Regulation of Phosphoinositide-specific Phospholipase C Isozymes*

The hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), 1 by a specific phospholipase C (PLC) is one of the earliest key events in the regulation of various cell functions by more than 100 extracellular signaling molecules (1–4). This reaction produces two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which mediate the activation of protein kinase C (PKC) and intracellular Ca release, respectively. Furthermore, a decrease in the amount of PIP2 itself in the cell membrane is likely an important signal because the activities of several proteins are modulated by this phospholipid (5). PIP2 is a cofactor for phosphatidylcholine-specific phospholipase D (PLD) and a substrate for phosphoinositide 3-kinase (PI 3-kinase), both of which are also receptor-activated effector enzymes. In addition, PIP2 modulates actin polymerization by interacting with various actin-binding proteins and serves as a membrane-attachment site for many signaling proteins that contain pleckstrin homology (PH) domains. Consequently, the activity of PLC is stringently regulated in cells through several distinct mechanisms that link multiple PLC isoforms to various receptors.

The three-dimensional structure of a PLC-␦1 molecule lacking the PH domain has recently been determined (6). As expected, the X and Y regions are tightly associated. The structure also revealed two accessory modules, an EF-hand domain and a C2 domain, the latter of which was previously suggested to mediate the Ca 2ϩ -dependent binding to lipid vesicles. On the basis of the structural information, a catalytic mechanism comprising two steps, tether and fix, was proposed. The PH domain of PLC-␦1 would tether the enzyme to the membrane by specific binding to PIP 2 , and the C2 domain would fix the catalytic domain in a productive orientation on the membrane. The EF-hand domain would serve as a flexible link between the PH domain and the rest of the enzyme. Calcium is required for the function of the C2 domain. Another Ca 2ϩ ion located at the active site, together with His 311 and His 356 , directly participates in catalysis, consistent with the fact that all eukaryotic PLC isozymes require Ca 2ϩ for activity, that the two histidines equivalent to His 311 and His 356 are completely conserved among all PLC isoforms, and that mutation of either of the two histidine residues results in enzyme inactivation (7).
The multidomain structure observed with PLC-␦1 is likely to be common to all mammalian PLC isoforms (Fig. 1). However, PLC-␤ and PLC-␥ isozymes contain additional regulatory COOH-terminal and SH domains, respectively. These regulatory domains are responsible for the fact that different PLC isozymes are linked to receptors through distinct mechanisms. Furthermore, the COOHterminal domain of PLC-␤ isozymes might contribute to the tethering of the enzyme to the membrane surface, given that truncation of this domain completely blocked membrane association of PLC-␤1 (8). The SH domains of PLC-␥ appear to play a critical role in mitogenic signaling independently of PLC activity; catalytically inactive mutants of PLC-␥ (containing mutations at the essential His residues) elicited a mitogenic response when microinjected into NIH 3T3 cells, and mitogenic activity was localized to the SH region (7,9).
The GTP␥S-activated G␣ q or G␣ 11 subunits stimulate PLC-␤ isoforms with the rank order of potency PLC-␤1 Ն PLC-␤3 Ͼ PLC-␤2 (4,5). PLC-␤4 is also activated by G q ␣ subunits; however, because the basal activity of this enzyme is inhibited by ribonucleotides, including GTP␥S, accurate estimation of the extent of activation is difficult (12). All four G q ␣ members are palmitoylated at residues Cys 9 and Cys 10 (13). Removal of the two palmitate groups affects neither the capacity of the proteins to activate PLC-␤1 nor their association with the cell membrane. G␣ 16 , which is detected only in hematopoietic cells and is distantly related to the more widely expressed G␣ q (amino acid sequence identity of 55%), activates PLC-␤1, -␤2, and -␤3 in a manner essentially indistinguishable from that of G␣ q (14). However, the ␣ subunits can be discriminated by certain receptors (11).
The receptor-mediated activation of PLC-␤ has been studied in detail by reconstituting the m1 muscarinic acetylcholine receptor, G protein, and PLC-␤ in lipid vesicles (15,16). The muscarinic agonist carbachol stimulated PLC activity 90-fold, and each member of the G q ␣ family mediated this activation. The intrinsic GTPase activity of purified G␣ q was low but was stimulated Ͼ50fold by the presence of PLC-␤1, that is PLC-␤1 is a GTPaseactivating protein for G␣ q (16). In the reconstituted system, PLC-␤1 also increased the rate of GTP hydrolysis by G␣ q up to 60-fold in the presence of carbachol, which alone stimulated activity 6 -10-fold (16). These results indicate that the receptor and PLC-␤1 coordinately regulate the amplitude of the PLC signal and the rate of signal termination.
The G␤␥ dimer also activates PLC-␤ isozymes (1)(2)(3)(4)15). The sensitivity of PLC-␤ isozymes to G␤␥ subunits differs from that to G q ␣ and decreases in the order PLC-␤3 Ͼ PLC-␤2 Ͼ PLC-␤1 (4,5). The ability of G␤␥ subunits to activate PLC-␤2 in response to ligation of the luteinizing hormone receptor, V2 vasopressin receptor, ␤ 1 -and ␤ 2 -adrenergic receptors, m2 muscarinic acetylcholine receptor, and the receptors for the chemoattractants interleukin 8 (IL-8), formyl-Met-Leu-Phe, and complementation factor 5a was demonstrated using a cotransfection assay system in COS cells (4,17,18). These receptors also stimulate PLC-␤ through G q ␣ subunits. Although the concentrations of G␤␥ required for maximal activation of PLC-␤ isoforms in vitro are much larger than those of G q ␣ subunits, the final extents of activation are similar. Thus, both G q ␣ and G␤␥ likely are transducers in PLC signaling. However, it was recently suggested that G␤␥ is the predominant transducer in the activation of Xenopus oocyte PLC and that the role of G q ␣ subunits is to specify the receptor coupled to the enzyme (19).
The region of PLC-␤ that interacts with G q ␣ differs from that responsible for interaction with G␤␥; whereas the COOH-terminal region downstream of the Y domain is essential for the activation of PLC-␤1 and PLC-␤2 by G q ␣ (4, 5), the site of interaction of PLC-␤2 with G␤␥ was localized to the region spanning Glu 435 to Val 641 (20). Thus, G q ␣ and G␤␥ subunits may independently modulate a single PLC-␤ molecule concurrently. Several positively charged residues important for interaction with G q ␣ have been identified in the COOH-terminal region of PLC-␤1 (8). The COOH-terminal 14 residues of the G␤ subunit were also shown to be important for PLC-␤ activation (21).
Mammalian cDNAs that encode five distinct G␤ subunits and 11 G␥ subunits have been isolated. Although certain subunits are expressed only in specific tissues and there is some selectivity in the interaction of ␤ and ␥, G␤␥ subunits are available in many different combinations. Among several permitted combinations tested, all except ␤1␥1 activated purified PLC-␤3 with similar potencies (4,5). In cells, however, not all the available G q ␣ members and G␤␥ combinations appear to be utilized to activate PLC. Experiments with antisense oligonucleotides directed against the mRNAs encoding various G protein subunits suggested that m1 muscarinic acetylcholine receptor interacts only with the G protein complexes composed of the subunits ␣ q , ␣ 11 , ␤ 1 , ␤ 4 , and ␥ 4 to activate PLC in RBL-2H3 cells, despite the fact that the subunits ␣ 14 , ␤ 2 , ␤ 3 , ␥ 2 , ␥ 3 , ␥ 5 , and ␥ 7 are also expressed in the cell (22).

