein regulators (Rebecchi, 2000). One of the two major roles the PH domain plays in the function (and most plausible) is to promote anchoring of phospholipase C to the inner leaflet of membranes enriched in PI(4,5)P2.  The PH domain weakly tethers itself to PI(4,5)P2 with high specificity.  The binding takes place specificially at the basic amino acid residues 30-43 of phospholipase C-d.  The hypothesis is that this binding along with several other factors activate phospholipase C-d as an allosteric regulator.  This activation allows the enzyme to hydrolyze multiple substrate molecules before it breaks the weak interactions and dissociates from the membrane surface (Garcia, 1995).

Figure 2 - Overall Structure for Phospholipase C from rat.  The C-terminal C2 Domain is shaded to clarify the domain boundaries.  The "top" view approximately corresponds to a view from the membrane surface.  The bound Calcium in the active site is shown as a sphere, and 1,4,5-IP3 in the active site is represented in ball-and-stick form.  The positions of calcium binding sites in the C2 domain are also indicated as spheres. (Kraulis, 1991)

The other major role the PH domain plays in the functioning of Phospholipase C is to serve as the docking point for the ß?-subunits of heterotrimeric G proteins.  Activation of the G protein subunits by the G protein-coupled receptors serve to regulate the activation of Phospholipase C.  See IV. Regulators.         

EF Hands Domain

Phospholipase C isozymes have up to 4 EF hands each consisting of a characteristic helix-loop-helix structure shown in Figures 1 and 2.  Its specific function is not known but its presence is essential for enzymatic activity (Nakashima, 1995).  The domain occurs from approximately amino acid residues 144-172 in Phosphorylase C-d and forms a two characteristic double lobes.  These EF hands are present in and show high structure homology to many other important proteins such as calmodulin and tropinin C.  At first it was thought that the EF hands served as a binding site for Ca²⁺ because its homologous domain in calmodulin does so (Katan, 1997).  But experiments have proved that Ca²⁺ does not bind to the structure and therefore is not a site of binding or regulation.  Also determined was that, specifically, the point mutations of amino acid residues Asp153, Asp157, and Glu164 are very sensitive to the activity of phospholipase C and affect its core structure.  (Nakashima, 1995).

Triose Isomerase (TIM) Domain

            The TIM Domain is made up of X, Y, and Z regions and functions as the catalytic center of phospholipase C.  See Figures 2, 3, and 4.  The X region is made up of 147 residues, the Y region is made up 118 residues, and the Z region separating the two is made up 43 residues.  The domain is constructed of alternating a helicies and ß-barrel structures that strongly resembles the structure of triose isomerase (Rebecchi, 2000).  The main components of the TIM domain are a hydrophobic ridge that promotes membrane penetration, a dished barrel containing catalytic residues, and a site for calcium binding.    

Figure 3 - The anatomy of core catalytic residues within Phospholipase C.

The hydrophobic ridge in the domain surrounds the barrel as a lip except for one side.  This side (called a spout in scientific literature) serves as an exchange point for substrates and products. (Rebecchi, 2000).  See Figure 4 and 5.  The ridge itself, specifically the hydrophobic ridge residues Leu-320, Tyr-358, Phe-360, Leu-529, and Trp-555 serve to promote the insertation of the TIM barrel into the acyl-chain region of the membrane surface.  By having these hydrophobic residues, the amount of work the enzyme must do to penetrate the membrane surface is lessened.  This penetration brings the active site into the membrane where it can interact with the PI(4,5)P2 substrate (Rebecchi, 2000).     

Catalytic residues of the active site are located at the bottom of the barrel.  It is the co-ordinated action of these residues that catalyze the hydrolysis of the O-P bond connecting phosphoinositol to diacylglycerol.  See Figure 2.  There are three major requirements for the activity of the catalytic site.  Firstly, the catalytic residues holding the substrate in place must be present.  Secondly, the catalytic residues co-ordinating the binding of calcium must be present.  And thirdly, the catalytic residues that co-ordinate hydrolysis of the phosphodiester bond must be present. 

