Some Articles Planned for FutureVolumes The RNA World in Plant Mitochondria STEFAN BINDER, MICHAELAHOFFMANN,JOSEPH KUtTN, AND KLAUSDASCHNER CTD Phosphatose: Role in RNA Polymerase II Cycling and the Regulation of Transcript Elongation MICHAEL E DAHMUS, NICK MARSIIALL,AND PATRICKLIN ATP Synthase: The Missing Link STANLEYD DUNN, D T MCLACHLIN,AND M j REVINGTON Functional Analysis of MUC1, a Carcinoma-Associated Mucin SANDRAJ GENI)LER HIV-1 Nucleoprotein: Retroviral/Retrotransposon Nucleoproteins JEAN-LUG DABLIX Biochemistry of Methiogenesis: Pathways, Genes, and Evolutionary Aspects UWE DEPPENMEIER Manipulation of Aminoacylation Properties of tRNAs by Structure-Based and Combinational in Vitro Approaches t/ICHARI) GIEGE AND JOEM PUETZ Shunting and Reinitiation: Viral Strategies to Control Initiation of Translation THOMAS HOHN Functions of Alphavirus Nonstructural Proteins in RNA Replication LEEV] KAARIAINENAND TERO AHOLA DNA-Protein Interactions Involved in the Initiation and Termination of Plasmid Rolling Circle Replication SALEEM A KAHN, T.-L CttANG, M.G KRAMER,AND M ESPINOSA Specificity and Diversity in DNA Recognition by E coil Cyclic AMP Receptor Protein JAMES C LEE Molecular Mechanisms of Error-Prone DNA Repair ZVI LIVNEH Catalytic Properties of the Translation Factors Necessary for mRNA Activation and Binding to 40S Subunits WILLIAM C MERRICK x SOME ARTICLES PLANNED FOR FUTURE VOLUMES Initiation of Eukaryotic DNA Replication and Mechanisms HEINZ-PETER NASHEUER FGF3: A Gene with a Finely Tuned Spatiotemporal Pattern of Expression during Development CHRISTIAN LAVIALLE Molecular Basis of Fidelity of DNA Synthesis and Nucleotide Specificity of Retroviral Reverse Transcriptase LUIS MENENDEZ-ARIAS Initiation of Eukaryotic DNA Replication and Mechanisms HEINZ-PETER NASHEUER, KLAUS WEISSHART, AND FRANK GROSSE A Growing Family of Guanine Nucleotide Exchange Factors Is Responsible for the Activation of Ras-FamilyGTPases LAWRENCE A QUILLIAM Translational Factors That Affect 5'-3' mRNA Interaction NAttUM SONENBERG AND FRANCIS POULIN HIV Transcriptional Regulation in the Context of Chromatin ERIc VERDIN The Molecular Biologyof the Group VIA Ca2+-Independent PhospholipaseA2 Z H O N G M I N MA AND JOHN TURK Division of Endocrinolof~y, Diabetes, and Metabolism Department of Medicine Washington University School of Medicine St Louis, Missouri 63110 I Introduction II Classification and Nomenclature iii Sequence and Structural Characteristics A Lipase Consensus Motif GXSXG B ATP-Binding Domain C Ankyrin-Repeat Domain D Bipartite Nuclei Localization Signal E Caspase-3 Cleavage Site F Proline-Rieh Region of Hnman Long Group VIA PLA2 lsoform G Other Features IV Gene Structure, Alternative Splicing, and Chromosomal Localization V Tissue Distribution and Expression VI Enzymology of Group VIA PLA2 A Phospholipase A2 and Phospholipase AI Aeti~dties of Group VIA PLA2 B Selectivity of Group VIA PLA2 for Phospholipids C Lysophospholipase, PAF Aeetylhydrolase, and Transaeylase Activities of Group VIA PLA~ VII Potential Celhdar Functions A Signaling Function in Insulin-Secreting Cells B Apoptosis (2 Membrane Phospholipid Remodeling D Membrane Homeostasis and Other Functions VIII Future Perspectives References S 10 14 15 15 16 17 20 20 20 20 21 22 22 24 25 "26 28 29 The group VIA PLA2 is a member of the PLAg superfamily This enzyme, which is cytosolie and Cag+-independent, has been designated iPLA2fl to distinguish it from another recently eloned Ca2+-independent PLA2 Features of iPLA2/3 moleeular strueture offer some insight into possible eellular funetions of the enzyme At least two catalytically active iPLAzfi/isoforms and additional 1To whom eorrespondenee should he addressed Progress in Nucleic Acid Research and Molecular Biology,Vo] 67 l Copyright O 2001 by Academic Press All rights of reproduction m any fonn reserved 0079-6603/01 $35,00 ZHONGMIN MA AND JOHN TURK splicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1 Potential tumor suppressor genes also reside at or near this locus Structural analyses reveal that iPLA2~ contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform, iPLA2~ is widely expressed among mammalian tissues, with highest expression in testis and brain, iPLA2~ prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase AI, lysophospholipase, PAF acetylhydrolase, and transacylase activities, iPLA~/3 may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonatc release, and exocytotic membrane fusion Structural features and the existence of multiple splicing variants ofiPLAg/~ suggest that iPLAg/3 may be subject to complex