From the Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
The hydrolysis of a minor membrane phospholipid,
phosphatidylinositol 4,5-bisphosphate
(PIP2),1 by a specific
phospholipase C (PLC) is one of the earliest key events in the
regulation of various cell functions by more than 100 extracellular
signaling molecules (1-4). This reaction produces two intracellular
messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which
mediate the activation of protein kinase C (PKC) and intracellular Ca2+ release, respectively. Furthermore, a decrease in the
amount of PIP2 itself in the cell membrane is likely an
important signal because the activities of several proteins are
modulated by this phospholipid (5). PIP2 is a cofactor for
phosphatidylcholine-specific phospholipase D (PLD) and a substrate for
phosphoinositide 3-kinase (PI 3-kinase), both of which are also
receptor-activated effector enzymes. In addition, PIP2
modulates actin polymerization by interacting with various
actin-binding proteins and serves as a membrane-attachment site for
many signaling proteins that contain pleckstrin homology (PH) domains.
Consequently, the activity of PLC is stringently regulated in cells
through several distinct mechanisms that link multiple PLC isoforms to
various receptors.
PLC Isoforms and Structural Organization The 10 mammalian PLC isozymes (excluding alternatively spliced
forms) identified to date are all single polypeptides and can be
divided into three types, Two regions of high sequence homology (40-60% identity), designated X
and Y, constitute the PLC catalytic domain (1-5) (Fig. 1). A PH domain is located in the
NH2-terminal region, preceding the X domain, in all three
types of PLC. Whereas PLC-
The three-dimensional structure of a PLC- The multidomain structure observed with PLC- Activation of PLC- The
The GTP The receptor-mediated activation of PLC- The G The region of PLC- Mammalian cDNAs that encode five distinct G Activation of PLC- Polypeptide growth factors, such as platelet-derived growth factor
(PDGF), epidermal growth factor, fibroblast growth factor, nerve growth
factor, and hepatocyte growth factor, induce PIP2 turnover
by activating PLC-
Phosphorylation of PLC- Nonreceptor PTKs also phosphorylate and activate PLC- Tyrosine phosphorylation of PLC- PTK-independent Activation of PLC- PLC- Ligation of a variety of receptors results in the activation of PI
3-kinase, which phosphorylates the D3 position of PIP2 to
produce phosphatidylinositol 3,4,5-trisphosphate (PIP3).
PIP3 activates purified PLC-
Although four distinct PLC- All PLC isozymes are activated by Ca2+ in vitro,
but PLC- PLC signaling also appears to occur in the nucleus (40). PLC- Inhibition of PLC via Protein Kinases A and C The activation of PKC or cAMP-dependent protein kinase
(PKA) attenuates the PLC signaling pathway in a variety of cells. The proposed targets for phosphorylation by these kinases include cell
surface receptors, G proteins, and PLC itself. PLC- The interaction of PLC and PKA was studied in COS cells transfected
with cDNAs encoding PLC- Genetic Mapping and Disruption of PLC Genes and the Relation of
PLC to Human Disease The
,
, and
, of which four PLC-
, two
PLC-
, and four PLC-
proteins are known (1-5). The
-type isozymes are smaller (Mr 85,000) than the
PLC-
and PLC-
(Mr 140,000-155,000)
isoforms. Lower eukaryotes such as yeast and slime molds contain only
-type isozymes, suggesting that
- and
-type isoforms in higher
eukaryotes evolved from the archetypal PLC-
.
and PLC-
isozymes contain a short
sequence of 50-70 amino acids that separates the X and Y regions,
PLC-
isozymes have a long sequence of ~400 amino acids that
contains Src homology (SH) (two SH2 and one SH3) domains. PLC-
isozymes contain an additional PH domain that is split by the SH
domains. PH (~100 residues), SH2 (~100 residues), and SH3 (~50
residues) domains are protein modules that are shared by many signaling
proteins; whereas PH domains mediate interaction with the membrane
surface by binding to PIP2, SH domains mediate interactions
with other proteins by binding to phosphorylated tyrosine residues
(SH2) or proline-rich sequences (SH3).
Fig. 1.
Linear representation of the various domains
identified in the three types of PLC isozymes. Catalytic domains X
and Y as well as PH, EF-hands, C2, and SH (SH2 and SH3) domains are indicated.
