JB Minireview- Signaling J. Bi ochem.131.293-299.2002,

Structure, Regulation, and Function of C Isozymes

Kiyoko Fukami1 Department of Biochemisin, The Institute of Medical Science , The University of Tokyo,4-6-1 Shirohanedai , Minatoku , Tokyo 108-8039; and CREST Japan Science and Technology Corporation

Received December 7, 2001; accepted December 26 , 2001

Key words: calcium, , PI(4 ,5)P2, Ras.

Phospholipase C (PLC) is a key in phosphatidyl ino (SH) domain in PLC-y and the Ras-associating (RA) domain sitol turnover and generates two second messengers , and Ras-GTPase exchange factor (RasGEF)-like domain in inositol 1,4,5-bisphosphate (IF( and diacylglycerol (DAG), PLC,, (Fig. 1). PLC is a soluble protein that is localized from phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P.,[ in mainly in the cytosol, and it is translocated to the plasma response to activation of receptors by hormones, neuro membrane, where it functions to hydrolyze PI(4,5(P, in transmitters, growth factors, and other molecules. IP., in response to receptor activation. Thus, targeting of PLC to duces calcium mobilization, and DAG induces activation of the plasma membrane is a critical event for transducing protein kinase C. PI(4,5)P., is a substrate for PLC, and PI 3- signals. kinase. In addition, PI(4,5(P, directly regulates a variety of PLCƒÀ is composed of the N-terminal PH domain, EF- functions, including cytoskeletal reorganization, exocy hand domain, catalytic X and Y domains, and C2 domain. tosis, and channel activity; therefore, strict regulation of In addition to these domains, ƒÀ-type PLC isozymes have C- PI(4,5)P, levels by PLC or other converting is nec terminal extensions of approximately 400 amino . The essary for homeostasis. PH domain of PLCƒÂ1, which comprises approximately 100 Eleven PLC isozymes have been identified in , residues, binds tightly to PI(4,5)P., and IP, (see below); and they are divided into four classes, (3(1-4)-, y(1,2)-, 6(1- however, that of ƒÀ-type PLC isozymes binds neither to 4)-, and c(1)-type, on the basis of structure and regulatory PI(4,5)P., nor to IP,. Instead, it binds specifically to PI(3)P, activation mechanism (1, 2). Each isozyme is composed of although the concentration of PI(3(P in mammalian cells is subtype-specific domains and conserved domains. Struc very low, even when P13-kinase is activated (4). The PH tural analyses of the domains (3) have revealed the detailed domain of PLC 32 and PLC 133 also binds the heterotrimeric mechanisms of protein-protein and protein-lipid interaction G protein subunit, GƒÀƒÁ (5). In contrast, GTP-bound Gƒ¿q needed for anchoring the enzymes to the plasma mem binds the C2 domain of PLC(31 and PLC(32 (6). Deletion brane. The regulatory mechanisms of ƒÀ-type and ƒÁ-type analysis showed that C-terminal basic residues in cluster PLCs have been analyzed extensively Association of het regions are responsible for this binding (7, 8). Comparison erotrimeric G proteins of the Gq family stimulates activity of the ligand-binding affinities of different PLC(3 isozymes of ƒÀ-type PLC, and -y-type isozymes are regulated primarily revealed that PLC(32 and PLC(33 are more sensitive to the by receptor and cytosolic tyrosine kinases. In contrast, ƒÂ- ƒÀƒÁ subunit than are PLC(31 and PLC(34. The affinities of type PLC isozymes are thought to be regulated by calcium. Gƒ¿q for PLC(31 and PLC(33 are higher than that for e-type PLC was recently identified as an effector of Ras pro PLC(32. These results suggest that each isozyme is regu tein and is regulated by Ras in a GTP-dependent manner. lated differently by both subunits of Gq. Interestingly, how- Molecular and genome-based technologies have provides ever, PLCƒÀ2 isozymes were not activated by various stimuli new insights onto the roles of PLC in and in platelets from Gƒ¿q-deficient mice, showing that Gƒ¿q is development. This review focuses on recent advances and essential and cannot be replaced by GƒÀƒÁ (9). All PLC-ƒÀ iso

physiological functions of PLC isozymes, highlighting a zymes contain a C-terminal PDZ-binding motif, (Ser/Thr)- novel type of PLC, PLCƒÃ, and the functional analysis of X-(Val/Leu)-COOH, and PLC[33 binds specifically to Na*/K* each enzyme by -disruption techniques. In addition, exchanger regulatory factors (NHERFs), which have two the mechanisms by which the isozymes act in concert and PDZ domains (10). These findings indicate that PLC(3 independently will be discussed . might be targeted to the plasma membrane through inter- actions with specific partners. The Caenorliabditis elegans

