From the Laboratory of Cell Signaling, NHLBI,
National Institutes of Health, Bethesda, Maryland 20892, the
§ Department of Medicine, Beth Israel Hospital, Harvard
Medical School, Boston, Massachusetts 02115, and the ¶ Division of
Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University
of Kentucky, Lexington, Kentucky 40506
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ABSTRACT |
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Signal transduction across cell membranes often
involves the activation of both phosphatidylinositol (PI)-specific
phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase).
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a
substrate for both enzymes, is converted to phosphatidylinositol
3,4,5-trisphosphate (PI(3,4,5)P3) by the action of PI
3-kinase. Here, we show that PI(3,4,5)P3 activates purified
PLC- isozymes by interacting with their Src homology 2 domains.
Furthermore, the expression of an activated catalytic subunit of PI
3-kinase in COS-7 cells resulted in an increase in inositol phosphate
formation, whereas platelet-derived growth factor-induced PLC
activation in NIH 3T3 cells was markedly inhibited by the specific PI
3-kinase inhibitor LY294002. These results suggest that receptors
coupled to PI 3-kinase may activate PLC-
isozymes indirectly, in the
absence of PLC-
tyrosine phosphorylation, through the generation of
PI(3,4,5)P3.
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INTRODUCTION |
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Activation of both PLC1 and PI 3-kinase often occurs in response to stimulation of cells by a variety of agonists. PLC catalyzes the hydrolysis of PI(4,5)P2 to generate the second messengers inositol 1,4,5-trisphosphate (I(1,4,5)P3) and diacylglycerol (1-3). PI 3-kinase phosphorylates the D-3 position of PI(4,5)P2 to produce PI(3,4,5)P3, which is then sequentially dephosphorylated to PI(3,4)P2 and phosphatidylinositol 3-phosphate (4-7). The activation of each of these two enzymes has been implicated in such diverse cellular processes as mitogenesis, chemotaxis, secretion, and cytoskeletal assembly (4-7).
The phosphoinositides PI(3,4)P2 and PI(3,4,5)P3 are not substrates of any known PLC (8) and are normally absent from resting cells; however, they appear within seconds to minutes of stimulation of cells with various growth factors or other cellular activators. In contrast, the concentration of phosphatidylinositol 3-phosphate does not change substantially in response to cell stimulation (4-7). It has thus been suggested that PI(3,4)P2 and PI(3,4,5)P3 might function as intracellular messengers (4-7). With regard to potential targets of these D-3-phosphorylated lipids, they have been shown to activate Ca2+-independent isoforms of protein kinase C (9, 10) as well as to bind the pleckstrin homology (PH) domain of the protein serine-threonine kinase Akt, thereby activating its kinase activity (11-13), and to the SH2 domains of the 85-kDa (p85) subunit of PI 3-kinase, thereby preventing its binding to tyrosine-phosphorylated proteins (14).
The 10 mammalian PLC isozymes identified to date are single
polypeptides and can be divided into three types: PLC-, PLC-
, and
PLC-
(1). All contain a PH domain in their NH2-terminal region. The
type isozymes differ from the other two types in that
they contain two SH2 domains, one SH3 domain, and an additional PH
domain that is split by the SH domains; these domains are arranged in
the order PH(N)-SH2-SH2-SH3-PH(C), where N and C in parentheses denote
NH2- and COOH-terminal locations, respectively. Upon
stimulation of cells with growth factors like platelet-derived growth
factor (PDGF) and epidermal growth factor, the SH2 domain of PLC-
binds to the autophosphorylated tyrosine residues of growth factor
receptors, leading to tyrosine phosphorylation and activation of
PLC-
(1). PLC isozymes can also be activated at least in
vitro by the presence of phosphatidic acid (15) or arachidonic
acid (16). Therefore, activation of PLC-
isozymes may occur
secondarily to receptor-mediated activation of phospholipase D and
cytosolic phospholipase A2, which results in the production
of phosphatidic acid and arachidonic acid, respectively. We now report
that another lipid-derived messenger, PI(3,4,5)P3,
activates PLC-
isozymes specifically by interacting with their SH2
domains.
