(Received for publication, August 5, 1996, and in revised form, December 17, 1996)
From the The cytoskeletal protein, tubulin, has been shown
to regulate adenylyl cyclase activity through its interaction with the
specific G protein Heterotrimeric GTP-binding proteins (G proteins) couple a wide
range of cell surface receptors to membrane-associated effector molecules, including adenylyl cyclase,
PLC The cytoskeletal GTP-binding protein tubulin activates or inhibits
adenylyl cyclase through its interaction with G Although an association of PLC with the turkey erythrocyte cytoskeleton
has been reported (18), the effect of tubulin on PLC Sf9 cells were infected in
different combinations with baculoviruses bearing the m1
muscarinic receptor, G cDNA encoding the
entire rat PLC Microtubule proteins were prepared as
described (25). Microtubule-associated proteins were removed by
phosphocellulose chromatography (PC-tubulin) as described (26).
Tubulin-GppNHp or tubulin-[32P]AAGTP were prepared from
PC-tubulin by the removal of GTP by means of charcoal pretreatment,
followed by incubation in the presence of 150 µM of
GppNHp (or [32P]AAGTP) for 30 min on ice as described
(27). Prior to use, tubulin-guanine nucleotide preparations were passed
through P6-DG columns twice in order to remove unbound nucleotide.
After this procedure 0.4-0.6 mol of nucleotide were bound per mol of
tubulin. This remained stable even after five desalting steps (27).
Tubulin-guanine nucleotide concentrations used throughout the study
were based on the protein concentration. [32P]AAGTP and
AAGTP were synthesized as described (28).
20 µg of Sf9 cell membranes,
containing indicated recombinant proteins, and 40 µl of substrate
mixture (PIP2 at a final concentration of 30 µM (1 µCi of [3H]PIP2),
evaporated under a stream of nitrogen and sonicated on ice for 5 min in
20 mM Tris maleate, pH 6.8, 6 mM
MgCl2, 4 mM ATP, EGTA, and CaCl2 to
give a final concentration of 50 nM Ca2+ (29)
and deoxycholate to give a final concentration of 1 mM) were incubated in a Branson water bath sonicator for 15 min at 4 °C.
Ten µl of GppNHp, tubulin-GppNHp, or tubulin, with or without 10 µl
of carbachol, were added at appropriate concentrations to a final
volume of 80 µl, and the tubes were incubated for 15 min at 37 °C
with constant shaking. Aqueous and lipid phases were separated as
described (30), and [3H]inositol trisphosphate
([3H]IP3) in the aqueous phase was quantified
by liquid scintillation counting.
Sf9 cells were infected with
G Sf9 cell
membranes, expressing recombinant proteins, were incubated with 1 µM tubulin-[32P]AAGTP in the presence or
absence of 1 mM carbachol in 100 mM Pipes
buffer, pH 6.9, 2 mM EGTA, 1 mM
MgCl2 (buffer A) for 10 min at 23 °C with constant
shaking. The tubes were UV-irradiated for 4 min on ice, and the
reaction was quenched with ice-cold Hank's buffer, 1 mM
MgCl2, 4 mM DTT. After centrifugation at
100,000 × g for 15 min, the membrane pellets and
concentrated supernatants (Amicon, Centriprep 10) were dissolved in 3%
SDS Laemmli sample buffer with 50 mM DTT. Membrane (70 µg
of protein) and supernatant fractions (20 µg of protein) were
subjected to SDS-PAGE (10% acrylamide and 0.133% bisacrylamide) as
described (31). Gels were either stained (Coomassie Blue) or subjected
to Western blotting, followed by autoradiography (Kodak XAR-5 film).
The radioactivity of the bands was measured with a PhosphorImager
(Molecular Dynamics).
Sf9 cells were infected separately with
baculoviruses bearing either the m1 muscarinic receptor,
G Homogeneous PIP2
micelles were prepared by suspending 100 µg of PIP2 in
0.5 ml of buffer A and sonicating on ice for 5 min. Tubulin was
incubated with PIP2 micelles in a Branson water bath sonicator for 15 min at 4 °C, followed by an additional 45 min on
ice. The samples were run on a 0.7 × 50-cm column of Ultrogel AcA
34 at 4 °C. The column was equilibrated with buffer A (flow rate = 20 ml/h), and fractions of 0.5 ml were collected and
assayed for protein. The data are given in arbitrary units since lipids quench the Bradford dye-binding assay (32).
