Hydrolysis of GTP by dynamin is essential for
budding clathrin-coated vesicles from the plasma membrane. Two distinct
domains of dynamin are implicated in the interactions with dynamin
GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to
the pleckstrin homology (PH) domain. In this study we tested the effect
of different phosphoinositides on dynamin GTPase activity and found
that the best activator is phosphatidylinositol 4,5-bisphosphate
followed by
1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains
of dynamin, PRD and PH, can be engaged simultaneously, and determined
the effects of dual occupancy on dynamin GTPase activity. We found that
Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the
dynamin GTPase activity up to 4-fold higher than that obtained by these
activators tested separately, and also reduced the dynamin
concentration required for half-maximal activities by 3-fold. These
results indicate that both stimulators can bind to dynamin
simultaneously resulting in superactivation of dynamin GTPase activity.
We propose that SH3-containing proteins such as Grb2 bind to the
dynamin PRD to target it to clathrin-coated pits and prime it for
superactivation by phosphoinositides.
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INTRODUCTION |
Dynamin is a GTPase required for membrane internalization during
synaptic vesicle recycling and receptor-mediated endocytosis (for
recent reviews, see Refs. 1-5). GTP hydrolysis is necessary for the
cellular functioning of dynamin since overexpression of inactive
dynamin mutants elicits a dominant inhibitory effect on host cell
endocytosis (6, 7). Therefore, the regulation of this enzymatic
activity has been under intense investigation. GTPase activity is
tightly coupled to dynamin self-assembly, which can occur in the
absence of other molecules (8-10), but is facilitated by multivalent
surfaces provided by microtubules (11-15) or anionic liposomes (15).
Specific activity is also increased by phosphorylation (16) and by
interaction with several SH3 domain-containing proteins (17, 18). The
mechanisms by which these interactions regulate dynamin GTPase activity
are poorly understood.
Until recently it was believed that all dynamin activators interact
with a carboxyl-terminal domain of approximately 100 residues designated PRD1 for its high
content of prolines (P) and arginines (R). Negatively charged
molecules, such as microtubules and phosphatidylserine-containing liposomes, bind to the PRD via ionic interactions which are disrupted at physiological ionic strength (14). SH3 domain-containing proteins,
including Grb2 and amphiphysin, bind tightly to specific proline-rich
motifs located in the PRD (18-20).
Another potential site for dynamin interactions is the pleckstrin
homology (PH) domain, a module found in numerous signaling and
cytoskeletal proteins (21, 22). The dynamin PH domain, consisting of
approximately 110 amino acid residues, has been expressed in bacteria
and its structure has been solved by x-ray crystallography (23, 24) and
NMR (25). Like other PH domains whose structures are known, the dynamin
PH domain contains seven amino-terminal
-strands arranged in two
antiparallel sheets and a carboxyl-terminal
-helical segment. Harlan
et al. (21) first showed that the PH domains of numerous
proteins can interact with phosphoinositides. Selectivity of distinct
PH domains for different phospholipids has been recently reported by
Rameh et al. (26). Two groups have now shown the binding of
phospholipids to recombinant dynamin PH domain (27, 28). Using NMR and
fluorescence spectroscopy, Zheng et al. (27) determined that
the dynamin PH domain binds to PI(4)P, PI(4,5)P2, and
phosphatidylserine with equilibrium dissociation constants of 1.8, 4.4, and 47 µM, respectively. Salim et al. (28)
found that a mutant form of dynamin lacking only the PH domain was not
activable by PI(4,5)P2, although it retained Grb2-stimulated GTPase activity. We performed the converse experiment, eliminating the carboxyl-terminal PRD (29). As expected, this truncated
dynamin was not activable by Grb2 or microtubules, but was stimulated
by PI(4,5)P2, presumably due to an interaction with the PH
domain.