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EXPERIMENTAL PROCEDURES |
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Materials-- PLC isozymes were purified from HeLa cells that had been transfected with recombinant vaccinia virus containing the entire coding sequence of the respective enzyme (17). Dipalmitoyl-PI(3,4)P2 and dipalmitoyl-PI(3,4,5)P3 were synthesized as described (18). An expression vector (pCMVp110-CAAX) that encodes a fusion protein consisting of Myc epitope and p110-CAAX was kindly provided by J. Downward (Imperial Cancer Fund, London, United Kingdom).
PLC Assay--
The activities of PLC-1, PLC-
1, PLC-
2,
and PLC-
1 were measured with a mixed micellar substrate containing
[3H]PI(4,5)P2, phosphatidylethanolamine, and
phosphatidylserine in a molar ratio of 1:3:3 together with various
amounts of synthetic dipalmitoyl-PI(3,4,5)P3 or
dipalmitoyl-PI(3,4)P2 in 0.1% deoxycholate. The final
assay mixture (100 µl) contained 10 µM
[3H]PI(4,5)P2 (26,000 cpm), 50 mM
Hepes-NaOH (pH 7.0),10 mM NaCl, 120 mM KCl, 2 mM EGTA, 0.05% deoxycholate, bovine serum albumin (5 µg/ml), 1 µM free Ca2+, and the indicated
concentrations of PI(3,4,5)P3 or PI(3,4)P2. After incubation for 10 min at 30 °C, the reaction was terminated by
addition of 200 µl of 10% (w/v) trichloroacetic acid and 100 µl of
10% (w/v) bovine serum albumin, followed by centrifugation. The amount
of radioactivity in the resulting supernatant, corresponding to
[3H]I(1,4,5)P3, was measured by liquid
scintillation spectroscopy. The amount of PLC isozymes (4-7 ng) was
adjusted to give similar basal activity.
Preparation of GST Fusion Proteins--
For each fusion protein
(denoted PH(N)-SH2-SH2-SH3-PH(C), SH2-SH2-SH3, SH2-SH2, N-SH2, C-SH2,
PH(NC), and SH3), the corresponding polymerase chain reaction product,
flanked by BamHI and EcoRI linkers, was inserted
into the BamHI and EcoRI sites of the glutathione S-transferase (GST) expression vector pGEX-2TK (Pharmacia
Biotech Inc.). Amino acid sequences of PH(N)-SH2-SH2-SH3-PH(C),
SH2-SH2-SH3, SH2-SH2, N-SH2, C-SH2, and SH3 correspond to residues
483-936, 533-851, 550-745, 550-657, 668-745, and 758-851 of
PLC-1, respectively. Polymerase chain reaction products
corresponding to residues 482-523 (PH(N)) and 865-936 (PH(C)) of
PLC-
1 were fused with a glycine codon insertion to yield the
combined PH(NC) construct. The 5' and 3' primers for PH(N) contained
BamHI and SmaI sites, respectively; thus, the
PH(NC) construct contained sequential BamHI,
SmaI, and EcoRI sites. Escherichia
coli cells were transformed with the various expression vectors
and cultured at 30 °C. Expression of the GST fusion proteins was
induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside, and the cells
were subsequently collected by centrifugation at 2000 × g
for 15 min, sonicated in phosphate-buffered saline, and centrifuged at
5000 × g for 15 min. The resulting supernatant was
mixed and incubated at room temperature for 30 min with 2 ml of a 50%
(v/v) slurry of glutathione-Sepharose 4B (Pharmacia) that had been
equilibrated with phosphate-buffered saline. After centrifugation of
the mixture at 5000 × g for 15 min, the supernatant was
removed and the pellet washed with 10 bed volumes of phosphate-buffered saline. Bound proteins were cleaved from GST by incubation of the beads
with thrombin (10 µg/ml) at room temperature for 6 h. The eluted
proteins were further purified by high performance liquid
chromatography on a Mono Q column and quantitated
spectrophotometrically with extinction coefficients at 280 nm
calculated on the basis of their amino acid composition.