Samples were taken from tubulin
polymerization reactions (2 mg/ml final protein concentration) carried
out in buffer A, containing 3 mM MgCl2, 1 mM GTP, and 30% glycerol, for 30 min at 37 °C with constant shaking (26). PIP2, when added, was sonicated in
buffer A as described. 10 µl of the reaction mixture was applied to a carbon/Formvar-coated grid for about 10 s. The grids were stained with several drops of 1% uranyl acetate and air-dried. Observations were made in a JEM 100CX (JEOL) electron microscope. For microtubule length determinations, at least 100 assembled structures were measured
in multiple fields from two different grids representing two different
experiments.
[ Simultaneous baculovirus-mediated expression of
G
Effect of GppNHp on IP3 generation in membranes from normal and
infected Sf9 cells
Department of Physiology and Biophysics and
the Committee on Neuroscience,
Laboratory of Cell
Signaling, NHLBI, National Institutes of Health,
Bethesda, Maryland 20892
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
subunits, G
s or
G
i1. Tubulin activates these G proteins by transferring
GTP and stabilizing the active nucleotide-bound G
conformation. To
study the possibility of tubulin involvement in
G
q-mediated phospholipase C
1
(PLC
1) signaling, the m1 muscarinic receptor, G
q, and PLC
1 were expressed in
Sf9 cells. A unique ability of tubulin to regulate PLC
1
was observed. Low concentrations of tubulin, with guanine nucleotide
bound, activated PLC
1, whereas higher concentrations
inhibited the enzyme. Interaction of tubulin with both
G
q and PLC
1, accompanied by guanine
nucleotide transfer from tubulin to G
q, is suggested as
a mechanism for the enzyme activation. The PLC
1
substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and
prevented microtubule assembly. This observation suggested a mechanism
for the inhibition of PLC
1 by tubulin, since high
tubulin concentrations might prevent the access of PLC
1
to its substrate. Activation of m1 muscarinic receptors by
carbachol relaxed this inhibition, probably by increasing the affinity
of G
q for tubulin. Involvement of tubulin in the
articulation between PLC
1 signaling and microtubule
assembly might prove important for the intracellular governing of a
broad range of cellular events.
1,1 and ion channels.
Receptor activation leads to GTP binding by, and activation of, G
protein
subunits. The activated
subunit, and in some instances
the
dimer of the G protein, transmits the receptor-generated
signal to the effector molecule (1, 2). Many hormones and
neurotransmitters elicit intracellular responses by activating
receptors linked to G
q-mediated stimulation of
PLC
1 (3-8). Although isoforms of PLC
have been
demonstrated to be activated by G protein
and
subunits,
PLC
1 appears to be activated by G
q
(9-11). PLC
1 hydrolyzes the lipid precursor phosphatidylinositol 4,5-bisphosphate (PIP2) to generate
two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate
(IP3). Diacylglycerol and IP3 trigger a variety
of cellular functions by activating protein kinase C and mobilizing
stored calcium (12).
s or
G
i1 (13-16). Tubulin binds to these proteins and
activates them via direct transfer of GTP. Both guanine nucleotide
transfer and the stabilization of the active G
conformation are
thought responsible for the sustained G protein activation by tubulin
(15, 16). Complexes of dimeric tubulin with G
s and
G
i1 exist in the synaptic membrane (17), and these
complexes provide the physical framework for the interface between G
protein-mediated signal transduction and the cytoskeleton. Previous
work has focused on the regulation of adenylyl cyclase by tubulin, but
the possibilities that other G protein-mediated signaling enzymes
suffered similar regulation waranted investigation.
