progress in nucleic acid research and molecular biology [vol 67] - k. moldave (ap, 2001) ww

Some Articles Planned for Future Volumes
The RNA World in Plant Mitochondria
STEFAN BINDER, MICHAELA HOFFMANN, JOSEPH KUtTN, AND KLAUS DASCHNER
CTD Phosphatose: Role in RNA Polymerase II Cycling and the Regulation of
Transcript Elongation
MICHAEL E. DAHMUS, NICK MARSIIALL, AND PATRICK LIN
ATP Synthase: The Missing Link
STANLEY D. DUNN, D. T. MCLACHLIN, AND M.
j.
REVINGTON
Functional Analysis of MUC1, a Carcinoma-Associated Mucin
SANDRA
J.
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] KAARIAINEN AND 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-Family GTPases
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 Biology of the
Group VIA Ca2+-Independent
Phospholipase A2
ZHONGMIN MA 1 AND JOHN TURK

Division of Endocrinolof~y, Diabetes,
and Metabolism
Department of Medicine
Washington University School of Medicine
St. Louis, Missouri 63110
I. Introduction 2
II. Classification and Nomenclature 3
iii. Sequence and Structural Characteristics 4
A. Lipase Consensus Motif GXSXG 5
B.
ATP-Binding Domain S
C. Ankyrin-Repeat Domain 10
D. Bipartite Nuclei Localization Signal 14
E. Caspase-3 Cleavage Site 15
F. Proline-Rieh Region of Hnman Long Group VIA PLA2 lsoform 15
G. Other Features 16
IV. Gene Structure, Alternative Splicing, and Chromosomal Localization 17
V. Tissue Distribution and Expression 20
VI. Enzymology of Group VIA PLA2 20
A. Phospholipase A2 and Phospholipase AI Aeti~dties of Group VIA PLA2. 20
B. Selectivity of Group VIA PLA2 for Phospholipids 20
C. Lysophospholipase, PAF Aeetylhydrolase, and Transaeylase Activities
of Group VIA PLA~ 21
VII. Potential Celhdar Functions 22
A. Signaling Function in Insulin-Secreting Cells 22
B. Apoptosis 24
(2. Membrane Phospholipid Remodeling 25
D. Membrane Homeostasis and Other Functions "26
VIII. Future Perspectives 28
References 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 Copyright O 2001 by Academic Press.
and Molecular Biology, Vo]. 67 l All rights of reproduction m any fonn reserved.
0079-6603/01 $35,00
2 ZHONGMIN MA AND JOHN TURK
splicing variants are derived from a single gene that consists of at least 17 ex-
ons 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 cleav-
age 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 ac-
tivities, iPLA~/3 may participate in signaling, apoptosis, membrane phospholipid
remodeling, membrane homeostasis, arachidonatc release, and exocytotic mem-
brane 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 hy-
drolyzed to generate intraeellular and intercellular messengers. Phospholipase
A-2 (PLA2) enzymes catalyze hydrolysis ofsn-2 fatty acid substituents from glyc-
erophospholipid substrates to yield a free fatty acid and a 2-1ysophospholipid
(1). This group of enzymes has been intensively studied because they play cru-
cial roles in diverse cellular responses, including phospholipid digestion and
metabolism, host defense and signal transduction, and production of proinflam-
matory mediators, such as prostaglandins and leukotrienes, through the release
of arachidonie acid (AA) from membrane phospholipids (2, 3). The lysophospho-
lipid 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 clas-
sified as group VIA PLA2 (5) and is designated iPLA2fl here to distinguish it
from another recently cloned Ca2+-independent PLA2 (6). iPLA2/~ was first pu-
rified 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
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 3
of its primary sequence have revealed structural characteristics that may pro-
vide 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 en-
zymes can be dMded into Ca'2+-dependent and Ca2+-independent classes. The
former includes several groups of secretory PLA2s (sPLA2), which require mil-
limolar Ca '2+ concentrations for catalytic activity, and group IV Ca2+-dependent
cytosolie PLA,2 isoenzymes (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 sub-
divided 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@hydro-
lases (1.5,
16).
A common feature of these Ca2+-independent PLA.2s is the pres-
ence 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 sub-

sequently 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 sub-
sequently 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 inter-
esting 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.2 that 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 ATP-
binding 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
4 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 ho-
mologous 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 prenyla-
tion 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 cat-
alyze 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 clas-
sification 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, myris-
toylated 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 sin-
gle 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

• • • 25 37
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
Asp 49, and an active site His 48
(28).
Asp 49 is located adjacent to the catalytic
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 ,5
R-iPLA2 I~
SH-iPLA2 I~
LH-iPLA2 ~
[
Caspase-3 cleavage site Lipase consensus motif
DVTDlS3y GTS~'rG
DVTDIg3y GTS619TG
Eight Ankyrin-repeats domain [] Bipartite nuclear localization signal
• ATP binding domain [] Proline-rich
region
FIG. 1. Schematic representation of the structure of iPLA:lfi. The upper bar represents the
rodent or human short isoform ofiPLA2fl, and the lower bar represents the human long isofnrm of
iPLA2/~. The position of the eight an lg, r in repeats, the putative ATP-binding domain, the bipartite
nuclear localization signal, the proline-rich region of HL-iPLA2/~, the caspase-3 cleavage site, and
the lipase consensus motif are shown.
His 4s, forming the so-called His/Asp dyad. Mechanistic studies indicate that
the sPLA.2 do 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 [Ca 2+] induced by ex-
tracellular signals
(29, 30).
The CaLB domain of ePLA2~ exhibits significant
homology with the C2 domains in proteins such as protein kinase C, GTPase-
activating 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<actors govern association of iPLA0fi with membrane phos-
pholipids, but this is a potentially important control point because iPLA2fl is a
eytosolie protein in resting cells. Analyses ofiPLA2/3 structural features may yield
some clues about potential mechanisms for regulating membrane association of
the enzyme.
A. Lipase Consensus Motif GXSXG
The amino acid sequence of iPLA2f contains a lipase consensus motif
GXS465XG (Ser 619 in long isoform of iPLA2/3) (Fig. 1) that is commonly found
in neutral lipases and is essential for enzymatic activity
(32, 33).
Indeed,
an iPLA2/3 mutant protein expressed in CHO cells
with
an iPLA2/3 eDNA
ZHONGMIN MA AND JOHN TURK
containing a mutation
at
Ser 465 to Ala completely abolished the catalytic ac-
tivity of iPLA2/~, supporting the participation of Ser 465 in catalysis (8). In the
case of cPLA2ot, mutation of Sel 2es to Ala at GLS22SGS, which is similar to