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RESULTS AND DISCUSSION |
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We investigated the effects of PI(3,4)P2 and
PI(3,4,5)P3 on the activities of PLC isozymes by measuring
the hydrolysis of PI(4,5)P2 with mixed micellar substrates
containing phosphatidylethanolamine, phosphatidylserine,
[3H]PI(4,5)P2, and various amounts of
synthetic D-3-phosphorylated lipids. PI(3,4,5)P3 increased
the activities of PLC-1 and PLC-
2 but had no effect on PLC-
1
or PLC-
1 (Fig. 1A). The
dependence of the activities of PLC-
1 and PLC-
2 on
PI(3,4,5)P3 concentration was sigmoidal, with maximal
activation (approximately 8-fold) apparent at 100 µM
lipid. In contrast, PI(3,4)P2 had no effect on the
activities of any of the PLC isozymes examined (Fig.
1B).
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We prepared various recombinant proteins containing different domains
of PLC-1 (Fig. 2, A and
B) and measured their effects on the activity of full-length
PLC-
1 in the presence of 100 µM PI(3,4,5)P3. All SH2 domain-containing proteins inhibited
the PI(3,4,5)P3-stimulated activity of PLC-
1 in a
concentration-dependent manner, whereas proteins corresponding to the
SH3 and PH(NC) (fusion of PH(N) and PH(C)) domains had no effect (Fig.
2C). The inhibition by SH2 proteins was apparent only in the
presence of PI(3,4,5)P3, the basal activity of PLC-
1
being unaffected (Fig. 2D). These results suggest that
PI(3,4,5)P3 activates PLC-
isozymes by binding to their
SH2 domains. Furthermore, the three proteins PH(N)-SH2-SH2-SH3-PH(C), SH2-SH2-SH3, and SH2-SH2, all of which contain two SH2 domains, inhibited PI(3,4,5)P3-dependent PLC-
1
activity to a greater extent than did the NH2-terminal SH2
(N-SH2) or COOH-terminal SH2 (C-SH2) domains alone. This result,
together with the sigmoidal response of PLC-
isozyme activities to
PI(3,4,5)P3, indicates that the two SH2 domains bind
PI(3,4,5)P3 with positive cooperativity or that the two
PI(3,4,5)P3-bound domains mediate enzyme activation synergistically. The ability of PI(3,4,5)P3 but not
PI(3,4)P2 to activate PLC-
1 via its SH2 domains is
consistent with the previous observation that p85, Src, and Abl SH2
domains show higher affinity for PI(3,4,5)P3 than for
PI(3,4)P2 or PI(4,5)P2 (14). PLC-
isozymes
contain an additional PH domain near their NH2 terminus.
Recent results by Falasca et al. (19) suggest that PI(3,4,5)P3 may also bind to the PH domain (19).
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The concentration of PI(3,4,5)P3 required for activation of
PLC- isozymes in vitro is relatively high. However, it
has been suggested that the intracellular concentration of
PI(3,4,5)P3 can achieve values of up to 200 µM in neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP) (20). Specific
generation of PI(3,4,5)P3 at sites of PLC-
localization
might be one means of ensuring effective activation of PLC-
isozymes. The sigmoidal response to PI(3,4,5)P3 also
suggests that PLC-
activation would be minimal until the lipid
concentration exceeds a certain threshold.
To determine whether activation of PLC by PI(3,4,5)P3 could
be detected in intact cells, we transiently expressed in COS-7 cells
the 110-kDa subunit (p110) of PI 3-kinase with c-Myc epitope and
farnesylation signal (CAAX) sequences located at the
NH2 and COOH termini, respectively (21), and measured the
release of inositol phosphates resulting from the hydrolysis of
PI(4,5)P2. Expression of the Myc-tagged
p110-CAAX protein was detected by immunoblot analysis with
antibodies specific to the Myc sequence (Fig.
