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INTRODUCTION |
Vital cellular responses including cell metabolism, proliferation,
and differentiation are regulated by specific interactions between
extracellular ligands and their plasma membrane-anchored receptors.
Accessibility of ligand to receptor, is therefore, an important facet
of biological regulation. Receptor expression on the cell surface is
dictated, at least in part, by vesicle trafficking via regulation of
receptor internalization. Receptor internalization (also termed
endocytosis) is implicated in receptor resensitization,
down-regulation, and more recently signal transduction (1, 2).
Endocytosis of G protein-coupled receptors and receptor tyrosine
kinases is dependent on the invagination, constriction, and fission of
vesicles (clathrin-coated and caveolae) from the plasma membrane into
the cytosol. Dynamin, a large GTPase, plays a crucial role in these
steps of receptor-mediated endocytosis (3, 4).
The GTPase activity of dynamin is stimulated 5-10-fold over basal
levels by self-assembly (5). In the native state, dynamin exists as a
homotetramer (6). At low ionic strength conditions (7), or in the
presence of GDP and 
-phosphate analogues at physiological salt
conditions (8), concentrated dynamin tetramers spontaneously
self-assemble into spiral stacks of rings, similar to the structure
which appears as an electron dense "collar" around the necks of
endocytic pits observed at synaptic nerve terminals (9, 10). Purified
dynamin also assembles onto phospholipid liposomes to generate
dynamin-coated helical tubes that constrict and vesiculate upon GTP
addition (11, 12). On the other hand, dynamin-decorated phospholipid
nanotubes undergo nucleotide-dependent conformational
changes causing extension in pitch along the dynamin helix without
constriction or fragmentation (13, 14).
Recently, the GTPase effector domain of dynamin was shown to play a
role in self-assembly and subsequent increase in GTPase activity (6,
15). The GTPase effector domain has an intrinsic GTPase activating
protein activity and interacts with the N-terminal GTPase domain to
stimulate GTP hydrolysis. Although the molecular details governing the
action of dynamin in the fission of vesicles from the plasma membrane
have been controversial, it is, nonetheless, clear that GTP binding and
hydrolysis play critical roles (3, 4).
Tyrosine phosphorylation plays an important, if poorly understood, role
in the internalization of cell surface receptors. Exposure of cells to
tyrosine kinase inhibitors profoundly attenuates B cell receptor (16)
and asialoglycoprotein receptor (17) endocytosis. Furthermore,
inhibition of the non-receptor tyrosine kinase c-Src attenuates stem
cell factor-induced c-Kit internalization in hematopoietic cells (18),
antibody-induced internalization of neural cell adhesion molecule L1 in
neuroblastoma cells (19), and
EGFR1 endocytosis (20).
Conversely, overexpression of c-Src causes an increase in EGFR
internalization rate following EGF treatment (21).
Some components of the cellular endocytic machinery have been shown to
undergo regulated tyrosine phosphorylation. In the case of EGFR
internalization, one such target is clathrin. Stimulation with EGF
increases c-Src-mediated tyrosine phosphorylation of clathrin, which
regulates clathrin translocation to the plasma membrane (20). Another
target is dynamin, which can directly interact with c-Src (22) and
becomes tyrosine-phosphorylated in response to insulin (23) and
lysophosphatidic acid (24) stimulation. The c-Src-mediated
tyrosine phosphorylation of dynamin is required for agonist-induced
endocytosis of 
2-adrenergic (25) and M1 muscarinic
acetylcholine (26) receptors. However, unlike clathrin, the mechanisms
whereby phosphorylation of dynamin regulates receptor-mediated
endocytosis are unclear. Here, we show that tyrosine phosphorylation of
dynamin induces its self-assembly and subsequent increase in GTPase
activity, and is required for agonist-induced EGFR internalization.
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EXPERIMENTAL PROCEDURES |
Expression Plasmids--
Expression vectors for wild type and
K44A dynamin have been described before (25). To generate single
tyrosine mutants (Y231F, Y354F, and Y597F), each tyrosine residue of
the wild type or K44A dynamin I was mutated to phenylalanine by
overlapping polymerase chain reactions (UAC or UAU (Y) 
UUC or UUU
(F)). The double-tyrosine mutant (Y231F/Y597F) dynamin was
constructed by recombination of the two single tyrosine mutant
constructs. All dynamin I cDNAs were subcloned in pcDNA3
expression vector. Baculovirus transfer vector containing dynamin I
cDNA (wild type or Y231F/Y597F) was constructed in
EcoRI/NotI sites of pVL1393 plasmid. The plasmid encoding the FLAG epitope-tagged EGFR was generated by polymerase chain
reactions. The FLAG epitope coding sequences were introduced at the 5'
end of EGFR cDNA in pBK expression vector.
Cell Culture and Transfection--
LipofectAMINE and tissue
culture reagents were from Invitrogen. COS-7 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 100 µg ml
1 gentamicin at
37 °C in a humidified 5% CO2 atmosphere. 70-80% confluent COS-7 cells in 100-mm plates were transfected with a total of
7 µg of DNA and 35 µl of LipofectAMINE according to the manufacturer's instructions. One day after transfection, cells were
detached by trypsin and seeded in either two 100-mm plates (for
immunoprecipitation) or 24-well dishes (for enzyme-linked immunosorbent
assay). All assays were performed ~60 h after transfection.
Dynamin Immunoprecipitation and
Immunoblotting--
Serum-starved (overnight) dynamin-expressing cells
in 100-mm plates were pretreated with the appropriate concentrations of chemicals as indicated in the figure legends. The Src kinase inhibitor PP2 and the EGFR kinase inhibitor tyrphostin AG1478 were from Calbiochem. Sodium orthovanadate (Na3VO4) was
from Sigma. Cells were stimulated with 10 ng ml1 EGF
(Calbiochem) at 37 °C for the indicated times, washed once with
ice-cold phosphate-buffered saline, lysed in 1 ml of glycerol lysis
buffer (5 mM HEPES, pH 7.4, 250 mM NaCl, 10%
(v/v) glycerol, 0.5% Nonidet P-40, 2 mM EDTA, protease
inhibitor tablets (Roche Molecular Biochemicals), 1 mM
phenylmethylsulfonyl fluoride, 100 µM
Na3VO4) on ice, and clarified by
centrifugation. Cell lysates were mixed with 2 µg of a monoclonal
anti-dynamin antibody (Hudy-1; Upstate Biotechnology) and 70 µl of
30% slurry of Protein G plus/Protein A-agarose beads (Oncogene) and
rotated for 4 h at 4 °C. Immune complexes were washed three
times with ice-cold glycerol lysis buffer containing 0.1% (w/v) SDS
and denatured in Laemmli sample buffer followed by boiling.