Structure and regulation of PLC isozymes genome project revealed that encoding transcription All PLC isozymes contain catalytic X and Y domains as factors and G-protein-coupled receptors are the most well as various regulatory domains. Several domains, in numerous ones. Through a database search, we identified cluding the C2 domain , EF-hand domain, and pleckstrin several types of PLCs in C. elegans: two PLC(3 types, and homology (PH) domain , are conserved among the PLCs. one each of PLCƒÁ, PLCƒÂ, and PLCƒÃ type. Therefore, PLCƒÀ- Subtype-specific domains contribute to the specific regula mediated signaling may be a common pathway in C. ele tory mechanisms . These domains include the src homology gans, as well as mice and . PLCƒÁ isozymes are characterized by two SH2 domains ' For correspond and one SH3 domain that are located between the X and Y ence: E-mail: [email protected], Fax: 81-3- domains. When growth factors such as platelet-derived 5449-5417, Tel: 81-3-5449-5417 growth factor (PDGF), epidermal growth factor (EGF), and (C) 2002 by The Japanese Biochemical Society . nerve growth factor (NGF) engage their respective recep-

, No. 3, 2002 293 J.131 Biochem. Vol. 294 K. Fukami

Fig. 1. Domain structure of each type PLC. Catalytic and regulatory domains and their interacting molecules are shown. PH, pleckstrin homology domain; EF, EF-hand do- main,; X and Y domain, PLC catalytic do- main; C2,C2 domain; PDZ, PDZ-binding motif; SH, src homology domain; RasGEF, Ras GTPase exchange factor-like domain; RA, Ras associating domain; Tyr-P, phosphoty rosine residues; pro-rich, proline-rich se quence.

tors, which are intrinsic tyrosine kinase, increases in cellu domain, EF-hand domain, X-domain, Y-domain, and C-ter lar calcium and activation of multiple protein cascades are minal C2 domain. Recent molecular analyses revealed that observed (11, 12). The SH2 domains of PLCƒÁl are required PH domain of PLCƒÂd associates tightly with PI(4,5)P2 and to target PLCƒÁ1 to tyrosine-autophosphorylated receptors IP3, and the binding ability of the former is correlated with in this process. PLCƒÁ1 is then tyrosine-phosphorylated and PLCƒÂ1 activity (15, 16). Three-dimensional crystal struc activated. This phosphorylation is necessary but not suffi ture analysis supported the following interaction mecha cient for full activation. A recent study indicated that nism (17, 18). The 4-phosphoryl groups of IP3 interact with PDGF-induced generation of PI(3,4,5)P3, a product of PI3- the Lys32 and Lys57 residues, and the 5-phosphoryl groups kinase, contributes to translocation and activation of PLCƒÁ interact with the Lys30, Arg4O, and Lys57 residues. These (13, 14). Membrane anchoring is mediated by an interac basic amino acids are needed to form the inositol phos tion between PI(3,4,5)P., and the PH domain or C-terminal phate-binding pocket; however, they are not well conserved SH2 domain of PLCƒÁl. The PH domain of PLCƒÁ has a high in PLC(3 and -y subtypes, suggesting that the PH domain is affinity for PI(3,4,5)P3, which appears to be responsible for specific for different and the GƒÀƒÁ subunit. Lemmon et this membrane targeting. In contrast, PI(3,4,5)P,3 is de al. proposed the "tether and fix" theory, by which the bind- ing to plasma membrane and activation of PLCƒÂ1 was phosphorylated by PTEN or other polyphosphoinositide specific soon after receptor stimulation. So described (19). First, PLCƒÂ1 is tethered to the plasma PLCƒÁ1 activity is strictly regulated by generation and elim membrane through interactions of the PH domain and ination of partners that interact. The SH3 domain of PI(4,5)P2. This is an inactive form, which does not catalyze PLCƒÁl has been shown to bind to SOSI via a proline-rich a high rate of . Further interactions with the domain and to stimulate the guanine nucleotide exchange membrane mediated by the C2 domain and catalytic do- activity. mains expose the . This fixed (active) form hydro Comparison of DNA sequences suggests an evolutionary lyzes PI(4,5)P2 efficiently. PLCƒÂ1 is then released from the relationship in which PLCƒÂ appeared in primitive eukary membrane depending on production of IP3 . otes (Fig. 2). Yeasts, slime molds, and plants contain only Although all PLC isozymes require calcium for activity, PLCƒÂ-related PLCs. The PLCƒÀ and y subtypes arose later. the PLCƒÂ type is the most sensitive to calcium, suggesting PLCƒÂ is archetypal and contains only the N-terminal PH that it may be regulated by calcium (20) . There are several

J Biochem, Structure, Regulation, and Function of Phospholipase C Isozymes 295

Fig. 2. Evolutionary appearance of PLC.

