From the Howard Hughes Medical Institute and
Department of Cell Biology, Yale University School of Medicine, New
Haven, Connecticut 06510, and § Howard Hughes Medical
Institute and Department of Pathology, Harvard Medical School, Boston,
Massachusetts 02115
Received for publication, September 29, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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Amphiphysin 1 is a phosphoprotein expressed at
high levels in neurons, where it participates in synaptic vesicle
endocytosis and neurite outgrowth. It is a substrate for
cyclin-dependent kinase (cdk) 5, a member of the
cyclin-dependent protein kinase family, which has been
functionally linked to neuronal migration and neurite outgrowth via its
action on the actin cytoskeleton. The yeast homologue of amphiphysin,
Rvs167, functions in endocytosis and actin dynamics, is phosphorylated
by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit
Pcl2. We show here that amphiphysin 1 interacts with the
cdk5-activating subunit p35 and that this interaction is mediated by
the conserved NH2-terminal region of amphiphysin.
Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and
at actin-rich peripheral lamellipodia in transfected fibroblasts.
Amphiphysin is phosphorylated by cdk5 in a region including serines
272, 276, and 285. Amphiphysin 1 is also phosphorylated by the
cdc2/cyclin B kinase complex in the same region and undergoes mitotic
phosphorylation in dividing cells. These data indicate that
phosphorylation by members of the cyclin-dependent kinase
family is a conserved property of amphiphysin and suggest that this
phosphorylation may play an important physiological role both in
mitosis and in differentiated cells.
Amphiphysin 1 and 2 are SH3 domain-containing proteins
concentrated in nerve terminals of mature neurons (1, 2). They belong
to a protein family conserved from yeast to humans, whose members play
pleiotropic roles in endocytosis, actin function, and regulation of
growth control. Studies of the yeast amphiphysin homologue Rvs167 have
shown that this protein is phosphorylated by the Pho85 kinase and binds
Pcl2, an activator of this kinase (3). The Pho85 kinase is a member of
the cyclin-dependent kinase family and functions in
cellular growth control as well as endocytosis and actin function. The
homologue of the Pho85 kinase in mammalian cells is
cdk51 (4, 5). Its two
activators, p35 and p39 (6-8), which represent functional homologues
of Pcl2, are expressed only in neurons and developing muscle cells (6,
7, 9). Consistent with these homologies, amphiphysin 1 is a substrate
for cdk5 and is a component of a high molecular weight complex in brain
that also contains cdk5/p35 (10). In addition to its role in neurons,
amphiphysin is likely to function outside the nervous system because it
is expressed in other cell types (11, 12). In this study we have further characterized the relationship between amphiphysin 1 and the
cdk5/p35 kinase complex. We also show that amphiphysin 1 is phosphorylated by cdc2, another cyclin-dependent kinase
family member, and undergoes mitotic phosphorylation.
Antibodies--
Amphiphysin polyclonal antibodies CD5 and CD9
and monoclonal antibodies have been described previously (11, 13). p35
antibodies were obtained from Santa Cruz Biologicals (antibody C-19) or
were generated as described previously (14). Anti-cdc2 antibody was obtained from Transduction Laboratories, and anti-cdk5 antibody was
obtained from Upstate Biotechnology. Nonimmune control rabbit IgG was
obtained from Sigma.
Glutathione S-Transferase (GST) Fusion
Proteins--
cDNAs encoding sequences of p35, p25, cdk5, or
amphiphysin 1 were subcloned into either pGEX2T, pGEX4T, or pGEX6P
(Pharmacia) as described previously (15, 16). Fusion proteins were
prepared according to the manufacturer's instructions.
Baculovirus-expressed human GST-cdc2/cyclin B1 with activating T14A and
Y15F mutations as described previously (17) was a kind gift of Dr.
Graham Warren (Yale University, New Haven, CT).