[View Larger Version of this Image (20K GIF file)]
1 molecule lacking the PH
domain has recently been determined (6). As expected, the X and Y
regions are tightly associated. The structure also revealed two
accessory modules, an EF-hand domain and a C2 domain, the latter of
which was previously suggested to mediate the
Ca2+-dependent binding to lipid vesicles. On
the basis of the structural information, a catalytic mechanism
comprising two steps, tether and fix, was proposed. The PH domain of
PLC-
1 would tether the enzyme to the membrane by specific binding to
PIP2, and the C2 domain would fix the catalytic domain in a
productive orientation on the membrane. The EF-hand domain would serve
as a flexible link between the PH domain and the rest of the enzyme.
Calcium is required for the function of the C2 domain. Another
Ca2+ ion located at the active site, together with
His311 and His356, directly participates in
catalysis, consistent with the fact that all eukaryotic PLC isozymes
require Ca2+ for activity, that the two histidines
equivalent to His311 and His356 are completely
conserved among all PLC isoforms, and that mutation of either of the
two histidine residues results in enzyme inactivation (7).
1 is likely to be common
to all mammalian PLC isoforms (Fig. 1). However, PLC-
and PLC-
isozymes contain additional regulatory COOH-terminal and SH domains,
respectively. These regulatory domains are responsible for the fact
that different PLC isozymes are linked to receptors through distinct
mechanisms. Furthermore, the COOH-terminal domain of PLC-
isozymes
might contribute to the tethering of the enzyme to the membrane
surface, given that truncation of this domain completely blocked
membrane association of PLC-
1 (8). The SH domains of PLC-
appear
to play a critical role in mitogenic signaling independently of PLC
activity; catalytically inactive mutants of PLC-
(containing
mutations at the essential His residues) elicited a mitogenic response
when microinjected into NIH 3T3 cells, and mitogenic activity was
localized to the SH region (7, 9).
by G Proteins
subunits (
q,
11,
14, and
16) of all four members of the
Gq subfamily of heterotrimeric G proteins activate PLC-
isozymes but not PLC-
1 or PLC-
1 (1-4, 10) (Fig.
2). The receptors that activate this
Gq
-PLC-
pathway include those for thromboxane A2, bradykinin, bombesin, angiotensin II, histamine,
vasopressin, acetylcholine (muscarinic m1 and m3),
1-adrenergic agonists, thyroid-stimulating hormone, C-C
and C-X-C chemokines, and endothelin-1 (4, 11).
Fig. 2.
Receptor-induced activation of PLC-
isozymes by Gq
and G
subunits.
[View Larger Version of this Image (26K GIF file)]
S-activated G
q or G
11 subunits
stimulate PLC-
isoforms with the rank order of potency PLC-
1
PLC-
3 > PLC-
2 (4, 5). PLC-
4 is also activated by
Gq
subunits; however, because the basal activity of this
enzyme is inhibited by ribonucleotides, including GTP
S, accurate
estimation of the extent of activation is difficult (12). All four
Gq
members are palmitoylated at residues
Cys9 and Cys10 (13). Removal of the two
palmitate groups affects neither the capacity of the proteins to
activate PLC-
1 nor their association with the cell membrane.
G
16, which is detected only in hematopoietic cells and
is distantly related to the more widely expressed G
q (amino acid sequence identity of 55%), activates PLC-
1, -
2, and
-
3 in a manner essentially indistinguishable from that of G
q (14). However, the
subunits can be discriminated
by certain receptors (11).
has been studied in detail
by reconstituting the m1 muscarinic acetylcholine receptor, G protein,
and PLC-
in lipid vesicles (15, 16). The muscarinic agonist
carbachol stimulated PLC activity 90-fold, and each member of the
Gq
family mediated this activation. The intrinsic GTPase activity of purified G
q was low but was stimulated
>50-fold by the presence of PLC-
1, that is PLC-
1 is a
GTPase-activating protein for G
q (16). In the
reconstituted system, PLC-
1 also increased the rate of GTP
hydrolysis by G
q up to 60-fold in the presence of
carbachol, which alone stimulated activity 6-10-fold (16). These
results indicate that the receptor and PLC-
1 coordinately regulate
the amplitude of the PLC signal and the rate of signal termination.