Immunoprecipitated proteins were resolved on 4-20% SDS-polyacrylamide
gels (Invitrogen) and transferred to nitrocellulose membranes
(Bio-Rad), immunoblotted with a 1:2,000 dilution of a horseradish
peroxidase-conjugated anti-phosphotyrosine antibody (PY20H;
Transduction Laboratories) to detect tyrosine phosphorylation signals.
Before immunoprecipitation, 50 µl of the cell lysate was aliquoted to
provide samples for dynamin expression determined by immunoblotting
with a 1:2,000 dilution of an anti-dynamin antibody (Hudy-1) followed
by a 1:5,000 dilution of a horseradish peroxidase-conjugated anti-mouse
secondary antibody (Jackson ImmunoResearch). Proteins were visualized
using Supersignal chemiluminescence reagent (Pierce) and quantified
using Fluor-S MultiImager (Bio-Rad).
Dynamin Isolation Using GST-Grb2 Affinity Binding--
Isolation
of endogenous dynamin II for analysis of its tyrosine phosphorylation
content was performed by affinity purification with full-length
GST-Grb2 fusion protein as described previously (24, 25). Briefly,
serum-starved cells in 100-mm plates were stimulated with 10 ng
ml1 EGF at 37 °C for the indicated times in the
presence of 100 µM Na3VO4, washed
once with ice-cold phosphate-buffered saline, and lysed in 1 ml of
glycerol lysis buffer on ice. Cell lysates were clarified by
centrifugation and incubated with the GST-Grb2 fusion protein (10 ng
for 1 µg of cell lysates), immobilized on glutathione-conjugated agarose beads, for 3 h at 4 °C. Protein complexes were
extensively washed with ice-cold glycerol lysis buffer containing 0.5 M NaCl and 0.1% (w/v) SDS, resuspended in Laemmli sample
buffer, and resolved on SDS-polyacrylamide gels. Tyrosine
phosphorylation of dynamin II was detected by immunoblotting as
described above. The amount of isolated dynamin II was determined by
immunoblotting with a 1:250 dilution of an anti-dynamin II antibody
(Transduction Laboratories).
Sequestration of EGF Receptor--
Agonist-induced
internalization of FLAG epitope-tagged EGFR was determined by
enzyme-linked immunosorbent assay as described (27). COS-7 cells
transiently expressing FLAG-EGFR in 24-well dishes were washed once
with phosphate-buffered saline and incubated with Dulbecco's modified
Eagle's medium containing EGF (10 ng ml1) for 30 min at
37 °C. All subsequent steps were performed at room temperature.
Stimulation was terminated by removing media and fixing the cells with
3.7% formaldehyde in TBS (20 mM Tris, pH 7.5, 150 mM NaCl) for 10 min. Cells were washed three times with TBS
and incubated in a blocking solution (TBS containing 1% bovine serum
albumin) for 45 min. A M1 anti-FLAG monoclonal antibody (Sigma) was
added to the cells at a dilution of 1:1,000 in the blocking solution
supplemented with 1 mM CaCl2 for 1 h. After three washes with TBS, cells were reblocked for 15 min and incubated with an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Sigma) diluted 1:1,000 in the blocking solution for
1 h. Cells were washed three times with TBS, and 200 µl of a
colorimetric alkaline phosphatase substrate (Bio-Rad) was added to each
well. When adequate color change was achieved, 100-µl reaction
solutions were transferred to 96-well plates containing 100 µl of 0.4 M NaOH, and absorbance was determined using a microplate reader (Bio-Rad) at 405 nm. Background signal was concurrently determined using empty vector-transfected cells and subtracted from
each of the values for the receptor-transfected cells. Receptor sequestration was defined as the fraction of total cell surface receptors that were removed from the plasma membrane following agonist
treatment and thus were not accessible to antibody from outside of the cell.
Purification of Recombinant Rat Dynamin I--
Dynamin
expression baculoviruses were generated by co-transfection of
recombinant baculovirus transfer vector (pVL1393), containing either
wild type or Y231F/Y597F rat dynamin I cDNA, together with BaculoGold DNA using the BaculoGold Transfection Kit (BD PharMingen). Purification of dynamin was carried out essentially as described (5).
Briefly, 3 liters of suspension culture of Sf9 cells (1 × 106 cells ml1) were infected with high titer
dynamin I-encoding recombinant virus stocks and harvested 48 h
later by centrifugation at 2,500 × g for 15 min.
Pellets were washed in ice-cold phosphate-buffered saline and
resuspended in 50 ml of ice-cold HCB (20 mM HEPES, pH 7.0, 2 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, protease inhibitor tablets (Roche
Molecular Biochemicals), and 1 mM phenylmethylsulfonyl fluoride) containing 100 mM NaCl (HCB100). Cells were
disrupted by ultrasound sonication (7× 10-s pulses on ice), diluted
1:1 with HCB containing 50 mM NaCl (HCB50) and clarified by
centrifugation at 50,000 rpm in TFT 50.38 rotor (Sorvall) for 1 h.
Ammonium sulfate ((NH4)2SO4) was
slowly added to the supernatant fraction up to 30% saturation, and the
mixture was stirred for 30 min at 4 °C followed by centrifugation
for 15 min at 10,000 × g. Pellets were resuspended in
50 ml of HCB50 using a loose fitting Dounce homogenizer and centrifuged
again for 10 min at 10,000 × g. Solubilized 30% (NH4)2SO4 was applied to a
5-ml Q-Sepharose anion exchange column (Amersham Biosciences)
pre-equilibrated with HCB50. The column was washed with 100 ml of
HCB100, and dynamin was then eluted with HCB containing 150 mM NaCl (HCB150). 2-ml fractions were collected at 1 ml
min1, and dynamin-containing fractions, identified by
immunoblotting, were combined. Purified dynamin in HCB150 was
concentrated up to 2 mg ml1 using Vivaspin 20 (Vivascience). Protein assays and SDS-PAGE were used to assess protein
yield and purity, which was greater than 90% as judged by Coomassie
Blue staining.