In Vitro Binding Assay--
p35 was
[35S]methionine-radiolabeled using a coupled in
vitro transcription/translation kit (Promega). 5 µl of the total
reaction mixture was combined with 10 µg of recombinant GST protein
fused to amino acids 1-161, 1-246, 1-306, 262-435, 411-581, and
545-695 of the human amphiphysin 1 sequence in 1 ml of buffer A (150 mM NaCl, 50 mM Tris-HCL, 5 mM EDTA,
and 1% Triton X-100). These reactions were incubated for 1 h at
room temperature, combined with 50 µl of 50% slurry of
glutathione-Sepharose in buffer A, incubated for 30 min at room
temperature, washed four times with 1 ml of buffer A, separated by
SDS-PAGE, and dried to Whatman filter paper. Radioactive protein bands
were detected by fluorography.
In Vitro Kinase Assays--
Kinase assays were performed as
described previously (18), with the following modifications: 1 µM purified histone H1 (Roche) or amphiphysin fusion
protein cleaved from the GST tag according to the manufacturer's
instructions was included in a total reaction volume of 50 µl
containing either 10 µg of purified GST-cdk5 and 10 µg of purified
GST-p25, anti-p35 immunoprecipitate from rat brain extract, or 1 µg
of GST-tagged human cyclin B1/cdc2. [ Tryptic Digestion and Identification of Phosphorylated
Peptides--
50 µg of recombinant amphiphysin 1 was
32P-radiolabeled in vitro using p35
immunoprecipitates from rat brain as described above. The reaction
products were separated by SDS-PAGE and stained with Coomassie
Blue, and the amphiphysin band was excised. In conjunction with
the Keck Foundation Biotechnology Resource Laboratory at Yale
University, this sample was trypsin-digested, and peptides were eluted
from the gel and separated by HPLC as described previously (19, 20).
Radioactive fractions were detected by Cerenkov counting, and
constituent peptides were determined by matrix assisted-laser desorption ionization mass spectrometry as described previously (21)
and Edman sequencing on a Procise cLC instrument (PerkinElmer Life
Sciences) as per the manufacturer's protocols.
Mutagenesis--
Constructs containing the mutations
T260L, S262A, S268A (Mut 1), S272A, S276A, S285A (Mut 2), T260L, S262A,
S268A, S272A, S276A, and S285A (Mut 3) in the human amphiphysin 1 sequence were generated by polymerase chain reaction, digested with
restriction enzyme DpnI, and transformed into competent
bacteria as described previously (22).
Mitotic Synchronization of CHO Cells--
CHO cells stably
transfected with amphiphysin 1 were synchronized with nocodazole as
described previously (23). Mitotic cells were harvested and homogenized
or allowed to progress to G1 phase before harvesting.
Proteins were analyzed by SDS-PAGE and Western blotting.
Cell Culture and Transfections--
Culture of primary rat
cortical neurons was performed as described previously (24, 25). An
amphiphysin stable cell line was generated by transfecting Chinese
hamster ovary cells maintained in Dulbecco's modified Eagle's
medium/10% fetal bovine serum with human amphiphysin cDNA in
pRc/RSV vector (Invitrogen). Transfected cells were selected by
neomycin resistance, and serial dilutions were performed to obtain
single clones. Cos7 cells were maintained in Dulbecco's modified
Eagle's medium/10% fetal bovine serum. Transient transfections were
performed using p35 or amphiphysin 1 in vector pcDNA3 and
LipofectAMINE Plus (Life Technologies, Inc.) according to the
manufacturer's instructions.
Miscellaneous--
Immunoprecipitation, immunostaining, and
SDS-PAGE were performed as described previously (15). Triton X-100
extracts of whole rat brain were prepared as described previously
(15).
Amphiphysin and p35 Interact in Brain and in Transfected
Cells--
Recently, amphiphysin has been identified as a substrate
and interactor of the cdk5 kinase complex (10). To further investigate this interaction, we performed immunoprecipitation experiments from rat
brain extracts. Anti-amphiphysin antibodies purified the amphiphysin
1-2 heterodimer and coprecipitated p35, the activating subunit of the
cdk5 kinase complex. Likewise, anti-p35 antibodies coprecipitated
amphiphysin (Fig. 1). cdk5 was not
detected in anti-amphiphysin immunoprecipitates, indicating
that amphiphysin does not interact directly with the kinase and that a
ternary complex of amphiphysin, p35, and cdk5 is not present at
significant levels (data not shown).