1
signaling has not been examined. The current study was designed to
determine whether tubulin plays a role in the regulation of PLC
1, and, conversely, how phosphoinositides might
affect the ability of tubulin to assemble into microtubules or to
interact with G proteins. This was approached using Sf9 cells
expressing various combinations of m1 muscarinic receptors,
G
q, and PLC
1. It was determined that
tubulin does indeed bind to G
q and activates that
molecule via the direct transfer of GTP. It is also demonstrated that
PLC
1 may enjoy a dual regulation by dimeric tubulin, the molecule inhibiting PLC
1 by binding to the enzyme
substrate PIP2. This represents another arena in which cell
structure and cell signaling share a complex regulatory liaison.
Baculovirus-directed Protein Expression in Sf9 Cells, Membrane
Preparation, and Western Blotting
q, or PLC
1
cDNAs at a ratio of 1:1:1 and multiplicity of infection of 5. The
construction of the recombinant baculoviruses was described earlier
(19-21). Cells were harvested after 65 h, sonicated in ice-cold
20 mM Hepes, pH 7.4, 1 mM MgCl2,
100 mM NaCl, 1 mM DTT, 0.3 mM
phenylmethylsulfonyl fluoride. After centrifugation at 500 × g, the supernatant was collected and centrifuged at
100,000 × g for 30 min at 4 °C. The membrane pellet
was washed, resuspended in the same buffer, and frozen in aliquots in
liquid nitrogen. Protein concentrations were determined by the Bradford
dye-binding assay (22), using bovine serum albumin as a standard. The
expression of recombinant proteins was verified by immunoblotting.
Membrane proteins transferred to nitrocellulose were probed with
antisera specific for the m1 muscarinic receptor (number
71, from G. Luthin, Philadelphia), G
q/11 (number 0945, from D. Manning, Philadelphia) or PLC
1 (anti-holoenzyme) at a dilution of 1:500. Biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate were used for detection, and a Molecular Dynamics densitometer was used to assess expression levels, which varied by no more than 10% for a given recombinant polypeptide. Receptor binding studies using [3H]QNB
(0.02-4.00 nM) as a ligand were also performed to assess the number and affinity of the m1 muscarinic receptors
(23). The nonspecific binding, measured in the presence of 1 µM atropine, was less than 10%. Saturation isoterms were
analyzed using the LUNDON 1 program.
1
1 amino acid sequence was cloned into the
pSC11 vaccinia virus expression vector and HeLa cells were infected
with recombinant virus as described (24). The enzyme was extracted from
the membrane with 2 M KCl for 2 h. The extracted
PLC
1 was consecutively subjected to preparative HPLC on
phenyl-5PW (150 × 21.5-mm column), analytical HPLC on heparin-5PW
(75 × 7.5-mm column), and ion-exchange HPLC on Mono-Q FPLC
(60 × 7-mm column) chromatography as described (24).
1 Assay
q and
PLC
1
q baculovirus and extracted after nitrogen cavitation
(500 p.s.i. for 20 min) with 0.1% CHAPS in 20 mM Hepes, pH
7.5, 1 mM EDTA, 3 mM MgCl2, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride,
and 2 µg/ml each of aprotinin, leupeptin, pepstatin, and benzamidine
at 4 °C for 1 h with constant stirring. The tubes were
centrifuged at 100,000 × g for 30 min at 4 °C.
G
q-containing extracts (protein concentrations as
indicated) were reconstituted into lipid vesicles
(phosphatidylethanolamine:PIP2 at a 4:1 molar ratio) by
means of sonication in 20 mM Tris maleate, pH 6.8, 6 mM MgCl2, 30 mM NaCl, 120 mM KCl, and EGTA and CaCl2 to give a final
concentration of 50 nM Ca2+ (29). The
PIP2 final concentration was 30 µM (1 µCi
of [3H]PIP2). 40 µl of this mixture was
transferred to each experimental tube; 20 µl of purified recombinant
PLC
1 (protein concentration as indicated) in 100 µg/ml
bovine serum albumin was added, and the tubes were sonicated in a
Branson water bath sonicator for 15 min at 4 °C as described above.
Ten µl of GppNHp, tubulin-GppNHp, or tubulin, stripped of nucleotide,
was added at appropriate concentrations to a final volume of 90 µl.