In Vitro Dynamin Phosphorylation--
Purified c-Src
(recombinant human c-Src (
N85), lacking the first 85 amino acid
residues) was the kind gift of Y-C. Ma (28) and M. J. Eck (29).
Purified recombinant dynamin I and c-Src were mixed in 50 µl of
kinase reaction buffer (10 mM Pipes, pH 7.0, 10 mM MnCl2, 50 mM NaCl, 100 µM ATP, 10 µCi of [-32P]ATP) on ice.
Reactions were performed at either 37 °C or room temperature for the
indicated times and terminated by addition of Laemmli sample buffer
followed by 5 min boiling. Phosphorylated dynamin was resolved by
SDS-PAGE, visualized by autoradiography, and quantified using a Storm
PhosphorImager (Amersham Biosciences) to calculate
phosphorylation stoichiometry.
In Vitro Dynamin GTPase Assay--
Dynamin GTPase activity was
determined by measuring the release of 32Pi
from [-32P]GTP-dynamin as described (30). Purified
recombinant dynamin I in kinase reaction buffer (routinely 2 µg of
dynamin in 12.5-µl solution) was added to a final volume of 75 µl
of GTPase assay buffer (20 mM HEPES, pH 7.0, 10 mM MgCl2) on ice. Reactions were initiated by
addition of 25 µl of 1 mM [-32P]GTP
mixture (~200 cpm pmol1) in GTPase assay buffer
followed by incubation at 30 °C for the indicated times. The
reactions were terminated by addition of 1 ml of isobutyl
alcohol/benzene (v/v, 1:1) and 0.25 ml of 4% tungstosilic acid in 3 N H2SO4 followed by brief mixing.
Next, 0.3 ml of 10% ammonium molybdate was added followed by vigorous vortexing and brief centrifugation. 500 µl of the aqueous phase solution, containing released 32Pi from
[-32P]GTP, were counted using a -counter (Packard).
In Vitro Dynamin Assembly Assay--
Dynamin self-assembly was
monitored by a simple sedimentation assay (5). Purified recombinant
dynamin I (4 µg in 25 µl of kinase reaction buffer) was diluted
into 125 µl of GTPase assay buffer on ice and mixed with 50 µl of 1 mM GTP in GTPase assay buffer, yielding a final
concentration of 20 ng ml1 dynamin. Mixtures were
incubated for 2 min at 30 °C and then centrifuged at either 3,000 or
150,000 × g for 10 min. Pellets were dissolved in
Laemmli sample buffer and resolved by SDS-PAGE. Dynamin in the pellets
was visualized by Coomassie Blue staining and quantified using Fluor-S
MultiImager (Bio-Rad).
Negative Staining Electron Microscopy--
Purified recombinant
dynamin I was resuspended in phosphorylation reaction buffer as
described. The concentrations of salt and dynamin in the reaction
mixture were 50 µM and 0.16 mg ml1,
respectively. The mixture was adsorbed to carbon-coated EM grids that
had been glow-discharged using a Hummer X glow discharge unit (Technics
West Inc.) just prior to use as described (31), and negatively stained
with 2% aqueous uranyl acetate before air-drying. Images were obtained
using a Philips 301 electron microscope at 80 kV with 50-µm objective aperture.
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RESULTS |
EGFR Stimulation Induces Tyrosine Phosphorylation of
Dynamin--
c-Src-mediated tyrosine phosphorylation of clathrin (20)
and dynamin (25) are required for EGFR and 2-AR
internalization, respectively. However, the involvement of tyrosine
phosphorylation of dynamin in the process of EGFR endocytosis has not
been reported. To test this possibility, we examined EGF-induced
tyrosine phosphorylation of dynamin I transiently expressed in COS-7
cells after pretreatment with the tyrosine phosphatase inhibitor sodium
orthovanadate. Stimulation with EGF caused a 5-10-fold increase in the
tyrosine phosphorylation of dynamin (Fig.
1A). Interestingly, we found that the tyrosine phosphate content of K44A dynamin was 2-3-fold higher than that of wild type dynamin (Fig. 1A). After
targeting to the plasma membrane, K44A dynamin remains there because of its impaired ability to bind and hydrolyze GTP (32, 33). This may
suggest that tyrosine phosphorylation of dynamin was stabilized at the
plasma membrane.
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Fig. 1.
EGFR-regulated tyrosine phosphorylation of
transiently expressed dynamin I. A, EGF promotes
phosphorylation of wild type and K44A dynamin. COS-7 cells transiently
expressing wild type or K44A dynamin were serum-starved overnight and
pretreated with 100 µM Na3VO4 for
1 h followed by stimulation with EGF (10 ng ml1) for
an additional 1 h. Dynamin immunoprecipitates (IP) were
resolved by SDS-PAGE, transferred to nitrocellulose filters, and
immunoblotted (IB) with anti-phosphotyrosine
(pTyr) antibody (PY20H). Values shown are expressed
as percent of the EGF-induced tyrosine phosphorylation signal of K44A
dynamin. B, time-dependent tyrosine
phosphorylation of K44A dynamin by EGF. COS-7 cells expressing K44A
dynamin were serum-starved, exposed to Na3VO4
for a total of 2 h, and treated with EGF at 37 °C for the
indicated times. Tyrosine phosphorylation content of K44A dynamin was
determined as above. Values are presented as percent of maximal signal
obtained after 2 h stimulation. C, EGF-regulated
tyrosine phosphorylation of dynamin involves Src kinase. Serum-starved
K44A dynamin-expressing cells were treated with
Na3VO4 for 30 min before adding either PP2 (5 µM) or AG1478 (250 nM). After an additional
30-min incubation, cells were stimulated with EGF for 1 h and
analyzed for dynamin tyrosine phosphorylation. Data shown are expressed
as percent of EGF-induced tyrosine phosphorylation of K44A dynamin from
control cells, treated with the vehicle dimethyl sulfoxide only. A
representative immunoblot is shown on the right of each
panel. In each box, the lower panel shows total
dynamin expression in lysates from the corresponding sample. Each data
point represents the mean ± S.E. from at least three independent
experiments.