We probed the interaction of amphiphysin with p35 by reconstituting it
in a transfected cell system. To this end, a CHO cell line stably
transfected with amphiphysin 1 was transiently transfected with either
p35 or empty control vector. As shown in Fig.
2, immunoprecipitation with antibodies
directed against either amphiphysin or p35 precipitated a complex
containing both proteins. cdk5 was endogenously expressed in this cell
line and could be immunoprecipitated by anti-p35 antibodies (Fig.
2B, bottom panel). However, in agreement with our
observation in brain extracts, amphiphysin immunoprecipitates did not
contain cdk5.
Amphiphysin 1 and p35 Colocalize in Vivo--
Based on the results
of coprecipitation experiments, we investigated whether p35 and
amphiphysin colocalize in cells. Both p35/cdk5 and amphiphysin have
been implicated in neurite outgrowth and neuronal migration (14,
26-29). As shown by Fig. 3, A
and B, endogenous amphiphysin 1 and p35 colocalize in the
growth cones of rat cortical neurons in culture, consistent with a
possible role for their interaction in growth cone navigation.
Similar observations were made in Cos7 cells cotransfected with
amphiphysin 1 and p35 cDNAs. In these cells, a large fraction of
both proteins had a diffuse cytosolic distribution. In addition, pools
of amphiphysin 1 and p35 were colocalized in ruffles and lamellipodia
at the cell periphery (Fig. 3, C and D). These
structures were also positive for filamentous actin, as demonstrated by
phalloidin immunostaining. These findings are consistent with previous
observations that both amphiphysin and the p35/cdk5 complex are present
at actin-rich lammellipodial structures in neuronal growth cones (14,
28).
p35 Binds the NH2-terminal Region of Amphiphysin
1--
To map the domain of amphiphysin 1 that binds to p35, we
radiolabeled p35 by coupled in vitro
transcription/translation and performed in vitro binding
experiments with glutathione S-transferase-tagged fragments
of amphiphysin 1. As shown in Fig. 4, the
first 306 amino acids of amphiphysin 1 were sufficient to retain
in vitro translated p35 on glutathione-Sepharose beads.
These data confirm the interaction of amphiphysin 1 with p35 and
demonstrate that the binding site for p35 resides in the
NH2-terminal domain of the molecule.
Amphiphysin 1 is Phosphorylated by cdk5 between Amino Acids 254 and
320--
We next investigated cdk5 phosphorylation of amphiphysin 1. To this end, we prepared immunoprecipitates of the p35/cdk5 complex from rat brain using anti-p35 antibodies. These immunoprecipitates were
then used in in vitro kinase reactions with recombinant
amphiphysin 1 or histone H1, a known cdk5 substrate. As shown
in Fig. 5, lanes a-d, p35
immunoprecipitates, but not control IgG immunoprecipitates, incorporated radiolabeled phosphate into both histone H1 and
amphiphysin 1. In vitro kinase reactions performed with
bacterially expressed recombinant p35/cdk5 instead of
immunoprecipitates gave similar results, indicating that amphiphysin 1 is phosphorylated by cdk5 and not by a contaminating kinase from brain
extracts (data not shown).
To determine what region of amphiphysin 1 is phosphorylated by cdk5,
recombinant peptides corresponding to overlapping regions of the
protein were tested in in vitro kinase reactions. This strategy demonstrated the presence of a major phosphorylation site or
sites selectively in amphiphysin 1 fragments comprising amino acids
1-306 and 262-435 (Fig. 5). Strikingly, the region of overlap between
these two peptide fragments, but not other regions of the two
fragments, contains several potential consensus phosphorylation motifs
for cdk5, as indicated in Fig.
6A.
To confirm that the region between amino acids 262-306 contained the
phosphorylation site for cdk5, tryptic digestion of in vitro
32P-phosphorylated amphiphysin 1 was performed, and the
resulting peptide fragments were separated by HPLC chromatography.
Chromatographic fractions were then assayed for radioactivity, and the
radioactive peptides were identified by matrix-assisted laser
desorption mass spectroscopy and subsequent microsequence analysis. By
this method, we identified a phosphorylated peptide corresponding to
amino acids 260-292 (arrowheads in Fig. 6A), in
agreement with the results shown in Fig. 5.