calcium-binding sites in PLCƒÂ type. Structual analysis of of other proteins containing this domain. At that time a the X and Y domains of PLCƒÂ1 in the presence of calcium PLC-like protein was proposed to have this PA domain. and IP3 revealed that the G1u341, Asp343, G1u390, and Kataoka's group. using a yeast two-hybrid system, first Asn312 residues of PLCƒÂ1 are involved in calcium-binding identified this novel PLC, PLC210, in a C. elegans cDNA (17). The C2 domain of PLCS1 is predicted to contain three library (28). The mammalian homologue, PLCƒÃ, was then calcium-binding sites. Recently Lomasney et al. reported isolated (29-31). Sequence analysis revealed that PLCƒÃ that calcium binding to the C2 domain of PLCƒÂ increases contains two RA domains (RA1 and RA2) in the C-termi PLC activity by producing a ternary complex of protein nus and a Ras-GEF-like domain at the N-terminus as well phosphatidyl -calcium, which facilitates enzyme ac as the catalytic domains, PH domain, EF hand, and C2 do- cess to PI(4,5)P2 vesicles (21). In addition, calcium binding main. Therefore, PLCƒÃ is predicted to bind activated Ras. to the EF-hand domain of PLCƒÂ1 is required for association In fact, the interaction between Ras or Ras-related protein of the PH domain with PI(4,5)P, (22). This calcium depen Rapl and RA of PLCƒÃ was confirmed to occur in a GTP dency appears to be related to the hypothesis that PLCƒÂ - dependent manner (29, 30). Furthermore, a complete corre type is activated secondarily to the increase in intracellular lation was found between the mutational effect on Ras calcium induced by PLCƒÀ or PLCƒÁ type activation (23). binding to the RA2 domain and the resulting ability of Ras However, its evolutionary appearance shows that PLCƒÂ has to stimulate PLCƒÃ activity. This finding suggests that the roles even when no other PLC type exists. PLCƒÂ type may binding of activated Ras to the RA2 domain stimulates be a relatively primitive form that can be activated by con PLCƒÃ activity. Evolutionary comparison of all known PLC centrations of calcium that are too low to activate PLC(3 isozymes indicated that PLCƒÃ is most similar to the PLC(3 and -y. Finally, it has been reported that the a subunit of a type, which is regulated by the G protein Ga and GƒÀƒÁ sub- novel class of G protein, tissue transglutaminase TGII units. PLCƒÃ has a long C-terminal extension similar to that (Gƒ¿h), activates PLCƒÂ1 directly in vitro (24). Gh has trans of PLC(3, which is needed for interaction with Ga. There- glutaminase activity as well as the ability to bind and fore, Lopez et al. examined the effect of Go on PLCƒÃ activity hydrolyze GTP Recent evidence suggests that PLCƒÂ1 asso (31). Among the various Gƒ¿ subunits, Ga,, stimulated PLC ciated with Gh is inactive and becomes active when re- activity most effectively. In addition to Ga, GƒÀƒÁ was re leased from the complex by GTP in response to receptor cently reported to activate PLCƒÃ by Wing et al. (32). Be- activation (25). Furthermore, PLCƒÂ1 acts as a guanine nu cause the PH domains of PLCƒÀ2 and PLC(33 interact with cleotide exchange factor for TGII in a coupling system in GO-y, they investigated whether GƒÀƒÁ modulated the activity volving TGII and ƒ¿1B-adrenoreceptor (26), suggesting that of PLCƒÃ. When PLCƒÃ and GƒÀ1ƒÁ2 were co-transfected into PLCƒÂ1 plays an important role in a,B adrenoreceptor func COS-7 cells, PLC activity increased remarkably. This in- tion. crease was observed even when a RA-domain deletion mu tant of PLCƒÃ was used, indicating that the effect of GƒÀƒÁ on PLCƒÃ, as Ras regulatorleffector PLCƒÃ activity is not mediated by Ras. Furthermore, ectopi Recently a novel PLCƒÃ was identified as an effector of cally expressed PLCƒÃ is translocated to the plasma mem Ras protein. Ras is a monomeric GTP-binding protein that brane in response to epidermal growth factor stimulation is involved in a variety of cell functions including cell (29), suggesting the importance of interactions mediated by growth, differentiation, and oncogenesis. The ras gene is RA, C-terminal, and PH domains for activation of PLCƒÃ. mutated in approximately 15170 of all human tumors; there- Because PLCƒÃ contains a Ras-GEF-like domain, it is fore, an understanding of signaling pathways mediated by interesting to examine if PLCƒÃ works as Ras-GEF and acti Ras is important. In 1996, Ponting and Benjamin (27) pro- vates Ras. Jin et al, used an in vitro assay to study the posed the existence of a motif of approximately 100 amino specificity of PLCƒÃ-RasGEF activity and found that PLCƒÃ acids, known as the RA region, that is conserved in Ras has an activity toward Rap1, but not to Ras, Rap2A or Rho effectors RalGDS and AF-6. They also identified a variety (33). Lopez et al. (31) reported that transfection of cells with