3A). The farnesylation signal
sequence causes the constitutive activation of p110 by targeting it to
the cell membrane. As expected from the fact that most mammalian cells
contain a relatively high concentration (>20 µM) of
inositol phosphates even before stimulation (22), a substantial amount
of 3H-labeled inositol phosphates was detected in COS-7
cells labeled with [3H]inositol to equilibrium (Fig. 3).
Expression of Myc-p110-CAAX induced a 45% increase in the
amount of inositol phosphates, and this effect was blocked by
pretreatment of cells with LY294002, a specific inhibitor of PI
3-kinase (23). LY294002 had no effect on the amount of inositol
phosphates in cells not expressing Myc-p110-CAAX. These
results suggest that PI(3,4,5)P3 generated by the activated p110 subunit was able to activate PLC. The increase in PLC activity was
smaller than that in cells stimulated with PLC-activating agonists,
probably because in the local area where PLC- was activated by
PI(3,4,5)P3, PI(4,5)P2 (a common substrate for
PLC and PI 3-kinase) had been depleted by Myc-p110-CAAX. It
has been shown previously that availability of PI(4,5)P2 is
a limiting factor for PLC activity (24).
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Further evidence that PI(3,4,5)P3 activates
PI(4,5)P2 hydrolysis in intact cells is provided by
previous studies with wortmannin, a potent (median inhibitory
concentration (IC50), 3 nM), irreversible inhibitor of PI 3-kinase (23). Wortmannin inhibits
I(1,4,5)P3 formation and Ca2+ mobilization in
bovine adrenal glomerulosa cells stimulated by angiotensin II (25), rat
basophilic leukemia (RBL-2H3) cells stimulated by cross-linking of high
affinity immunoglobulin E (IgE) receptors (26), and human neutrophils
stimulated by fMLP (27). At the time of the studies with
neutrophils and adrenal glomerulosa cells, wortmannin was known to
inhibit myosin light chain kinase (MLCK), but its effect on PI 3-kinase
was not known. The inhibition of I(1,4,5)P3 and
Ca2+ responses by 20 nM wortmannin in adrenal
glomerulosa cells was thus speculated to result from inhibition of
MLCK. However, it is now known that the concentration of wortmannin
required for inhibition of MLCK is 100 times that required for
inhibition of PI 3-kinase (23), so that MLCK was likely not inhibited
by 20 nM wortmannin. Our data indicate that they are likely
attributable to prevention of PI(3,4,5)P3-induced
activation of PLC-. PLC-
1 is a widely expressed and abundant
enzyme, whereas PLC-
2 is abundant in cells of hematopoietic
origin.
Recently, type III PI 4-kinase was shown to be inhibited by wortmannin
(IC50, 50 nM) (23). Because PI 4-kinase is
required for PI(4,5)P2 synthesis, it is possible that the
reduced activity of PLC observed in wortmannin-treated cells was
attributable to the diminished supply of substrate. Thus, we studied
the effect of the more specific inhibitor LY294002 (IC50: 2 and 100 µM for PI 3-kinase and type III PI 4-kinase,
respectively; Ref. 23) on PI(4,5)P2 hydrolysis induced by
PDGF in NIH 3T3 cells. Whereas PDGF induced an 8-fold increase in PLC
activity in control cells, pretreatment of cells with 10 µM LY294002 reduced this response by 40% (Fig.
4A). The presence of the
inhibitor affected neither the basal amount of inositol phosphates
(Fig. 4A) nor the tyrosine phosphorylation of PLC-1 and
PDGF receptor (Fig. 4B). In a similar experiment with COS-7
cells, epidermal growth factor induced a 3-fold increase in PLC
activity and pretreatment of cells with 10 µM LY294002
reduced the response by 34% (data not shown).
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Characterized mechanisms for the activation of PLC isozymes include the
phosphorylation of PLC- isoforms by protein-tyrosine kinases (PTKs)
and the interaction of PLC-
isozymes with G proteins (1-3).
