From the Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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We have recently shown that phospholipase C- Activation of phosphoinositide-specific phospholipase C
(PLC)1 is a key event in
cellular signal transduction. PLC catalyzes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2), generating
two second messengers, inositol 1,4,5-trisphosphate (IP3)
and 1,2-diacylglycerol. To date, a total of 10 different isozymes of
PLC have been identified in mammalian cells, which can be classified
into three major subfamilies, Several lines of evidence suggested alternative mechanisms for PLC- We have also shown that variously spliced forms of the
microtubule-associated protein tau (6) stimulate PLC- Here, we report that non-neuronal cells also contain a protein that can
activate PLC- Materials
Phosphatidylserine (PS) and phosphatidylethanolamine (PE) were
purchased from Avanti Polar Lipids. AA and cholesterol were purchased
from Calbiochem. PIP2 was obtained from Roche Molecular Biochemicals. [inositol-2-3H]PIP2
and [2-3H]myo-inositol were purchased from NEN
Life Science Products. PLC isozymes (PLC- Purification of PLC- All manipulations were performed at 4 °C unless otherwise
indicated. During purification, PLC- The purification procedure consisted of the following steps.
Preparation of Bovine Lung Cytosolic Extracts--
Fresh bovine
lungs (3.0 kg) were obtained from a local slaughterhouse and
homogenized in 10 liters of a solution containing 20 mM
Hepes-NaOH (pH 7.2), 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, leupeptin (1 µg/ml), and aprotinin (1 µg/ml) with a
Waring blender. The homogenate was centrifuged at 1000 × g for 10 min, and the resulting supernatant was centrifuged further at 13,000 × g for 1 h.
Heat Treatment--
Collected supernatant was heated at 80 °C
for 10 min and centrifuged at 13,000 × g for 30 min.
The resulting supernatant was filtered through Whatman No. 1 paper to
eliminate lipids.
Heparin-Sepharose CL-4B--
The heat-treated supernatant (5 g
of protein) was applied to a heparin-Sepharose CL-4B column (5 × 30 cm, Amersham Pharmacia Biotech) that had been equilibrated with 20 mM Hepes-NaOH (pH 7.2), 1 mM EDTA, and 0.1 mM dithiothreitol. After washing with the equilibration
buffer, bound proteins were eluted with 2 liters of a linear gradient
of 0-1.0 M NaCl in the buffer at a flow rate of 10 ml/min.
Fractions (20 ml) were collected and assayed for PLC- HPLC on DEAE-5PW Column--
The dialyzed protein (12 mg) from
the previous step was applied to a TSKgel DEAE-5PW HPLC column
(21.5 × 150 mm) that had been equilibrated with 20 mM
Tris-HCl (pH 8.5), 1 mM EDTA, and 0.1 mM
dithiothreitol. Proteins were eluted at a flow rate of 5 ml/min with
equilibration buffer for 5 min followed by a linear gradient of 0-1.0
M NaCl in equilibration buffer over 40 min (see Fig.
1B). Fractions (5 ml) were collected and assayed for
PLC- Electroelution of Proteins from SDS-Polyacrylamide
Gels--
Purified proteins (~700 µg) from the DEAE-5PW column
were separated by preparative SDS-PAGE on 8% gel (3-mm thickness,
single-well comb). The gel was stained lightly with Coomassie Brilliant
Blue, and visualized protein bands were excised from the gel with a razor blade. The proteins were then eluted with an Electro-Eluter (C.B.S. Scientific, Del Mar, CA). Coomassie Brilliant Blue and SDS were
extracted with isobutyl alcohol, and proteins were precipitated by cold acetone.
Tryptic Digestion and Amino Acid Sequencing
Electroeluted proteins (10 µg each) from three different bands
(bands 1-3 in Fig. 1C) were digested with trypsin for
24 h and subjected to HPLC analysis on a Vydac C18 column
(4.6 × 250 mm) equilibrated with 0.1% (w/v) trifluoroacetic
acid. Peptides were eluted at a flow rate of 1 ml/min by a linear
acetonitrile gradient of 0-70% (v/v) in 0.1% trifluoroacetic acid
over 70 min. The peptides isolated were subjected to amino acid
sequencing analysis.
PLC Assay
The mixed micellar substrate was prepared as follows. Chloroform
solutions of lipids (PIP2, PS, cholesterol, and PE in a
molar ratio of 1:1:1:1:4 plus [3H]PIP2 as the
tracer, together with various amounts of fatty acids where stated) were
mixed and dried under N2 stream. Dried lipids were
dispersed by sonication in a buffer composed of 50 mM
Hepes-NaOH (pH 7.0), 120 mM KCl, 10 mM NaCl,
and 0.067% (w/v) sodium deoxycholate. The assay was started by mixing
50 µl of the micellar substrate with 50 µl of a solution containing
PLC and the samples to be tested. The final assay mixture (100 µl)
typically contained 10-20 ng of PLC- In the case of the kinetic analysis, the micellar substrate was
prepared with [3H]PIP2 without other lipids
in a buffer containing 10 mM octyl glucoside instead of
sodium deoxycholate.
Detection of Complex Formation of AHNAK and PLC- Wells of microtiter plates (Immuno Plate MaxiSorp, Nunc) were
coated with AHNAK by incubating with 100 µl of a 10 µg/ml protein solution in 50 mM Hepes-NaOH (pH 7.0) at 4 °C overnight.