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Using K44A dynamin I transiently expressed in COS-7 cells, we
determined the time course of its EGF-induced tyrosine phosphorylation. In the presence of sodium orthovanadate, tyrosine phosphorylation of
K44A dynamin was significantly increased within 5 min and reached a
plateau 30 min after EGF stimulation (Fig. 1B). The signal
of K44A dynamin tyrosine phosphorylation was attenuated by the EGFR kinase inhibitor AG1478 as well as the Src kinase inhibitor PP2 (Fig.
1C), indicating that EGF-induced tyrosine phosphorylation of
dynamin was EGFR- and Src kinase activity-dependent.
Because the data shown in Fig. 1 were obtained using transient
expression of exogenous dynamin I, we confirmed these results by
examining the EGF-induced tyrosine phosphorylation of endogenous dynamin. Endogenous dynamin II was isolated using GST-Grb2 affinity binding (24, 25) from COS-7 cells after stimulation with EGF for the
indicated times (Fig. 2A),
following pretreatment with sodium orthovanadate. Tyrosine
phosphorylation of endogenous dynamin II was significantly increased
within 1 min after stimulation, similar to the previously reported
isoproterenol-stimulated signal (25). Unlike the situation for K44A
dynamin I (Fig. 1B), the EGF-induced tyrosine
phosphorylation of endogenous dynamin II was transient; the signal was
significantly reduced 30 min after stimulation. Maximal EGF-induced
tyrosine phosphorylation of endogenous dynamin II (10 min stimulation)
was attenuated by PP2 and AG1478 (Fig. 2B), like that of
transiently expressed dynamin I (Fig. 1C). Taken together,
these results resemble our previous finding that 2-AR
stimulation induces c-Src-mediated tyrosine phosphorylation of dynamin
(25), and suggest that this event is a general cellular response to
receptor activation.
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Fig. 2.
EGF-induced tyrosine phosphorylation of
endogenous dynamin II. A, time-dependent
tyrosine phosphorylation of endogenous dynamin II by EGF. COS-7 cells
were serum-starved overnight, exposed to Na3VO4
for a total of 2 h, and then stimulated with EGF at 37 °C for
the indicated times. Endogenous dynamin II was isolated using GST-Grb2
affinity beads and the tyrosine phosphorylation content of isolated
dynamin II was determined by immunoblotting (IB) as outlined
under Fig. 1. Values are presented as EGF-induced fold increases in
tyrosine phosphorylation over unstimulated samples. B,
EGF-induced tyrosine phosphorylation of endogenous dynamin II requires
Src activity. Serum-starved cells were treated with
Na3VO4 for a total of 2 h. PP2 (5 µM) or AG1478 (250 nM) were added 30 min
prior to stimulation with EGF for 10 min. The tyrosine phosphorylation
of endogenous dynamin II was determined as described above. Data shown
are expressed as EGF-induced fold increases in tyrosine phosphorylation
over unstimulated samples. A representative immunoblot is shown on the
right of each panel. In each box, the lower
panel shows the amount of dynamin II isolated on the GST-Grb2
beads in the corresponding samples. Each data point represents the
mean ± S.E. from five independent experiments. pTyr,
phosphotyrosine.
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Dynamin Is a Direct Substrate of c-Src Kinase--
Available
evidence demonstrates that c-Src directly interacts with dynamin (22)
and that c-Src kinase activity is required for tyrosine phosphorylation
of dynamin (25). To determine whether c-Src can directly phosphorylate
dynamin, we employed an in vitro kinase assay using purified
proteins. Baculovirus encoding rat dynamin I was generated and used to
infect Sf9 cells. The recombinant dynamin protein was purified
to homogeneity using ammonium sulfate fractionation and ion-exchange
Q-Sepharose chromatography. Purified dynamin was competent in GTP
hydrolysis and was not tyrosine phosphorylated (data not shown). As
shown in Fig. 3A, dynamin
could serve as a direct substrate of c-Src. Maximum stoichiometry of
phosphorylation achieved was calculated to be 0.39 mol of
Pi/mol of dynamin. Because the basic unit of the dynamin
oligomer is a homotetramer (6), it is reasonable to assume that only a
fraction of dynamin molecules are phosphorylated by c-Src in
vitro.
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Fig. 3.
c-Src phosphorylates dynamin.
A, recombinant c-Src phosphorylates purified
recombinant dynamin I in vitro. Upper panel, a
fixed amount of dynamin (500 ng) was mixed with increasing amounts of
c-Src. Lower panel, increasing amounts of dynamin were mixed
with a fixed amount of c-Src (0.5 µg). Reactions were carried out at
37 °C for 30 min, and products were then visualized by
autoradiography. B, c-Src phosphorylates wild type and
Y231F/Y597F dynamin proteins in vitro. Purified dynamin
proteins were mixed with c-Src (molar ratio of c-Src/dynamin = 1/5) in kinase reaction buffer at room temperature for the indicated
times. At the end of each time point, a fixed portion of the reaction
mixture was taken and analyzed for phosphorylation content by
autoradiography. Control samples were similarly prepared, but without
c-Src. Note that purified dynamin was minimally phosphorylated even in
the absence of added c-Src (A and B), presumably
by a kinase that was co-purified with dynamin. Because this
phosphorylation was not c-Src-dependent, this basal
phosphorylation signal was subtracted to calculate stoichiometry of the
c-Src-dependent tyrosine phosphorylation of dynamin.
Stoichiometry of phosphorylation of both wild type and Y231F/Y597F
dynamin was obtained from two independent experiments (left
panel). A representative autoradiograph is shown on the
right. C, c-Src phosphorylates dynamin I on
Tyr354 and Tyr597. Schematic summary of dynamin
structure and identification of phosphorylated tyrosine residues
in vitro by c-Src and in cells following receptor activation
are shown. GED, GTPase effector domain; PRD,
proline-rich domain.