regulatory mechanisms that differ among cell types Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function © 2001 Academic Press I Introduction In response to cellular stimulation, membrane phospholipids are often hydrolyzed to generate intraeellular and intercellular messengers Phospholipase A-2(PLA2) enzymes catalyze hydrolysis ofsn-2 fatty acid substituents from glycerophospholipid substrates to yield a free fatty acid and a 2-1ysophospholipid (1) This group of enzymes has been intensively studied because they play crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction, and production of proinflammatory mediators, such as prostaglandins and leukotrienes, through the release of arachidonie acid (AA) from membrane phospholipids (2, 3) The lysophospholipid generated in PLA2 hydrolysis serves as a precursor for the proinflammatory molecule platelet-activating factor (PAF), and lysophosphatidie acid is a potent mitogen (4) PLA2 enzymes are a rapidly growing superfamily of diverse enzymes that have been classified into at least 11 groups (5) Recent advances in DNA and protein databases that permit BLAST analyses and EST searches have permitted cloning of new PLA2 species This chapter summarizes the molecular biology of a recently cloned intracellular Ca2+-independent PLA2 that has been classified as group VIA PLA2 (5) and is designated iPLA2fl here to distinguish it from another recently cloned Ca2+-independent PLA2 (6) iPLA2/~ was first purified from the nmrine P338D1 macrophage-like cells as an 80-kDa protein on sodium doeeeyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) (7) The enzyme was subsequently isolated from chinese hamster ovary (CHO) cells (8), which led to the cloning of its cDNAs from several sources (8-12) Analyses GROUPVIACa2+-INDEPENDENTPHOSPHOLIPASEA2 of its primary sequence have revealed structural characteristics that may provide clues about the roles of the enzyme in cellular processes Determination of the structure of the human iPLA2~ gene has yielded insight into the geneses of multiple iPLA,2~ splice variants (11, 12), and the gene has been found to reside in a chromosomal location that contains loci for genes associated with human diseases II Classification and Nomenclature Based on their dependence on Ca 2+ for their enzymatic activity, PLA2 enzymes can be dMded into Ca'2+-dependent and Ca2+-independent classes The former includes several groups of secretory PLA2s (sPLA2), which require millimolar Ca '2+ concentrations for catalytic activity, and group IV Ca2+-dependent cytosolie PLA,2isoenzymes (cPLA2~ and -fl), which require submieromolar Ca 2+ concentrations to associate with membrane substrates The Ca'2+-independent PLA2s appear to represent a diverse group of enzymes that can be further subdivided into several categories: group VIA intraeellular Ca2+-independent PLA,2 (iPLA2fl) (8-12), membrane-associated Ca%-independent PLA.)(iPLA2F) (6), 61-kDa group IV cytosolic PLA2F (ePLA2F) (13, 14), and PAF ace@hydrolases (1.5, 16) A common feature of these Ca2+-independent PLA.2s is the presence of the lipase consensus motif GXSXG These enzymes exhibit no other similarities iPLAafi was initially identified and purified from murine P388DI maerophage-like cells (7, 17) and classified as group VI PLA2 (1, 18) and subsequently as group VIA PLA,2 (5) In the remainder of this chapter, the group VIA PLA,~will be designated iPLA.2fl for simplicity, unless otherwise indicated The eDNA encoding this enzyme was first cloned from CHO cells (8) and subsequently from other sources (9-12) iPLA2fi has two recognized enzymatically active isoforms (12) and exhibits lysophospholipase activity in addition to PLA2 activity (7, 8, 19) Sequence analyses reveal that iPLA.~fl contains several interesting structural features that may be related to its functions ill cells Analyses of a predicted 40-kDa protein identified by the human genome project and a TBLASTN database search of GenBank led to the cloning of a novel Ca2+-independent, membrane-associated PLA.2that has been designated iPLA.