This paper addresses two related issues. First, how do specific
phosphoinositides affect the GTPase activity of dynamin? The inositol
rings of phosphoinositides can be modified at multiple positions by
specific kinases and phosphatases, some of which have been implicated
in membrane trafficking events (30). If there is specificity in the
activation of dynamin by phosphoinositides, then these lipid modifying
enzymes are potential regulators of the endocytic process. Second,
since dynamin has two interaction sites, the PRD and the PH domain, can
these sites be occupied simultaneously and, if so, what is the
consequence of simultaneous occupancy for GTPase activation?
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EXPERIMENTAL PROCEDURES |
Materials--
Phosphocellulose (P11) and
diethylaminoethylcellulose (DE52) were from Whatman. Methyl sulfonate
(SP)-Sepharose and glutathione-Sepharose were from Pharmacia Biotech.
PI(4,5)P2, PI(4)P, and phosphatidylcholine were from
Calbiochem.
1-O-(1,2-Di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate ((PI(3,4,5)P3) and
1-O-(1,2-di-O-palmitoyl-sn-glycero-3-benzyloxyphosphoryl)-D-myo-inositol 3,4-bis-phosphate (PI(3,4)P2) were gifts from Dr. C-S.
Chen, University of Kentucky. Protease inhibitors, taxol, papain,
thrombin, and GTP were from Sigma. [
-32P]GTP was from
Amersham.
Purification of Proteins--
Dynamin I was purified from bovine
brains following a procedure described previously (31). Briefly, fresh
bovine brains were homogenized in an Omnimixer with 3 volumes of buffer
A, which contains 0.1 M MES, pH 7.0, 1 mM EGTA,
1 mM MgSO4, 1 mM dithiothreitol, 1 mM sodium azide, and a mixture of protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride and 10 µg/ml each of
N
-benzoyl-L-arginine
methyl ester,
N
-p-tosyl-L-arginine
methyl ester,
N
-p-tosyl-L-lysine
chloromethyl ketone, leupeptin, and pepstatin A. The extract following
centrifugation was chromatographed on three consecutive ion-exchange
columns: DE52-cellulose, P11 phosphocellulose, and SP-Sepharose.
Fractions enriched in dynamin were then mixed with microtubules and
ultracentrifuged. Dynamin, which co-sediments with microtubules, was
released by addition of 10 mM GTP. The supernatant was
finally passed through a DE52 column to remove any traces of tubulin.
All purification steps were carried out using buffer A and columns were
eluted with buffer A containing NaCl.
To remove the carboxyl-terminal PRD, dynamin was proteolyzed with
papain at 30 °C for 20 min at an enzyme to dynamin ratio of 1:1000
(w/w). Papain was first activated by incubation on ice for 15 min in a
solution containing 0.5 M NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 5 mM dithiothreitol.
Digestion was terminated by adding iodoacetic acid to a final
concentration of 5 mM and the digest was mixed with
GST-Grb2 coupled to glutathione-Sepharose to remove PRD fragments and
any traces of undigested dynamin. This treatment yields fragments of 53 and 32 kDa which are noncovalently associated to each other (29).
GST-Grb2 was expressed in Escherichia coli as a fusion
protein with glutathione S-transferase (GST) in a pGEX-2T
vector and purified on glutathione-Sepharose 4B using a standard
procedure. Grb2 was obtained by thrombin cleavage of GST-Grb2 in a
solution containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2.5 mM CaCl2 for
4 h at room temperature. Approximately 130 NIH units of thrombin were used to digest 4 mg of GST-Grb2 attached to glutathione-Sepharose 4B beads. The identity of released Grb2 was confirmed by immunoblotting with anti-Grb2 antibodies and anti-GST antibodies.
NH2-terminal sequencing confirmed that GST-Grb2 was cleaved
at the expected site, producing full-length Grb2. Tubulin was purified
according to the procedure of Williams and Lee (32) but MES rather than PIPES buffer was used. Stable microtubules were obtained by
polymerization of tubulin in the presence of taxol at a 2-fold molar
excess to tubulin dimer.