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Previously, two dynamin tyrosine residues (Tyr231 and
Tyr597) were identified as being phosphorylated following
2-AR stimulation in HEK293 cells (25). To determine
whether the tyrosine-phosphorylated residues by c-Src in
vitro are the same as those identified in cultured cells, we
compared the in vitro phosphorylation pattern of recombinant
wild type and Y231F/Y597F dynamin proteins. The mutant protein was
impaired by ~40-50% in its tyrosine phosphorylation compared with
wild type protein (Fig. 3B). Mass spectrometric analysis of
trypsin-digested fragments of in vitro c-Src-phosphorylated dynamin and immunoblotting of the phosphorylated dynamin with an
antibody generated specifically against phospho-Tyr597
revealed phosphorylation of two residues, Tyr354 and
Tyr597. Tyr597 was previously identified to be
phosphorylated in response to 2-AR activation in
cultured cells, whereas Tyr354 was not detected in the
prior in vivo analysis (25) (Fig. 3C). Tyr231, another residue that becomes phosphorylated
following 2-AR stimulation in cultured cells (25), was
not found to be phosphorylated by c-Src in vitro, suggesting
that Tyr231 could be phosphorylated by another kinase
rather than c-Src. Together, these data suggest the relevance of
c-Src-mediated phosphorylation of Tyr597 in the in
vivo function of dynamin.
EGF-induced Tyrosine Phosphorylation of Dynamin Is Required for
EGFR Internalization--
To identify the physiological significance
of the tyrosine phosphorylation sites in dynamin following receptor
activation, we examined EGF-induced tyrosine phosphorylation content of
the single tyrosine mutant dynamins, Y354F and Y597F, as well as the double tyrosine mutant dynamin Y231F/Y597F, transiently expressed in
COS-7 cells. In addition, we examined the tyrosine phosphorylation of
the K44A dynamin version of each tyrosine mutant, because K44A dynamin
showed a higher tyrosine phosphorylation signal compared with wild type
dynamin following EGF stimulation (Fig. 1A). EGF stimulation
induced tyrosine phosphorylation of both wild type and K44A dynamin
(Fig. 4A), consistent with the
data shown in Fig. 1A. Replacement of Tyr354
with Phe showed no substantial effect on the tyrosine phosphorylation level of either wild type or K44A dynamin in response to EGF
stimulation (Fig. 4, A and B). In contrast, the
Y597F mutation resulted in 50-70% reduction in the EGF-induced
dynamin tyrosine phosphorylation signal. Furthermore, replacement of
both Tyr231 and Tyr597 with Phe caused a more
dramatic attenuation (75-90%) in the tyrosine phosphorylation of
dynamin than the Y597F single mutation following EGF stimulation. These
results suggest that EGF stimulation induces phosphorylation of both
Tyr597 and Tyr231, but not Tyr354,
in COS-7 cells.
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Fig. 4.
Identification of the biologically relevant
tyrosine residues in dynamin. A, EGF induces
phosphorylation of specific tyrosine residues in wild type and K44A
dynamin in COS-7 cells. Cells transiently expressing each of the
indicated mutant dynamin I proteins were stimulated with EGF and
analyzed for tyrosine phosphorylation content, exactly as outlined
under Fig. 1. The lower panel shows equivalent dynamin
expression in lysates among different samples. NS,
non-stimulated. B, the relative amount of tyrosine
phosphorylation of each dynamin in A was determined by
densitometry scanning and values are shown as percent of EGF-induced
tyrosine phosphorylation of K44A dynamin. Each data point represents
mean ± S.E. from seven independent experiments.
C, tyrosine phosphorylation of dynamin is required for
EGFR endocytosis. FLAG-EGFR was transiently co-transfected with either
empty pcDNA3 vector (CN) or each of the indicated
dynamin I expression plasmids in COS-7 cells. The amount of
internalized receptor was measured by enzyme-linked immunosorbent assay
after exposure of cells to EGF (10 ng ml1) for 30 min at
37 °C. Values shown are the mean ± S.E. obtained from ten
independent experiments done in triplicate and expressed as percent
loss of EGF-induced cell surface receptor over unstimulated
cells.
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To establish the physiologic relevance of dynamin tyrosine
phosphorylation in signaling receptor endocytosis, we tested the effect
of the tyrosine mutant proteins on agonist-induced EGFR internalization
in COS-7 cells. Expression of Y597F dynamin caused 60% reduction in
EGF-induced EGFR internalization, whereas expression of the Y354F
dynamin had no effect (Fig. 4C), as would be predicted from
the lack of Tyr354 phosphorylation following EGF
stimulation. Furthermore, expression of the Y231F/Y597F dynamin
exhibited additional attenuation (80-90%) of EGF-induced EGFR
internalization, which was equivalent to the degree of inhibition
observed with GTPase-deficient K44A dynamin. Taken together, these
results demonstrate that phosphorylation of both Tyr597 and
Tyr231 was required for EGFR internalization following EGF
stimulation, whereas phosphorylation of Tyr354 was not
involved in EGFR endocytosis.
Tyrosine Phosphorylation of Dynamin Potentiates Its GTPase Activity
in Vitro--
Tyrosine phosphorylation of dynamin (Fig. 4C
and Refs. 25 and 26) and GTP hydrolysis (3, 4) are both required for ligand-induced endocytosis of signaling receptors. To test whether c-Src-mediated tyrosine phosphorylation regulates dynamin GTPase activity, we compared the rate of GTP hydrolysis by in vitro
tyrosine-phosphorylated and unphosphorylated recombinant dynamin
proteins. Fig. 5A shows that
preincubation of dynamin with c-Src and ATP induced a 4-5-fold increase in steady-state GTP hydrolysis rate compared with the unphosphorylated control protein. Assays performed using dynamin alone,
dynamin mixed with ATP or c-Src, or dynamin mixed with non-hydrolyzable
ATP analogues (App(NH)p or ATPS) and c-Src, all failed to stimulate
GTPase activity of dynamin (Fig. 5A), demonstrating that
tyrosine phosphorylation of dynamin is required for the enhanced GTP
hydrolysis.
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Fig. 5.
Tyrosine phosphorylation of dynamin
potentiates the rate of GTP hydrolysis. A,
c-Src-catalyzed phosphorylation of dynamin increases its GTPase
activity. Purified recombinant rat dynamin I was co-incubated with
c-Src (molar ratio of c-Src/dynamin = 1/5) and ATP for 6 h at
room temperature to generate tyrosine-phosphorylated dynamin protein.