but slightly different from the classical serine lipase consensus, also completely
eliminates both PLA2 and lysophospholipase activities of ePLA2~ without sig-
nificantly altering its structure
(34).
Like cPLA2oe, iPLA.2/3 also can be inhib-
ited by arachidonyl trifluoromethyl ketone (AATFMK, also referred to as
AACOCF3)
and methyl arachidonyl fluorophosphonate (MAFP)
(17, 18),
per-
haps because both the cPLA2a and iPLA2/~ appear to use a central Ser in
similar catalytic mechanisms. The activities of iPLA2/3 and cPLA2a are dif-
ferentially affected by a bromoenol lactone (BEL) suicide substrate at a con-
centration as low as 1 #M. At such low concentrations, BEL inhibits iPLA2/~
activity but not sPLA2s or cPLA2a activities
(35).
BEL has been found to bind
covalently to iPLA2/3 (17), although the site of attachment has not been de-
termined. This covalent modification inactivates the enzyme irreversibly. Iden-
tification of the BEL binding site could be useful in relating iPLA2]3 struc-
tural features to its catalytic activities. A partial lipase consensus GPS'252GF
also occurs in hamster iPLA2/~ (8). A similar sequence in cPLA2ot contains the
active site serine, but site-directed mutagenesis Ser 2'52 to Ala in iPLA2/~ does
not alter catalytic activity when the enzyme is expressed in CHO cells (8). The
Ser 25'2 in hamster iPLA.2/~ is also not conserved in iPLA2]3 cloned from other
species.
cPLA2F (group IVC) contains a GvsS'2GS motif that also occurs in cPLA2~
(13, 14).
Mutagenesis of Ser "22s, Arg a°°, Asp 549, or Arg 566 also abolishes cPLA,)c~
activity

(14,
36). These amino acid residues are conserved in cPLA2F as Arg 54,
Asp asS, and Arg 4°'2
(13, 14).
Mutagenesis of these residues to Ala greatly re-
duces cPLA2F activity, indicating that Arg 54, Asp 3s5, and Arg 4°2 are required for
catalysis in addition to the central serine in the GVSS2GSsequence
(14).
Analysis of the deduced amino acid sequence of iPLA2F (group VIB PLA2)
revealed that this novel enzyme, like iPLAeJ3, also contains a Gxs4SaXG motif
(6). This conserved sequence motif occurs in two translational variants with
apparent molecular masses of 77 and 63 kDa. These proteins may arise from use
101 221
of alternative translational initiation sites at Met and Met ' , respectively. It
is likely that the GXSXG consensus motif contains the active serine required for
catalysis. There is also a partial lipase consensus sequence (GDS2°3FY) in the
deduced amino acid sequence of iPLA2F (6). This motif is probably included
in the 77-kDa protein but not in the 63-kDa protein based on the proposed
translation initiation sites. Further studies by site-directed mutagenesis will be
required to determine whether the Gxs4S3XG or GDS2°3Fy sequences contain
an active site Ser that is required for catalysis by iPLA2F.
Group VII enzymes include a secreted plasma form
(25)
and an intracellu-
lar isoform II (26). Group VIII includes the/3 and F subunits of intracellular
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A,~ 7'
isoform Ib
(16, 37, 38).
The cloning of a eDNA encoding the plasma form of
PAF aeetylhydrolase was reported in 1995 (25), and the deduced amino acid

sequence contained a GHS 273FG motif (Table I). Using site-directed mutage-
nesis, Set 273 was found to be essential for catalysis. Both Asp 296 and His 351 are
also essential for catalytic activity (25). The orientation and spacing of these cat-
alytic residues are consistent with an
ot/fl
hydrolase conformation that has been
observed in other lipases (25). The deduced amino acid sequence ofisoform II
of PAF acetylhydrolases (group VIIB) exhibits 41% identity with that of plasma
PAF acetylhydrolase (group VIIA) and also has a GHSFG consensus motif that
occurs in plasma PAF aeetylhydrolase (group VIIA). The group VIIB enzyme
is inactivated by diisopropyl fluorophosphate consistent with involvement of a
Ser residue in catalysis, and the substrate specificity of the group VIIB enzyme
is similar to that of plasma PAF aeetylhydrolase (39). The I3 and y subunits of
isoform Ib (group VIII) exhibit 63.2% identity in deduced amino acid sequence
overall. There is 86% identity in the catalytic and PAF-receptor-homologous
domains. There is no overall homology with other PAF aeetylhydrolases
(38).
This group of enzymes contains a GXSXV motif instead of GXSXG (Table I).
48 48
Mutagenesis of Ser of the GXS XV consensus motif of the y-subunit of the
group VIII enzyme abolishes enzymatic activi~, indicating that Ser 4s is involved
in catalysis
(38).
The consensus sequence of GXSX(G/V) is a common feature of all
Ca2+-independent PLA.2 so far cloned, and the observations summarized above
TABLE
I
o+
CtIARACTERISTICS OF THE CLONED Ca- -INDEPENDENT PHOSPI1OLIPASE A2
Name Location Source Size (kDa) Active site References

iPLA2fl (group VIA PLA2) Intraeelhdar Islet, CHO, P338D, 85 and 88 GTSTG
8-12
B lymphoe~es
Patatins and Patatin-like Intraeelhdar Potato tubes 40 GTSTG
(5, 20, 21
(iPLA2a)
iPLA2F (group VIB PLA,2) Membrane Human heart and 88.5, 77, GVSTG 6
-associated skeletal nmscle and 63
ePLA2y (group IVC PLA,2) Iutracellular Brain EST 61 GVSGS
13, 14
PAF aee~lhydrolase Secreted Plasma 45 GttSFG 2,5
(group VIIA)
PAF aeetylhydrolase Intraeellular Liver and kidney 40 GHSFG 26
Isoforms II (group VIIB)
PAF acetylhydrolase Ib, Intracellular Brain 30 GDSLV
16,
27
/J subunit (group VIIIA)
PAF acetylhydrolase Ib, Intraeellular Brain 29 GDSMV
16, 27
F subunit (group VIIIB)
8 ZHONGMIN MA AND JOHN TURK
indicate that the central serine in this motif participates in catalysis in these
enzymes (Table I).
B. ATP-Binding Domain
When the cloned rat islet iPLA2fi eDNA is transiently expressed in COS-7
or CHO cells, its actMty is stimulated about 2-4-fold by adenosine triphosphate
(ATP) (9). This is similar to the effect of ATP on the iPLA2fl activity isolated
from P388D1 macrophage-like cells (7). Furthermore, iPLA2fl overexpressed in
S. frugiperda (Sfg)

cells adsorbs to both ATP-agarose and ealmodulin-agarose
matrices, and this can be exploited in affinity chromatographic purification of
the enzyme
(40, 41).
Recently, Yang
et al. (42)
reported the purification of an
80-kDa Ca2+-independent PLA2 from rat brain using the same strategy. Amino
acid sequencing of several peptides from tryptie digests of the purified rat brain
iPLA2 protein indicated that it corresponds to the iPLA2fl cloned from rat pan-
ereatie islets by Ma
et al. (9).
These observations suggest that an ATP-binding
motif exists within the iPLA2fi molecule.
Surveys of the protein kinase family reveal that their catalytic domains ex-
hibit a highly conserved G-X-G-X-X-G sequence motif that appears to par-
tieipate in ATP binding
(43).
The consensus motif the G-X-G-X-X-G is also
found in many nueleotide binding proteins in addition to the protein kinases
(43, 44).
Alignment of the iPLA2fl sequence with the ATP-binding domain of
protein kinases revealed that the sequence from amino acid residue 431 to 457
of iPLA2fl exhibits similarity to the region of protein kinases involved in ATP
binding (Fig. 2). A model for the ATP-binding site of the protein kinase
v-src,
based on the three-dimensional structures from other nucleotide-binding pro-
teins, shows that the G-X-G-X-X-G residues form an elbow around ATR with
the first glyeine in contact with the ribose moiety and the second glyeine ly-
ing near the terminal pyrophosphate. Mutagenesis analyses of the ATP-binding