We mapped which amino acid residues of amphiphysin 1 within this region
are targets for phosphorylation by cdk5 by generating full-length
amphiphysin 1 constructs in which threonine 260, serine 262, serine
268, serine 272, serine 276, and serine 285 were mutated to
nonphosphorylatable residues. These recombinant proteins were then
tested in in vitro kinase reactions using the p35/cdk5
complex immunoprecipitated from rat brain. This strategy demonstrated that mutation of threonine 260, serine 262, and serine 268 (Fig. 6,
Mut 1) did not affect amphiphysin phosphorylation, whereas constructs harboring mutations of serine 272, serine 276, and serine
285 (Fig. 6, Mut 2 and 3) showed reduced
phosphorylation. The cdk5 phosphorylation site or sites in amphiphysin
therefore reside in one or more of these three amino acids.
Amphiphysin Is a Substrate for the cdc2 Kinase and Is
Phosphorylated during Mitosis--
Members of the
cyclin-dependent family of kinases require similar sequence
motifs in their respective substrates (30). Two other proteins
implicated in clathrin-mediated endocytosis and actin function, epsin
and eps15, are substrates for cyclin-dependent kinases in
mitotic cells (23, 31). Whereas amphiphysin 1 is expressed at high
concentration in neurons, it is also expressed at lower levels in other
cells, including dividing cells (11, 12). In addition, it is
overexpressed in some breast tumors and breast cancer cell lines and
was identified as a paraneoplastic syndrome autoantigen in patients
with breast cancer (2, 11, 32). These considerations prompted us to
investigate whether amphiphysin undergoes mitotic phosphorylation.
Based on substrate sequence preference similarities among
cyclin-dependent kinases, we tested whether amphiphysin 1 is phosphorylated in vitro by the mitotic
cyclin-dependent kinase cdc2/cyclin B1. To this end, we
used a purified recombinant cdc2/cyclin B1 complex in which cdc2
harbors an activating mutation (17). As shown in Fig.
7A, full-length amphiphysin 1 was phosphorylated by this complex. Using amphiphysin fragments, we
mapped the major cdc2/cyclin B1 phosphorylation site(s) to the region
between amino acids 262 and 306, i.e. the same region that
is phosphorylated by cdk5 (Fig. 7A). Both cdc2/cyclin B1 and
cdk5 showed reduced phosphorylation of the amphiphysin mutant (Mut 2)
harboring serine to alanine substitutions at positions 272, 276, and
285, indicating that amphiphysin is phosphorylated at these residues by
both of these kinases (Fig. 7B).
We next tested phosphorylation of amphiphysin in mitotic cells. CHO
cells stably transfected with amphiphysin 1 were mitotically synchronized using nocodazole, and the electrophoretic mobility of
amphiphysin 1 was analyzed by Western blotting in the mitotic and
G1 stages. As shown by Fig. 7C, amphiphysin 1 undergoes a mobility shift in mitotic cells. This slower mobility,
which has been observed for several other mitotic phosphoproteins,
could be reversed by alkaline phosphatase treatment of the mitotic
extract, as expected for a shift due to phosphorylation (Fig.
7C, lane 2). Interestingly, cdc2/cyclin B1 could
induce an electrophoretic shift in recombinant amphiphysin 1 in
in vitro kinase reactions (Fig. 7D). This shift
was reduced in the Mut 2 amphiphysin construct harboring serine to
alanine mutations at residues 272, 276, and 285. Note, however, that
even the Mut 2 variant of amphiphysin runs slightly slower than wild
type amphiphysin in SDS-PAGE after the in vitro kinase
reaction with cdc2/cyclin B1 complex. This observation suggests that
under these in vitro conditions, cdc2 can phosphorylate
additional sites.