Controls included incubation of dynamin alone (CN), or together with
ATP, c-Src, or c-Src and the non-hydrolyzable ATP analogues App(NH)p
and ATPS. At the end of reaction, dynamin (0.2 µM)
from each sample was incubated in GTPase assay buffer at 30 °C for
15 min, and GTP hydrolysis rates were then determined as described
under "Experimental Procedures." B, GTP hydrolysis
by tyrosine-phosphorylated wild type and Y231F/Y597F dynamin proteins.
Wild type and Y231F/Y597F-phosphorylated dynamin proteins were
generated as outlined in A. Control samples
( c-Src) were prepared by incubating each dynamin protein
alone in kinase reaction buffer. The rate of GTP hydrolysis was
determined after incubation in GTPase buffer at 30 °C for 2 min. The
asterisk indicates p < 0.05 versus tyrosine-phosphorylated wild type dynamin. Data
represent mean ± S.E. from five independent experiments done in
triplicate (A) or duplicate (B).
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Our results demonstrate that the Tyr597 residue of dynamin
is phosphorylated by c-Src in vitro (Fig. 3), and that this
phosphorylation regulates ligand-induced EGFR endocytosis (Fig.
4C), albeit by undefined mechanisms. To determine the role
of Tyr597 phosphorylation on the function of dynamin, we
compared the GTPase activities of in vitro
tyrosine-phosphorylated wild type and Y231F/Y597F recombinant dynamin
proteins. Because Tyr231 was not phosphorylated by c-Src
in vitro, mutation of this residue (Y231F) would not be
expected to influence the tyrosine phosphorylation-regulated GTPase
activity of dynamin. After 2 min incubation with GTP at 30 °C,
Y231F/Y597F dynamin showed ~40% reduction in its tyrosine phosphorylation-induced initial rate of GTP hydrolysis compared with
the wild type protein (Fig. 5B). Unphosphorylated wild type and Y231F/Y597F dynamin had equally low basal GTPase activities. These results demonstrate that in vitro tyrosine
phosphorylation of dynamin stimulates its GTPase activity, and that
phosphorylation of Tyr597 significantly controls this process.
Tyrosine-phosphorylated Dynamin Spontaneously Self-assembles to
Generate Spiral Stacks of Interconnected Rings--
At low ionic
strength, purified dynamin self-assembles into structures composed of
rings and spiral stacks, which are sedimentable by high-speed
centrifugation (>100,000 × g) (5, 7). Because self-assembly of dynamin is known to regulate its GTP hydrolysis rate
(5, 6, 15), we hypothesized that the increased GTPase activity of
tyrosine-phosphorylated dynamin involves its enhanced self-assembly.
Purified recombinant dynamin protein was phosphorylated, diluted in
GTPase reaction buffer, and incubated at 30 °C, exactly as described
for the GTPase assay. Self-assembled dynamin was collected by
sedimentation. Initial results, utilizing high-speed centrifugation
(150,000 × g for 10 min), showed no difference in
self-assembly of in vitro tyrosine-phosphorylated or
unphosphorylated dynamin proteins (Fig.
6A, small gel). However, when
centrifugal force was reduced to 3,000 × g, a striking
difference was noted; dynamin incubated with c-Src and ATP was
sedimentable at 3,000 × g, whereas unphosphorylated
proteins precipitated in trace amounts only (Fig. 6A).
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Fig. 6.
Tyrosine phosphorylation promotes dynamin
self-assembly. A, c-Src-phosphorylated dynamin
precipitates at low centrifugal force. Tyrosine-phosphorylated dynamin
and appropriate controls were prepared as outlined under Fig.
5A, and diluted in GTPase buffer to exactly mimic conditions
used in the GTPase assay. After incubating at 30 °C for 2 min, each
dynamin sample was centrifuged at 3,000 × g for 10 min. Pelleted dynamin was visualized by Coomassie Blue staining and
quantified. Values shown are expressed as fold-increase over control
(CN) in which dynamin was incubated alone during the
in vitro phosphorylation reaction. Additional controls
included the high speed centrifugation (150,000 × g
for 10 min) of dynamin proteins prepared in the absence or presence of
c-Src with ATP. B, tyrosine phosphorylation-promoted
self-assembly of wild type and Y231F/Y597F dynamin proteins. Wild-type
and Y231F/Y597F dynamin proteins were subjected to in vitro
phosphorylation by c-Src kinase, diluted in GTPase buffer, and
incubated at 30 °C for 2 min, exactly as described under Fig.
5B. In each sample, precipitated dynamin was visualized by
Coomassie Blue staining and quantified after centrifugation at
3,000 × g for 10 min. Values are expressed as fold
increase over the wild type control (c-Src). The
asterisk indicates p < 0.05 versus tyrosine-phosphorylated wild type dynamin. A
representative gel is shown for each panel. Each data point represents
mean ± S.E. from five independent experiments.
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To further establish a relationship among dynamin GTPase activity,
self-assembly, and tyrosine phosphorylation, we examined the effect of
tyrosine phosphorylation of dynamin on its self-assembly using wild
type and Y231F/Y597F recombinant proteins that were phosphorylated by
c-Src. Fig. 6B shows that the Y231F/Y597F dynamin has
~40% diminution in its tyrosine phosphorylation-promoted
sedimentation compared with wild type protein, exactly paralleling the
effect on GTPase activity (Fig. 5B). Unphosphorylated
Y231F/Y597F dynamin showed the same extent of precipitation as wild
type control. Taken together, these data demonstrate that
tyrosine-phosphorylated dynamin polymerizes into large molecular mass
structures. Because dynamin self-assembly enhances its GTPase activity,
these results also suggest that c-Src-mediated phosphorylation of
dynamin regulates the GTPase activity by controlling self-assembly.
To confirm that tyrosine phosphorylation induces formation of real
self-assembled structures of dynamin large enough to be precipitated at
relatively low (3,000 × g) centrifugal force, we
employed negative-staining electron microscopy. As shown in Fig.
7A, clustered stacks (50-300
nm length) of tightly interconnected dynamin rings were observed in
solutions of tyrosine-phosphorylated samples obtained by incubation
with c-Src and ATP. High magnification of a large stack showed a well
ordered spiral array of dynamin rings (Fig. 7B). In
contrast, unphosphorylated control dynamin was composed of short curved
structures, some of which developed partial rings and globules (Fig.
7C).
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Fig. 7.