Activation of PLC-␥ by Protein Tyrosine Kinases
Polypeptide growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor, fibroblast growth factor, nerve growth factor, and hepatocyte growth factor, induce PIP 2 turnover by activating PLC-␥ in a wide variety of cells. Binding of these growth factors to their receptors results in activation of the intrinsic protein tyrosine kinase (PTK) activity of the receptor and the consequent tyrosine phosphorylation of numerous proteins, including the receptor itself and PLC-␥ (1-4) (Fig. 3). Receptor autophosphorylation creates high affinity binding sites for several SH2 domain-containing proteins, including PLC-␥1. A specific autophosphorylated site (for example, Tyr 1021 of the ␤-type PDGF receptor) is recognized by one of the SH2 domains of PLC-␥. Mutation of the PLC-␥-binding Tyr residue to Phe in the receptors for PDGF, epidermal growth factor, and nerve growth factor prevents association of the receptor with PLC-␥ and abolishes the growth factor-dependent production of inositol 1,4,5-trisphosphate (4).
Phosphorylation of PLC-␥1 by all growth factor receptors occurs at identical sites: tyrosines 771, 783, and 1254. Phe substitution at Tyr 783 completely blocks the activation of PLC by PDGF in NIH 3T3 cells (1)(2)(3)(4)(5). Tyrosine phosphorylation of PLC-␥1 appears to promote its association with unidentified components of the cytoskeleton; the SH3 domain of PLC-␥1 is responsible for targeting the enzyme to the actin microfilament network. Whether this cytoskeletal association serves to bring the enzyme into contact with its substrate or whether it promotes interaction with another protein component essential for its activation is unknown. Autophosphorylation of growth factor receptors and subsequent tyrosine phosphorylation of substrate proteins, including PLC-␥1, require the presence of H 2 O 2 , whose concentration increases transiently and which appears to function as an intracellular messenger in growth factor-stimulated cells (23). This requirement probably reflects that the activation of a receptor PTK by the binding of a growth factor is insufficient to increase the steady-state level of protein tyrosine phosphorylation. Concurrent inhibition of protein tyrosine phosphatases by H 2 O 2 is also necessary.
Nonreceptor PTKs also phosphorylate and activate PLC-␥ isozymes in response to the ligation of certain cell surface receptors. Such receptors include the T cell antigen receptor, membrane immunoglobulin (Ig) M, the high affinity IgE receptor, the IgG receptors, the IgA receptor, CD20, CD38, the ␣ 2 -macroglobulin receptor, integrins, and several receptors for cytokines such as ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, IL-1, IL-4, IL-6, and IL-7 (4, 24 -26). These receptors, most of which comprise multiple polypeptide chains, do not themselves possess PTK activity, but they activate a wide variety of nonreceptor PTKs such as the members of Src, Syk, and Jak/Tyk families. The activated PTKs often phosphorylate one of the receptor com-