site of cAMP-dependent protein kinase indicated that replacing the third Gly 5'5
had minimal effects on steady-state kinetic parameters, whereas replacement
of either Gly 5° or Gly '52 had major effects on both K,,, and kc~t values con-
sistent with the predicted importance of the tip of the glyeine-rieh loop for
catalysis
(45).
A nearly invariant Val residue lies within subdomain I of
v-src
(Fig. g) and is located just two positions on the carboxyl-terminal side of the
G-X-G-X-X-G consensus. This residue may contribute to the positioning of con-
served glyeines
(43).
In addition, several amino acid residues in subdomain II of
v-src,
such as alanine '29s, lysine 3°°, and leucine 3°2, are represented at analogous
positions in the sequence ofiPLA2fi Lys 3°° in
v-src
appears to be directly involved
in the phospho-transfer reaction (46). Since iPLA2CJ adsorbs to ATP-agarose and
is desorbed by ATR it is possible that ATP directly binds to the ATP-binding do-
main of iPLA.2JL Sueh binding may modulate the function of iPLA~, although
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 9
Serine/threonine protein kinase family "r "r'r
cAPK-0~ 40
cAPK-~ 41
SRA3 92
TPKI 84
PKC-~ 336
PKC-~ 339
PKC-y 332

CDC28 5
Tyrosine protein kinase 1
Src 267
EGFR 685
INS.R 1020
PDGFR 565
iPLA2 ~ 420
Consensus
FIr'. 2. Alignment of the sequence of iPLA2fl with the ATP-binding domain of some of
serine/threonine protein kinases and of tyrosine protein kinases. The sequence of iPLA2fl and
the consensus of ATP-binding dolnain are shown at the bottom of the figure. Hyphens represent
gaps introduced fbr optim~d alignment. The kinase subdolnains are indicated by roman nmnerals
at the top of the alignment Residues conserved in all or nearly dl protein kinases are enclosed in
black boxes.
this possibility has not been verified experimentally. Interestingly, analyses of
hmnan iPLA2fl gene strneture revealed that the ATP-binding domain and lipase
eonsensus lnotif are encoded by the same exon
(12),
and this structural location
suggests that these two domains eould interact to regulate the iPLA2fi fimetion.
ATP could affect iPLA2fl function by at least three mechanisms. First, ATP could
bind to the ATP-binding domain of iPLA2fi to stimulate its intrinsie enzymatic
aetivity. Second, ATP could induce association of iPLA2fl monomers to form
muhimerie aggregates. Third, ATP could stimulate transloeation ofiPLA2fl from
eytosol to membranes, where its substrates are located. In many eases, iPLA2fi
activity has been found to be stimulated by ATE as well as other di- and triphos-
phate nueleosides (•
9, 12).
In some eases
(8, i0, 19),

ATP has been observed not
to stimulate activity of purified or partially purified iPLA2fl. Interestingly, activity
of the recombinant short isoforIn of human iPLA,2fi expressed in
Sf9
cells is not
affected by ATR but activity of the long isoform of human iPLA2fi increases about
3-fold in the presence of ATP
(12).
A study by Kio and Dennis (19) suggested that
ATE stabilizes iPLA2fl and protects it from denaturation. In cells, iPLA2fi might
interact with other proteins that negatively regulate its activity. ATP may induce
dissociation of iPLA2fi from such negative modulators and result in the activa-
tion of iPLA2fi. Nevertheless, the faet that iPLA2fi can bind to an ATP-affinity
column suggests that ATP might interact with iPLA.~fi in vivo; this possibility
deserves further study, for example,
by
deletion and mutagenesis analyses.
10 ZHONGMIN MA AND JOHN TURK
iPLA2F, like iPLA2fl, contains an ATP-binding motif at amino acid sequence
449GGGTRGW457. All glycines in this motif are conserved between iPLA2/3 and
iPLA2F, as is the Val residue (Ile in human iPLA.2fl) two positions after Gly 4'55.
In contrast to iPLA2fi, both the ATP-binding motif and the lipase consensus
sequence are encoded by two adjacent exons in the iPLA2F gene (6).
C. Ankyrin-Repeat Domain
Examination of the iPLA2fi deduced amino acid sequence by dot matrix anal-
ysis revealed that the domain between amino acid residues 150 and 414 (Fig. 1)
is composed of eight strings of a repetitive sequence motif of approximately 33
amino acid residues (Fig. 3) (9). This repeating-sequence motif is highly homolo-
gous to that of an 89-kDa domain ofankyrin
(8, 9).

In 1987, Breeden and Nasmyth
(47)
reported an "~33-residue repeating motif in the sequence of two yeast cell-
cycle regulators, SWi6 and cdcl0, and in the Notch and LIN-12 developmental
regulators from
Drosophila melanogaster
and
Caenorhabditis elegans.
Subse-
quently, the discovery of 24 copies of this sequence in the cytoskeletal protein
ankyrin led to the designation of this motif as the ankyrin repeat
(48).
Ankyrin-
repeat-containing proteins perform a wide variety of biological functions and
have been detected in organisms ranging from viruses to humans. The motif has
now been recognized in >400 proteins, and the number of repeats within any
one protein is highly variable
(49).
Molecules that contain such repeats include
ankyrin proteins that link integral membrane proteins to cytoskeletal elements
DNK ~ Q ~ D N]p~-IIr, Q
3 NN Q L QMG K~Q_~M~RV
4 - GP KFSQKGCIAIEM
5 PRY
~N~aM
8 ND F E K I S K[Q]- -ILIQ D
• . .G.TPLH. A E.V.
i I
QY-CH Q
DVT

GKNAS G NQV
-L-CN R NIM
SMDSNQIHSKD
KRG C - - DHD S T
TYGANIG R
- V-FG~E~D T P
PV S R A R K~A F I
L L A Consents
FIG. 3. Alignment of the eight strings of ankyrin-repeat sequences ofiPLA2fl. Identical amino
acid residues are enclosed in black boxes and conservative changes are enclosed in open boxes.
The ankyrin-repeat consensus and defined structure are shown at the bottom of the figure. Arrows
represent the fl hairpins, and cylinders represent the u helices. Ankyrin repeat was defined as a fl2c~2
motif. [Reprinted with permission from Z. Ma, S. Ramanadham, K. Kempe, X. S. Chi, J. Ladenson,
andJ. Ture, J.
Biol. Chem.
272, 11118-11127 (1997).]
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2
11
(48, 50),
developmental regulators in
Drosophila
and
C. elegans,
cell-cycle con-
trol proteins in yeast, transcriptional factors, toxins, and viral proteins (Table II)
(49-5l).
Recently, a novel family of postsynaptic-density proteins,
Shank,
has
been reported to contain seven ankyrin repeats at the N terminus