Negative-staining electron micrographs of
dynamin. Purified recombinant rat dynamin I was incubated in
kinase reaction buffer with (A and B) or without
(C) c-Src, as outlined under Fig. 5. The concentrations of
dynamin and salt in the reaction buffer were 0.16 mg ml1
and 50 mM, respectively. After incubation for 6 h at
room temperature, each sample was adsorbed to carbon-coated EM grids
and negatively stained as described under "Experimental
Procedures." A, tyrosine-phosphorylated dynamin shows
clustered large stacks of interconnected rings. B, a high
magnification image of a large single spiral stack of assembled dynamin
in which each ring was visibly preserved. C,
unphosphorylated control dynamin consists of curved structures
(large arrow), globules (small arrow), or partial
rings (arrowheads). The images in A and
C are shown at the same magnification. Scale
bars, 100 nm.
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Helical stacks of dynamin have been observed upon dilution into low
ionic strength buffer (<50 mM salt) (7) or in the presence of GDP and -phosphate analogues at physiological salt conditions (8). However, those structures were prevalent only at high dynamin
protein concentrations (>1 mg ml1), whereas the tyrosine
phosphorylation-induced assembled structures shown here were generated
at much lower protein concentrations (0.16 mg ml1).
Furthermore, the helical stacks appeared tightly ordered (Fig. 7,
A and B), similar to spirals obtained using a C
terminus truncated 90-kDa fragment of dynamin, which was compactly
arranged compared with intact dynamin protein in the presence of GDP
and -phosphate analogues at physiological salt conditions (8).
Dimensions of the stacks (~50 nm) were comparable not only to the
structures obtained at high dynamin protein concentrations in
vitro, but also to the collars observed at the neck of
invaginated coated pits that accumulate at synaptic nerve terminals (9,
10). It has also been shown that helically coated phospholipid tubes generated from purified dynamin and liposomes in vitro have
the same diameter (11). Taken together, these results demonstrate that
tyrosine phosphorylation of dynamin induces its spontaneous self-assembly into large helical structures composed of interconnected rings.
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DISCUSSION |
Several models exist to describe the role of dynamin in the
fission of vesicles from the plasma membrane to the cytosol, including dynamin being a pinchase (7, 11, 12), a poppase (13, 14), or an adapter
(15) to recruit downstream effectors, which mediate the fission step.
Regardless of which model is correct, it is an indisputable fact that
control of dynamin GTPase activity is the key for endocytic vesicle
formation (3, 4). Similarly, it is well established that dynamin
self-assembles to form spiral structures on the neck of invaginated
pits during endocytosis (9, 10, 33), which has been demonstrated to
stimulate its GTPase activity (5, 6, 15). Although a certain
conformation of GTP-bound dynamin was proposed to favor self-assembly
(8), regulatory mechanisms inducing and stabilizing the assembled
dynamin structures remain unclear. The present data show that
c-Src-mediated tyrosine phosphorylation promotes significant increases
in dynamin self-assembly into helically interconnected rings and also
stimulates the rate of GTP hydrolysis, both of which are hallmarks for
dynamin execution of vesicle budding from the plasma membrane.
Agonist stimulation triggers recruitment of c-Src and dynamin into the
vicinity of activated receptor on the plasma membrane. Some receptors,
such as EGFR (34) and 3-AR (35), bind c-Src directly,
whereas others, such as 2-AR (36), form complexes with
c-Src via bridging adapter proteins. Dynamin translocates to the plasma
membrane by direct interaction with phospholipids (37) and adapter
proteins, such as amphyphisin (3, 38). Once in close proximity to each
other on the plasma membrane, c-Src phosphorylates dynamin. In support
of this hypothesis is our finding that K44A dynamin, which is competent
to translocate to invaginated pits on the plasma membrane following
receptor activation, but which does not recycle to the cytosol because of its impaired ability to bind and hydrolyze GTP (32, 33), displays a
significant increase in tyrosine phosphorylation compared with wild
type dynamin (Fig. 1A). Furthermore, disruption of c-Src recruitment to activated 2-AR inhibits tyrosine
phosphorylation of dynamin (39). These data suggest that
dephosphorylation of dynamin occurs in the cytosol following GTP
hydrolysis-induced disassembly.
Phosphoamino acid analysis of in vitro c-Src-phosphorylated
dynamin revealed two tyrosine residues, Tyr354 and
Tyr597, to be phosphorylated (Fig. 3C). However,
in vivo analysis shows that Tyr597, but not
Tyr354, becomes phosphorylated in response to EGF
stimulation. Furthermore, phosphorylation of dynamin on
Tyr597, but not Tyr354, appears to be required
for EGF-induced EGFR internalization (Fig. 4). Tyr597 in
dynamin also needs to be phosphorylated to mediate 2-AR
internalization (25) and expression of Y231F/Y597F dynamin inhibits M1
muscarinic acetylcholine receptor internalization (26). Replacement of Tyr597 with Phe reduces the tyrosine
phosphorylation-induced GTPase activity (Fig. 5B) and
self-assembly of dynamin (Fig. 6B), suggesting that
phosphorylation of Tyr597 regulates dynamin function in
endocytosis of ligand-activated receptors by controlling self-assembly
and subsequent GTP hydrolysis.
Does tyrosine phosphorylation of dynamin play a role in
clathrin-mediated endocytosis of constitutively recycling nutrient receptors, similar to ligand-induced internalization of signaling receptors? Vallis et al. (40) showed that expression of
bovine Y596F mutant dynamin I has no effect on transferrin uptake,
suggesting that phosphorylation of Tyr596, which
corresponds to the Tyr597 residue of rat dynamin I, is not
required for constitutive endocytosis of nutrient receptors. It is
possible that the function of dynamin is regulated differently for
constitutive endocytosis of nutrient receptors, in the absence of
tyrosine phosphorylation. In support of this hypothesis, the existence
of two functionally and biochemically distinct subpopulations of
clathrin-coated pits that mediate agonist-regulated internalization of
the 2-AR and constitutive endocytosis of the transferrin
receptor has been demonstrated (41). Alternatively, the tyrosine
phosphorylation-induced assembled structure of dynamin may have an
unidentified specific role for endocytosis of activated signaling
receptors, which is not required for constitutive endocytosis of
recycling receptors. In the case of EGFR endocytosis, one possibility is that tyrosine phosphorylation-induced dynamin self-assembly leads to
recruitment of the ligand-activated receptor to the endocytic machinery. It has been suggested that biochemical differences between
EGF and transferrin uptake mostly reflect specific requirements for the
recruitment of the EGFR to coated pits (42). We also cannot exclude the
possibility that the effect of Y597F dynamin on EGFR endocytosis may be
indirect, such as by interfering with receptor activation, because
expression of K44A dynamin was shown to alter high affinity binding of
EGF to the receptor (43). Currently, however, what determines this
specificity of tyrosine phosphorylation-regulated function of dynamin
in signaling receptor internalization is not known.