Preparation of Phospholipid Vesicles--
Phospholipid vesicles
were prepared by dissolving phospholipids in chloroform and drying them
under a stream of argon. Dried lipids were dissolved in 0.1 M MES, pH 7.0, and sonicated for 10 min at maximum power
(Bath sonicator model W185; Heat System Ultrasonics, Inc., Farmingdale,
NY). Phosphoinositide vesicles were prepared as mixtures with
phosphatidylcholine (PC) at a 1:9 molar ratio (33).
Sedimentation Equilibrium--
Sedimentation equilibrium was
performed on a Beckman XLA Analytical Ultracentrifuge. The data
(absorbances at 280 nm) were collected from cell radii of 6.8-7.2 cm,
with a step size of 0.001 cm. Five scans were averaged in the final
output. The initial concentrations of GST-Grb2 and Grb2 were 0.2 and
0.5 mg/ml, respectively. Sedimentation analyses of GST-Grb2 and Grb2
were performed in the same buffer (0.1 M MES, pH 7.0, 1.0 mM EDTA, 1.0 mM MgSO4, 1.0 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, and 1 mM NaN3). GST-Grb2 was
centrifuged at 10,000 rpm, and Grb2 at 15,000 rpm in rotor An-60ti
using a 12-mm double-sector centerpiece. The background absorbance was
estimated by overspeeding at 40,000 rpm until a flat baseline was
obtained. The overspeed absorbances were 0.019 and 0.018 for Grb2 and
GST-Grb2, respectively. The partial specific volume of Grb2 was
calculated to be 0.73 ml/g by the method of Cohn and Edsall (34). The
actual molecular masses of Grb2 and GST-Grb2 were calculated to be 26 and 52 kDa, respectively.
GTPase--
GTPase activities were measured by the release of
32Pi from [
-32P]GTP (35) after
incubation at 37 °C in buffer A containing additionally 1 mM MgGTP. The reaction times varied from 2 to 30 min,
depending on dynamin or lipid concentrations, to ensure that less than
15% of GTP was hydrolyzed. Dynamin was preincubated with lipids and/or
Grb2 for 10 min at room temperature prior to initiation of the reaction
by addition of 5 mM MgGTP. In some experiments (as
indicated in the figure legends) buffer A contained additionally 0.1 M NaCl. Although a high concentration of MES was used in
these assays, this zwitterionic buffer contributes little to the ionic
strength of the solution. Except in the case where the effect of salt
was examined (Fig. 3), all GTPase assays were carried out at low ionic
strength, allowing a comparison of our results with those of other
investigators, who also employ low salt assay conditions (16-18, 28,
36).
Turbidity Measurements--
Samples were placed in a 1-cm path
length cuvette and absorbance at 340 nm was determine using a Beckman
DU 650 spectrophotometer.
Other Methods--
Protein concentration was determined as
described by Bradford (37) using bovine serum albumin as a standard.
SDS-polyacrylamide gel electrophoresis was carried out according to the
method of Laemmli (38) as modified by Matsudaira and Burgess (39).
 |
RESULTS |
Specificity of Dynamin Activation by
Phosphoinositides--
Inositol phospholipids, which have been
implicated in the regulation of dynamin GTPase activity, are subject to
phosphorylation and dephosphorylation at multiple positions of the
inositol ring. Here we show that the site of inositol phosphorylation
is a key determinant for dynamin activation. The order of efficacy is
PI(4,5)P2 > PI(3,4,5)P3 > PI(4)P (Fig.
1). At concentrations below 1 µM, PI(4)P is somewhat more effective than
PI(3,4,5)P3. However, maximal PI(4)P stimulated activity is
only about 50 min
1, less than half the value obtained
with PI(3,4,5)P3 and about one-third of
PI(4,5)P2 stimulated activity. PI(3,4,)P2,
which has the same charge as PI(4,5)P2, is by far the least
effective activator, providing no stimulation even at concentrations as high as 40 µM.