dimer also activates PLC-
isozymes (1-4, 15). The
sensitivity of PLC-
isozymes to G
subunits differs from that to Gq
and decreases in the order PLC-
3 > PLC-
2 > PLC-
1 (4, 5). The ability of G
subunits to
activate PLC-
2 in response to ligation of the luteinizing hormone
receptor, V2 vasopressin receptor,
1- and
2-adrenergic receptors, m2 muscarinic acetylcholine receptor, and the receptors for the chemoattractants interleukin 8 (IL-8), formyl-Met-Leu-Phe, and complementation factor 5a was demonstrated using a cotransfection assay system in COS cells (4, 17,
18). These receptors also stimulate PLC-
through Gq
subunits. Although the concentrations of G
required for maximal
activation of PLC-
isoforms in vitro are much larger than
those of Gq
subunits, the final extents of activation
are similar. Thus, both Gq
and G
likely are
transducers in PLC signaling. However, it was recently suggested that
G
is the predominant transducer in the activation of
Xenopus oocyte PLC and that the role of Gq
subunits is to specify the receptor coupled to the enzyme (19).
that interacts with Gq
differs
from that responsible for interaction with G
; whereas the
COOH-terminal region downstream of the Y domain is essential for the
activation of PLC-
1 and PLC-
2 by Gq
(4, 5), the
site of interaction of PLC-
2 with G
was localized to the
region spanning Glu435 to Val641 (20). Thus,
Gq
and G
subunits may independently modulate a
single PLC-
molecule concurrently. Several positively charged residues important for interaction with Gq
have been
identified in the COOH-terminal region of PLC-
1 (8). The
COOH-terminal 14 residues of the G
subunit were also shown to be
important for PLC-
activation (21).
subunits and 11 G
subunits have been isolated. Although certain subunits are expressed
only in specific tissues and there is some selectivity in the
interaction of
and
, G
subunits are available in many different combinations. Among several permitted combinations tested, all except
1
1 activated purified PLC-
3 with similar potencies (4, 5). In cells, however, not all the available Gq
members and G
combinations appear to be utilized to activate PLC.
Experiments with antisense oligonucleotides directed against the
mRNAs encoding various G protein subunits suggested that m1
muscarinic acetylcholine receptor interacts only with the G protein
complexes composed of the subunits
q,
11,
1,
4, and
4 to activate
PLC in RBL-2H3 cells, despite the fact that the subunits
14,
2,
3,
2,
3,
5, and
7 are also expressed in the cell (22).
by Protein Tyrosine Kinases
in a wide variety of cells. Binding of these
growth factors to their receptors results in activation of the
intrinsic protein tyrosine kinase (PTK) activity of the receptor and
the consequent tyrosine phosphorylation of numerous proteins, including
the receptor itself and PLC-
(1-4) (Fig. 3).
Receptor autophosphorylation creates high affinity binding sites for
several SH2 domain-containing proteins, including PLC-
1. A specific
autophosphorylated site (for example, Tyr1021 of the
-type PDGF receptor) is recognized by one of the SH2 domains of
PLC-
. Mutation of the PLC-
-binding Tyr residue to Phe in the
receptors for PDGF, epidermal growth factor, and nerve growth factor
prevents association of the receptor with PLC-
and abolishes the
growth factor-dependent production of inositol 1,4,5-trisphosphate (4).
Fig. 3.
Phosphorylation and activation of PLC-
isozymes by a receptor PTK (left), a nonreceptor PTK
coupled to a multichain receptor (middle), and a
nonreceptor PTK coupled to a heptahelical receptor (right).
PY and YP, phosphotyrosine.
[View Larger Version of this Image (33K GIF file)]
1 by all growth factor receptors occurs at
identical sites: tyrosines 771, 783, and 1254. Phe substitution at
Tyr783 completely blocks the activation of PLC by PDGF in
NIH 3T3 cells (1-5). Tyrosine phosphorylation of PLC-
1 appears to
promote its association with unidentified components of the
cytoskeleton; the SH3 domain of PLC-
1 is responsible for targeting
the enzyme to the actin microfilament network. Whether this
cytoskeletal association serves to bring the enzyme into contact with
its substrate or whether it promotes interaction with another protein
component essential for its activation is unknown. Autophosphorylation
of growth factor receptors and subsequent tyrosine phosphorylation of
substrate proteins, including PLC-
1, require the presence of
H2O2, whose concentration increases transiently
and which appears to function as an intracellular messenger in growth
factor-stimulated cells (23). This requirement probably reflects that
the activation of a receptor PTK by the binding of a growth factor is
insufficient to increase the steady-state level of protein tyrosine
phosphorylation. Concurrent inhibition of protein tyrosine phosphatases
by H2O2 is also necessary.