FIG. 1. Linear representation of the various domains identified in the three types of PLC isozymes.
Catalytic domains X and Y as well as PH, EF-hands, C2, and SH (SH2 and SH3) domains are indicated. ponents to which PLC-␥ then binds via its SH2 domains and becomes phosphorylated by the PTK. PLC-␥1 associates directly with Src and Syk in cells, and in vitro it is phosphorylated by various soluble PTKs including Src, Fyn, Lck, Lyn, and Hck (4,5). Tyrosine phosphorylation of PLC-␥1 has also been shown to be elevated in cells that express SV40 middle T antigen, that are strained mechanically, or that are exposed to electroconvulsive shock (27)(28)(29).
Tyrosine phosphorylation of PLC-␥ has also been observed in response to the ligation of several heptahelical, G protein-coupled receptors, including m5 muscarinic acetylcholine receptor in Chinese hamster ovary cells, the angiotensin II and thrombin receptors in vascular smooth muscle cells, and platelet-activating factor (4,30,31). Src appears to be responsible for the phosphorylation of PLC-␥1 in vascular smooth muscle cells and platelets; electroporation of antibodies to Src inhibited the tyrosine phosphorylation of PLC-␥1 elicited by angiotensin II or platelet-activating factor. Although activation of Src family PTKs in response to stimulation of a variety of G protein-coupled receptors has been demonstrated (32), the mechanism by which the enzymes are coupled to the receptors is not clear. One possible mechanism is through a member of the recently identified proline-rich PTK (Pyk) family; stimulation of receptors coupled to the G proteins G i or G q in neuronal cells resulted in tyrosine phosphorylation of Pyk-2, binding of the SH2 domain of Src to the phosphorylated Pyk-2, and activation of Src (33).

PTK-independent Activation of PLC-␥
PLC-␥ isozymes can be activated directly by several lipid-derived second messengers in the absence of tyrosine phosphorylation. Phosphatidic acid produced by the action of PLD activates purified PLC-␥1 by acting as an allosteric modifier (34). PLC-␥ isozymes are also stimulated by arachidonic acid (AA) in the presence of the microtubule-associated protein tau (in neuronal cells) or tau-like proteins (in non-neuronal cells) (35). The effect of tau and AA was specific to PLC-␥ isozymes and was markedly inhibited by phosphatidylcholine (PC). These observations suggest that the activation of PLC-␥1 by tau or tau-like proteins might be facilitated by a concomitant decrease in PC concentration and an increase in AA concentration, both of which occur in cells upon activation of an 85-kDa cytosolic phospholipase A 2 (cPLA 2 ). This enzyme is coupled to various receptors and preferentially hydrolyzes PC containing AA. Therefore, activation of PLC-␥ isozymes may occur secondarily to receptor-mediated activation of cPLA 2 . Several studies are consistent with the notion that stimulation of PLC by endogenously released AA occurs in cells.
Ligation of a variety of receptors results in the activation of PI 3-kinase, which phosphorylates the D3 position of PIP 2 to produce phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). PIP 3 activates purified PLC-␥ isozymes specifically by interacting with their SH2 domains. 2 In addition, incubation of NIH 3T3 cells with PIP 3 resulted in a transient increase in the intracellular Ca 2ϩ concentration, an effect that was blocked in the presence of a PLC inhibitor. Thus, receptors coupled to PLD, cPLA 2 , or PI 3-kinase may activate PLC-␥ isozymes indirectly, in the absence of tyrosine phosphorylation, through the generation of lipid-derived second messengers (Fig. 4).