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Fig. 1.
Activation of dynamin I GTPase activity by
various phosphoinositides. A, dynamin GTPase activity as a
function of phosphoinositide concentrations. GTPase of dynamin at 0.1 µM concentration was assayed as described under
"Experimental Procedures." GTPase activities decrease at high
concentrations of PI(4,5)P2 and PI(3,4,5)P3, presumably due to the inhibition of dynamin-dynamin interactions previously shown to occur in the presence of high concentrations of
phosphatidylserine or microtubules (14). B, data from
panel A were replotted as a bar graph to
highlight the differences at maximal activation. Lipid concentrations
giving maximal activation were: 4 µM
PI(4,5)P2 and PI(3,4,5)P3, 16 µM
PI(4)P, and 40 µM PI(3,4)P2. Data represent
the mean ± S.E. from three experiments, each done in duplicate
for an n of 6.
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We then asked if PI(3,4)P2 binds to dynamin despite its
inability to stimulate GTPase activity. In a competition assay,
PI(4,5)P2 stimulated activity was measured in the presence
of various concentrations of PI(3,4)P2. If
PI(3,4)P2 binds to dynamin, it should displace PI(4,5)P2 from its binding site and thus inhibit GTPase
activity. However, no inhibition by PI(3,4)P2 was observed,
even at a 10-fold molar excess over PI(4,5)P2 (Fig.
2). Therefore, PI(3,4)P2 has a much lower affinity for dynamin than PI(4,5)P2, or binds
at a different site on the molecule.

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Fig. 2.
PI(4,5)P2-stimulated dynamin
GTPase is not inhibited by PI(3,4)P2. The GTPase
activity of dynamin (0.1 µM) was assayed in the presence
of 4 µM PI(4,5)P2 and 40 µM
PI(3,4)P2. The bars indicate average values of
duplicate measurements.
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Dynamin contains two potential sites for phosphoinositide binding, the
PH domain and the highly basic carboxyl-terminal PRD. Ionic charges
promote the associations of anionic phospholipids with the PRD and
interactions are significantly reduced at physiological ionic strength
(14). Therefore, by measuring lipid-stimulated GTPase activity in the
presence of 100 mM NaCl, as in Fig.
3, it is likely that interactions with
the PH domain predominate. In Fig. 3 we show the dependence of GTPase
specific activity on dynamin concentration, at fixed concentrations of
phosphoinositides. Unlike most enzymes, the specific activity of
dynamin is not invariant as a function of dynamin concentration, but
increases in a highly cooperative manner (15). This presumably reflects
the well established propensity of dynamin to self-associate and the
consequent increase in GTPase activity that is coupled to this
self-association. We had previously shown that
PI(4,5)P2-containing vesicles provide a multivalent surface
that allows cooperative increases in GTPase activity even at relatively
low dynamin concentrations (29). Fig. 3 indicates that
PI(4,5)P2 retains its ability to support high specific
activity at 100 mM NaCl whereas salt greatly diminishes the
efficacies of PI(3,4,5)P3 and PI(4)P. Therefore, under
conditions more closely resembling those in the cell, only
PI(4,5)P2 appears to be a potent stimulator of dynamin
activity.

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Fig. 3.
Specific GTPase activities in the presence of
4 µM phosphoinositides as a function of dynamin
concentration. Experiments were performed in buffer A containing
additionally 0.1 M NaCl. Data represent the mean ± S.E. from two experiments, each done in triplicate for an n
of 6.