How does tyrosine phosphorylation induce dynamin self-assembly? It is
well established that phosphotyrosine residues primarily serve as
docking sites for proteins containing Src homology (SH) 2 or
phosphotyrosine-binding domains (44, 45). Indeed, some dynamin-binding
proteins stimulate the GTPase activity of dynamin (3, 38). However, our
in vitro results using purified dynamin show that tyrosine
phosphorylation per se increases the GTPase activity of
dynamin. Additionally, dynamin itself does not contain SH2 or
phosphotyrosine-binding domains, indicating a lack of potential intramolecular SH2 or phosphotyrosine-binding domain-mediated interactions in tyrosine-phosphorylated dynamin. Several lines of
evidence have suggested that tyrosine phosphorylation of proteins may
stimulate their enzymatic activity by inducing a local conformational change rather than by creating binding sites for other partners. For
example, most protein-tyrosine kinases, including receptor tyrosine
kinases and non-receptor tyrosine kinases such as Src, are activated by
phosphorylation of tyrosine residues in the activation loop (45). The
SH2 domain-containing tyrosine phosphatase SHP-2 is
tyrosine-phosphorylated in growth factor-stimulated cells, which leads
to its activation (46). Furthermore, it has been reported that c-Src
phosphorylates the G
s, Gi,
Go, and Gq/11 GTPases (47, 48). In
in vitro reconstitution assays, tyrosine-phosphorylated Gs shows an increased rate of binding to GTPS as well
as an increased rate of receptor-stimulated GTP hydrolysis (47).
Together, the present results suggest the possibility that tyrosine
phosphorylation may induce a conformational change of dynamin, which
favors self-assembly.
Tyr597 is well conserved among dynamin isoforms and resides
in the PH domain. This domain is required for endocytosis (49, 50),
presumably to recruit dynamin to the plasma membrane via binding to
phospholipids (37). The contribution of the PH domain to dynamin
self-assembly is not clear. In vitro, the PH domain was
shown either not to participate (51, 52) or to even be a negative
regulator (6, 53) of dynamin-dynamin interaction. However, it is not
clear whether the dynamin proteins used in these studies were tyrosine
phosphorylated. Interaction of the PH domain phosphorylated on
Tyr597 with other domains of dynamin remains to be
determined. On the other hand, whereas phosphorylation of
Tyr354 in dynamin appears to be physiologically irrelevant,
at least with regard to EGFR endocytosis, the in vitro
results demonstrate that phosphorylation of this residue exerts a
positive effect on dynamin self-assembly and GTPase activity. Thus, it
will be of interest to test whether phosphorylation of
Tyr354 alters other functions of dynamin, such as rapid
endocytosis of synaptic vesicles in nerve terminals (3, 38), because the Tyr354 residue is exclusively found in neuronal dynamin I.
In addition to being phosphorylated on tyrosine residues, neuronal
dynamin I is also phosphorylated on a serine residue by protein kinase
C in intact synaptosomes (54). In vitro, protein kinase
C-mediated phosphorylation stimulates dynamin I GTPase activity (55),
and increases binding to calcium (56), but blocks binding to
phospholipids (57). Upon synaptic membrane depolarization,
serine-phosphorylated dynamin I was dephosphorylated by the
calcium-dependent phosphatase calcineurin (58), which was
required for endocytosis in nerve terminals (59). Calcineurin-mediated dephosphorylation restores the ability of dynamin to bind phospholipids (57) and inhibits its GTPase activity (58) in vitro. Thus, it will be of interest to examine the relationship between tyrosine and
serine phosphorylation of dynamin I in nerve terminals and their impact
on endocytosis.
The effect of GTP binding on dynamin self-assembly is not entirely
clear. In vitro, addition of GDP with -phosphate
analogues induces dynamin self-assembly to generate spiral stacks (8), and treatment of isolated nerve terminals with GTPS promotes tubular
membrane invaginations decorated with electron-dense dynamin rings
(10). However, the binding of guanine nucleotides GTPS, GTP, or GDP
destabilizes assembled dynamin structures (5), and GTP binding-impaired
K44A dynamin self-assembles, like wild type protein under low ionic
strength conditions (5). Our data show that tyrosine phosphorylation
controls dynamin self-assembly (Fig. 6) even in the absence of guanine
nucleotides (Fig. 7). Furthermore, stimulation with EGF promotes
tyrosine phosphorylation of GTP binding-deficient K44A dynamin (Fig.
1), suggesting that nucleotide binding was not required for tyrosine
phosphorylation. It is not clear at this juncture whether
c-Src-regulated tyrosine phosphorylation exerts any effect on the
affinity of dynamin to bind these nucleotides. Dynamin can also
generate helically assembled structures around phospholipid liposomes
(11, 12) or nanotubes (13, 14) in vitro, which were shown to
undergo conformational changes in a nucleotide-dependent
manner (11-14). Furthermore, the recently resolved crystal structure
of the dynamin GTPase domain (60) and three-dimensional reconstruction
of dynamin in the constricted state (12) provides additional evidence
for the requirement of dynamin self-assembly and conformational change in endocytic vesicle formation. Thus, it remains to be determined how
tyrosine phosphorylation collaborates with the guanine nucleotide in
the context of dynamin self-assembly and conformational change.
Together, our results establish the novel paradigm that
post-translational tyrosine phosphorylation of dynamin serves as a regulator of dynamin function in endocytosis of signaling receptors via
control of self-assembly. Furthermore, the
agonist-dependent tyrosine phosphorylation of dynamin
provides a mechanism by which tyrosine kinase receptors regulate their
own accessibility to external stimulation and, as a result, cellular response.
§,
,
TOP
ABSTRACT
INTRODUCTION