Vol. 131, No. 3, 2002 296 K. Fukami

PLCƒÃ or the PLCƒÃ-GEF domain alone stimulates MAP tochemical analyses are summarized in Table I. kinase, a downstream target of Ras; activation of Ras, how- The expressions of PLC(3 isozymes have been well char ever, resulted in decreased PLC activity. This finding differs acterized. PLC(31 is the most widely expressed, especially from those of Kelly et al. and Song et al., who reported that in brain. PLC(31 is expressed at high level in the cerebral Ras activates PLC,, activity. This may because of differ cortex and hippocampus, whereas PLC(34 levels are the ences in assay systems or types of cells used for expression highest in the cerebellum and retina and are almost negli of PLCƒÃ. Thus PLCƒÃ acts as both a positive regulator and gible in the cerebral cortex and hippocampus (35). The positive/negative effector of Ras. Drosophila PLC(34 homologue, NorpA, was identified as an Schmidt et al. clarified a novel pathway from adenyl eye-specific gene, and PLC(34 was first isolated from cere cyclase-coupled receptors to calcium mobilization mediated bellum and retina (36). Expression of PLC(32 is restricted to by cAMP, Rap GTPase, and PLCƒÃ. The cAMP- and phos hematopoietic cells (37). PLC(33 is distributed in brain, phatidylinositol-mediated pathways are common intracel liver, and , but its expression is low through- lular signaling systems for cell surface receptors (34). Ex out the brain. tracellular signals are integrated differentially. In certain PLCƒÁ1 is expressed ubiquitously, especially in brain and signaling systems, cAMP inhibits PLC activity through lung (38). In adult rat brain, expression of PLCƒÁl protein is PKA-dependent phosphorylation of PLCs. In contrast, highest in neurons, followed by oligodendrocytes and astro cAMP-mediated signaling stimulates PLC activity in some cytes (39). Significantly high level of expression is observed cases, where both signaling systems function coordinately in embryonic cortical structure. It is worth noting that The mechanism by which this occurs has not been clarified. expression of PLCƒÁ1 is enhanced and phosphorylation of A transient increase in calcium was observed when HEK- PLCƒÁl on tyrosine residues is detected in primary human 293 cells expressing ƒÀ2-adrenergic receptor (ƒÀ2-AR) were breast or colorectal carcinoma cells (40). On the other hand, treated with adrenalin or the adenyl cyclase activator, PLCƒÁ2 is abundant in hematopoietic cells (38), a finding forkolin, indicating that this increase was mediated by that supports the theory that PLCƒÁ2 is essential for B cell cAMP. Furthermore, co-expression of an inactive Rap2B development (41). mutant reduced PLC activity, whereas co-expression of PLCƒÂ1 is the most abundant and widely expressed PLCƒÂ PLCƒÃ with (3._ AR enhanced the calcium level in cells. These isozyme. PLCƒÂ1 is distributed abundantly in brain, heart, results indicate that PLCƒÃ functions as a novel cross-talk lung, skeletal muscle, and testis (42). In rat brain, it is con signaling pathway that may be involved in a specific func centrated in astroglial cells and to a lesser extent in neu tion. Therefore, PLCƒÃ may be a target for new medicines rons. A remarkable aberrant accumulation of PLCƒÂ1 is that limit the function of adrenalin. observed in brain from Alzheimer-disease patients (43), but its role in the brain remains unclear. PLCƒÂ2 was isolated Mammalian tissue distribution and involvement in from bovine brain library, and although its localization in diseases other tissues has not been analyzed, it is thought to be The distributions of PLC isozymes often reflect their involved in cancers including intestinal metaplasia and physiological functions, which have been confirmed in stud adenocarcinoma (44). PLCƒÂ3 is detected abundantly in ies of knockout mice. The distribution of different isozymes brain, skeletal muscle, and heart (45), whereas expression as determined by Northern blot, Western blot, and his of PLCƒÂ4 is restricted to testis, brain, and skeletal muscle

TABLE I. Mammalian PLC isozymes. Species: (H), human; (B), bovine; (R), rat; (MI, mouse.

J Biochem. Structure, Regulation, and Function of Phospholipase C Isozvmes 297

(46, 47). contributes mainly to chernoattractant-mediated produc- The mRNA encoding PLCƒÃ is most abundant in the tion of superoxide and regulation of protein kinases but not heart, followed by the lung and kidney , but it is not found to chemotaxis. They also indicated that large amounts of in brain. This remarkable expression in the heart , along antigen-specific antibodies were produced when PLCƒÀ3?? with correlation of adrenalin function , suggests that PLCƒÃ mice or PLCƒÀ2/3?? mice were immunized with T cell-inde has important physiolosical functions in this organ . pendent antigen, suggesting that PLC(32 and PLC(33 play defined roles in immune function. Functional analysis with knockout mice With respect to PLCƒÁ, Ji et al. generated PLCƒÁ-deficient Null of PLCƒÀ1, 2, 3, and 4 have been gener mice (51). Homologous disruption of the PLCƒÁ1 gene re ated, and analyses of the resulting phenotypes support sulted in lethality at approximately embryonic day 9. The their roles in specific signaling and distinct biological func gross morphology of PLCƒÁ1?? embryos at day E8.5 is nor tions. PLCƒÀ4 gene--deficient mice were first reported in mal, but their growth and development at day E9.5 is 1996 (48). As mentioned above, PLC(34 is localized in retina markedly retarded in compared with wild-type embryos. and shares homology with NorpA, which mediates the pho Fibroblasts derived from these embryos were used to show totransduction cascade in Drosophila photoreceptors , and it that growth factor-induced DNA synthesis, cell growth, was therefore expected to be involved in mammalian visual and cell migration were unaffected , However, the PLCƒÁl?? processes. Jiang et al. reported that PLC(34/ mice have embryo fibroblasts did not mobilize in response to EGF. defective visual responses, whereas they retain their audi PLCƒÁl is widely expressed in embryos, especially in the tory capabilities. In addition, a reduction in the maximal dorsal aorta and limbs. However, the relation of its distri amplitude of the rod a- and b-wave components of elec bution to embryonic lethality is unclear. troretinograms was observed in PLCƒÀ4?? mice . However, PLCƒÁ2-deficient mice have a number of defects in signal- there was no obvious change in rod morphology, suggesting ing through immunogloblin superfamily receptors. PLCƒÁ2 that PLC(34 plays a role in distal rod-mediated signaling in?? mice have decreased numbers of mature B cells because of the retina. blocked pro-B cell differentiation (41). IgM receptor-in Kim et al. generated PLC(31- and PLCƒÀ4-deficient mice , duced calcium increase and proliferation of B cell mitogens and reported that PLCƒÀ1?? mice develop epilepsy and that are also absent. The phenotype is similar to those of Btk PLCƒÀ4?? mice have ataxia (35). PLCƒÀ4?? mice showed a and Blnk-deficient mice, suggesting that PLCƒÁ2 is down- waddling gait, with the rear body swinging left-right. The stream of Btk/Blnk signaling. In addition, collagen-induced