(52).
These
molecules are present in the nucleus, cytoplasm, and membranes, as well as the
extracellular milieu
(49).
The role of ankyrin repeats in mediating protein-protein interactions is well
documented, and their presence is often interpreted as an indicator of a sin>
ilar filnction in otherwise uncharacterized systems. The presence of ankyrin
repeats in iPLA2fl suggests that intra- or intermolecular protein-protein interac-
tions may regulate its function. Ankyrin is a linker molecule between membrane
and cytoskeletal proteins
(50).
Its C-terminal domain binds to cytoskeletal pro-
teins such as spectrin and tubulin, while its N-terminal 89-kDa ankyrin-repeat
domain binds to integral membrane proteins, such as ion channels and cell
adhesion/signaling molecules (Table III)
(50).
The ion channels that ankyrin-
repeat domains bind include the Na+,K+-ATPase of renal basolateral men>
branes, a renal amiloride-sensitive Na + channel, the red cell anion exchanger, a
cerebellar inositol triphosphate receptor, and voltage-dependent Na + channel in
myelinated neurons
(50).
The binding of ankyrin repeats to integral membrane
proteins raises two possible roles for ankyrin repeats in the iPLA2 protein.
TABLE II
PROTEINS WITII ANKYRIN REPEATS
Groups Examples Number of repeats
Ankyrin proteins
Developmental proteins

Transcriptional factors
and cell-cycle regulato D'
proteins
Synaptie proteins
Toxins
Viral host range proteins
Other proteins
Ankyrins
24
une-44
(C. elegans)
24
in-12, glp-1, ibm-1
(C. elegans) 6
Notch
(D. melanogaster)
6
TAN-1
(human, Notch-like)
6
53BP'2, c' lx
Bs,"
NF-x B preeursor( 110 kDa), 2-8
Lyt-10, CDK
inhibitors,"
GABPo~//~, a ode10,
SWI4, SWI6"
(yeast)
Shank
proteins

7
Latrotoxin/latroinsectotoxin (black widow spider)
19
Vaeeina 32-1d (vaeeinia
vires) 3
Cowpox HRP (cowpox virus)
Fowlpox 47-kd (fbwlpox virus)
Group
VI PLA2 (iPLA,2fl) 8
Myotrophin a
4
PYK-2"
(Nonreceptor tyrosine kinase)
4
2-5A RNAse 9
"Tile
three-dimensional structures of the
an~,rin repeat have
been determined
by X-ray and/or NMR.
12
ZHONGMIN MA AND JOHN TURK
TABLE III
INTEGRAL MEMBRANE PROTEINS THAT BIND TO ANKYRIN REPEAT
Integral membrane proteins Ankyrins
Ion channels
Bed cell anion exchanger (AE1)
AE1 (kidney isofbrm)
IP3 receptor (270 kDa)
Voltage-sensitive Na + channel

(homologous to c¢ subunits of voltage-
dependent Ca 2+ channel)
Na+,K+-ATPase (~ subunit)
Amiloride-sensitive Na + channel
Cell adhesion/signaling molecules
Lymphocyte adhesion antigen CD44
(putative hyaluronic acid receptor)
GP116 (CD44-1ike endothelial protein)
Ankyrin-binding glycoprotein 205 (AB-GP205)
Erythroid ankyrin (Ankl)
Epithelial anlgwin(s)
Brain ankyrin (Ank2)
Brain ankyrin (Ank2)
Epithelial ankyrin(s)
Epithelial ankyrin(s)
Lymphocyte ankyrin
Erythroid ankyrin (Ankl)
Brain ankyrin (Ank2)
(1) Although cPLA2 has a CaLB domain that shares homology with the C2
domain in the conventional isoforms of PKC, phospholipase CF, synaptotamin,
and so forth (29,
30),
iPLA,213 has no similar sequence that might mediate associ-
ation with membrane phospholipids. The translocation iPLA213 from cytosol to
membrane is likely to be important for its function in cells because its phospho-
lipid substrates are in membranes. It is possible that iPLA213 is able to associate
with membrane phospholipids through the binding of its ankyrin-repeat domain
to integral membrane proteins. Recently, we demonstrated that iPLA213 can be
induced to associate with a membrane fraction of INS-1 insulinoma cells upon
cell stimulation (manuscript in preparation). The long isoform of human iPLA.213

was also found to be associated with membrane when overexpressed in COS-7
cells
(53).
Interestingly, Western blot analysis of iPLA213 in adult rat ventrieular
myocytes revealed that full-length iPLA213 is detected only in the membrane
fraction
(54).
(2) Regulation of ionic fluxes is critical to the function of pan-
creatic islet 13 cells, neurons, and muscle cells, and proteins containing ankyrin
repeats associate with a number of ion transporters (Table III). Arachidonic acid
(AA) and other polyunsaturated fatty acids affect many ion channels
(55).
The
concentrations of free AA and other polyunsaturated acids within cells are very
low. It is possible that, upon activation, iPLA213 might transloeate to associate
with membrane ion channels via its ankyrin-repeat domain. Hydrolysis of AA
and other polyunsaturated acids from phospholipids catalyzed by iPLA213 could
then yield high regional concentrations of polyunsaturated acids that could affect
ion channel functions. It was reported that Na + flux induced by angiotension II
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 13
required the activation of a BEL-sensitive iPLA2 in LLC-PK1 cells
(56),
consis-
tent with the possibility that iPLAefl might interact with ion channels.
The first three-dimensional structure of an ankyrin-repeat-containing mol-
ecule was determined by X-ray crystal structural analysis of 53BP2 bound to the
p53 cell-cycle tumor suppressor
(57).
Subsequently, the ankyrin-repeat struc-
tures of several proteins, including cyclin-dependent kinase (CDK) inhibitors,

GABPa/fl, myotrophin, IKBa-NF4cB, Swi6, and PYK2, were determined by
X-ray and/or NMR methods (Table II)
(58, 59).
The ankyrin repeat consists of
pairs of antiparallel a helices stacked side-by-side and connected by a series
of intervening fl-hairpin motifs. The extended fl sheet projects away from the
helical pairs almost at right angles to them, resulting in a characteristic L-shaped
cross section. This assembled structure has been likened to a cupped hand: the
fi hairpins form the fingers, and the concave, inner surface of the anlcyrin groove,
which is made up of solvent-exposed residues from the a-helical bundle, Ibrms
the palm (,58, 59).
The crystal structure analyses revealed that ankyrin repeats play a critical
role in forming fimctional complexes. It has been reported that iPLA2 exists as a
multimerie complex of 270-350 kDa (8). Deletion of the N-terminal 150-amino
acid residues plus the eight ankyrin repeats of iPLA,2fl results in loss of catalytic
actMty (8). This could indicate that the ankyrin-repeat domain is important for
formation of a multimeric complex of iPLA2fl and that this is the catalytic active
ibrm. A recent report by Larsson
et al. (11)
demonstrated that cells cotrans-
feeted with full-length iPLA2fl and with a deletion mutant that contained the
ankyrin-repeat domain but not the catalytic domain (ankyrin-iPLA2-1) exhib-
ited decreased activity compared to cells transfected with full-length iPLA2fl
alone. This suggests that multimerie complexes of iPLA,2/? represent the func-
tional forms. The fact that there is residual iPLA2fi activity in the cotransfectants
could reflect the existence of a subpopulation of homomultimeric complexes.
Alternatively, heteromultimeric complexes might retain some activity. In either
case, these results suggest that the ankyrin-repeat domain participates in forma-
tion of fimctional of iPLA2/? complexes.
Inflamed tissue expresses a host of proteins that are not normally expressed.