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The sensitivity of the PI(3,4,5)P3-dynamin interaction to
ionic strength raised the possibility that this phosphoinositide binds
primarily to the PRD. However, this proved not to be the case. To
examine this, we deleted the PRD by papain treatment of dynamin, which
yields two non-covalently associated fragments of 53 and 32 kDa. The
53-kDa fragment contains the GTP-binding site (thus originating from
the NH2-terminal portion of dynamin), whereas the 32-kDa
fragment contains the PH domain and extends to the beginning of the PRD
(Fig. 4, see also Ref. 29). As we showed
in a prior study, the 53/32-kDa fragments are activated by
PI(4,5)P2, but not by microtubules or Grb2. Activation of
the papain fragments by phosphoinositides follows the basic pattern observed with intact dynamin: PI(4,5)P2 and
PI(3,4,5)P3 are the best activators, PI(4)P is much less
effective, and PI(3,4)P2 fails to activate. Therefore, at
low ionic strength, PI(3,4,5)P3 binds at least as well as
PI(4,5)P2 to a truncated dynamin lacking the PRD. This
result agrees with that of Salim et al. (28) who demonstrated by surface plasmon resonance that PI(3,4,5)P3
can bind to expressed dynamin PH domains at low ionic strength but not
in the presence of 1 µM Ca2+ and 2 mM Mg2+, which more closely reflect
physiological conditions. Our results also suggest that a phosphate at
the 5-position of the inositol ring is necessary for high levels of
activation, whereas a phosphate at the 3-position reduces the
phospholipid activation of dynamin.

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Fig. 4.
Activation of dynamin proteolytic fragments
by phosphoinositides. A, scheme of the dynamin digestion
pattern. Digestion of dynamin with papain, as described under
"Experimental Procedures," yields a 53-kDa fragment which is
noncovalently associated with a 32-kDa fragment. B, GTPase
assays of the 53/32-kDa fragments. Protein concentration in the GTPase
assay was 0.12 µM. Each point represents the average of
duplicate measurements.
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Superactivation of Dynamin GTPase Activity by Grb2 and
PI(4,5)P2--
Most in vitro activators of
dynamin GTPase activity, including microtubules and SH3-containing
proteins, interact solely with the carboxyl-terminal PRD (14, 17, 18).
Instead, activation by phosphoinositides involves interactions outside
the PRD, presumably with the PH domain (Ref. 29, see also Fig. 4). This
raises the question of whether or not phosphoinositides and
PRD-binding proteins can interact simultaneously with dynamin and, if
so, how simultaneous binding influences GTPase activation. To address
this question we set out to determine the effect of Grb2, an
SH3-containing protein, on phosphoinositide-stimulated GTPase
activity.
First it was necessary to determine the extent to which Grb2 alone
could stimulate dynamin activity. All previous studies have utilized
expressed fusion proteins of Grb2 and GST. However, since GST has a
tendency to dimerize (40), these GST-Grb2 fusion proteins are also
predominantly dimeric, as we verified by sedimentation equilibrium
(Fig. 6A). This multivalency poses a particular problem when
considering dynamin GTPase activation, which can even occur upon
cross-linking with anti-dynamin antibodies (36). Therefore, we prepared
nonfusion Grb2 by thrombin cleavage of GST-Grb2 (Fig. 5), and verified its monomeric nature by
sedimentation equilibrium (Fig.
6B). Fig.
7 shows that the nonfusion Grb2
stimulates dynamin activity in a dose-dependent manner
which plateaus at greater than 2 µM Grb2. In contrast,
GST-Grb2 yields a biphasic activation which peaks at 0.5 µM GST-Grb2. These differences may be a consequence of
the different complex-forming abilities of Grb2 versus
GST-Grb2. A low speed co-sedimentation assay revealed that both
molecules can cross-link dynamin into large networks (Fig.
8A). Under conditions wherein
dynamin alone, GST-Grb2 alone, and Grb2 alone remain soluble (13,000 × g, 15 min), mixtures of dynamin with either
GST-Grb2 or Grb2 are pelletable. The formation of a large complex is
also demonstrated by a rapid increase in turbidity when Grb2 or
GST-Grb2 are added to dynamin. The traces shown in Fig. 8B
were obtained using 0.1 µM dynamin and 2 µM
Grb2 or GST-Grb2. Clearly, GST-Grb2, which is tetravalent, is a more
potent dynamin cross-linker than the divalent Grb2. The nature of these
differences is still unclear, although it is possible to draw an
analogy with the antigen-antibody precipitin reaction (41) (see
"Discussion").