ataxia waaaaaaaaaaasnot due to bone deformation or muscle weak- platelet aggregation is defective, indicating that PLCƒÁ2 ness. Histological analysis of PLCƒÀ4?? mice revealed that plays a critical role in signaling through a receptor requir the cerebellar development was retarded, with an aberrant ing the FcRƒÁ chain. pattern of folia and incompletely migrated external granule Recently, we reported that PLCƒÂ4 gene-disrupted male cells at P15. They further clarified that PLC(34 is involved mice either produced a few smaller litters or are sterile in metabotropic glutamate receptor-mediated signal trans (52). In vitro fertilization studies showed that insemination duction in the cerebellum. Yoshioka's group reported that. with PLCƒÂ4?? sperm resulted in significantly fewer eggs PLC(34 is involved in induction of long-term depression becoming activated and that the calcium transients associ through glutamate receptor 1 in the cerebellum (49). ated with fertilization are absent or delayed. These results In contrast to the above findings in PLC34, mice, most suggest that PLCƒÂ4 in sperm plays an essential role in an PLCƒÀ1?? mice die suddenly from epileptic-like seizures early step of fertilization. Histochemical analysis of testes starting the third week after birth. The seizures were of a revealed that PLCƒÂ4 is concentrated in the anterior acroso

generalized type characterized as tonic-clonic or by tonic mal region of sperm. Furthermore, PLCƒÂ4?? sperm were extension of the entire body. Behavior data obtained after unable to initiate the acrosome reaction, an exocytotic administration of pentamethylenetetrazole, which blocks event required for fertilization that is induced by interac the inhibitory neuronal pathway, or kainic , which acti tion with the egg coat, the zona pellucida. These data dem vates the excitatory neuronal pathway, suggested that onstrate that PLCƒÂ4 functions in the zona pellucida PLC(31 is essential for normal function of the neuronal induced acrosome reaction during mammalian fertilization. inhibitory pathway In addition, carbachol-induced phos

phoinositide hydrolysis is markedly attenuated in PLCƒÀ1?? Conclusion hippocampus, indicating that PLCƒÀ1 is required for musca The subtype-specific regulations of PLC isozymes by rinic acetylcholine receptor signaling. tyrosine kinase, heterotrimeric G proteins, and calcium PLC(33 is reported to be involved in ƒÊ-opioid-mediated have been extensively analyzed; however, recent advances responses. Me et al. reported that mice lacking PLCƒÀ3 have provided additional information regarding specific exhibit a 10-fold decrease in morphine sensitivity in pro regulators. For activation, agonist-induced anchoring of ducing antinociception (50). They also showed that the p. PLCs to the plasma membrane is the first step. This is fol opioid-induced increase in intracellular calcium does not lowed by conformational changes. These associations are occur in PLC1ƒÀ3?? cells, whereas the bradykinin- or bombe mediated by protein-protein or protein-lipid interactions sin-induced calcium increase, which is mediated by the through relatively large molecular domains and small mol Gaq pathway, is not affected. Because PLCƒÀ2 and PLCƒÀ3 ecules such as lipids and phosphotyrosine-containing pep- are potently activated by GƒÀƒÁ in vitro, these data indicate tides. that PLC 133 participates in a pathway involved in inhibi The most remarkable recent finding is the identification tion of opioid responses mediated by GƒÀƒÁ. of a novel mammalian PLC, PLCƒÃ. Although it is not yet Li et al. carried out further analyses of the in vivo func clear if PLCƒÃ is a regulator or effector of Ras, PLC and P13- tions of PLC(32 and PLC(33 (37). They found that PLCƒÀ2 kinase appear to have similar regulators, G protein and