Activation of PLC-␦
Although four distinct PLC-␦ isoforms are known, the mechanism by which these isozymes are coupled to membrane receptors remains unclear. A new class of GTP-binding protein, termed G h and containing 75-80-kDa ␣ and ϳ50-kDa ␤ subunits, has been shown to be associated with agonist-bound ␣ 1 -adrenergic receptors (␣ 1 -AR). The G h ␣ subunit, a multifunctional protein that also possesses tissue transglutaminase activity (37), activates purified PLC-␦1 and forms a complex with PLC-␦1 in cells stimulated via ␣ 1 -AR (38). Furthermore, overexpression of G h ␣ in COS cells enhanced the activation of PLC induced by ligation of the ␣ 1 -AR (37). These results suggest that G h ␣ directly couples ␣ 1 -AR to PLC-␦1. It is not yet known whether other PLC-␦ isozymes are also activated by G h ␣, what other receptors couple to G h ␣, and how the tissue transglutaminase activity of G h ␣ is related to its PLC-␦1-activating function. The GTPase-activating protein for the small GTP-binding protein RhoA (RhoGAP) also activates purified PLC-␦1; PLC-␦1 activation was thus suggested to occur downstream of RhoA activation (39).
All PLC isozymes are activated by Ca 2ϩ in vitro, but PLC-␦ isozymes are more sensitive to Ca 2ϩ compared with the other isozymes. Furthermore, PLC-␦ can be tethered to PIP 2 -containing membranes via its PH domian in the absence of other signals. An increase in the intracellular concentration of Ca 2ϩ to a level sufficient to fix the C2 domain of PLC-␦ might therefore trigger its activation. Thus, activation of PLC-␦ isozymes might occur secondarily to receptor-mediated activation of other PLC isozymes or Ca 2ϩ channels.

Nuclear PLC
PLC signaling also appears to occur in the nucleus (40). PLC-␤1 is the major PLC isoform that has been detected in the nucleus of various cells. The amount of nuclear PLC-␤1 protein, which appears to be activated independently of its plasma membrane counterpart by an unknown mechanism, increases during cell growth and decreases during differentiation (41)(42)(43)(44). The changes in the amount of nuclear PLC-␤1 correlate with changes in the amount of PIP 2 hydrolyzed in the nucleus. Studies of cells lacking PLC-␤1 as a result of gene ablation revealed that it is essential for the onset of DNA synthesis in response to insulin-like growth factor I (45). The COOH-terminal region downstream of the Y domain was also shown to be necessary for translocation of PLC-␤1 to the nucleus (8).

Inhibition of PLC via Protein Kinases A and C
The activation of PKC or cAMP-dependent protein kinase (PKA) attenuates the PLC signaling pathway in a variety of cells. The proposed targets for phosphorylation by these kinases include cell surface receptors, G proteins, and PLC itself. PLC-␤1 is rapidly phosphorylated in cells treated with phorbol ester and is phosphorylated at Ser 887 by PKC in vitro; however, phosphorylation had no effect on either the basal or G q ␣-stimulated activities of PLC-␤1 (1). In human Jurkat T cells, activation of PKA or PKC results in an increase in phosphorylation of Ser 1248 and a concomitant decrease in the tyrosine phosphorylation of PLC-␥1, the latter of which might be responsible for the decreased PLC activity apparent in Jurkat cells treated with PKA-or PKC-stimulating agonists (1).
The interaction of PLC and PKA was studied in COS cells transfected with cDNAs encoding PLC-␤2, G protein subunits, and PKA (46). Expression of the catalytic subunit of PKA specifically inhibited G␤␥ stimulation of PLC-␤2 activity, without affecting G␣ qinduced activation. The effect of PKA was not mimicked by PKC isozymes. Furthermore, PKA directly phosphorylated serine residues of PLC-␤2 both in vivo and in vitro.

Genetic Mapping and Disruption of PLC Genes and the Relation of PLC to Human Disease
The ␦-type PLC, which is the only known PLC in yeast and slime mold, has been disrupted in these lower eukaryotes by gene targeting (47,48). Both mutants were viable. Whereas the yeast mutant showed increased sensitivity to various stresses, the slime mold mutant appeared normal, including with regard to such phenotypic aspects as growth, development, and chemotaxis. Chromosome positions for 10 mouse PLC genes and 8 human homologs were determined (49). The genes encoding PLC-␤1, PLC-␤4, and PLC-␥1 have been targeted in mouse. The homozygous mutants for PLC-␤1 or PLC-␤4 are born normal but subsequently manifest postnatal dwarfism; the PLC-␤4 mutants also show a defect in motor coordination and aberrant cerebellar development. 3 The PLC-␥1 mutation was lethal at early mid-gestation (around embryonic day 9) (36). Finally, platelets from a patient with a mild inherited bleeding disorder as well as abnormal platelet aggregation and secretion were shown to have one-third the amount of PLC-␤2 compared with normal platelets (50).