In this study we report evidence for a functional link between
amphiphysin 1 and the cdk5 kinase complex. We have demonstrated the
in vivo occurrence of an interaction between amphiphysin and the cdk5 regulatory subunit p35 by immunoprecipitation and
immunofluorescence. This interaction is analogous to the Pcl2-Rvs167
interaction in yeast. We have also shown that p35 binds to the
evolutionary conserved NH2-terminal region of amphiphysin
1, in agreement with the reported binding of Pcl2 to the
NH2-terminal moiety of Rvs167 (3). Thus, our findings point
to a further similarity between the mammalian p35/cdk5/amphiphysin 1 and the yeast Pcl2/Pho85/Rvs167 protein networks. We also mapped the
main cdk5 phosphorylation site to a small fragment containing three
serine residues in the central region of amphiphysin 1, just upstream
of the binding sites for clathrin and the clathrin adaptor
AP-2, although phosphorylation of this site did not appear to
affect AP-2 or clathrin
binding.2 Because Rvs167,
like several other members of the amphiphysin family, does not contain
recognizable clathrin or AP-2 binding sites (33-35), it is not
unexpected that cdk5 phosphorylation may not represent a conserved
mechanism to regulate these interactions.
Studies of p35/cdk5 have revealed a key role of this complex in the
regulation of actin function at the leading edge of neuronal processes
with important functional implications for neuronal migration and
growth cone navigation (14, 26, 36). Likewise, independent studies have
suggested a role of amphi- physin and its binding partners in
growth cone dynamics (28). Amphiphysin has also been implicated in
endocytosis, a process for which a key role of actin is emerging (37,
38). Although a function of cdk5 phosphorylation in endocytosis has not
been reported, in yeast, Pho85, Plc2, and Rvs167 mutations have
strikingly similar effects on both actin function and endocytosis (3).
We suggest therefore that amphiphysin, p35, and cdk5 may be
interrelated in their physiological functions in vivo. Our
demonstration that amphiphysin 1 and p35 colocalize with each other and
with actin in lamellipodia of transfected cells supports this
possibility. We note that an interaction between amphiphysin 2 and the
cAbl kinase has been reported (39). cAbl has recently been shown to
phosphorylate and activate cdk5 and to bind cdk5 through the bridging
protein cables (40). In addition, cAbl is known to have regulatory
actions on the actin cytoskeleton and participate in neuronal
development (41, 42).
Finally, we have shown that amphiphysin 1 undergoes mitotic
phosphorylation. The same region that is a target for cdk5
phosphorylation can also be phosphorylated by the mitotic cdc2/cyclin
B1 complex. Thus, phosphorylation of amphiphysin 1 in this region may
have a similar conserved function in both neurons and dividing cells. One of the critical events that correlates with mitosis is the dramatic
rearrangement of the peripheral cytoplasm that results in the partial
dissociation of cells from the substratum. Cyclin-dependent kinase phosphorylation of amphiphysin may help to produce local changes
in the actin cytoskeleton that are crucial for the dynamic properties
of dividing cells, neuronal growth cones, the leading edge of migrating
cells, and the function of the mature synapse.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
was included at a final specific activity of 1-10 Ci/mol in a total
concentration of 200 µM. The reaction mixtures were incubated at room temperature for 30 min, and the reaction was stopped
by adding sample buffer and boiling for 1 min. The proteins were
separated by SDS-PAGE, the gels were dried to Whatman filter paper, and
radioactive protein bands were detected by autoradiography. Radioactive
bands were quantitated on a STORM 860 PhosphorImager (Molecular
Dynamics) or by scanning on a Gel Doc 2000 densitometer (Bio-Rad). For
SDS-PAGE mobility assays, 1 µM amphiphysin 1 or mutant 2 was incubated with 200 µM ATP in the presence or absence of 1 µg of GST cyclin B1/cdc2 overnight at room temperature in a
total volume of 50 µl. Proteins were then separated on 6% SDS-PAGE gels and probed by Western blot with monoclonal amphiphysin 1 antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amphiphysin and p35 exist together in a
complex in rat brain. A, immunoprecipitate from rat
brain using control anti-cdc2 (lane 4) or anti-amphiphysin
monoclonal 8 (lane 5) antibodies Western blotted with
anti-amphiphysin 1 and 2 CD9 (top panel) and p35 C-19
(bottom panel) antibodies indicates that p35 coprecipitates
with amphiphysin. One-tenth of the starting material used for
immunoprecipitation is loaded in lane 1, and unbound
fractions from control and amphiphysin immunoprecipitation are loaded
in lanes 2 and 3, respectively. B,
immunoprecipitate from Triton X-100 extract of total rat brain
homogenate using nonspecific rabbit immunoglobulin (lane 4)
or anti-p35 antibodies (14) (lane 5) Western blotted with
anti-amphiphysin 1 monoclonal antibody 3 (top panel) or
anti-p35 C-19 antibodies (bottom panel) shows that
amphiphysin 1 coprecipitates with p35 from rat brain. One-tenth of
starting material is loaded in lane 1, and unbound fractions
from control and p35 immunoprecipitation are loaded in lanes
2 and 3, respectively.