Tyrosine phosphorylation of PLC-
requires SH2 domain-mediated association with a tyrosine-phosphorylated PTK. Because
PI(3,4,5)P3 competes with tyrosine-phosphorylated proteins
for binding to SH2 domains, an increase in PI(3,4,5)P3
concentration might attenuate PTK-dependent activation of
PLC-
. PI 3-kinase exists in two types: one that is activated by PTKs
and one activated by G proteins (4-7). The PTK-activated PI 3-kinase
is a heterodimer of an 85-kDa regulatory subunit (p85
,
) and a
110-kDa catalytic subunit (p110
,
), whereas the G
protein-activated PI 3-kinase consists of a single polypeptide of 110 kDa (p110
) (28-30).
More than 60 different receptors are known to stimulate PLC (1-3).
Although only 30 receptors are currently known to stimulate PI 3-kinase
(4-7), most of these also activate PLC. Therefore, the amount of
I(1,4,5)P3 generated in a variety of cells in response to
an agonist may reflect the sum of that produced as a result of direct
activation of PLC and that produced by indirect activation of PLC-
through the PI 3-kinase pathway. Given that LY294002, at a
concentration sufficient for inhibition of PI 3-kinase but not for
inhibition of PI 4-kinase, inhibits 40% of PDGF-dependent PLC activity, the ratio of direct to indirect activation appears to be
3 to 2 in NIH 3T3 cells stimulated with PDGF. The indirect activation
of PLC-
is likely not limited to PTK receptors such as the PDGF
receptor and PTK-coupled receptors such as the IgE receptor. The fact
that wortmannin inhibits PLC activation induced by G protein-coupled
receptors such as those for angiotensin II and fMLP suggests
that PLC-
might be activated indirectly in response to the occupancy
of such receptors. However, the activation of PI 3-kinase appears not
always to result in activation of PLC-
, because there is no evidence
that insulin or colony-stimulating factor-1, both of which activate PI
3-kinase, elicits the production of I(1,4,5)P3 (31, 32).
One possible explanation is that PLC-
isozymes may not be located at
the sites where PI(3,4,5)P3 is generated in the cells
activated with colony-stimulating factor or insulin.
Finally, our results may explain how engagement of the FcRIIB
inhibitory receptor in mast cells reduces the IgE-induced increase in
intracellular Ca2+ concentration (33). Fc
RIIB binds
SHIP, a phosphatase that dephosphorylates the D-5 position of
PI(3,4,5)P3 or I(1, 3,4,5)P4. A decrease in the
concentration of PI(3,4,5)P3 could reduce
I(1,4,5)P3 production by PLC-
isozymes and thereby
reduce long term release of intracellular Ca2+ and
ultimately Ca2+ influx.
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ACKNOWLEDGEMENTS |
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We thank J. Downward and A. Toker for the Myc-tagged p110-CAAX expression vector and Lewis Cantley for valuable discussions and comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK48871 (to L. G. C.) and GM53448 (to C.-S. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry, Chungbuk National
University Medical School, Chongju 361-763, Korea.
** To whom correspondence should be addressed: National Institutes of Health, Bldg. 3, Rm. 122, 3 Center Dr., MSC 0320, Bethesda, MD 20892-0320. Tel.: 301-496-9646; Fax: 301-480-0357.
1 The abbreviations used are: PLC, phosphatidylinositol-specific phospholipase C; PI 3-kinase, phosphoinositide 3-kinase; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; I(1,4,5)P3, inositol 1,4,5-trisphosphate; SH2, Src homology 2; PH, pleckstrin homology; p85, the 85-kDa regulatory subunit of PI 3-kinase; p110, the 110-kDa catalytic subunit of PI 3-kinase; GST, glutathione S-transferase; fMLP, formylmethionyl-leucyl-phenylalanine; PDGF, platelet-derived growth factor; MLCK, myosin light chain kinase; PTK, protein-tyrosine kinase; DMEM, Dulbecco's modified Eagle's medium.
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REFERENCES |
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