After further incubation with 300 µl of 1% BSA for 1 h, wells
were washed twice with 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.02% (v/v) Tween 20. Incubation with PLC- Purification of PLC-
We screened various tissues and selected bovine lung cytosol for large
scale purification of the PLC- Identification of the PLC- Activation of PLC-
The combined effects of the purified lung AHNAK and AA on
PIP2 hydrolysis by PLC- Activation of PLC-
In addition, the stimulation of PLC- Activation of PLC- Kinetic Analysis of the Activation by AHNAK--
The effect of
AHNAK and AA on PLC- Direct Binding of AHNAK to PLC-
We could also precipitate the complex of GST-AHNAK-R4 and
PLC- Stimulation of PLC in Crude Cell Extract by AA--
Increasing
amounts of cytosolic fractions of HeLa cells, which are rich in AHNAK
and PLC-
Since AHNAK and AA stimulated PLC- [3H]Inositol-labeled Membrane as the Substrate of
PLC- In the present study, we find that, like tau, AHNAK activates
PLC-
(PLC-
) is activated by tau, a neuronal cell-specific
microtubule-associated protein, in the presence of arachidonic acid. We
now report that non-neuronal tissues also contain a protein that can
activate PLC-
in the presence of arachidonic acid. Purification of
this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (~700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in
length. Two AHNAK fragments containing one and four of the repeated
motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins
activated PLC-
1 at nanomolar concentrations in the presence of
arachidonic acid, suggesting that an intact AHNAK molecule contains
multiple sites for PLC-
activation. The role of arachidonic acid was
to promote a physical interaction between AHNAK and PLC-
1, and the activation by AHNAK and arachidonic acid was mainly attributable to
reduction in the enzyme's apparent Km toward the
substrate phosphatidylinositol 4,5-bisphosphate. Our results
suggest that arachidonic acid liberated by phospholipase A2
can act as an additional trigger for PLC-
activation, constituting
an alternative mechanism that is independent of tyrosine phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(
1 to
4),
(
1 and
2),
and
(
1 to
4) isozymes, based on their primary structures (1).
Their structural differences correlate with varying mechanisms for
their activation. Stimulation of
-isozymes by many agonists occurs
through receptors coupled to heterotrimeric G-proteins and is mediated
by the
-subunits of the Gq subfamily members and by
-subunits. In contrast, the
-isozymes are activated when
phosphorylated by various receptor-coupled protein-tyrosine kinases
(1).
activation in the absence of tyrosine phosphorylation. Jones and
Carpenter (2) reported that phosphatidic acid could activate both
tyrosine-phosphorylated and -unphosphorylated forms of PLC-
to a
similar extent. Since phosphatidic acid is the immediate product of
phosphatidylcholine hydrolysis by phospholipase D, activation of
phospholipase D in cells may lead to subsequent activation of PLC-
.
We (3, 4) and others (5) have recently shown that the product of
phosphatidylinositol 3-kinase, phosphatidylinositol 3,4,5-trisphosphate
(PIP3), is an activator of PLC-
. A considerable portion
(30-50%) of IP3 generated in response to platelet-derived growth factor was not a consequence of tyrosine phosphorylation of
PLC-
but rather a secondary event following PIP3
generation by platelet-derived growth factor-stimulated
phosphatidylinositol 3-kinase (3, 5).
activity independently of tyrosine phosphorylation in the presence of
unsaturated fatty acids, such as arachidonic acid (AA). Although the
concentration of AA in resting cells is quite low, a large quantity of
AA can be liberated from phosphatidylcholine by the action of cytosolic phospholipase A2 (cPLA2) upon cell activation
(7). Therefore, it is likely that certain stimuli that elicit
cPLA2 activation may indirectly cause the activation of
PLC-
if the tau proteins are present. Tau proteins are exclusively
expressed in neuronal cells (8).
in concert with AA, and we identify it as AHNAK. Our
finding further bolsters the thesis that indirect activation of PLC-
can occur in the absence of tyrosine phosphorylation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, -
1, -
2, and -
1)
were purified from HeLa cells that had been transfected with
recombinant vaccinia virus containing the entire coding sequence of the
respective enzyme as described (9).
Activator
1-activating activity was
measured at 30 °C for 10 min in 100 µl of a reaction mixture
containing 36,000 cpm of [3H]PIP2, 30 µM PIP2, 120 µM PE, 30 µM PS, 30 µM cholesterol, 30 µM arachidonic acid, PLC-
1 (10 ng), 3 mM
CaCl2, 2 mM EGTA, 0.033% (w/v) sodium
deoxycholate, 50 mM Hepes-NaOH (pH 7.0), and a source of
activator. To maintain the stimulated activity in the linear range of
the assay, we adjusted the amount of PLC to obtain an unstimulated,
basal activity in the range of a 500-1200 cpm of [3H]IP3 generated.
1-activating
activity (see Fig. 1A). Peak fractions (fractions 59-61)
were pooled and dialyzed against equilibration buffer.
1-activating activity. Peak fractions (numbers 15-18) were
pooled and dialyzed against equilibration buffer.
1; a 30 µM
concentration each of [3H]PIP2 (8-10 × 103 cpm), PS, and cholesterol; 120 µM PE in
50 mM Hepes-NaOH (pH 7.0); 120 mM KCl; 10 mM NaCl; 0.033% deoxycholate; 1 mM
MgCl2; 2 mM EGTA; and 1 µM free
Ca2+ ions, unless otherwise stated. After incubation for 10 min at 30 °C, the reactions were terminated by the addition of 200 µl of 10% (w/v) trichloroacetic acid and 100 µl of 10% (w/v)
bovine serum albumin (BSA), followed by centrifugation. The amount of radioactivity in the resulting supernatant, corresponding to liberated [3H]IP3, was measured by a liquid
scintillation counter.