isozymes in
response to the ligation of certain cell surface receptors. Such
receptors include the T cell antigen receptor, membrane immunoglobulin (Ig) M, the high affinity IgE receptor, the IgG receptors, the IgA
receptor, CD20, CD38, the
2-macroglobulin receptor,
integrins, and several receptors for cytokines such as ciliary
neurotrophic factor, leukemia inhibitory factor, oncostatin M, IL-1,
IL-4, IL-6, and IL-7 (4, 24-26). These receptors, most of which
comprise multiple polypeptide chains, do not themselves possess PTK
activity, but they activate a wide variety of nonreceptor PTKs such as
the members of Src, Syk, and Jak/Tyk families. The activated PTKs often
phosphorylate one of the receptor components to which PLC-
then
binds via its SH2 domains and becomes phosphorylated by the PTK.
PLC-
1 associates directly with Src and Syk in cells, and in
vitro it is phosphorylated by various soluble PTKs including Src,
Fyn, Lck, Lyn, and Hck (4, 5). Tyrosine phosphorylation of PLC-
1 has
also been shown to be elevated in cells that express SV40 middle T
antigen, that are strained mechanically, or that are exposed to
electroconvulsive shock (27-29).
has also been observed in response
to the ligation of several heptahelical, G protein-coupled receptors,
including m5 muscarinic acetylcholine receptor in Chinese hamster ovary
cells, the angiotensin II and thrombin receptors in vascular smooth
muscle cells, and platelet-activating factor (4, 30, 31). Src appears
to be responsible for the phosphorylation of PLC-
1 in vascular
smooth muscle cells and platelets; electroporation of antibodies to Src
inhibited the tyrosine phosphorylation of PLC-
1 elicited by
angiotensin II or platelet-activating factor. Although activation of
Src family PTKs in response to stimulation of a variety of G
protein-coupled receptors has been demonstrated (32), the mechanism by
which the enzymes are coupled to the receptors is not clear. One
possible mechanism is through a member of the recently identified
proline-rich PTK (Pyk) family; stimulation of receptors coupled to the
G proteins Gi or Gq in neuronal cells resulted
in tyrosine phosphorylation of Pyk-2, binding of the SH2 domain of Src
to the phosphorylated Pyk-2, and activation of Src (33).
isozymes can be activated directly by several
lipid-derived second messengers in the absence of tyrosine
phosphorylation. Phosphatidic acid produced by the action of PLD
activates purified PLC-
1 by acting as an allosteric modifier (34).
PLC-
isozymes are also stimulated by arachidonic acid (AA) in the
presence of the microtubule-associated protein tau (in neuronal cells)
or tau-like proteins (in non-neuronal cells) (35). The effect of tau
and AA was specific to PLC-
isozymes and was markedly inhibited by
phosphatidylcholine (PC). These observations suggest that the activation of PLC-
1 by tau or tau-like proteins might be facilitated by a concomitant decrease in PC concentration and an increase in AA
concentration, both of which occur in cells upon activation of an
85-kDa cytosolic phospholipase A2 (cPLA2). This
enzyme is coupled to various receptors and preferentially hydrolyzes PC containing AA. Therefore, activation of PLC-
isozymes may occur secondarily to receptor-mediated activation of cPLA2.
Several studies are consistent with the notion that stimulation of PLC by endogenously released AA occurs in cells.
isozymes specifically by
interacting with their SH2 domains.2 In
addition, incubation of NIH 3T3 cells with PIP3 resulted in a transient increase in the intracellular Ca2+
concentration, an effect that was blocked in the presence of a PLC
inhibitor. Thus, receptors coupled to PLD, cPLA2, or PI 3-kinase may activate PLC-
isozymes indirectly, in the absence of
tyrosine phosphorylation, through the generation of lipid-derived second messengers (Fig. 4).
Fig. 4.