Vol. 131. No. 3, 2002 298 K.Fukami

tyrosine kinase, and effector, Ras. In addition, these 25316-25326 enzymes have a common substrate, PI(4,5(P.,. The mecha 16. Yagisawa, H., Sakuma, K., Paterson, H.F., Cheung, R., Allen, V., nism by which these enzymes act cooperatively in signal Hirata, H., Watanabe, Y., Hirata, M., Williams, R.L., and Katan, M. (1998) Replacement of single basic amino acids in transduction pathways needs to be elucidated. the pleckstrin homology domain of phospholipase C-Si alter the Gene-targeting technologies have contributed signifi ligand binding, phospholipase activity and interaction with the cantly to our understanding of physiological functions of plasma membrane. J. Biol. Chem. 273,417-424 PLC isozymes. Each PLC isozyme apparently plays a deci 17. Essen, L.O., Perisic, 0., Cheung, R., Katan, M., Williams, R.L. sive but specific role in a particular function, even when (1996) Crystal structure of a mammalian phosphoinositide-spe different isozymes show a similar distribution. The iso cific phospholipase C delta. Nature 380, 595-602 zymes act in concert, each contributing to a specific aspect 18. Ferguson, K.M., Lemmon. M.A., Schlessinger, J., and Sigler, P.B. (1995) Structure of the high affinity complex of inositol of the cellular response. This combination of individual but trisphophate with a phospholipase C pleckstrin homology do coordinated functioning may be why so many PLC iso main. Cell 83,1037-1046 zymes exist in mammalian cells. Further analysis of tar- 19. Lemmon, M.A., Falasca, M., Ferguson, K.M., and Schlessinger, geted mice should provide additional data to aid in J. (1997) Regulatory requirement of signaling molecules to the clarifying the function of PLC isozymes. cell membrane by pleckstrin-homology domains. Trends Cell Biol. 7, 237-242 20. Banno,Y. Okano, Y., and Nozawa, Y. (1994) Thrombin-mediated REFERENCES phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-deltal. J Biol. Chem. 269, 1. Rhee, S.G. (2001) Regulation of phosphoinositide-specific phos 15846-15852

pholipase C.Annu. Rev. Biochem. 70, 281-312 21. Lomasney, J.W., Ceng, H.F., Roffler, S.R., and King, K.(1999( 2. Katan, M. (1998) Families of phosphoinositide-specific phospho Activation of phospholipase C deltal through C2 domain by

lipase C: structure and function. Biochem. Bioph ,ys. Acta 1436, Ca2+-enzyme-phosphatidylserine ternary complex. J Biol, 5-17 Chem. 274, 21995-22001 3. Williams, R.L. (1999) Mammalian phosphoinositide-specific 22. Yamamoto, T., Takeuchi, H., Kanematsu, T, Allen, V., Yagisawa,

phospholipase C. Biochem. Biophvs. Acta 1441, 255-267 H. Kikkawa, U., Watanabe, Y., Nakasima, A., Katan, M., and 4. Razzini, G., Brancaccio, A., Lemmon, M.A., Guarnieri, S., and Hirata, M. (1999) Involvement of EF hand motifs in the Ca2+- Falasca, M. (2000) The role of the pleckstrin homology domain dependent binding of the pleckstrin homology domain to phos

in membrane targeting and activation of phospholipase C-ƒÀ1. J. phoinositides. Eur: J. Biochem. 265, 481-490 Biol. Chem. 275, 14873-14881 23. Kim, Y.H., Park, T.J., Lee, Y.H., Baek, K.W., Suh, PG., Ryu, 5. Wang, T., Dowal, I.,., EI-Maghrabi, M.R., Rebecchi, M., and S.H., and Kim, K.T. (1999) Phospholipase C-ƒÂ1 is activated by Scarlata, S. (2000) Pleckstrin homology domain of PLC-beta2 capacitative calcium entry that follows phospholipase C-ƒÀ1 confers G-ƒÀƒÁ activation to catalytic core. J Biol. Chem. 275, activation upon bradykinin stimulation. J Biol. Chem. 274, 7466-7469 6127-26134 6. Wang, T., Pentyala, S., Elliott, J, Dowal, L., Gupta, E., Rebecci, 24. Feng, J.F., Rhee, S.G., and Im, M.J. (1996) Evidence that phos

M.J., and Scarlata, S. (1999) Selective interaction of the C2 do pholipase deltal is the effector in the Gh (transglutaminase II)- main of phospholipase C-betal and beta2 with activated Gq. mediated signaling. J Biol. Chem. 271, 16451-16454 Proc. Natl. Acad. Sri. USA 96, 7843-7846 25. Murthy, S.N., Lomasney, J.W, Mak, E.C., and Lorand, L. (1999) 7. Park, D., Jhon, D.Y., Lee, C.W., Ryu, S.H., and Rhee. S.G. (1993) Interaction of Gh/transglutaminase with phospholipase CM Removal of the carboxyl-terminal region of phospholipse C- and with GTP Proc. Natl. Acad. Set. USA 96, 11815-11819. betal by calpain abolishes activation by G alpha q. J Biol. 26. Baek, K.J., Kang, S.K., Damron, D.S., and Im, M.J. (2001) Phos

Chem. 268,3710-3714 pholipase CƒÂ1 is a guanine nucleotide exchange factor for 8. Wu, D.Q., Jiang, H.P., Katz, A., and Simon, MI. (1993) Identifi transglutaminase II (Gƒ¿h) and promotes ƒ¿18-adrenoreceptor cation of critical regions on phospholipse C-betal required for mediated GTP binding and intracellular calcium release. J

activation by G-proteins. J Biol. Chem. 268, 3704-3709 Biol. Chem. 276, 5591-5597 9. Offermanns, S., Toombs, C.F., Hu, Y.H., and Simon, M.I. (1997) 27. Ponting, C.PP and Benjamin, D.R. (1996) A novel family of Ras Defective platelet activation in G alpha)q)-deficient mice. binding domains. Trends Biochem. Sci. 21, 422-425.