Many of the genes encoding such proteins are activated by NF-x B, a transcrip-
tional factor that is normally in an inactive form in the cytoplasm. NFKB can be
activated by a variety of proinflammatory and noxious stimuli
(60, 61).
Under
resting conditions, NF-gB is tightly associated with IKBs, a class of specific in-
hibitory proteins that prevent nuclear transloeation and DNA binding of NF-K B.
The structural hallmark of the various IicB proteins is an ankyrin-repeat domain
containing six or seven closely adjacent repeats
(6,2).
Crystal structure analyses
of the bc B/p65/p50 complex indicate a fundamental role of ankyrin repeats in
the formation of inactive IKB-NF4cB complexes. The ankyrin-repeat domain
14 ZHONGMIN MA AND JOHN TURK
of IK Ba forms a slightly bent cylinder with five loops protruding from the packed
arrangement of stacked a helices. The loops between the repeats contain residues
that specifically recognize NF-KB. The appearance of IxB in the NF-xB com-
plex is reminiscent of a backbone lying between two lungs, where each ankyrin
repeat is a vertebra
(62-64).
Interestingly, ankyrin repeats 1 and 2 interact with
sequences encompassing the nuclear localization signal (NLS) of p65
(62).
Re-
peats 3 to 5 bind tightly over a large surface to the C-terminal Ig-fike domains
of both p50 and p65 Rel homology domains (RHDs)
(62).
Structural analyses
ofiPLA2fi reveal that the enzyme contains a bipartite nuclear localization signal
sequence (NLS), as discussed below. This raises the possibility that the ankyrin-

repeat domain might bind the NLS of iPLA.2/~ intramolecularly to regulate the
translocation of iPLA2/3 from cytoplasm to nucleus. This would be analogous to
the binding of the ankyrin repeats of IKBot to the NLS of NF-KB in IxB-NF-KB
complexes.
D. Bipartite Nuclei Localization Signal
Using the ExPaSy (Expert Protein Analysis System) profileScan to scan
the iPLA2/3 amino acid sequence against protein profile databases (including
PROSITE), only two domains in iPLA2J3 yielded significant matches with con-
sensus domains in the databases. These are the ankyrin-repeat domain and a
bipartite nuclear localization signal sequence (Fig. 1). The two best defined
NLSs are that of SV40 large T antigen (SV40TAg), which has a simple basic
NLS (PKKKRKV) sequence
(65),
and that of nncleoplasmin
(66),
which has
a bipartite basic NLS sequence (KRPAATKKAGQAKKKK), in which two in-
terdependent clusters of basic amino acids are separated by a flexible spacer
(66, 67).
Usually, the first two adjacent basic amino acids (Arg or Lys) in this
sequence are followed by a spacer region of any ten residues and at least three
basic residues (Arg or Lys) in the five positions after the spacer region
(67).
Pro-
teins containing NLS are transported into the nucleus in a process that involves
NLS binding to the nuclear import receptor importin/~ together with members
of the importin a family
(68).
The sequence511KREFGEHTKMTDV KKPK 52'
of rodent iPLA2fl (or 565KREFGEHTKMTDV ff-KPK

TM
of the long is~orm of
human iPLA2/3) perfectly matches the bipartite nuclear localization signal in nu-
cleoplasmin
(66),
suggesting that iPLA2fl might have the ability to translocate to
the nucleus. Recently, we found that iPLA2fl protein can be identified immuno-
chemically in nuclei isolated from iPLA2fl-overexpressing IN S-1 insulinoma cells
(manuscript in preparation). We found that a small percentage of iPLA2fi was
detected in nuclei compared with cytosol under resting conditions. We are ex-
ploring the possibility that the translocation ofiPLA2fl to nuclei can be stimulated
under some conditions. As suggested by crystal structures of IKB-NF-xB com-
plexes
(63, 64),
we propose that the ankyrin-repeat domain of iPLA2fi might
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 15
regulate nuclear translocation through its binding with the NLS of iPLA2fi in-
tramolecularly. The ankyrin repeats might form a slightly bent cylinder like those
of IxBce. The NLS might fold back to contact the ankyrin-repeat domain and
thereby block the NLS of iPLA2fl and prevent recognition by nuclear import
receptors. Upon stimulation, modulatory factors might interact with the ankyrin-
repeat domain to release the NLS of iPLA0fi and lead to nuclear translocation.
E. Caspase-3 Cleavage Site
Recently, Atsumi
et al. (69, 70)
reported that treatment of human promono-
eytie U937 cells with apoptosis-indncing agents, such as anti-Fas antibody or
TNFa/cyeloheximide (CHX), was accompanied by a time-dependent increase
in [3H]araehidonie acid (AA) release from prelabeled cells. The time-dependent
[3H]AA release paralleled the accumulation of apoptotie cells. By immunoblot-

ting analyses of U937 cells with anti-iPLA2j~ antibody, this group observed that, in
addition to an intact 85-kDa iPLA2J? protein, another immunoreaetive band with
an estimated molecular mass of 70 kDa became visible 6-12 h after treatment
with TNFot/CHX. During this time period, TNFo~/CHX-indueed AA release
and easpase-3 activity increased significantly
(70),
suggesting that the 70-kDa
immunoreactive band might be produced by caspase-3 action. Indeed, a po-
ls3
tential caspase-3 cleavage site, DXXDIX
(71),
occurs in iPLAafl around Asp ',
which is located near the N-terminal end of the first ankyrin repeat. Moreover;
183 183
ifiPLA2fl is cleaved at this site (DVTD Y in humans, DVTD N in rodents),
the predicted size of the resulting C-terminal fragment would be consistent
with the size of the cleaved fragment observed in this study
(70).
Caspase-3,
one of the key executioners of apoptosis, participates in proteolytic cleavage of
many key proteins, such as the nuclear enzyme poly(ADP-ribose} polymerase
(PARP), during apoptosis (
72}.
A survey of many protein substrates of easpase-3
indicates that each contains a common cleavage motif DXXDSX
(71).
The fact
that iPLA2fl is a substrate for caspase-3 was further confirmed by eotransfection
ofcaspase-3 and iPLA2fi. Cleavage at Asp is'3 resulted in the activation ofiPLA2fi
activity