~F (6) The deduced amino acid sequence fi'om this transcript showed no homology to known Ca2+-independent PLA9 enzymes except the putative ATPbinding and GXSXG lipase consensus motifs that also occur in iPLA2fl Both of these motifs also exist in a 40-kDa enzyme from potato with Ca'e+-independent phospholipase A2 activity (20, 21) that has been designated iPLA,)oe (6) The classification sehelne of Six and Dennis designates iPLA,~fl as group VIA and iPLA_gF as group VIB PLA2, respectively (5) iPLA2F also contains a C-terminal ZHONGMIN MA AND JOHN TURK peroxisomal targeting sequence (SKL) (22, 23) Because iPLA2F is tightly bound to membrane fractions in cell homogenates, it may be that the major subcellular location of iPLA2F is in the peroxisomal matrix enclosed within the peroxisomal membrane (6) By combined BLAST and EST database searches and 5'-RACE methods, a largely membrane-bound PLA2 with a calculated molecular mass of 60.9 kDa homologous to ePLA20t (group IVA PLA2) was cloned (13, 14) This protein, which exhibits Ca'2+-independent PLA2 activity, has been designated cPLA2F (13, 14) According to the scheme of Six and Dennis, this enzymes is classified as group VIC PLA2 (5) The deduced amino acid sequence indicates that the cPLA.2F protein lacks the C2 domain of cPLA2a, and accordingly has no dependence upon Ca ~+ for membrane association or catalytic activity This enzyme is thus a Ca'2+-independent PLA2 cPLA2F protein contains a pren~lation motif(-CCLA) (24) at the C terminus (13) The isoprenoid precursor [ H]mevalonolactone is incorporated into the prenylation motif ofcPLA2F when expressed in COS cells, and the mutagenesis of CCLA to SSLA at the C terminus of cPLA_gF prevents the ['3H]mevalonolactone incorporation, suggesting that the consensus prenylation site is indeed utilized This may account for the membrane localization of cPLA2F (13) Platelet-activating factor (PAF) aeetylhydrolases are also Ca2+-independent PLA2 (16) PAF acetylhydrolases are structurally diverse isoenzymes that catalyze hydrolysis of the sn-2 aeyl group of choline glycerolipids containing an sn-1 alkyl ether linkage and a short-chain or oxidized sn-2 substituent (16) The classification scheme of Six and Dennis (5) places these enzymes into two groups The group VII enzymes have molecular masses of 40-45 kDa and include both secreted isozymes found in plasma (group VIIA) (25) and intracellular, myristoylated enzymes found in lung and kidney (group VIIB) (26) The group VIII enzymes are intraeeIlular, have molecular mass of 29-30 kDa, and are found in brain (16, 27) III Sequence and Structural Characteristics The iPLA2fl cDNAs have been cloned from several sources (8-12) Rodent iPLA2fi and the human short isoform of iPLA2fi eDNA species encode a single 752-amino acid protein with calculated molecular mass of about 85 kDa The long isoform of human iPLA2fi eDNA encodes an 807-amino acid protein which has a 55-amino acid residue insertion at position 395 (Fig 1) (11, 12) The iPLA2fl enzymes share no sequence similarity with other known PLA2 enzymes Among the consensus structural features of sPLA2 enzymes are a Ca2+-binding -loop with the typical glyclne-neh sequence Y ' -G-C-X-C-G-X-G-G-X-X-X-P (the number of amino acid residues is based on Type I PLA2) and the residue Asp49, and an active site His48 (28) Asp49 is located adjacent to the catalytic • • • 25 37 GROUPVIACa2+-INDEPENDENTPHOSPHOLIPASEA2 Caspase-3 cleavage site DVTDlS3y R-iPLA2 I~ SH-iPLA2 I~ ,5 Lipase consensus motif GTS~'rG LH-iPLA2 ~ [ DVTDIg3y Eight Ankyrin-repeats domain • GTS619TG [ ] Bipartite nuclear localization signal ATPbindingdomain [] Proline-rich region FIG Schematicrepresentationof the structure of iPLA:lfi.The upper bar represents the rodent or human short isoformofiPLA2fl,and the lowerbar represents the humanlongisofnrmof iPLA2/~.The positionof the eight anlg,rin repeats, the putativeATP-bindingdomain,the bipartite nuclear localizationsignal,the proline-richregionof HL-iPLA2/~,the caspase-3cleavagesite, and the lipaseconsensusmotifare shown His 4s, forming the so-called His/Asp dyad Mechanistic studies indicate that the sPLA.2 not form a classic aeyl enzyme intermediate that is characteristic of serine esterases Instead, they utilize the catalytic site His, assisted by Asp, to polarize a bound water molecule that then attacks the substrate earbonyl group The Ca '2+ ion, bound in the conserved Ca2+-binding loop, stabilizes the transition state Serine esterases such as iPLA,2 employ a mechanism for catalysis that is different from that of sPLA2 enzymes The group IVA cytosolic Ca'2+-dependent PLA.2 (ePLA2ot) has a Ca2+-dependent lipid-binding (CaLB) domain at its N terminus that is responsible for transloeation of cPLA2 from cytosol to membranes in response to rises in cytosolic [Ca2+] induced by extracellular signals (29, 30) The CaLB domain of ePLA2~ exhibits significant homology with the C2 domains in proteins such as protein kinase C, GTPaseactivating protein (GAP), synaptotagmin, and phospholipase C Such domains bind to phospholipid membranes in a Ca'2+-dependent manner (30, 31) It is not yet certain what t