1
was carried out in a total volume of 100 µl of a buffer containing 50 mM Hepes-NaOH (pH 7.0), 120 mM KCl, 10 mM NaCl, 1 mM dithiothreitol, 1 mM
MgCl2, 2 mM EGTA, 1 µM free
Ca2+ ions, 0.1 mg/ml BSA, and 100 µM
arachidonic acid plus various amounts of PLC-
1 and proteins to be
tested at ambient temperature for 1 h. Wells were then washed
twice with 200 µl of the same buffer containing 0.02% Tween 20. Bound materials were eluted by incubating with 50 µl of SDS-PAGE
sample buffer for 30 min, subjected to SDS-PAGE, and transferred to
nitrocellulose membranes. PLC-
1 on the membrane was detected by
using a specific monoclonal antibody (F-7-2) (10) and alkaline
phosphatase-conjugated anti-mouse IgG goat antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-activating Proteins from Bovine
Lung--
We previously noticed that the addition of crude
cytosol of bovine brain or HeLa cells to purified PLC-
markedly
enhanced PIP2-hydrolyzing activity measured in the presence
of AA. We then identified the bovine brain activator as tau (6). Since
tau is exclusively expressed in neuronal cells, we presumed that the stimulating activity seen in HeLa cell extract was due to a protein with functional similarity to tau and initiated the isolation of this
presumed non-neuronal PLC-
activator.
activator. The increase in PLC-
activity toward a mixed micellar substrate containing PIP2
and AA was monitored throughout the purification. Treatment of bovine
lung cytosol at 80 °C for 10 min resulted in a great enrichment of
PLC-
activator (data not shown). The heat-treated cytosol fraction
was subjected to sequential chromatographies on heparin-Sepharose CL-4B
and DEAE-5PW columns to yield a sharp activity peak that coincided with
a discrete protein peak (Fig. 1,
A and B). SDS-PAGE of the peak fraction showed
closely spaced protein bands with apparent molecular masses ranging
from 70 to 130 kDa (Fig. 1C, lane T).
Further attempts at purification including gel filtration on TSKgel
G3000-SW or ion exchange chromatography on Mono Q failed to separate
these proteins. Thus, proteins were fractionated on a preparative
SDS-polyacrylamide gel, and four major protein bands between 70 and 130 kDa were excised and electroeluted (Fig. 1C). After the
removal of SDS, each eluted protein was tested for PLC-
-activating
activity. All of the eluted proteins activated PLC-
1 (Fig.
1D).
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Fig. 1.
Purification of
PLC- 1-activating protein. Heat-treated
bovine lung cytosolic proteins were subjected to a heparin-Sepharose
CL-4B column (A) and then to an HPLC DEAE-5PW (B)
column. Fractions were assayed for PLC-
1-activating activity
(closed circles) as described under "Experimental
Procedures." The peak fractions (700 µg) pooled from the DEAE-5PW
column chromatography step were separated on a preparative
SDS-polyacrylamide gel. Four protein bands (bands 1-4, beginning with
the largest protein) with apparent molecular size of 130, 110, 85, and
70 kDa were excised and electroeluted from the gel. A recombined
mixture of the four bands (lane M), band 1 (lane
1), band 2 (lane 2), band 3 (lane 3), band 4 (lane 4), and the pooled peak fraction from the DEAE-5PW
column (lane T) were then subjected to SDS-PAGE on an 8%
gel and visualized by staining with Coomassie Brilliant Blue
(C). The PLC-
1-activating activity of the electroeluted
proteins was measured in the absence (control bar) and in
the presence of ~200 ng of band 1 (bar 1), band 2 (bar 2), band 3 (bar 3), band 4 (bar
4), or a recombined mixture of the four bands (bar M)
using an assay mixture containing 50 ng of PLC-
1 (D). The
positions of molecular size standards are shown on the left
in C.
Activator as AHNAK--
Three of the
electroeluted proteins (bands 1-3 in Fig. 1C) were
individually digested with trypsin and analyzed on a reverse phase HPLC
column. The peptide maps for bands 1, 2, and 3 were very similar (data
not shown), suggesting that these three proteins were related. Seven
peptides including two that were common to all three electroeluted
proteins were sequenced to yield 1) LKGPK, 2) VDIDVPDVDVQGPDWHL, 3)
FSMPGFK, 4) VPDVNIEGPDAK, 5) VKGDVDVSLPK, 6) ADIEISGPK, and 7)
GDVDVSLPK. A search of data bases revealed that the sequence of an
unusually large protein named AHNAK (meaning "giant" in Hebrew;
Ref. 11) carries exact coding sequences for all seven tryptic peptides;
the sequences of peptides 1, 3, 4, 5, and 7 were found 29, 17, 9, 10, and 9 times, respectively, in the different regions of AHNAK, whereas
the sequence of peptide 2 was found to match AHNAK residues 3923-3939
and 4387-4403, and peptide 6 corresponded only to AHNAK residues
4096-4104. AHNAK is a 5643-membered protein composed of highly
conserved repeated motifs (see below). Thus, the purified activator
proteins are probably fragments derived from AHNAK protein.
by the Purified AHNAK Fragments--
The
ability of the purified AHNAK fragments to activate PLC-
, measured
with a mixed micellar substrate containing PIP2 plus PE,
PS, cholesterol, and sodium deoxycholate, was dependent on unsaturated
fatty acids. As shown in Fig.
2A, various unsaturated fatty
acids were all somewhat effective in stimulating PLC-
in the
presence of AHNAK when tested at a concentration of 30 µM, whereas these lipids alone had no effect. However,
corresponding saturated fatty acids were ineffective even at higher
concentrations (up to 100 µM). Among those tested, AA was
the most potent stimulant of PLC-
activity in the presence of the
activator, and half-maximal stimulation was observed at 25 µM (Fig. 2B). AA was equally effective when
added to preformed micelles as when incorporated into the substrate
micelles (data not shown).
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Fig. 2.