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Fig. 5.
Electrophoretic analysis of GST-Grb2 and
Grb2. The left lane of each panel shows GST-Grb2, the
right lane shows Grb2. Molecular mass markers designated on
the left are 200, 116, 96, 66, 45, 31, 21.5, 14.4, and 6.5 kDa. Panel A, Coomassie Blue-stained gel. Panel
B, immunoblot with anti-Grb2 antibody. Panel C,
immunoblot with anti-GST antibody.
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Fig. 6.
Sedimentation equilibrium analysis of
GST-Grb2 and Grb2. A, the lower panel presents
data obtained at 10,000 rpm, 4 °C for GST-Grb2 as well as predicted
profiles for monomer (M), dimer (D), and
monomer-dimer equilibrium (M-D) model. Initial GST-Grb2 concentration was 0.2 mg/ml. B, the lower panel
presents data obtained at 15,000 rpm, 4 °C for Grb2 and the
predicted profiles for monomeric and dimeric species. Initial Grb2
concentration was 0.5 mg/ml. Residual plot for the monomer-dimer fit of
GST-Grb2 data and the monomer fit of Grb2 data are shown in the
upper panels. The analysis indicates that Grb2 alone behaved
as a single species of about 28,000 Mr while
GST-Grb2 gave a more complex equilibrium profile consistent with a
monomer/dimer equilibrium and an association constant
(Ka) of 3 × 106
M 1.
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Fig. 7.
Activation of dynamin GTPase by GST-Grb2 and
Grb2. GTPase activities were assayed at 0.1 µM
dynamin in the presence of GST-Grb2 or Grb2. Data represent the
mean ± S.E. from two experiments, each done in duplicate, for an
n of 4.
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Fig. 8.
Dynamin cross-linking by Grb2 and GST-Grb2.
A, co-sedimentation assay. Dynamin (0.3 µM)
was mixed with 1 µM of either Grb2 or GST-Grb2, incubated
for 10 min at room temperature, and centrifuged at 13,000 × g for 15 min. Pellets were resuspended to equal volumes as
supernatants and aliquots of each were electrophoresed. The gel was
stained with Coomassie Brilliant Blue. Molecular weights of markers are
shown on the left. B, turbidity was measured as absorbance
at 340 nm. Cuvettes contained 0.1 µM dynamin I. At a time
designated by the arrow, 2 µM Grb2 or GST-Grb2
were added. In the absence of dynamin, neither Grb2 nor GST-Grb2 gave
any increase in absorbance.
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Grb2 activates dynamin GTPase to maximal levels of only 50-60
min
1. Therefore, if Grb2 competes with
PI(4,5)P2 for dynamin binding, it should inhibit
PI(4,5)P2 stimulated activity. Instead, dynamin specific
activity in the presence of both Grb2 and PI(4,5)P2 is considerably higher than the additive activities obtained if the two
activators were introduced separately. At 4 µM Grb2,
PI(4,5)P2-stimulated GTPase activity was about double that
obtained in the absence of Grb2 (Fig. 9).
This synergistic effect of Grb2 and phospholipids on GTPase activation
provides strong evidence that these two molecules can bind
simultaneously to dynamin.

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Fig. 9.
Grb2 potentiates GTPase activity of dynamin
stimulated by PI(4,5)P2. The GTPase activity of
dynamin (0.1 µM) was assayed as a function of
PI(4,5)P2 concentration in the absence or presence of 4 µM Grb2. Dotted line, theoretical curve
showing expected activities if stimulation by PI(4,5)P2 and
Grb2 were simply additive. Data represent the mean ± S.E. from
two experiments, each done in duplicate, for an n of
4.