Receptor-induced activation of PLC-
isozymes by tau and AA generated by cPLA2
(left), PIP3 generated by PI 3-kinase
(middle), and phosphatidic acid (PA) generated
by PLD (right).
[View Larger Version of this Image (30K GIF file)]
isoforms are known, the mechanism
by which these isozymes are coupled to membrane receptors remains unclear. A new class of GTP-binding protein, termed Gh and
containing 75-80-kDa
and ~50-kDa
subunits, has been shown to
be associated with agonist-bound
1-adrenergic receptors
(
1-AR). The Gh
subunit, a multifunctional
protein that also possesses tissue transglutaminase activity (37),
activates purified PLC-
1 and forms a complex with PLC-
1 in cells
stimulated via
1-AR (38). Furthermore, overexpression of
Gh
in COS cells enhanced the activation of PLC induced
by ligation of the
1-AR (37). These results suggest that
Gh
directly couples
1-AR to PLC-
1. It
is not yet known whether other PLC-
isozymes are also activated by
Gh
, what other receptors couple to Gh
,
and how the tissue transglutaminase activity of Gh
is
related to its PLC-
1-activating function. The GTPase-activating protein for the small GTP-binding protein RhoA (RhoGAP) also activates purified PLC-
1; PLC-
1 activation was thus suggested to occur downstream of RhoA activation (39).
isozymes are more sensitive to Ca2+ compared
with the other isozymes. Furthermore, PLC-
can be tethered to
PIP2-containing membranes via its PH domian in the absence of other signals. An increase in the intracellular concentration of
Ca2+ to a level sufficient to fix the C2 domain of PLC-
might therefore trigger its activation. Thus, activation of PLC-
isozymes might occur secondarily to receptor-mediated activation of
other PLC isozymes or Ca2+ channels.
1
is the major PLC isoform that has been detected in the nucleus of
various cells. The amount of nuclear PLC-
1 protein, which appears to
be activated independently of its plasma membrane counterpart by an
unknown mechanism, increases during cell growth and decreases during
differentiation (41-44). The changes in the amount of nuclear PLC-
1
correlate with changes in the amount of PIP2 hydrolyzed in
the nucleus. Studies of cells lacking PLC-
1 as a result of gene
ablation revealed that it is essential for the onset of DNA synthesis
in response to insulin-like growth factor I (45). The COOH-terminal
region downstream of the Y domain was also shown to be necessary for
translocation of PLC-
1 to the nucleus (8).
1 is rapidly
phosphorylated in cells treated with phorbol ester and is
phosphorylated at Ser887 by PKC in vitro;
however, phosphorylation had no effect on either the basal or
Gq
-stimulated activities of PLC-
1 (1). In human Jurkat T cells, activation of PKA or PKC results in an increase in
phosphorylation of Ser1248 and a concomitant decrease in
the tyrosine phosphorylation of PLC-
1, the latter of which might be
responsible for the decreased PLC activity apparent in Jurkat cells
treated with PKA- or PKC-stimulating agonists (1).
2, G protein subunits, and PKA (46).
Expression of the catalytic subunit of PKA specifically inhibited
G
stimulation of PLC-
2 activity, without affecting G
q-induced activation. The effect of PKA was not
mimicked by PKC isozymes. Furthermore, PKA directly phosphorylated
serine residues of PLC-
2 both in vivo and in
vitro.
-type PLC, which is the only known PLC in yeast and slime
mold, has been disrupted in these lower eukaryotes by gene targeting
(47, 48). Both mutants were viable. Whereas the yeast mutant showed
increased sensitivity to various stresses, the slime mold mutant
appeared normal, including with regard to such phenotypic aspects as
growth, development, and chemotaxis. Chromosome positions for 10 mouse
PLC genes and 8 human homologs were determined (49). The genes encoding
PLC-
1, PLC-
4, and PLC-
1 have been targeted in mouse. The
homozygous mutants for PLC-
1 or PLC-
4 are born normal but
subsequently manifest postnatal dwarfism; the PLC-
4 mutants also
show a defect in motor coordination and aberrant cerebellar
development.3 The PLC-
1 mutation was
lethal at early mid-gestation (around embryonic day 9) (36). Finally,
platelets from a patient with a mild inherited bleeding disorder as
well as abnormal platelet aggregation and secretion were shown to have
one-third the amount of PLC-
2 compared with normal platelets
(50).