A network of hydrogen bonds and salt-bridges orient the PI(4,5)P2 within the active site by its phosphate ring substituents at inositol positions 4 and 5 while hydrolysis takes place at position 1.  Lys-438 and Lys-440, interact with the phosphomonoester at position 4 of the inositol-(4,5)-diphosphate ring and Ser-522 and Arg-549 interacts with the phosphomonoester at position 5 (Essen, 1997).  It is the co-ordination of these residues that are partly responsible for the enzymes substrate preference.  These residues are highly conserved within the classes of isozymes (Rebecchi, 2000).  Arg-549 not only orients the PI(4,5)P2 in place but acts as a substrate determiner.  Amino acid substitutions of the positively charged amino acid Arg-549 to progressively non-polar amino acids change the substrate preference from PI(4,5)P2 to PI (Essen, 1997).  See Figure 3.     

The binding of a single calcium ion is to the active site is also essential for catalytic activity.  Its binding is co-ordinated by the side chains of Asn-312, Glu-341, Asp-343, and Glu-390 (Rebecchi, 2000).  The electron withdrawing nature of the calcium appears to lower the pK_a of the hydroxyl at insitol position 2 and to stablize the negative charge of the transition state of catalysis (Essen, 1997).  See III. Mechanisms of Catalytic Function.    

And finally, there are a few main catalytic residues that are directly involved in and essential for the functioning of the active site.  These residues, determined using in vitro single point mutations, were determined to be His-311, Arg-338,   Glu-341, His-356, Asp-343, and Glu-390 based on the reduction or abolishment of activity (Cheng, 1995).  See Figure 6 for the results of point mutations.  The action of these residues, the action of calcium binding residues, and the orientating residues all act in unison to hydrolyze the PI(4,5)P2 substrate.  The hydrolysis mechanism for phospholipase C is explained in detail later.  See Figure 8. 

The C2 Domain

            The C2 domain has been characterized in many other proteins and ranges from 120-130 residues in length (Riza, 1998).  The main structure is made up of 8 antiparallel ß-pleated sheet strands in three superimposable layers with three loops at the top that form the binding sites for up to 3 Ca²⁺(Rebecchi, 2000).  See Figure 7 for a schematic of the C2 domain.  The calcium binding sites are found between residues 643-657, 675-680, and 706-714.  These sequences are very well conserved throughout the classes and isozymes of phospholipase C and suggest that they serve some important function.   However, despite this conservation which indicates an important function, the disruption of these calcium binding site residues do not affect the calcium dependant activity of the enzyme when the PI(4,5)P2 is not the limiting factor.  The only calcium bound site that is a requirement for enzymatic activity is the one found in the active site (Rebecchi, 2000).  Therefore the hypothesized role of C2 in the function of phospholipase C is to activite the enzyme by Ca²⁺ binding when the PI(4,5)P2 substrate concentration in the membrane is very low.  It was found experimentally that when the PI(4,5)P2 concentration is at or below 1 mol %, high concentrations of Ca²⁺ would promote anchoring of phospholipase c to PI(4,5)P2 in the membrane.  This tethering would orient and fix the the catalytic core to the membrane surface and thus increase activity (Rebecchi, 2000).            

The d and ? isozymes have short C2 domain extensions whereas ß isozymes have large extensions of around 400 amino acids.   These extentions contain sequences that function as a docking point for activation by G protein subunits ß? (Kim, 1996).   Phospholipase C d and ? do not have these extensions and are also not regulated specifically by G protein subunits ß? but by various other means.  See III.  Regulators.     

III.              MECHANISMS OF CATALYTIC FUNCTION

The mechanism of catalytic function for phospholipase C serves describe to how the enzyme hydrolyzes various phosphatidylinositol substrates into diacylglycerol and various forms of inositol phosphate.   A definitive mechanism is not fully understood yet but a very good hypothetic model has been proposed by Essen based on what is know about the structure and the interactions within the catalytic site (Essen, 1997).   The reaction template is shown below by the hydrolysis of PI to IP and DAG. 