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The experiments shown in Fig. 9 were carried out at a fixed
concentration of dynamin (0.1 µM). However, as shown
above, there is a highly cooperative increase in
PI(4,5)P2-stimulated GTPase activity as a function of
dynamin concentration. In view of the synergy between Grb2 and
PI(4,5)P2, we checked if the cooperative dynamin dependence
of activity was also affected by the joint presence of both activators.
Fig. 10 shows that Grb2 activates GTPase slightly, compared with the large increase obtained with PI(4,5)P2 alone. However, in the presence of both Grb2 and
PI(4,5)P2, there is a marked shift in the dynamin
concentration dependence from that obtained in the presence of
PI(4,5)P2 alone. At 4 µM PI(4,5)P2, half-maximal activities occur at 0.11 µM dynamin. The effect of adding Grb2, also at 4 µM, reduces these dynamin concentration dependences
approximately 3-fold, to 0.032 µM. The synergistic activation by Grb2 and phosphoinositides is particularly prominent at
suboptimal levels of dynamin. For example, at 0.05 µM
dynamin the GTPase activity is 4-fold higher in the presence of both
activators than in the presence of PI(4,5)P2 alone. Since
Grb2 has two SH3 domains and dynamin has multiple potential SH3-binding
motifs within its PRD, a plausible explanation for the synergistic
activation is that Grb2 cross-links dynamin molecules into a cluster by
virtue of its two SH3 domains and thus increases its effective
concentration on the phospholipid vesicles surface.

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Fig. 10.
Stimulation of dynamin GTPase activity by
Grb2 and PI(4,5)P2. GTPase activity was assayed as a
function of dynamin concentration at fixed concentrations (4 µM) of Grb2 and phosphoinositide. The values obtained
with Grb2 alone were subtracted. In the absence of
PI(4,5)P2 or Grb2, increases in activity as a function of
dynamin concentration were negligible throughout the concentration
range tested. Data represent the mean ± S.E. from two
experiments, each done in duplicate, for an n of 4.
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 |
DISCUSSION |
In this paper we present two observations pertinent to the
regulation of dynamin: first, at physiological ionic strength dynamin GTPase activity is stimulated by PI(4,5)P2, less so by
PI(3,4,5)P3, but not at all by PI(4)P or
PI(3,4)P2, and second, another dynamin activator, Grb2,
binds to dynamin simultaneously with PI(4,5)P2, resulting
in synergistic rather than additive stimulation of activity.
Previously, we had shown that the GTPase activity of dynamin is
stimulated by liposomes containing PI(4,5)P2, even in the absence of its COOH-terminal proline and arginine-rich domain (29).
This domain was earlier thought to participate in all interactions
leading to GTPase activation. It is likely that PI(4,5)P2 stimulates GTPase activity by interacting with the dynamin PH domain,
because recombinant PH domains bind to phosphoinositides and dynamin
mutants lacking the PH domain are not activated by these phospholipids
(27, 28). In the present study we found that PI(3,4,5)P3 is
also a potent stimulator of dynamin, but only at low ionic strength. In
contrast, PI(4)P and PI(3,4)P2 are poor activators under
all conditions examined. These results prompt us to speculate that in
cells, dynamin activity can be reduced or terminated by the action of a
phosphatidylinositol-5-phosphatase, such as synaptojanin, which
converts PI(4,5)P2 or PI(3,4,5)P3 to PI(4)P and
PI(3,4)P2, respectively (42, 43). Synaptojanin is enriched
at clathrin-coated pits and interacts with amphiphysin, a
dynamin-binding protein also enriched at the coated pit (42, 44).
Grb2, an adaptor protein that consists of two SH3 domains flanking a
central SH2 domain, binds exclusively to the dynamin PRD (17, 18).