Effects of various fatty acids on
PLC- 1 activity. A, the
PIP2-hydrolyzing activity of PLC-
1 (20 ng/assay) was
measured in the absence (control) or presence of the
indicated fatty acid. The assay mixture contained 10 µg/ml AHNAK
purified from lung and mixed micellar substrates including the
indicated fatty acids at a final concentration of 30 µM.
B, the PIP2-hydrolyzing activity of PLC-
1 was
measured in the absence (open circles) or presence
(closed circles) of 10 µg/ml purified activator protein
with substrate vesicles containing the indicated concentrations of AA.
Results are expressed as -fold activation over the activities obtained
in the absence of fatty acids.
1, -
1, -
2, and -
1 were
compared in the presence of an excess amount of AHNAK (Fig.
3). Activation by AHNAK and AA were
apparent with both PLC-
1 and PLC-
2 (7-9-fold). Stimulation of
the activity of PLC-
1 was less apparent, and PLC-
1 activity was
not affected at all.
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Fig. 3.
Combined effects of AA and the AHNAK
fragments purified from lung on the PIP2-hydrolyzing
activity of various PLC isozymes. The PIP2-hydrolyzing
activities of the indicated PLC isozymes (20-100 ng/assay) were
measured in the absence or presence of 10 µg/ml AHNAK purified from
lung with substrate vesicles containing 50 µM AA. Results
are expressed as -fold activation over unstimulated activities obtained
in the absence of AHNAK.
1 by Glutathione S-Transferase (GST)-AHNAK
Fusion Protein--
AHNAK contains approximately 30 repeats of a
highly conserved motif, most of which are 128 amino acids in length
(see Fig. 13). We investigated if one or several units of this repeated
motif are capable of stimulating PLC-
1. We prepared two GST-AHNAK
fusion proteins (Fig. 4A), one
containing only one repeat (GST-AHNAK-R1) corresponding to
AHNAK residues 3470-3882 and the other containing three full repeats
and two split one-half repeats (GST-AHNAK-R4) corresponding
to residues 3817-4412 (see Fig. 13B). The two GST-AHNAK fusion proteins from E. coli could stimulate the
PIP2-hydrolyzing activity of PLC-
1 in a
dose-dependent manner in the presence of AA, whereas GST
alone in concentrations up to 100 nM had little effect on
the activity (Fig. 4B). GST-AHNAK-R1 was
somewhat less potent than the longer form, but both stimulated apparent
PLC-
1 activity more than 10-fold at concentrations as low as 10 nM. Half-maximal stimulation by the longer form was
observed at 1.5 nM in the presence of 100 µM
AA. In the absence of AA, both forms were ineffective in stimulating
PLC-
1, consistent with the behavior of the AHNAK fragments purified
from bovine lung. We also reevaluated the sensitivity of PLC-
1,
-
1, and -
1 isozymes using the GST-AHNAK fusion protein (Fig.
5). Activation of PLC-
1 was again most
efficient. PLC-
1 was also activated by the combination of AA and
GST-AHNAK-R4 but to a lesser extent compared with PLC-
1,
and the activity of PLC-
1 was not affected at all, thus confirming
the result with AHNAK fragments purified from bovine lung (Fig. 3).
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Fig. 4.
Activation of PLC- 1
by GST-AHNAK fusion proteins. A, SDS-PAGE of purified
GST fusion proteins. B, the percentage of PIP2
hydrolyzed by PLC-
1 (20 ng/assay) was measured in the presence of
indicated concentrations of GST-AHNAK-R4 alone
(closed diamonds), GST-AHNAK-R1 alone
(open diamonds), GST-AHNAK-R4 plus 100 µM AA (closed circles), or
GST-AHNAK-R1 plus 100 µM AA (open
circles) with assay mixtures containing 1 µM free
Ca2+ ions and 1 mM Mg2+ ions. The
percentage of PIP2 hydrolyzed in the presence of 100 nM GST and 100 µM AA is indicated by the
closed triangle.
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Fig. 5.
Effects of various concentrations of
recombinant AHNAK protein on the PIP2-hydrolyzing activity
of PLC isozymes. The PIP2-hydrolyzing activities of
PLC- 1 (closed diamonds), PLC-
1 (closed
circles), and PLC-
1 (open circles) were measured in
the presence of 100 µM AA plus the indicated
concentrations of GST-AHNAK-R4. Other conditions were as
described in the legend to Fig. 4. Results are expressed as -fold
activation over unstimulated activities obtained in the absence of
AHNAK.
1 activity was partially
dependent on Mg2+ ions. At a free Ca2+
concentration of 10
6 M, the stimulation was
maximum at an Mg2+ concentration of 1 mM, which
is close to physiological concentrations, and declined at higher
concentrations (Fig. 6A). The
mechanism by which Mg2+ ions potentiated AHNAK-stimulated
activity was not clear, but the basal activity of PLC-
1 in the
absence of AHNAK and AA was not significantly affected by the addition
of Mg2+ ions. Stimulation of PLC-
1 activity by
GST-AHNAK-R4 plus AA was also investigated at different
concentrations of free Ca2+ ions (Fig. 6B).
Activities of all PLC isozymes are dependent on Ca2+ ions
(12), and the activity of PLC-
1 increases rapidly from 10
8 M of Ca2+ to reach a plateau
at 10
6 M of Ca2+ (13). The
PLC-
1 activity stimulated by GST-AHNAK-R4 plus AA showed
a similar Ca2+ dependence. AA alone had some stimulative
action at 10
4 M of Ca2+ (Fig.
6B).
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Fig. 6.
