JB Minireview-Lipid Signaling J. Bi ochem.131.293-299.2002, Structure, Regulation, and Function of Phospholipase 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, phospholipase C, PI(4 ,5)P2, Ras. Phospholipase C (PLC) is a key enzyme 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- cell 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 enzymes is nec terminal extensions of approximately 400 amino acids. The essary for homeostasis. PH domain of PLCƒÂ1, which comprises approximately 100 Eleven PLC isozymes have been identified in mammals, 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 cell signaling 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 gene-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 genes 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 human. 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 lipids 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 phosphatases 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 hydrolysis. 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 active site. 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.