The samples were incubated for 10 min at 30 °C with constant
shaking. The reaction was stopped and [3H]IP3
was quantified as described above.
q, or PLC
1 cDNA. Each of the three
different membrane preparations was extracted with 1% sodium cholate
in buffer A for 1 h at 4 °C with constant stirring. The tubes
were centrifuged at 20,000 × g for 15 min at 4 °C.
Membrane extracts (0.5 mg/ml membrane protein) were incubated with 1 µM tubulin-[32P]AAGTP, as described above.
After UV irradiation and preclearing (Calbiochem Standardized Pansorbin
cells, manufacturer's instruction), each membrane extract was
incubated overnight with appropriate specific or nonspecific antiserum
(1:20 dilution) at 4 °C with constant shaking. Immune complexes were
precipitated with pansorbin cells, and 100 µg of protein of each
immunoprecipitate were subjected to SDS-PAGE and autoradiography. The
antisera used showed no cross-reactivity to tubulin.
-32P]GTP was from ICN
Biomedicals, Inc. [3H]PIP2 was from American
Radiolabeled Chemicals Inc. [3H]QNB was from Amersham
Corp. GppNHp, GDP, and GDP
S were from Boehringer Mannheim.
Carbachol, atropine sulfate, PIP2, phosphatidylcholine, and
phosphatidylethanolamine were from Sigma.
p-Azidoaniline was synthesized by Dr. William Dunn
(University of Illinois, Chicago). Ultrogel AcA 34 was from Pharmacia
Biotech Inc. P6-DG was from Bio-Rad. All other reagents were of
analytical grade.
Receptor-independent Regulation of PLC1 by
Tubulin
q and PLC
1 was performed in Sf9 cells,
and the effects of GppNHp and tubulin with GppNHp bound
(tubulin-GppNHp) on G
q-regulated PLC
1
activity were studied in membranes prepared from infected cells (Fig.
1). GppNHp increased the already high basal
PLC
1 activity in a concentration-dependent and saturable manner (Fig. 1A), confirming the functional
coupling between the recombinant G
q and
PLC
1. GppNHp (at 100 µM) did not affect
PLC
1 activity in membranes from cells expressing only PLC
1 (Table I), thus indicating that
neither G
nor any G
resident in Sf9 cell membranes is capable
of activating the expressed PLC
1. Expressed
G
q did not couple to any endogenous PLC activity. Endogenous PLC activity was also undetectable in the presence or
absence of GppNHp. Tubulin-GppNHp had been shown to activate adenylyl
cyclase in membranes from either COS 1 or C6 glioma cells (16, 17) in a
manner similar to GppNHp. Surprisingly, it appeared that in membranes
prepared from Sf9 cells expressing G
q and
PLC
1 tubulin-GppNHp inhibited PLC
1 under
the same conditions that GppNHp stimulated the enzyme (Fig.
1B).
Fig. 1.
Regulation of receptor-independent
PLC1 activity by tubulin. Sf9 cells were infected
with baculoviruses bearing G
q and PLC
1
cDNAs and cultured as described under "Experimental Procedures." Cells were harvested at 65 h postinfection, and
membranes were prepared as described. 20 µg of Sf9 cell membranes
containing the indicated recombinant proteins and 40 µl of substrate
mixture were incubated for 15 min at 4 °C. GppNHp or tubulin-GppNHp
(TubGppNHp) were added at appropriate concentrations to
a final volume of 80 µl and incubated for 15 min at 37 °C as
described. A, concentration response to GppNHp.
B, comparison of the effects of GppNHp and tubulin-GppNHp,
both at a concentration of 5 µM, on
receptor-independent PLC
1 activity. The
experiments shown are representative of four with similar results. Each
value is mean ± S.D. of duplicate determinations.