Effect of Mg2+ and
Ca2+ ions on the AHNAK- and AA-dependent
PLC- 1 activity. A, the
percentage of PIP2 hydrolyzed by PLC-
1 (20 ng/assay) was
measured at the indicated Mg2+ concentrations in the
absence (open circles) or in the presence of 10 nM GST-AHNAK-R4 alone (closed
diamonds), 100 µM AA alone (open
diamonds), or 10 nM GST-AHNAK-R4 plus 100 µM AA (closed circles). The assay mixture
contained 1 µM free Ca2+ ions. B,
the effect of Ca2+ ions on the percentage of
PIP2 hydrolyzed by PLC-
1 was assessed using assay
mixtures containing 1 mM Mg2+ ions and
indicated concentrations of free Ca2+ ions. The
symbols used to represent the different combinations of 100 µM AA and 10 nM GST-AHNAK-R4 are
the same as in A.
1 by AHNAK in the Presence of
Tau--
Because the tau protein also stimulates PLC-
1 and PLC-
2
rather specifically among various PLC isozymes in the presence of AA
(6), we asked whether tau and AHNAK share a common activation mechanism. As shown in Fig. 7, AHNAK was
no longer effective at enhancing the PLC-
1 activity in the presence
of a saturating concentration of tau. This result indicates that the
tau interaction site on PLC-
overlaps with that of AHNAK.
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Fig. 7.
Effect of tau on the
PLC- 1 activation by AHNAK. The percentage
of PIP2 hydrolyzed by PLC-
1 was measured in the absence
(open circles) or the presence of 20 µg/ml tau
(closed circles) using assay mixtures containing 100 µM AA and the indicated concentrations of
GST-AHNAK-R4.
activity at various concentrations of
PIP2 was evaluated using a micellar substrate system composed of PIP2 and octyl glucoside. In the presence of
2-20 mM octyl glucoside, the PIP2-hydrolyzing
activity of PLC-
was comparable with that measured with the mixed
micellar substrate containing PIP2 plus PE, PS,
cholesterol, and sodium deoxycholate (data not shown). With this
simplified substrate system, AHNAK and AA activated PLC-
activity
only 3-fold, which compares with the 7-9-fold activation observed in
Figs. 2-4 with the substrate consisting of PIP2, PE, PS,
cholesterol, and sodium deoxycholate. However, the effective
concentrations of AHNAK and AA were unchanged. The initial rate of
PIP2 hydrolysis was measured with varying concentration of
PIP2 in the presence and absence of
GST-AHNAK-R4 and AA. A plot of these data according to the
Eaddie-Hofstee equation gave fairly straight lines (Fig.
8). It appeared that the apparent Vmax was not affected by the presence of
saturating concentrations of GST-AHNAK-R4 and AA, whereas
the apparent Km for PIP2 was
considerably reduced from 24 ± 4 µM (mean ± S.E., n = 4) to 8 ± 1 µM
(n = 4).
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Fig. 8.
Eddie-Hofstee plot of
PLC- 1 reaction measured in the absence and
presence of GST-AHNAK-R4 plus AA. The hydrolysis of
substrate PIP2 by PLC-
1 (20 ng/assay) was followed in
the absence (open circle) and presence of 20 nM
GST-AHNAK-R4 plus 100 µM AA (closed
circle) using micellar substrates containing 5 mM
octyl glucoside. The initial velocities (V) monitored over 5 min were plotted against V/[PIP2] according to
the Eddie-Hofstee equation (V =
Km × V/[PIP2] + Vmax).
The Vmax values that correspond to the intercept
at the y axis were nearly identical in the absence and
presence of the activators. The Km values,
represented by the slope, were 24 ± 4 and 8 ± 1 µM, respectively, in the absence and presence of the
activators. Data are expressed as means of four independent
determinations.
1 in the Presence of
AA--
Because AHNAK could activate PLC-
1 efficiently even at
concentrations as low as 1 nM (Fig. 4B), we
presumed that AHNAK might form a tight complex with PLC-
1. To prove
this, GST-AHNAK-R4 was immobilized onto the wells of
microtiter plates, and PLC-
1 was incubated in the coated wells
containing AA. Bound PLC-
1 was eluted from the wells with a
SDS-containing buffer and detected by immunoblot analysis. Binding of
PLC-
1 to immobilized AHNAK was clearly detected, while no binding
was seen with a control well that had been coated with BSA (Fig.
9A). The binding to
GST-AHNAK-R4 was completely abolished by the addition of
GST-AHNAK-R4, but not by the addition of GST, to the well
containing PLC-
(Fig. 9A). Moreover, we found that the
binding was dependent on AA. As is shown in Fig. 9B, in the
presence of AA the binding was detectable at PLC-
1 concentrations as
low as 1 µg/ml and increased in a PLC-
1
concentration-dependent manner. However, in the absence of
AA, the binding was hardly seen even at 30 µg/ml PLC-
1. These results indicate that PLC-
1 directly interacts with AHNAK, and the
role of AA is to potentiate the interaction.
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Fig. 9.
Direct interaction of AHNAK with
PLC- 1. A, wells of microtiter
plates were coated with GST-AHNAK-R4
(R4) or BSA. After blocking unoccupied surface
with BSA, a solution of 10 µg/ml PLC-
1 was added and incubated in
the presence of 100 µM AA with 100 nM
GST-AHNAK-R4 or 100 nM GST or without
additional proteins. After washing, proteins bound to the wells were
eluted and subjected to immunoblot analysis with a monoclonal antibody
to PLC-
1. B, PLC-
1 at the indicated concentrations was
incubated in the absence or presence of 100 µM AA in the
wells coated with GST-AHNAK-R4, and the amount of bound
PLC-
was measured by immunoblot analysis as in A.
1 in the presence of AA using glutathione-conjugated agarose beads (data not shown). However, this experiment suffered from high
background, probably due to nonspecific binding of PLC-
1 to the beads.
1, were incubated with a mixed micellar substrate containing
[3H]PIP2 and octyl glucoside. Hydrolysis of
[3H]PIP2 was stimulated by the addition of
AA, but a saturated fatty acid (stearic acid) was ineffective (Fig.