Nature 389,183-186 28. Shibatohge, M., Kariya, K. Liao, Y., Hu, C.D., Watari, Y., 10. Hwang, J.L, Heo, K., Shin, K.J., Kim, E., Yon, C., Ryu, S.H., Goshima, M., Sh ima, F., and Kataoka, T, (1998) Identification Shin, H.S, and Suh, PG. (2000) Regulation of phospholipase C- of PLC210, a Caenorhabditis elegans phospholipase C, as a

ƒÀ3 activity by Na+/H+ exchanger regulatory factor 2. J Biol. putative effectors of Ras. J Biol. Chem. 273, 6218-6222. Chenz. 275,16632-16637 29. Song, C., Hu, C.D., Masago, M., Kariya, K., Kataoka, Y.Y., Shi 11. Rhee, S.G. and Bae, Y.S. (1997) Regulation of phosphoinositide hatohge, M., Wu, D., Satoh, T, and Kataoka, T. (2001) Regula specific phospholipase C isozymes. J. Biol. Chem. 272, 15045- tion of a novel human phospholipase C, PLC c, through mem 15048 brane targeting by Ras. J Biol. Chem. 276, 2752-2757

12. Rebecchi, M.J. and Pentyala, S.P. (2000) Structure, function, 30. Kelley, G.G., Reks, S.E., Ondrako, J.M., and Smrcka, A.V. (2001) and control of phosphoinositide-specific phospholipase C. Phys. Phospholipase Cc: a novel Ras effector. The EMBO J 20, 743- iol. Pen 80, 1291-1335 754 13. Bae, Y.S., Cantley, L.G., Chen, C.S., Kim, S.R., Kwon, K.S., and 31. Lopez, I., Mark, E.C., Ding, J., Hamm, H.E., and Lomasney, Rhee, S.G. (1998) Activation of phospholipase C-gamma by J.W. (2001) A novel bifunctional phospholipase C that is regu

phosphatidylinositol 3,4,5-trisphosphate. J Biol. Chem. 273, lated by Gƒ¿12 and stimulates the Ras/mitogen-activated kinase 4465-4469 pathway J Biol. Chem. 276,2758-2765 14. Falasca, M., Wallace, M.A., Lehto, V.P., Baccante, G., Lemmon, 32. Wing, M.R., Houston, D., Kelley, G.G., Der, C.J., Siderovski, M.A., and Schlessinger, J. (1998) Activation of phospholipase C D.P., and Harden, T.K. (2001) Activation of phospholipase C-e

gamma by PI 3-kinase-induced PH domain-mediated mem by heterotrimeric G protein ƒÀƒÁ-subunits. J Biol . Chem. 276, brane targeting. EMBO J 17, 414-422 48257-48261 33. Jin, T.G., Satoh, T., Liao, Y., Song 15. Lomasney, .J.W., Ceng, H.F., Wang, L.P., Kuan, YS., Liu, S.M., , C., Gao, X., Kariya, K., Hu, Fesik, S.W., and King, K. (1996) Phosphatidylinositol 4,5-bis C.D., and Kataoka, T (2001) Role of the CDC25 homology do

phosphate binding to the pleckstrin homology domain of phos main of phospholipase Cc in amplification of Rapl-dependent pholipase C-ƒÂ1 enhances enzyme activity. J Biol. Chem. 271, signaling. J Biol. Chem. 276, 30301-30307