[View Larger Version of this Image (24K GIF file)]
Baculovirus-directed expression
GppNHp added
IP3
production
nmol/min/mg
protein
None (normal Sf9
cells)
None
0.14 ± 0.01
100 µM
0.15 ± 0.02
G
q
only
None
0.15 ± 0.03
100 µM
0.16
± 0.03
PLC
1 only
None
0.60 ± 0.01
100 µM
0.57 ± 0.02
To study this further, extracts of Sf9 cells expressing
Gq were reconstituted with purified recombinant
PLC
1 and exogenous phospholipid vesicles and assayed
under conditions similar to those used for Sf9 cell membranes
(Fig. 2). The effects of tubulin-GppNHp, nucleotide-free tubulin, and GppNHp were compared. Although GppNHp stimulated PLC
1 activity at concentrations higher
than 1 µM, 30 nM tubulin-GppNHp activated the
enzyme. However, at higher tubulin-GppNHp concentrations inhibition of
PLC
1 was observed. Nucleotide-free tubulin did not
activate but only inhibited PLC
1 at concentrations
higher than 300 nM.
Tubulin Modulation of m1 Muscarinic Receptor Activation of PLC
Involvement of tubulin in
receptor-triggered PLC1 regulation was studied with
membranes from Sf9 cells expressing m1 muscarinic receptors, G
q, and PLC
1. The
m1 muscarinic receptor expression level was estimated by
receptor binding studies with [3H]QNB as a ligand. When
all three recombinant proteins were expressed, the m1
muscarinic receptor binding capacity (Bmax) was
240 ± 21 fmol/mg membrane protein and Kd = 0.162 ± 0.010 nM (n = 3). The ability
to construct a complete, receptor-activated and G protein-mediated
system allowed a comparison of the effects of GppNHp, tubulin-GppNHp,
and tubulin, denuded of exchangeable nucleotide, on
carbachol-stimulated PLC
1 activity (Fig.
3). Although GppNHp was able to activate
PLC
1 without receptor stimulation, the effect was
potentiated by carbachol (Fig. 3A). The response of
PLC
1 to GppNHp (up to 1 mM) under these
conditions was concentration-dependent, saturable, and
potentiated by carbachol throughout the concentration range (50.5 ± 9.2% at 100 µM GppNHp). However, the effect of
tubulin-GppNHp on PLC
1 activity was again biphasic,
stimulatory at the lower (30 nM) and inhibitory at the
higher concentrations of tubulin (Fig. 3A). Carbachol
potentiated the tubulin-GppNHp-evoked PLC
1 activation.
At 30 nM, tubulin-GppNHp was more efficacious than GppNHp,
independent of the addition of carbachol. The effect of carbachol was
receptor-mediated since, at tubulin concentrations (30 nM)
that stimulated PLC
1, atropine inhibited
carbachol-induced PLC
1 activity by about 80% (Fig.
3B). However, the m1 muscarinic receptor
stimulation was able to overcome the inhibitory effect of higher
tubulin concentrations, resulting in an effective PLC
1 activation. Note that the highest tubulin concentrations used were
below those where tubulin-GppNHp forms polymers. To clarify the
mechanism of this dual regulation of PLC
1 by tubulin,
the effect of nucleotide-free tubulin on enzyme activity was studied (Fig. 3C). Tubulin (without nucleotide) inhibited
PLC
1 activity in a concentration-dependent
manner. No enzyme activation was observed at any tested tubulin
concentration. Furthermore, unlike tubulin-GppNHp, nucleotide-free
tubulin did not potentiate the PLC
1 activation induced
by carbachol. Tubulin-GDP or tubulin-GDP
S (at 30 nM)
also failed to potentiate carbachol-triggered PLC
1 activation or to affect the basal enzyme activity. Thus, it appears that tubulin activates PLC
1 only when GTP or GTP analog
occupies the exchangeable GTP-binding
site.2
Effect of m1 muscarinic receptor
stimulation on PLC1 activity regulated by tubulin.
Sf9 cells expressing recombinant m1 muscarinic receptors,
G
q, and PLC
1 were harvested 65 h
after infection, and membranes were prepared as described under
"Experimental Procedures." PLC
1 activity was assayed
using 20 µg of membrane protein. A, effect of carbachol
(carb) on GppNHp or tubulin-GppNHp (Tub-GppNHp)
regulated PLC
1 activity. Indicated concentrations of
carbachol, GppNHp, or tubulin-GppNHp were added to the membranes, and
PLC
1 activity was assayed as described above.