10). The addition of recombinant AHNAK
to the reaction mixture did not further the effect of AA. Thus, it
appeared that these cells contained a saturating amount of
AA-dependent activator for PLC and also that no other components in the soluble fraction of HeLa cells significantly inhibited this activation process. Background activity in the absence
of AA was probably due to PLC-
1 as well as other PLC isozymes like
PLC-
1, PLC-
3, and PLC-
1 that are known to exist in HeLa cells
(HeLa cells do not contain PLC-
2
isozyme).2
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Fig. 10.
Effect of AA and AHNAK on PLC activity in
crude cell lysate. A confluent culture of HeLa cells grown in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
was harvested; washed with phosphate-buffered saline; suspended in 50 mM Hepes-NaOH (pH 7.0), 1 mM EDTA, 1 mM EGTA; and then lysed by sonication. The lysate was
centrifuged at 100,000 × g for 15 min.
PIP2 hydrolyzed by indicated amounts of the resulting
supernatant was measured by incubating with mixed micellar substrates
for 5 min at 30 °C in the absence (open circles) or
presence of 100 µM stearic acid (open
diamonds), 100 µM AA (closed diamonds),
or 100 µM AA plus 20 nM
GST-AHNAK-R4 (closed circles).
1 isozyme to a smaller but
significant extent (Figs. 3 and 5), we evaluated the contribution of
PLC-
1 to the AHNAK/AA-stimulated activity by employing TV-1 embryonic fibroblasts derived from the PLC-
1 null mouse (14). The
TV-1 cells expressed no detectable amount of PLC-
1 and very low
levels of PLC-
2 (14, 15). PLC-
1-expressing cells were obtained by
infecting TV-1 cells with vaccinia virus harboring the PLC-
1 gene.
Expression of PLC-
1 by the viral vector was detected by immunoblot
analysis (Fig. 11, inset).
Cytosolic fractions were obtained from either control TV-1 cells
infected with empty virus or the PLC-
1-expressing TV-1 cells, and
the PIP2-hydrolyzing activity of the cytosolic fractions
was measured in the presence and absence of AA (Fig. 11). The PLC
activity in the control cells was low and not affected by the addition
of AA. In contrast, the PLC activity in the cytosol from
PLC-
1-expressing cells was higher than that from control cells and
substantially enhanced by the addition of AA. These results indicate
that the
-isozyme is the main target of AA stimulation.
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Fig. 11.
Identification of PLC-
as the major target of activation by AHNAK and AA in fibroblast
extracts. PLC-
1 null TV-1 cells, which were established from
embryonic fibroblasts of Plcg1 (
/
) mouse (14), were
infected with vaccinia virus harboring PLC-
1 gene or with empty
virus and cultured for 2 days. Cytosolic fractions were obtained from
these cells as described in the legend to Fig. 10. PIP2
hydrolyzed by the indicated amounts of the cytosolic fractions from
PLC-
1-expressing cells (circles) and control cells
(diamonds) were measured by incubating with the mixed
micellar substrates for 10 min at 30 °C in the absence (open
symbols) or in the presence of 100 µM AA
(closed symbols). Inset, the presence of PLC-
1
in the cytosolic fractions of control and PLC-
1-expressing cells was
detected by immunoblot analysis with a monoclonal antibody to
PLC-
1.
1 Activated by AHNAK and AA--
Membranes were isolated from
HeLa cells that had been metabolically labeled with
[3H]myo-inositol for 24 h, washed, and
used as substrate for exogenously added PLC-
1. AA again augmented
the release of inositol phosphates by PLC-
1, but the addition of
recombinant AHNAK did not cause further augmentation (Fig.
12A). In a separate
experiment, the labeled membranes were extracted with organic solvents
to remove proteins and then used as the substrate. This time, AA alone
did not cause much stimulation of PLC-
activity, whereas the
addition of AHNAK rendered AA capable of stimulating PLC-
activity.
(Fig. 12B). These results suggest that the buffer-washed
membrane preparation contained enough AHNAK to activate PLC-
1 in the
presence of AA and that AHNAK and AA were capable of stimulating the
activity of PLC-
1 toward cell membrane phosphoinositides.
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Fig. 12.
Isolated cell membrane as the substrate of
PLC- 1 activated by AHNAK and AA.
A, HeLa cells were labeled with
[3H]myo-inositol (10 µCi/10-cm dish) for
24 h in inositol-free Dulbecco's modified Eagle's medium;
suspended in 50 mM Hepes-NaOH (pH 7.0), 3 mM
EDTA, 3 mM EGTA; and lysed by sonication. Membranes were
isolated by centrifugation (100,000 × g, 15 min);
washed once with the sonication buffer and then with the sonication
buffer containing 3 M KCl; and suspended in 50 mM Hepes-NaOH (pH 7.0), 120 mM KCl, 10 mM NaCl, and 0.067% deoxycholate. Labeled membranes (4000 cpm/assay) thus prepared were incubated with the indicated amounts of
PLC-
1 at 30 °C for 10 min, and liberation of
[3H]inositol phosphates ([3H]IPn)
was measured in the absence of both AA and GST-AHNAK-R4
(open circles) or in the presence of 100 µM AA
alone (closed diamonds) or 20 nM
GST-AHNAK-R4 plus AA (closed circles).
B, one volume of the membrane suspension from A
was extracted with 6 volumes of a solution containing chloroform,
methanol, and concentrated HCl (50:50:0.3, v/v/v) and then centrifuged.