J Biochem. Structure, Regulation, and Function of Phospholipuse C Isozymes 299

34. Schmidt,H M., Eveliin, S., Weernink, P.A.O ., Dorp. F., Rehmann , 44. Marchisio, M., Di Baldassarre ., Lomanskney, J.W., and Jakobs, K.H , A., Angelucci, D., Caramelli, E., . (2001) A new phospho Cataldi, A., Castorina, S ., Antonucci, A., Di Giovannantonio, L., lipase C-calcium signaling pathway mediated by cyclic AMP Schiavone, C., Di Biagio and a Rap GTPase. Nat. Cell Biol. 3 , R., Falconi, M., Zauli, G., and Miscia, .1020-102,4 S. (2001) Phospholipase C 12 expression characterizes the neo 35. Kim, D., Jun, K.S., Lee, S.B., Kang, N.G., Min, D.S., Kim, Y.H., Ryu. S.H., She, P.G., and Shin plastic transformation of the human gastric mucosa. Am. J , H.S. (1997) Phospholipase C Pathol.159,803-808 isozymes selectively couple to specific neurotransmitter recep 45. Lin, F.G., Cheng , H.F, Lee, I.F, Kao, H.J., Lo, S.H., and Lee, tors. Nature 389, 290-293 W.H. (20011 Down regulation of phospholipase C 13 by cAMP 36. Ferreira, P.A., Shortridge, R.D., and Park , W.L. (1993) Distinc and calcium. Biochem. Biophvs. Res. Commum. 286,274-280 tive subtypes of bovine phospholipase C that have preferential 46. Liu, N., Fukami, K., Yu, H., and Takenawa, T (1996) A new expression in the retina and high homology to the norpA gene phospholipase C 14 is induced at S-phase of the cell cycle and product of Drosophilae. Proc. Natl. Acad. Sci. USA 90, 6042- appears in the nuclei. •J. Biol. Chem. 271, 355-360 6046 47. Lee, S.B. and Rhee, S.G. (1996) Molecular cloning, splice vari 37. Li, Z., Jiang, H., Xie, W., Zhang, Z., Smrcka, A.V., and Wu , D. ants, expression, and purification of phospholipase C 14. J Biol. (2000) Roles of PLC-ƒÀ2 and -(33 and PI3KƒÁ in chemoattractant- Chem. 271.25-31 mediated signal transduction. Science 287. 1046-1049 48. Jiang, H., Lyubarsky, A., Dodd, R., Pugh, E., Baylor, D., Simon, 38. Homma, Y., Takenawa, T., Emori, Y., Sorimachi, H., and Suzu M.I., and Wu, D. (1996) Phospholipase C 134 is involved in mod ki, K. (1989)Tissue- and type-specificexpression of mRNAs for ulating the visual response in mice. Proc. Natl. Acad. Sci. USA four types of inositol -specificphospholipase C. 93,14598-14601 Biochem.Biophvs. Res. Commun. 164,406-412, 49. Hirono, M., Sugiyama, T., Kishimoto, Y., Sakai, I., Miyazawa, 39. Mizuguchi, M., Yamada, M., Kim, S.U., and Rhee, S.G. (1991) T, Kishio, M., Inoue, H., Nakao, K., Ikeda, M., Kawahara. S., Phospholipase C isozymes in neurons and glia cells in culture, Kirino, Y., Katsuki, M., Horie, H., Ishikawa, Y., and Yoshioka, T. an immunoeytochemical and immunochemical study Braun (2001( Phospholipase CƒÀ4 and protein kinase Cƒ¿ and/or protein Res. 548. 35-40 kinase CƒÀI are involved in the induction of long term depres 40. Arteaga, A.L., Johnson, M.D., Todderud, G., Coffey, R., Carpen sion in cerebellar purkinje cells. J Biol. Chem. 276, 45236- ter, G., and Page, D. (1991) Elevated content of tyrosine kinase 45242 substrate phospholipase C-ƒÁl in primary human breast carci 50. Xie, W., Gary, M., McLaughlin, J.P., Romoser V.A., Smrcka, A., nomas. Proc. Natl. Acad. Sci. USA 88, 104353-10439 Hinkie, P.M., Bidlack, J.B., Gross, R.A., Jiang, H., and Wet, D. 41. Wang, D., Feng, J., Wen, R., Marine,J.C., Sangster, MY, Parga (1999) Genetic alteration of phospholipase C (33 expression nas, E., Hoffineyer, A., Jackson, C.W., Cleveland, J. L., Murray, modulates behavioral and cellular responses to ƒÊ opioids. Proc. P.J., and Ihle, J.N., (2000) Phospholipase C-ƒÁ2 is essential in Natl. Acad. So. USA 96, 10385-10390 the functions of B cell and several Fc receptors. Inmmun ity 13, 51. Ji, Q.S., Winnier, G.E., Niswender, K.D., Horstman, D., Wis 25-35 dom, R., Magnuson, M., and Carpenter G. (1997) Essential role 42. Lee, W.K., Kim, J.K., See, M.S., Cha, J.H., Lee, K.J., Rha, H.K., of the tyrosine kinase substrate phospholipase C-ƒÁl in mam Min, D.S., Jo, Y.H., and Lee, K.H. (1999) Molecular cloning and malian growth and development. Proc. Nat!. Acad. Sci. USA expression analysis of mouse phospholipase CƒÂl. Biochenn. Bio 94,2999-3003

phys.Res. Commun.261,393-399 52. Fukami, K., Nakao, K., Inoue, T. Kataoka, Y., Kurokawa, M., 43. Shimohama, S., Homma, Y., Suenaga, T., Fujimato, S., Takie - Fissore, R.A., Nakamura, K., Katsuki. M., Mikoshiba, K., chi, T, Perry, G., Whitehouse, PJ., and Kimura, J. (1991) Aber Yoshida, N., and Takenawa, T (2001) Requirement of Phospho rant accumulation of phospholipase C-delta in Alzheimer lipase C-ƒÂ4 for zone pellucida-induced acrosome reaction. Sci brains. Am.J Pathol. 139. 2629-2634 ence 292, 920-923

Vol. 131, No. 3, 2002