{70}.
cPLA2ol also contains a caspase-3 cleavage motif DELD5225A, and
cleavage of cPLA2a at Asp 5~2 leads to inactivation
(69),
snggesting that cPLA2a
and iPLA2~ activities are differentially modulated in apoptosis.
F. Proline-Rich Region of Human Long
Group VIA PLA2 Isoform
Cloning of human islet iPLA2fl eDNA species from pancreatic islets and
insulinoma cells
(12)
revealed two isoforms of different lengths (Fig. 1). The
short iPLA2/3 isoform (SH-iPLA,2fi) corresponds to rodent iPLA.2]~, and the
long iPLA2/3 isoform (LH-iPLA2fl) corresponds to that cloned from human
B-lymphocyte-derived cell lines
(11).
The amino acid sequence for the long
16
ZHONGMIN MA AND JOHN TURK
E P DAF-3
LH-iPLA2~
P H N~N G]HILIQ~.PJM~PIP [H P GIH Y~V H ~A
Smad4
p x ¢ . _G ¢ P . . Q n x ¢ ¢ . I:! !t P F_ x , . 1~ ¢ Q_p _p ¢ ¢ S . N . L . ¢ Q Consensus
Fie,. 4. Alignment of proline-rich region of LH-iPLA2 with the proline-rich middle linker
domains of the Smad proteins DAF-3 and Smad4. Identical residues are enclosed in black boxes
and conservative changes are enclosed in open boxes. In the consensus sequence, amino acid residues
that are identical between at least two of the three sequences are indicated by underlined capihdized
letters. Conservative changes are based on functional groups of amino acids. Basic residues (H, K,
and R) by/~; hydrophobie residues (A, F, I, L, M, P, V, and W) by qb; polar residues (C, G, N, Q,

S, T, and Y) by n. Positions at which there is no similarity are denoted by dots. [Reprinted with
permission from Z. Ma, X. Wang, W. Nowatzke, S. Ramanadham, and J. Turk, J.
Biol. Chem.
274,
9607-9616 (1999).]
isoform differs from the short isoform by the presence of a 54-amino acid in-
sert in the region of the eighth ankyrin repeat. This insert corresponds exactly
to the amino acid sequence encoded by exon 8 of the human iPLA2fl gene
(12, 53).
This insert is proline-rich, and a BLAST search revealed similarities
to the proline-rich middle linker domain of the DAF-3 Smad protein from
C. elegans (73),
which is most closely related to mammalian Smad4 (Fig. 4)
(74).
Smad4 is a Mad-related protein and has been identified as the product of
the tumor suppressor gene
dpc4.
This gene is deleted or mutated in a propor-
tion of human pancreatic
(75),
breast, ovarian
(76),
and co!orectal tumors
(77).
The tumor suppressor activity of Smad4 is probably attributable to its participa-
tion in the signaling pathway of a family of cytokines that includes TGF-fl
(78).
The proline-rich middle linker region of Smad4 shares a PXsPXsHHPX12NX4Q
motif with the corresponding region of DAF-3 and the proline-rich region in the
long human iPLA2fl isoform. The Smad4 middle linker domain mediates pro-

tein interactions with signaling partners
(74),
is located near the center of the
protein, and separates an N-terminal MH1 domain with DNA binding activity
from a C-terminal MH2 domain with transcriptional activity
(79).
The proline-
rich region in the long iPLA2fl isoform is also located near the center of the
protein and separates an N-terminal domain with protein binding activity from
a C-terminal catalytic domain
(12).
Smad proteins participate in controlling cell
proliferation and apoptosis and form heterooligomers with signaling partners,
via the proline-rich middle linker domain in the case of Smad4
(79).
G. Other Features
Another feature of iPLA2/8 is its ability to bind calmodulin, a regulatory pro-
tein involved in a variety of cellular calcium-dependent signaling pathways. Both
iPLA~fl expressed in
Sf9
cells from the rat eDNA and native iPLA2fl in rat brain
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 17
can be purified by ealmodulin-affinity column
(41, 42).
These results suggest
that iPLA~2~ is able to bind calmodulin in a Ca2+-dependent manner. Removal
of Ca 2+ leads to the dissociation of iPLA2fi from ealmodulin-affinity matrices.
In the presence of Ca ")+, the aetivi~ of iPLA2fi is inhibited by calmodulin ill
a concentration-dependent manner
(41),

suggesting that Ca '2+ and ealmodulin
negatively regulate iPLA2~ activity. Cahnodulin is a protein capable of recog-
nizing positively charged, amphiphilie oe-helical peptides rather than a clearly
defined amino acid sequence motif (80); thus, it is relatively difficult to identify
cahnodulin-binding domains from sequence analyses. The amino acid sequences
of a number of cahnodulin-binding proteins have been determined, and in many
cases the loeations of the binding domains have been mapped by deletion nm-
tagenesis or chemical methods
(80).
Similar studies might permit identification
of the eahnodulin-binding domain of iPLA2~.
Phosphorylation is an important posttranslational modification for regulat-
ing the function of proteins. To date, no phosphorylation of iPLA2~ has been
reported, although PROSCAN results indicate that iPLA.2~ contains eonsensns
phosphorylation sites for calcium/calmodulin-dependent protein kinase II, pro-
tein kinase A, protein kinase C, protein kinase G, and casein kinase I!.
IV. Gene Structure, Alternative Splicing, and
Chromosomal Localization
Recently, we reported the cloning of the human iPLA2~ gene by screening
a human Lambda FIX II genomic library and determination of its structure
by combining sequencing and PCR approaches
(12).
Subsequently, Larsson
et al. (53)
reported the analysis of human iPLA2fi gene from two genomie
clones (H $228A9 and H $447C4, accession numbers AL022322 and AL021977,
Sanger Center, Hinxton, Cambridgeshire CB10 1SA, UK). The human iPLA2fi
gene spans about 70 kb and consists of at least 17 exons, ranging from 74 to
811 bp in size, and 16 introns, ranging from 0.2 kb to 23 kb (Fig. 5). The
5'-untranslated region was identified as exon la and part of lb. Exon la is con-

tained in done HS447C4, while exon lb and the rest of the exons are contained
in clone HS228A9. The translational stop codon of iPLA2~, the 3'-untranslated
region, and the polyadenylation signal were located in exon 16. Analysis of the
exon/intron boundary sequences indicated that the 5'-donor and 3~-aceeptor
sequences at splicing sites conform to the generally recognized consensus se-
quences
(12, 53).
Human islets express mRNA speeies encoding two iPLA2]~ isoforms, as do
human U937 promonocytie cells
(12).
The 162-bp in-frame insertion in the
~ tH
II I~ / /-
II llltt'//
,,,,~,~/
III
I~lltt /
IHmft /
I HU~l~ "i'i -~
q
1 U~
I Hill
I ttoo~
q
llH~ui~_~
"~ i Hu~.~-~ ~
III pUlI'I ~
"9 IU~
o~~o ,oqx
[ ,,

>
If!
II
!i
II
Cl
>
0
t",i
&
'T
"l:::l
<
E
©
i.
.~ <
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 19
eighth ankyrin repeat of S H-iPLA2/3 corresponds exactly to exon 8 of the human
iPLA2/~ gene. This indicates that mRNA encoding the SH-iPLA2/3 isoform arises
from an exon-skipping mechanism of alternative splicing
(81).
Severn splicing
variants of human iPLA2/3 have been identified from EST clones and reflect
insertions of 52, 53, and 168 bp, respectively, that do not occur in the transcripts
encoding LH- and S H-iPLA2/~ isoforms (11). Indeed, these insertions arise from
introns and are designated E8b, E9b, and E13a, accordingly. Analyses of the
exon/intron boundary sequences of these alternative-splice sites demonstrate
consensus splicing site sequences in the corresponding introns
(12, 53).