To the resulting supernatant, 2 volumes of 1 M HCl were
added, and the organic phase was separated. The extracted lipids in the
organic layer were dried under an N2 stream and suspended
in 50 mM Hepes-NaOH (pH 7.0), 120 mM KCl, 10 mM NaCl, 0.067% sodium deoxycholate. Using this extracted
lipid preparation, PLC-
1 reactions were carried out as in
A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in the presence of AA. AHNAK was initially isolated as a
680-kDa protein uniquely localized to the desmosomes of bovine muzzle
epidermal cells and termed desmoyokin (16). The same protein was also
independently identified by Bishop and colleagues as the product of a
gene whose expression is repressed in human neuroblastomas and several
other types of tumor cell lines and named AHNAK to convey its
exceptional size (11). Bishop's group showed that AHNAK was translated
from a 17.5-kb mRNA and isolated two cDNA clones of 5.5 kb
(GenBankTM accession number M80902) and 4.0 kb (accession
number M80899) that encode the N-terminal 1683 amino acids and the
C-terminal 1277 amino acids, respectively (Fig.
13A). From the amino acid sequence derived from the two cDNA clones, which represented about 50% of the entire AHNAK coding sequence, it was suggested that the
AHNAK protein can be divided into three structural regions: the
amino-terminal 251 amino acids, a large central region of about 4300 amino acids with multiple repeated motifs, and the carboxyl-terminal
1002 amino acids (Fig. 13B). Subsequently, the AHNAK gene
was localized to human chromosome band 11q12 (17). The region 11q12
that includes the sequences of clones M80902 and M80899 has recently
been established (accession number AC004230). The genomic sequence
predicts a length of 5643 amino acids for AHNAK. As suggested before,
the central region of AHNAK is composed of approximately 30 repeated
motifs. The repeated unit is 128 amino acids in length, but some of the
units are less than 128 residues. The 128-residue motifs are on average
approximately 80% identical to each other with respect to amino acid
sequence. Examination of the amino acid sequence within the repeated
unit reveals a number of recurrences of heptasequence (or octasequence) (D/E)
K(A/G)P(K), where
represents a hydrophobic residue,
represents a hydrophilic residue, and other letters correspond to
conventional one-letter representation of amino acid residues (Fig.
13C).
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Fig. 13.
AHNAK sequence. A, AHNAK
gene sequence derived from the sequence of chromosome band 11q12
(GenBankTM accession number AC004230) includes the
sequences of two cDNAs (accession numbers M80902 and M80899)
identified in the laboratory of Bishop (11). The nucleotide residue
numbers derived from AC004230 are indicated. B, amino acid
(aa) sequence deduced from the sequence of AC004230
indicates that AHNAK protein can be divided into three structural
regions, the amino-terminal 251 amino acids (black line), a
large central region of 4391 amino acids with multiple repeated motifs
(gray line), and the carboxyl-terminal 1002 amino acids
(black line). The regions included in the GST fusion
proteins, GST-AHNAK-R4 and GST-AHNAK-R1 are
indicated. R and 1/2R represent 1 and
1/2 unit, respectively, of the 128 amino acids motif.
C, repetitive units within the 128-amino acid motif of amino
acid residues 3746-3873 are shown.
The AHNAK genomic locus has no intron, suggesting that the 70-130-kDa activator proteins purified from bovine lung are probably proteolytic fragments of AHNAK rather than splice variants. To study this possibility, immunoblot analyses of HeLa cell extracts were carried out using rabbit serum prepared against the purified 110-kDa activator protein. A major protein band with an approximate size of 700 kDa was seen together with several minor bands of smaller size when extracts were freshly prepared in the presence of a mixture of protease inhibitors. However, when the extracts were prepared in the absence of protease inhibitors or the extracts were prepared with protease inhibitors but aged for several hours prior to immunoblot analysis, the 700-kDa band was not detectable, and instead the intensity of lower molecular bands was increased. This result suggests that AHNAK is readily susceptible to proteolysis.
AHNAK appears to be an abundant and ubiquitous protein found in various cellular compartments. AHNAK was located primarily (but not exclusively) in the nucleus of HeLa cells and phosphorylated on serine and threonine (18). In contrast, desmoyokin (AHNAK) was found mainly in the plasma membrane and to a lesser extent in the cytoplasm in squamous cell carcinomas, and it showed strong cytoplasm staining in melanomas (19). It was also claimed that different antibodies could yield different staining patterns in a given cell. In keratinocytes, AHNAK was mainly in the cytoplasm when cells were kept in low Ca2+ medium but translocated to the plasma membranes with a concomitant increase in the degree of phosphorylation upon an increase in extracellular Ca2+ concentrations or treatment with phorbol ester (20). The level of expression as well as the state of phosphorylation of the protein in several transformed cells was altered during growth and differentiation (18).
Despite these extensive studies on structure and cellular localization of AHNAK, no information has been available that might hint at the cellular function of AHNAK. Our current report is the first such insight.
Activation of PLC- isozymes by AHNAK was indistinguishable in many
aspects from activation by tau (6), although there is no similarity in
their primary structures except that both proteins are replete with
proline residues. These two activators competed against each other in
activating PLC-
, indicating that they may share a common mechanism
of activation. Recent reports have revealed that tau can interact with
all three of the activation reaction components, PLC-
(21), PIP2 (22), and AA (23). Tau was shown to
co-immunoprecipitate with PLC-
, but not with
- or
-isozymes of
PLC, from lysate of neuroblastoma cells (21). Tau was also shown to
interact with PIP2, since divalent cation-induced aggregation of PIP2 vesicles was modified by tau in a
manner similar to that caused by the known PIP2-binding
protein, gelsolin (22). Modulation of tau's function by fatty acids
was also evident from the fact that filament formation by tau was
stimulated by free fatty acids, with AA as the most effective and
saturated fatty acids much less potent (23).