Presumably, Grb2 stimulates dynamin GTPase activity as a consequence of
cross-linking the enzyme by virtue of its two SH3 domains. We found
that dynamin and Grb2 form large complexes, as monitored by light
scattering and low-speed centrifugation. Although the role of Grb2 in
endocytosis is uncertain, it has been speculated to serve as a linker
between activated tyrosine kinase receptors and dynamin (45). Until
now, all in vitro studies of Grb2-dynamin interactions have
employed bacterially-expressed fusion proteins with GST. We treated
GST-Grb2 with thrombin to obtain nonfusion Grb2 and confirmed that it
is monomeric, in contrast to GST-Grb2 which is predominantly dimeric.
Thus, GST-Grb2 contains four dynamin-binding SH3 domains whereas Grb2
itself has only two, presumably accounting for the greater efficacy of
GTPase stimulation by GST-Grb2 which we observed. The biphasic nature of GTPase activation by GST-Grb2 is analogous to the precipitin reaction between antigens and antibodies (41). In the presence of
excess GST-Grb2, single dynamin molecules are coated by multiple molecules of GST-Grb2, thus preventing the formation of a cross-linked dynamin lattice needed for GTPase activation. Monomeric Grb2, which is
bivalent, would be expected to yield a similar drop in GTPase activity
but at higher concentrations than those obtained with the tetravalent
GST-Grb2. Because dynamin GTPase activity is stimulated by
cross-linking, even with anti-dynamin antibodies (36), our results
suggest that previous reports of dynamin activation by GST-SH3 fusion
proteins should be re-evaluated and that only monomeric SH3-containing
species should be used in the future to avoid artifactual cross-linking
caused by GST dimerization.
Compared with PI(4,5)P2, Grb2 by itself is a relatively
weak stimulator of dynamin GTPase activity. Its major effect on
activity is the synergistic stimulation with PI(4,5)P2
which is most pronounced at low dynamin concentrations. Because there
is no evidence for an interaction between Grb2 and
PI(4,5)P2, the simplest explanation for this synergy
involves simultaneous binding of the two activators to dynamin. For
example, Grb2 may cluster dynamin molecules by cross-linking their SH3
binding domains, thus increasing the effective concentration of dynamin
bound to PI(4,5)P2-containing liposomes. In the absence of
Grb2, dynamin molecules would be dispersed on the liposome surface
until high enough concentrations were reached to promote the
dynamin-dynamin interactions required for high enzymatic activity.
The ability of dynamin to interact simultaneously with phospholipids
and Grb2 raises the possibility of simultaneous interaction with other
SH3-containing proteins such as amphiphysin (20). The importance of
amphiphysin for dynamin targeting was highlighted by the recent study
of Shupliakov et al. (46) who introduced expressed
amphiphysin SH3 domains into lamprey neurons, displacing dynamin from
the coated pit and blocking synaptic membrane recycling.
There is strong evidence that SH3-binding motifs in the PRD are
required to target dynamin to coated pit regions of the plasma membrane
(19). Likewise, it appears that phosphoinositides bind to the dynamin
PH domain and potently stimulate GTPase activity by this interaction.
Based on these results and our current observations, we propose the
following model for dynamin regulation: dynamin is first recruited to
the coated pit by SH3-containing protein, e.g. Grb2 or
amphiphysin. Once there, dynamin's interaction with the membrane is
strengthened by the binding of its PH domain with PI(4,5)P2
and, perhaps, PI(3,4,5)P3 which may also orient dynamin to
favor its polymerization. In a similar manner, binding of
-adrenergic receptor kinase to G
is favored by the
presence of both inositol phospholipids and G
on the
membrane (47). In the case of dynamin, these phospholipids also promote
self-association and GTPase stimulation, which could be reversed by the
enzymatic action of a phosphoinositide 5-phosphatase, e.g.
synaptojanin. This model is highly speculative, but it incorporates the
key observation presented in this paper, i.e. that
phospholipids and SH3-containing proteins bind simultaneously to
distinct domains of the dynamin molecule and synergistically activate
dynamin GTPase.
We thank Dr. Ching-Shih Chen for providing
phospholipids (PI(3,4,5)P3 and PI(3,4)P2). We
also thank Irma Rodman for expert technical assistance.