PLC
1 activity was 1.11 ± 0.03 nmol/min/mg protein
in the presence of carbachol alone. B, atropine inhibition
of carbachol-induced activation of PLC
1 in the presence
of tubulin-GppNHp. PLC
1 activity was studied in the
presence of 30 nM of tubulin-GppNHp, 1 µM
carbachol, and 100 nM atropine as indicated. Control
activity was 0.90 nmol IP3/min/mg protein. C,
comparison of the effects of tubulin-GppNHp and nucleotide-free tubulin
on PLC
1 activity in the presence and absence of
carbachol. Each experiment shown is representative of three with
similar results. Values are mean ± S.D. of duplicate determinations.
Activation of G
To study directly the ability of
tubulin-guanine nucleotide to interact with Gq, the
transfer of the hydrolysis-resistant photoaffinity GTP analog,
[32P]AAGTP, from tubulin to G
q, was
examined in Sf9 cells expressing the m1 muscarinic
receptor, G
q and PLC
1. Nucleotide
transfer from tubulin appears to be responsible for the activation of
G
s and G
i1, and this has been observed in
permeable cells, membrane preparations, and reconstituted systems
(13-17). It has been shown that G
proteins receive
[32P]AAGTP from tubulin under conditions when G
is
incapable of binding the free nucleotide. This appears to be due to the
formation of a complex between tubulin and G
. Under these
conditions, tubulin retains the nucleotide without releasing it to the
media (15). Carbachol (1 mM) increased (36 ± 7%) the
binding of tubulin-[32P]AAGTP to the membrane, and there
was commensurate loss (45 ± 8%) of
tubulin-[32P]AAGTP from the supernatant (Fig.
4). Transfer of [32P]AAGTP from tubulin to
G
q was also potentiated (42 ± 9%) by carbachol.
Atropine blocked carbachol-induced binding of tubulin to the membrane
as well as the increase in [32P]AAGTP
transfer.3 The manner in which the
activated m1 muscarinic receptor increases tubulin
association with the membrane to regulate PLC
1 is
currently under study. The increase in tubulin-GppNHp activation of
PLC
1 brought by carbachol may be explained partially by
this facilitated association of tubulin with the membrane.
These findings, together with the results in Figs. 2 and 3, support the
idea that nucleotide transfer from tubulin to Gq is a
potential mechanism for PLC
1 activation. Carbachol had
no effect on the membrane association of tubulin, nucleotide transfer to G
q, or PLC
1 activity in Sf9 cells
expressing only G
q and PLC
1. No effect of
carbachol, guanine nucleotide, or tubulin-guanine nucleotide was
observed in control Sf9 cells when measuring PIP2 hydrolysis or photoaffinity labeling.
To understand better the mode of interaction of
tubulin-GppNHp with the members of PLC1 signaling
cascade, coimmunoprecipitation studies with
tubulin-[32P]AAGTP were performed. As seen in Fig.
5, both G
q antiserum and, to a lesser
extent, PLC
1 antiserum coimmunoprecipitated tubulin when
membrane extracts of cells infected with viruses for G
q
or PLC
1, respectively, were studied. In the membrane extracts from cells expressing G
q,
[32P]AAGTP-labeling of G
q was also
observed. The source of [32P]AAGTP was tubulin. No
tubulin-[32P]AAGTP coimmunoprecipitation was observed
when extracts from normal Sf9 cells were tested with
anti-G
q or anti-PLC
1 antisera. Thus, it is suggested that the formation of a complex between G
q, PLC
1, and tubulin-GppNHp might be
responsible for the higher efficacy of tubulin-GppNHp, compared with
GppNHp, during the m1 muscarinic receptor stimulation.
PIP2 Binding to Tubulin
In order to clarify the
inhibitory action of tubulin on PLC1, the interaction
between tubulin and the PLC
1 substrate,
PIP2, was studied. It has been shown previously that
phosphatidylinositol inhibits microtubule assembly (34) and that the
binding of profilin (an actin-binding protein) to PIP2
micelles inhibits PLC
1-directed PIP2
hydrolysis (35). To test the possibility that tubulin binds PIP2, a gel filtration assay was performed. Two peaks of
tubulin were resolved, one represented a small portion of oligomeric
tubulin, and the second represented dimeric tubulin (Fig.