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Fig. 2.
Amphiphysin and p35 interact in transfected
cells in a complex that does not include cdk5. A, CHO
cells stably transfected with amphiphysin 1 were mock-transfected with
empty vector (lanes 2, 5, and 7) or vector
containing p35 (lanes 1, 3, 4, and 6). Triton
X-100 extracts of cells were prepared, and starting material
(lanes 1 and 2) or immunoprecipitates using
control IgG (lane 3), anti-amphiphysin monoclonal 3 (lanes 4 and 5), and anti-p35 (14) (lanes
6 and 7) antibodies were probed by Western blot with
amphiphysin monoclonal antibody 3 (top panel) or p35 C-19
antibody (bottom panel). Amphiphysin was detected in p35
precipitates, and p35 was detected in amphiphysin precipitates.
B, immunoprecipitates from Triton X-100 extracts of CHO
cells stably transfected with amphiphysin and transiently
transfected with p35 were prepared using amphiphysin polyclonal
antibody CD5 (lane 2), p35 polyclonal antibody (14)
(lane 3), or control IgG (lane 4). These
immunoprecipitates and one tenth of the starting material (Triton-100
extract, SM, lane 1) were probed by Western blot with
amphiphysin monoclonal antibody 3 (top panel), p35 C-19
(middle panel), or cdk5 (bottom panel)
antibodies. p35 coprecipitated cdk5, but amphiphysin did not,
indicating that amphiphysin does not bind directly to cdk5 and that a
ternary complex was not present at detectable levels.
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Fig. 3.
Amphiphysin and p35 colocalize in growth
cones of neurons in culture and at actin-rich structures in transfected
fibroblasts. A and B, P1 rat cortical
neurons were fixed and immunostained with anti-p35 (14) (A)
or anti-amphiphysin monoclonal 3 (B) antibodies and
visualized by confocal scanning microscopy. Scale bar in
B represents 2 µM. C F, light
micrographs of Cos7 fibroblasts transiently transfected with
amphiphysin 1 and p35 and then fixed and immunolabeled with anti-p35
C-19 polyclonal antibody (C), amphiphysin monoclonal
antibody 3 (D), and rhodamine phalloidin (E).
F shows a pseudocolored image with amphiphysin in
green, p35 in blue, and actin in red.
In cells coexpressing amphiphysin and p35, a pool of amphiphysin
is found at actin-rich structures near the cell periphery. Scale
bar in D represents 10 µM.
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Fig. 4.
The NH2 terminus of amphiphysin
binds p35 in vitro. GST-tagged overlapping
fragments of amphiphysin 1 corresponding to the indicated amino
acid numbers were incubated with radiolabeled in vitro
translated p35 and then isolated on glutathione-Sepharose beads.
Lane a shows the starting material for the binding reaction.
The NH2-terminal 306 amino acids of amphiphysin were able
to affinity-purify p35 (lane b), whereas neither the
remaining COOH-terminal fragments (lanes c-e) nor GST alone
(lane f) bound p35.
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Fig. 5.
Amphiphysin is phosphorylated by cdk5 between
amino acids 254 and 320. Immunoprecipitates from rat brain extract
using nonspecific rabbit IgG (lanes a and b) or
anti-p35 antibodies (lanes c-h) were combined with purified
recombinant histone H1 or recombinant amphiphysin 1 peptides as
indicated. These mixtures were combined with radiolabeled ATP,
incubated, separated by SDS-PAGE, and exposed to film. Histone H1
(lane c), full-length amphiphysin 1 (lane d), and
fragments corresponding to amino acids 1-306 (lane e) and
262-435 (lane f) show incorporation of radiolabeled
phosphate.