Depending on the substitutions for the orienting amino acid residues Lys-438, Lys-440, Ser-522, and Arg-549, the enzyme’s substrate preference can be changed (Essen, 1997).  See II.  Enzymology/Structure-TIM.  The major substrate in mammalian phospholipase C-d is PI(4,5)P2 so focus will be directed upon it rather than the other possible substrates.  Ca²⁺ bound to the active site by the side chains of Asn-312, Glu-341, Asp-343, and Glu-390 serves as a cofactor for enzymatic activity.  Ca²⁺ is used in the active site rather than other delocalizing metals such as magnesium because of its high variability in geometry within the action of the active site (McPhalen, 1991).  The high variability is needed for the penacovalent transistion state.  The actual catalytic process begins when PI(4,5)P2 is oriented within the active site by the phosphomonoester groups on positions 4 and 5 of the inositol sugar (Essen, 1997).  See II. Enzymology/Structure-TIM.  The hydroxyl group at position 2 on the inositol ring and the free oxygen on the phosphate both interact with the calcium ion.  This lowers the pK_a of the hydroxyl and stabalizes the negative charge on the transition state (Rebecchi, 2000).  The current model suggests that the basic amino acid residue Glu-390, in a charge relay system with His-392, strips the hydrogen from the 2-hydroxyl position.  See Figure 9 for a complete outline of the mechanism.  This causes the remaining oxygen to become very nucleophilic and promotes the attack of the oxygen to the phosphorus on the adjacent phosphorus.   The pentacovalent transition structure is very unfavourable and is stabalized by the hydrogen bonding from the nearby amino acid residue His-311.  Then His-356 donates a proton to the diacylglycerol leaving group in an acid/base catalysis reaction that generates a cyclic 1,2-phosphoinositol intermediate.  The basic His-356 residue then abstracts a proton from a water molecule to restore its orginal postive charge and the hydroxyl group attacks the cyclic phosphodiester intermediate (Essen, 1997). 

IV.               REGULATORS

The three different classes of phospholipase C are regulated differently.   The isozymes of phopholipase C- appear to be regulated by tyrosine or serine/threonine phosphorylation. Phospholipase C-ß isozymes are activated by the a and ß? subunits of the heterotrimeric G-proteins.  The regulatory mechanisms of phospholipase C-d isozymes remain unclear but it is thought that it is somehow linked to an atypical GTP-binding protein G_h (Pawelczyk, 1999;Rebecchi, 2000).  Other major environmental factors that may alter the activity of phospholipase C are the concentration of calcium, temperature, pH, the presence of activators and inhibitors, and the concentration of the PI(4,5)P2 substrate within the inner leaflet of the membrane.

Regulation of Phospholipase C-?

The isozymes of phopholipase C- are activated by tyrosine or serine/threonine phosphorylation by protein kinases.   The activation of these protein kinases are intrinsically regulated by receptor and non-receptor proteins.  These receptors are typically known in their control of cell growth, immunoglobins, and cytokines.  Their regulation of phospholipase C-? typically controls or helps to control many basic cellular functions such as cell division, transformation, differentiation, shape, motility, and apoptosis (Rebecchi, 2000).  Two critical tyrosine phosphoacceptor sites are located within the Z-region of the TIM a/ß barrel at positions Y771 and Y783.  Point mutation experiments on these sites concludes that just the Tyr at position Y783 must phosphorylated for the protein to be activated (Pei, 1998).  Some phosphorylated serine/theorine sites also activate phosphorylase C-?.   The site, Ser-1248, can be stimulated by either protein kinase A or C.  Interestingly, these in vivo studies do not reflect the results of those in vitro.  The tyrosine phosphorylated and serine phosphorylated phospholipase C-? isozymes have the same catalytic activity of nonphosphorylated isozymes in vitro.   This suggests that in vivo, the enzymes are also under the control of negative and positive modulators (Rhee, 1991).  The most important negative modulators have been found to be actin-binding proteins that suppress the activity of the non-phosphorylated forms more than the phosphorylated forms (Goldschmidt-Clermont, 1991).  The most signifigant positive modulator is the substrate anchor PI(4,5)P2 which is discussed later. 