6). When the same amount of tubulin was incubated with
PIP2 micelles and passed over an Ultrogel 34 column, an
increase in the higher molecular weight peak was observed along with a
corresponding decrease in the dimeric tubulin peak. When tested under
the same conditions, phosphatidylcholine micelles did not shift the
mobility of tubulin. Since PIP2 forms micelles of about 93 kDa in aqueous solutions (36), the increase in the higher molecular
weight fraction could be attributed to either PIP2-induced
tubulin oligomerization or to a binding of tubulin to PIP2
micelles, as has been observed in the case of profilin (32). To resolve
this, polymerization of tubulin in the absence (Fig.
7A) or presence (Fig. 7B) of
PIP2 micelles was studied by electron microscopy. Tubulin
failed to polymerize properly in the presence of PIP2 (Fig.
7B). Microtubules were considerably shorter (1.7 ± 1.0 µM, compared with 10.2 ± 4.8 µM in the
absence of PIP2) and 3-4 times thicker (75-100 nm versus 25 nm in the absence of PIP2). These
results suggest a direct interaction between tubulin and
PIP2 and support the hypothesis that increased
concentrations of dimeric tubulin might prevent access of
PLC
1 to its substrate. Since increased activation of PLC
1 might evoke localized increases in calcium which could increase tubulin dimer concentration, this might represent a feedback mechanism for the regulation of receptor-G protein-activated phospholipase C. It
is noteworthy in this regard that phosphorylation of
PLC
1 by EGF receptor tyrosine kinase appears to overcome
the inhibitory effect of profilin binding to PIP2,
resulting in an effective activation of the enzyme (35). Similarly,
m1 muscarinic receptor stimulation might reverse
tubulin-evoked inhibition of PLC
1 by increasing the
affinity of G
q for tubulin.
Feedback Inhibition of PLC
It is
suggested that the amount of dimeric tubulin normally accessible to the
membrane is low; thus, initially, stimulation of Gq by
tubulin would predominate. It is possible, however, that tubulin bound
to PLC
1 and/or PIP2 might not bind to
G
q. The observed muscarinic receptor-triggered increase
in G
q affinity for tubulin might be able to overcome
such inhibition. It is also possible that the down-regulation of
PLC
1 signaling would channel heterotrimeric
Gq to another signaling pathway (37, 38). Although complexes of tubulin with G
q, similar to those with
G
i1 or G
s (17), have not yet been shown
in the membrane, clearly the possibility exists for a system in which
membrane-associated tubulin, through reversible interaction with other
membrane proteins and/or lipids, could achieve a dual regulation of
phospholipase C signaling within the cell. It has also been suggested
that the activation of G protein
subunits by tubulin dimer may
provide an interface between G protein signaling through cAMP and G
protein signaling through calcium (39). These dynamic interactions
between G proteins and tubulin dimers at the synapse may also modify
synaptic microarchitecture.
A noteworthy feature of this study is that tubulin regulates
PLC1 signaling while the PLC
1 substrate,
PIP2, may regulate microtubule assembly. These reciprocal
events could be involved both in intracellular signaling and the
control of spindle morphogenesis and reorganization of microtubule
arrays during the cell cycle. Since alterations in the metabolism of
phosphoinositides (40, 41) or the expression of m1
muscarinic receptors (42) or G proteins (43, 44) have been implicated
in cellular transformation, this regulatory mechanism might prove
valuable in the control of cell proliferation as well.
We thank Gary Luthin, David Manning, and
Elliot Ross for their generous gifts of material and Richard Green and
Heidi Hamm for advice and criticism on this manuscript. Madhavi Talluri
is thanked for technical assistance and Sukla Roychowdhury for advice with electron microscopy. Meira Liang is thanked for constructing the
baculovirus containing Gq and Miller Jones is thanked
for G
q preparations.