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Fig. 6.
Amphiphysin 1 is phosphorylated at amino
acids 272, 276, and 285 in vitro. A,
amino acid sequence of the phosphorylated region from Fig. 5;
arrows indicate consensus phosphorylation sites for cdk5.
The boxed region indicates the overlap of peptides from Fig. 5, lanes
e and f. Arrowheads denote the peptide
sequence identified by matrix assisted-laser desorption ionization mass
spectrometry and Edman sequencing of tryptic digest of amphiphysin
in vitro kinase reaction using p35 immunoprecipitate from
rat brain. GST fusion proteins corresponding to full-length amphiphysin
1 with mutations at the indicated amino acid positions were prepared
using polymerase chain reaction-based mutagenesis. B, equal
molar amounts of these proteins were then tested in in vitro
kinase assays using immunoprecipitated p35/cdk5 for incorporation of
radiolabeled phosphate. Mutants 2 and 3 (Mut 2 and Mut
3) showed a significant decrease in phosphate incorporation. These
two constructs share mutations in serine 272, 276, and 285, indicating
that these residues are phosphorylated by cdk5.
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Fig. 7.
Amphiphysin is phosphorylated by cdc2/cyclin
B1 at amino acids 272, 276, and 285 in vitro and
undergoes mitotic phosphorylation. A, purified
recombinant human cdc2/cylcin B1 was combined with equal molar amounts
of purified recombinant amphiphysin 1 peptides and radiolabeled ATP as
indicated. Radioactive bands were quantitated by densitometry, and the
results shown are the mean of four experiments. Error bars
are the standard error of the mean. Full-length amphiphysin 1 and
fragments 1-306 and 262-435 show incorporation of radioactive
phosphate. B, full-length wild type (WT) or
mutant 2 (Mut2) amphiphysin constructs as described in the
Fig. 6 legend were tested in in vitro kinase reactions using
recombinant cdc2/cyclin B1 ( ) or p35/cdk5 (
). Radioactive bands
were quantitated by phophorimaging, and the results shown are the mean
of three experiments. Error bars are the standard error of
the mean. Mutation of serine 272, 276, and 285 to alanine reduced
incorporation of radiolabeled phosphate by both cdc2 and cdk5.
C, CHO cells stably transfected with amphiphysin 1 were
synchronized with nocodazole and separated into mitotic and
G1-phase pools. Triton X-100 extracts of these pools were
probed by Western blotting using anti-amphiphysin 1 monoclonal 3 antibody. Amphiphysin expressed in mitotic cells (lane 1)
migrates more slowly on SDS-PAGE than amphiphysin in
G1-phase cells (lane 3). Treatment of the
mitotic cell extract with alkaline phosphatase abolished this migration
shift (lane 2). D, recombinant wild type
amphiphysin 1 (lanes 4 and 5) or mutant 2 (lane 6) was incubated with ATP alone (lane 4) or
recombinant cyclin B1/cdc2 and ATP (lanes 5 and
6) and then probed by Western blot with an amphiphysin
monoclonal antibody. Incubation of wild type amphiphysin with cdc2 and
ATP resulted in a migration shift on SDS-PAGE. This shift was reduced
by serine to alanine mutations at amino acids 272, 276, and 285.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Graham Warren and Yanzhuang Wang for the generous provision of reagents and advice on cdc2 phosphorylation. We thank Warren T. Kim for aid with neuronal cultures and immunofluorescence.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants NS36251 and CA46128 and United States Army Medical Research and Development Command Grant DAMD17-97-7068 (to P. D. C.) and National Institutes of Health Grants NS37007 (to L.-H. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, Howard Hughes Medical Institute, 295 Congress Ave., New Haven, CT 06510.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008932200
2 S. R. Floyd, E. B. Porro, V. I. Slepnev, G.-C. Ochoa, L.-H. Tsai, and P. De Camilli, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: cdk, cyclin-dependent kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; CHO, Chinese hamster ovary.
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