These
alternative splicing events may yield three putative transcripts, as elucidated in
Fig. 5. These putative transcripts contain a polyadenylation signal encoded by
exon 16 and therefore acquire a poly(A) tail required for translational compe-
tence. The iPLA2-2 transcript includes E13a and encodes a C- terminally trun-
cated protein due to the introduction of a premature translational stop codon
x~thin E13a. This variant retains the GXSXG sequence. The ankyrin-iPLAe-1
transcript includes E9a and encodes a C-terminally trnncated protein due to
the introduction of a premature translational stop eodon within Ega. The tran-
script ankyrin-iPLA2-2 results from skipping of exon 2 and inclusion of ESa and
E9a. This transcript encodes a truncated protein that has a deletion close to the
N terminus and a C-terminal truncation
from a
premature stop codon within
the E9a. Neither variant ankyrin-iPLA2-1 nor -2 contains GXSXG sequence, and
both are catalytic inactive.
Since iPLA2/~ cDNAs cloned from rodents are similar to human SH-iPLA2¢I
(8-10, 12).
a question of interest is whether a long iPLA2/~ isoform also exists
in rodents. Larrson
et al. (53)
reported the identification a PCR fragment con-
taining a sequence corresponding to exon 8 of the hmnan iPLA2/~ gene from
the total RNA of rat vascular smooth muscle cells and concluded that the pres-
ence or absence of the exon 8 is a tissue-specific and not a species-specific
feature.
We have mapped the human iPLA2/~ gene to chromosome 22q13.1 us-
ing fluorescence
in situ
hybridization (FISH)

(12).
The two human genomic
clones HS228A9 and HS447C4 represent the DNA fragments from chromo-
some
22q12.3-13.32
between the genetic markers DS426 and DS272
(53).
Allelic losses of chromosome arm 22q are frequently observed in human menin-
giomas and carcinomas of the colon, ovary, and breast. Recent studies of loss
of heterozygosity (LOH) in human breast carcinomas revealed that 40-66% of
these tumors were associated with LOH on chromosome 22q13.1, suggesting
that one or more tumor suppressor genes associated with breast cancer reside
in this region
(82, 83).
Studies of allele loss in human head and neck carcinomas
also indicated the possibility that tumor suppressor genes reside on human chro-
mosome 22q13.1
(84).
These results raise the possibility that iPLA2/~ represents
a candidate for a tumor suppressor gene located on chromosome 22q13.1.
20 ZHONGMIN MA AND JOHN TURK
V. Tissue Distribution and Expression
The mRNA encoding iPLA2fl has been found in all human, mouse, and rat
tissues studied, with different relative abundance
(8-12, 53).
The tissue distri-
bution in rodents has been examined by Northern blot analysis using mouse/rat
multiple tissue blot (8). The data obtained from this study revealed that a single
3.2-kb iPLA2fl transcript could be identified in all examined tissues, with high-
est expression in testis followed by liver and kidney. Analyses of human tissues

identified multiple bands of different sizes
(53).
Among these, a 3.2-kb band,
which might represent the full-length human iPLA,2fl transcripts, was abun-
dantly expressed in testis, brain, spinal cord, and thyroid. On the other hand, the
3.2-kb band may contain multiple transcripts that include alternative splicing
variants because the "-~200-bp differences among these variants are difficult to
distinguish by Northern blots.
Characterization of the regional distribution of various iPLA2fi enzymes in
organs has been reported
(42, 85).
Although several of groups of PLA,2 are ex-
pressed in rat brain, Ca2+-independent PLA2 activity accounts for the dominant
PLA2 activity in all brain regions, including cerebral cortex, cerebellum, hip-
pocampus, hypothalamus, and striatum
(42).
Northern blot analyses indicated
that mRNA species of iPLA2fl, iPLA,2F, and cPLA2F are expressed in brain
(42).
Because iPLA2F and cPLA2F are largely associated with membranes, the
Ca2+-independent PLA2 activity measured in the cytosolic fraction of each brain
region probably represents the iPLA2fl activity
(42).
VI. Enzymology of Group VIA PLA2
A. Phospholipase A2 and Phospholipase A1 Activities
of Group VIA PLA2
Enzymatic activities of iPLA2fl have been characterized with the protein
purified from cultured cells (7, 8) and tissue
(42)
and with recombinant enzyme

expressed from iPLA2fl eDNA in various host cells
(40, 41 ).
Using singly labeled
1 14
(1-pa mitoyl-2-[1- C]palmitoyl-sn-glycerol-3-phosphorylcholine) and doubly
labeled substrates (1,2-[1-14C]palmitoyl-sn-glycerol-3-phosphorylcholine), pu-
rified iPLA2fl has been demonstrated to exhibit both phospholipase A2 and A1
activities. The enzyme preferentially hydrolyzes
sn-2
over
sn-1
fatty acid sub-
stituents by a factor of 5 for 1,2-dipalmitoyl phosphatidylcholine (PC) in mixed
micelles (7, 8).
B. Selectivity of Group VIA PLA2 for Phospholipids
The fatty acid selectivity of iPLA2fl was evaluated by using different sub-
strates. When purified iPLA2fl from P388D1 macrophage-like cells was assayed
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A:~ 21
using mixed micelles of 100 #M phospholipid and 400/zM Triton X-100, the
enzyme showed no preference for either
sn-2
arachidonic acid- or
sn-1
alkyl
ether-containing phospholipids (7). Under the same conditions, purified rat
brain iPLA2/~ revealed the following substrate preference toward the fatty acid
chain in the
sn-2
position of PC: lionleoyl > palmit@ > oleoyl > arachidonoyl
(42).

In the case ofiPLA2fl purified from CHO cells (8), the enzyme also failed
to exhibit any significant preference for particular fatty acid
sn-2
substituents,
although different rates of hydrolysis of unsaturated t~atty acids were observed
in the assay with different Triton X-100 concentrations. When the activity assay
was performed with 50 #M or 500 #M Triton X-100, different hydrolysis rates
were observed with some substrates. The overall rates of hydrolysis were, at
least for some substrates, sensitive to lipid presentation, e.g., 1,2-dipalmitoyl PC
was hydrolyzed four times more rapidly when substrate was dispersed in 500
vs.
50 #M Triton, whereas 1-hexadecyl-2-arachidonyl-PC was hydrolyzed eight
times more rapidly when substrate was presented in 50
vs.
500 #M Triton. These
findings indicate that the apparent fatty acid selectivity ofiPLA.~fl depends upon
substrate presentation (8).
Rat brain iPLA2fl has been reported to hydrolyze PC substrates with an
sn-2
linoleate residue five times more rapidly these with an sn-2 arachidonate sub-
stituent
(42).
Yang
et al. (42)
have proposed that iPLA2fl could be an important
enzyme in linoleate metabolism.
Purified recombinant iPLA2/~ exhibits little preference between substrates
containing choline
vs.
ethanolamine head groups (7,