Regulation of Phospholipase C-ß

The phospholipase C-ß isozymes are mainly controlled by the subunits of heterotrimeric G protein coupled with the hydrolysis of GTP.  The G protein subunits are activated by transmembrane rhodopsin receptors.  In the current model of phospholipase C-ß activation, the rhodopsin receptor is stimlulated by variety of ligands such as signalling proteins, odourant molecules, photons etc.  Once the receptor has been triggered, the GDP attached to the a-subunit of the heterotrimeric G-protein exchanges positions with free cellular GTP.  The a-subunit-GTP complex and ß? complex dissociate from both each other and the rhodopsin receptor into the plane of the membrane.  Then either the a or ß?-heterodimer subunit, or both, bind to phospholipase C-ß but the actual molecular basis has not yet been elucidated (Rebecchi, 2000). 

Regulation of Phospholipase C-d

The activation of phospholipase C-d isozymes are also not functionally well known.  What is known, however, is that that activation appears to be coupled to G_h, an atypical GTP-binding protein (Rebecchi, 2000).  The activation of G_h is controlled by a select set of heptahelical receptors (that are similar to the transmembrane rhodopsin receptors).   A proposed model of phospholipase C-d regulation involving GTP and G_h, a class II transglutaminase.  Transglutaminases can catalyze the transamination of glutamine residues with polyamines or hydrolyze lysine bridges.  Physiologically, the sequences 654-673 of G_h correspond with a small peptide region within the extended C2 region.  This the suspected site of activation (Feng, 1996). 

Regulation by Environmental Factors

Environmental factors that may affect the activity of phospholipase C include the cellular concentration of Ca²⁺, temperature, pH, and the concentration of substrate within the membrane (Rebecchi, 2000). 

As was described in II.  Enzymology/Structure-TIM/C2 Domain above, calcium binds to two separate sites within phospholipase C.  The absolute activity of phospholipase C is dependant on the binding of calcium to the TIM a/ß barrel (Lomasney, 1999).  The activity of phospholipase C is independent to the binding of 3 calcium ions to the C2 domain when the substrate is limiting (~1 mol %) (Lomasney, 1999).  Increasing the concentration of intercellular calcium is highly activating to the phospholipase C activity (Rebecchi, 2000).  A higher number of C2 domains become bound calcium as the concentration of Ca²⁺ increases.  It is proposed that the Ca²⁺ bound C2 domains increase the PH domain affinity for PI(4,5)P2.   This promotes anchoring of the free enzyme to the membrane and orients the catalytic core into the membrane surface (Essen, 1996).   Both of these alterations give phospholipase C more substrate engaging properties.  See Experimental Calcium Concentration Results in Figure 10.          

Like many other enzymes, phospholipase C is both temperature and pH sensitive.  However, since the enzyme strictly functions in vivo where these conditions are strictly moderated, they should not be of much concern as a regulatory factor.  However, in vitro, their effects are seen in the experimental data in Figures 11 and 12.  The in vitro pH and temperature optimums are found respectively to be 5.0 and 30^oC. 

Also like the classical enzyme, the concentration of its substrate affects the activity of phospholipase C.  As shown from the Lineweaver-Burk for in Figure 13, an increase in the concentration of the substrate increases the activity of the enzyme.  The kinetic parameters of phospholipase C are outlined in Figure 14.  The V_max was determined to be 28.5µmol·min^-1·mg^-1 and the K_m was found to be 105.3µM (Pawelcyzk, 1999).  The increase in activity due to the increase in phophotadic acid is proposed to stimulate the anchoring of the enzyme to the membrane(Rebecchi, 2000).

V.                  HOMOLOGY

The three classes of phospholipase C have the same common PH domain, EF hands, catalytic a/ß barrel, and C2 domain (Rebecchi, 2000).  Extensions and small differences in residues explain the small differences in function and regulation.  The major catalytic residues, His-311, His-356, Glu-341, Asp-343, and Glu-390 all appear to be well conserved throughout the classes of phospholipase C (Essen, 1997).  Comparisons of amino acid structure between isozymes and organisms are summarized in Figures 15, 16, and 17 (Rebecchi, 2000).  Also specific details concerning homology between structural domains within the classes of phospholipase C are discussed in II.  Enzymology/Structure.   

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