We have shown here that AHNAK can directly interact with PLC-1 in
the presence of AA. It seems that AHNAK and tau serve as the receptors
for unsaturated fatty acids, whereas PLC-
1 is not affected by fatty
acids, since the activity of the enzyme was unchanged by fatty acids in
the absence of the activator proteins. As a consequence of interaction
with the AA-bound AHNAK, the apparent Km for
PIP2 decreased. It should be noted that enzymes that
catalyze reactions on the water-lipid interface usually do not obey
Michaelis-Menten kinetics but rather obey "surface dilution kinetics" (24). This is also the case for PLC-
1 (13). The "apparent Km" values we obtained here actually
contain two parameters, the affinity for micellar surface and the
affinity for PIP2 molecule per se. More
experiments are needed to dissect the apparent Km
value into these two parameters. It is nevertheless apparent that the
effect of AHNAK plus AA results in more efficient recognition of the
micellar substrate, which can be caused by increase in the affinity for
hydrophobic surface, for PIP2, or for both. In this
connection, it is interesting to note that the activation of PLC-
via tyrosine phosphorylation is also attributable to an increase in the
affinity for substrate but not to an increase in the turnover number
(Vmax) (13). It is also important to note that
although the extent of activation somewhat varied, the AHNAK- and
AA-dependent activation was observed with vesicular
substrates prepared without detergents as well as with micellar
substrates in the presence of deoxycholate, octyl glucoside, or Tween 20.
Given that both tau and AHNAK are known to be phosphoproteins,
phosphorylation may play a role in regulation of their function as
PLC- activators. Tau is a substrate of various protein kinases, including cAMP-dependent protein kinase,
Ca2+/calmodulin-dependent protein kinase, and
Cdc2-like protein kinase (reviewed in Ref. 25), and can be
heavily phosphorylated. AHNAK is also shown to be phosphorylated
in vivo (18, 20).
In a classical scheme, binding of a variety of agonists to their
cognate receptors causes the activation of PLC- through tyrosine
phosphorylation. However, accumulating evidence shows that the
activation mechanism for PLC-
is not as simple as once thought.
Activation of phospholipase D may also lead to activation of PLC-
1
through accumulation of phosphatidic acid (2). We and others have
recently shown that PIP3 activates purified PLC-
and
that receptors coupled to phosphatidylinositol 3-kinase are capable of
activating of PLC-
indirectly in cells through the generation of
PIP3 (3-5, 26). Our previous (6) and present studies
suggest that intracellular accumulation of AA can be another trigger
for PLC-
activation in the presence of tau or AHNAK proteins. AA is
mainly released from phosphatidylcholine by the action of cPLA2 (7) and serves as the precursor of various
eicosanoids. The resulting eicosanoids in turn activate cells by
autocrine and/or paracrine mechanisms. AA is also known to modulate
several biological processes without conversion to eicosanoids. It can activate protein kinase C (27), guanylate cyclase (28), and neutral
sphingomyelinase (29, 30), while it inhibits
Ca2+/calmodulin-dependent protein kinase (31)
and GTP-binding to Gz
(32). Now PLC-
is added to the list of AA effectors.
Activation of cPLA2 requires intracellular Ca2+
mobilization (7), and it can thus be a secondary event following PLC
activation, either PLC- activation by G-protein-coupled receptors or
PLC-
activation by growth factor receptors. cPLA2
activation in turn may activate PLC-
if AHNAK or tau are present,
constituting a positive feedback loop in the hydrolysis of
PIP2. On the other hand, PIP2 is a potent
activator of cPLA2 (33). Therefore, hydrolysis of
PIP2 by PLC will attenuate the activity of
cPLA2, constituting a negative feedback loop in terms of AA mobilization.
In vitro results presented here all suggest that
intracellular accumulation of AA can induce PLC- activation, and we
have also shown that HeLa cells possess the necessary machinery for AA-induced activation. Several in vivo studies have
suggested that endogenously released AA stimulates PLC activity
independently of its conversion to eicosanoids. Incubation of human
trophoblasts with AA stimulates PLC activity, and neither
cyclooxygenase nor lipoxygenase inhibitors blocked this response (34).
They also found that activation of PLA2 was involved in the
stimulation of phosphoinositide metabolism and placental lactogen
release in these cells (35). AA stimulated phosphoinositide metabolism in and catecholamine release from bovine adrenal chromaffin cells, and
eicosanoid inhibitors were without effect (36). The fatty acid was also
shown to increase phosphoinositide breakdown and glutamate release in
rat hippocampal tissue (37) and to increase intracellular
Ca2+ ions by mobilizing an IP3-sensitive
Ca2+ pool in isolated rat pancreatic islets (38) and in a
human leukemic T cell line (39). The AA-induced Ca2+
release was shown to be independent of its metabolites (38). We believe
that most, if not all, of these earlier observations now can be
explained by the participation of AHNAK and tau.
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FOOTNOTES |
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* 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.
Japan Society for the Promotion of Science Fellow in Biomedical
and Behavioral Research at the National Institutes of Health.
§ These authors contributed equally to this work.
¶ Present address: Dept. of Biological Sciences, College of Natural Sciences, Ewha Womans University, Seoul, Korea 120-750.
Present address: Dept. of Food and Nutrition, College of Home
Economics, Chonnam National University, Kwangju, Korea 500-757.
** Present address: Dept. of Pulmonary and Critical Care Medicine, Ajou University Medical School, Suwon, Korea 442-749.
To whom correspondence should be addressed: Bldg. 3, Rm. 122, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9646; Fax: 301-480-0357; E-mail: sgrhee{at}nih.gov.
2 S. B. Lee and S. G. Rhee, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: PLC, phosphoinositide-specific phospholipase C; cPLA2, cytosolic phospholipase A2; AA, arachidonic acid; GST, glutathione S-transferase; BSA, bovine serum albumin; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PE, phosphatidylethanolamine; PS, phosphatidylserine; IP3, inositol 1,4,5-trisphosphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
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