8, 40),
although choline
substrates are hydrolyzed more rapidly than other head-group classes under
some assay conditions (8). Purified rat brain iPLA2fl also showed no significant
head-group preference
(42).
C. Lysophospholipase, PAF Acetylhydrolase,
and Transacylase Activities ol" Group VIA PLA2
Purified iPLA2fl from 388D1 cells and recombinant iPLA2fl expressed from
the CHO cell cDNA exhibited detectable lysophospholipase activity in a Triton
X-100 mixed-micelle assay (7, 8). The kinetic studies byWolfand Gross
(40)
using
the purified recombinant iPLA2fl overexpressed in
Sf9
cells from CHO cDNA
using L-or 1-palmitoyl-2- [ 1-14C]arachidonyl-PC as substrate demonstrated that
the recombinant protein exhibited calcium-independent phospholipase A1/A2
and lysophospholipase activities at similar levels when assayed in the absence
of Triton X-100. Assay conditions therefore have an important impact on the
apparent expression of various lipase activities of iPLAefl. With the combina-
tion of 50 #M Triton X-100 and 50% glycerol, the lysophospholipase activity
of iPLA2fl appeared equivalent to its PLA2 activity
(19).
The iPLA2fl activity
varied with Triton-X 100 concentration, and optimal activity was observed at a
Triton/phospholipid molar ratio of 4:1 (7).
22 ZHONGMIN MA AND JOHN TURK
The
sn-1

ether-linked substrate PAF can be hydrolyzed by iPLA2/3 in assays
employing 500/zM or 50/zM Triton X-100, indicating that the phospholipase
A2 activity of iPLA2/3 is not restricted to long-chain fatty acids
(7, 8).
The ob-
served PAF acetylhydrolase activity was about 5% of the corresponding to the
iPLA2 activity (7, 8). Like the lysophospholipase activity of iPLA2/3, the ap-
parent PAF acetylhydrolase activity was also influenced by assay conditions,
suggesting that the enzyme activity is strongly dependent on substrate presen-
tation
(19).
The iPLA2/3 also exhibited both lysophospholipid/transacylase and
phospholipid/transacylase activities that were susceptible to inhibition by BEL
(19).
VII. Potential Cellular Functions
A. Signaling Function in Insulin-Secreting Cells
Stimulation of islets with glucose leads to insulin secretion and hydrolysis
of arachidonate from/3-cell membrane phospholipids
(86, 87).
Nonesterified
arachidonate may participate as a second messenger in glucose-stimulated in-
sulin secretion (see Refs.
88-91).
The mechanism of glucose-stimulated arachi-
donate release from the membrane phospholipids is incompletely understood.
Many studies
(92-101)
have demonstrated that PLAz activation is involved
in insulin secretion since glucose and other secretagogues such as carbachol
and CCK-8 stimulate PLA2 activation in islets. Inhibition of PLA2 activities

suppresses insulin secretion, release of AA, and accumulation of lysophospho-
lipids. Recent studies
(102, 103)
in insulin-resistant mice indicate that islet P LA2
activation is potentiated in these animals, providing evidence that islet PLA2 ac-
tivation occurs in hyperinsulinemic mice. Studies in isolated islets, insulinoma
cells, and whole animals thus suggest that activation of PLA,2 is involved in
secretagogue-induced insulin secretion.
Several PLA2s have been identified in islets
(9, 104-107)
with various sen-
sitivities to PLA2 inhibitors. Any individual or multiple PLA2 enzymes might
play signaling roles in islet/3 cells. The suicide substrate BEL inhibits iPLA2/3 at
concentrations that do not inhibit Ca2+-dependent sPLA2s or cPLA2 activities
(17, 35).
BEL is thus a useful tool for distinguishing iPLA2/3 from other PLA2s
(18),
although BEL also inhibits the MgZ+-dependent phosphatidic acid phos-
phohydrolase (PAPH-1)
(108)
and iPLA2F (group VIB PLA2) (6). Interestingly,
glucose-induced release of arachidonate from islets requires glucose to be me-
tabolized (
88, 109, 11 O)
but can occur without calcium influx
(86, 88),
suggesting
that the PLA2 responsible for AA release in islet/3 cells may be activated by glu-
cose metabolism and is Ca2+-independent. Treatment of isolated islets or insuli-
noma cells with BE L suppresses both hydrolysis of arachidonate from membrane

phospholipids and glucose-stimulated insulin secretion
(111-116),
suggesting
GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 23
that iPLA2 may be responsible for glucose-stimulated AA release. These obser-
vations have led to the hypothesis that iPLA2 in fi cells may participate in glucose-
stimulated insulin secretion by releasing AA upon glucose stimulation
(88).
Characterization of islet iPLA2 activity led to the cloning of iPLA2fi from rat
and human islets
(9, 12).
BEL also inhibits the Mg2+-dependent PAPH-1 activ-
ity (108).
To circumvent this problem, Balsinde
et al. (117)
have used antisense
oligonucleotides to suppress iPLA2~ expression in P388D1 cells; however, sim-
ilar approaches have not been successful in insulinoma cells, perhaps because
of the high level of iPLA2fl expression in these cells
(118).
Nevertheless, the
PAPH-1 inhibitor propranolol failed to suppress glucose-induced arachidonate
release, as measured by isotope dilution gas chromatography/mass spectrometry
(GC/MS), from isolated islets under conditions where such release was effec-
tively suppressed by BEL
(118).
This indicates that PAPH-1 is not the BEL-
sensitive target involved in glucose-stimulated arachidonate release and insulin
secretion, but that iPLA2fl might be. Substantial evidence indicates that PLA2
is involved in secretagogue-induced insulin secretion and AA release, and the

studies with BEL suggest that iPLA2fl participates in these processes. In order
to examine the role of iPLA2fi further, we have achieved stable overexpression
of iPLAefl in INS-1 cells, an insulinoma cell line
(119).
We found that iPLA2fl-
overexpressing INS-1 cells exhibit increased secretory responses to insulin see-
retagogues compared to parent IN S-1 cells or INS-1 cells transfected with vector
only
(119a).
This enhanced insulin secretory response is inhibited by BEL but
not by propranolol. These data support the hypothesis that iPLA2/~ participates
in glucose-stimulated insulin secretion. Islet PLA2 activation also has been re-
ported to be involved in insulin secretion induced by cholecystokinin-8 (CCK-8)
(95, 98).
CCK-8 was found to induce accumulation of lysophosphatidylcholine
(LPC) and AA in a Ca2+-independent manner. Inhibition of islet iPLA2 activity
with BEL reduced CCK-8-induced AA release and insulin secretion, suggest-
ing that iPLA2 contributes to the insulinotropie action of eholecystokinin-8 in
rat islets
(99).
It is also possible that PLA2 enzymes in addition to the group VIA PLA2s are
also involved in insulin secretion. Our hypothesis is that secretagogue-induced
activation of iPLA2 in islet fl ceils results in accumulation of nonesterified AA
and that this amplifies the glucose-induced rise in cytosolic [Ca2+]. This could
result in activation of cPLA2, which is also expressed by islet fl cells, and cPLA2
could further amplify the release of AA. Islets also express group IB sPLA2 in
their secretory granules, and this enzyme might participate in the process of
fusion of insulin secretory granules and plasma membranes in the final steps
in exocytosis
(120).

It is not known whether islets express the recently cloned
membrane-associated iPLA2F (group VIB PLA2) (6), but this enzyme is also
BEL-sensitive and is another candidate for the BEL-sensitive target(s) that
participate in glucose-induced insulin secretion.