(Received for publication, January 14, 1997)
From the The major constitutive
phosphatidylinositol-3,4,5-P3 (PtdIns) 5-phosphatase
activity was purified and subjected to peptide sequence analysis
providing extensive amino acid sequence which was subsequently used for
cloning the cDNA. Peptide and cDNA sequences revealed that the
purified PtdIns(3,4,5)P3 5-phosphatase was identical to a
splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed synaptojanin. Since synaptojanin is not known to
possess PtdIns(3,4,5)P3 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P3 5-phosphatase activity
and synaptojanin are identical by Western blot using specific
antibodies raised against synaptojanin sequences. Immunoprecipitation
from crude lysates of rat brain tissue showed that synaptojanin
accounts for the major part of the active PtdIns(3,4,5)P3
5-phosphatase activity. It is also shown that the protein is localized
to the soluble fraction. Expression of a truncated recombinant protein demonstrates that the conserved 5-phosphatase region of the
synaptojanin gene expresses PtdIns(3,4,5)P3 5-phosphatase
activity. However, immunological analysis demonstrates that the
PtdIns(3,4,5)P3 5-phosphatase activity expressed from the
synaptojanin gene in brain is due to a particular splice variant which
contains a 16-amino acid insert as shown by immunoprecipitation using a
specific antibody raised against this particular splice variant.
The regulation of inositol phospholipid metabolism has provided a
cornerstone to at least two signal transduction pathways, one involving
the phospholipase C-dependent phosphodiesterase cleavage of
phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2)1 (1) and the
second the phosphorylation of this lipid by PtdIns 3-kinases yielding
PtdIns(3,4,5)P3 (2, 3). This latter lipid second messenger
has been implicated in a number of signaling pathways (PKB/akt,
p70S6k, PKC, PRK) as has its metabolite
PtdIns(3,4)P2 (4-9). It has been suggested that the
5-phosphatase responsible for the transformation of
PtdIns(3,4,5)P3 to PtdIns(3,4)P2 (10) is
due to a recently cloned 5-phosphatase termed SIP (11) or SHIP
(p145SHIP) (12-14) which has specificity for
3-phosphorylated inositol phosphates (11-14). However, other
PtdIns(3,4,5)P3 5-phosphatases have been identified (15,
16) indicating that this issue is still not resolved.
We have previously identified and purified the major constitutive
PtdIns(3,4,5)P3 5-phosphatase from rodent brain tissue
(16). Here we demonstrate that this protein is encoded by a particular splice variant of the recently described synaptojanin gene. It is
further shown that a recombinant synaptojanin protein has intrinsic PtdIns(3,4,5)P3 5-phosphatase activity. Thus, we are able
to show that synaptojanin which to date has not been associated with
PtdIns 3-kinase signaling could play a major role in PtdIns
3-kinase-dependent pathways.
The 5-phosphatase was purified (16), further
fractionated by SDS-polyacrylamide gel electrophoresis, and stained
with Coomassie Brilliant Blue R250. The bands were excised and digested
with lysyl endopeptidase (Wako Chemicals) or with endoproteinase Asp-N (Boehringer Mannheim). Peptides were extracted for 2 h in a
sonicating water bath. After concentration the peptides were resolved
using an Aquapore AX-300 (30 × 2.1 mm) and OD-300 (150 × 2.1 mm) columns connected in series on a Hewlett-Packard 1090M high
performance liquid chromatography system. The columns were developed
with a linear acetonitrile gradient in 0.1% trifluoroacetic acid.
Peptide elution was monitored by means of diode-array detection
(200-600 nm). Peptides were sequenced using an ABI 494 HT Procise
sequencer employing fast cycles as described previously (17). Initial yields were in the 2-10-pmol range.
Details
of the cloning of the PIP3 5-phosphatase will described
elsewhere. Briefly, primers were designed based on the obtained peptide
sequence and then used to amplify the corresponding cDNA sequence
using the polymerase chain reaction (PCR) from a rat brain library
(Stratagene). The cDNA produced was then used as a probe to search
for PIP3 5-phosphatase clones.
For expression of the PIP3 5-phosphatase in COS7 cells, a
cDNA encoding residues 401-1308 (Syn N-del; including the 16-amino acid insert (18)) and residues 401-989 (Syn core; excluding C terminus
and splice insert) of synaptojanin were amplified by PCR from the
isolated cDNA clone using oligonucleotides containing an
EcoRI site (5 Antibodies were
raised against the C terminus of synaptojanin (C-term) or the unique
splice sequence "GVGAPPSPGVTRREME"(insert) in rabbits. The
corresponding peptide antigen was included in immunoprecipitation
experiments or Western blots as a control. Antibodies raised against
the unique insert region (insert), the C terminus (C-term), or the myc
epitope were bound to protein A beads. Antibody-loaded beads were then
incubated for 3 h with rat brain fractions at 4 °C. The
supernatant was separated by centrifugation, and the beads were washed
several times using phosphate-buffered saline and phosphate-buffered
saline containing 0.5 M NaCl. Supernatant and washed beads
were then assayed for PtdInsP3 5-phosphatase activity as
described (16). For Western blots, the extracts were subjected to
SDS-gel electrophoresis transferred onto PVDF or nitrocellulose
membranes and incubated with the indicated antibodies. Proteins were
visualized by ECL (Amersham). All other methods were done as described
previously (16).
The 145-kDa protein previously purified and identified as a
PtdIns(3,4,5)P3 5-phosphatase was subjected to proteolytic
fragmentation and amino acid sequence analysis. A total of 23 peptide
sequences covering 361 amino acids were determined (not shown). During
the course of the cDNA cloning of this protein, the sequence of
synaptojanin was reported (18), and it became clear that the 145-kDa
protein sequenced was in fact a particular splice variant of
synaptojanin containing a defined 16-amino acid insert (see Fig.
1). For simplicity the purified protein will be termed
hereafter p145Isynj (145-kDa species of synaptojanin
containing the 16-amino acid insert); the insertless protein is denoted
p145synj.
Synaptojanin is a component of synaptic vesicles and is thought to play
a key role in vesicle traffic at the synapse (18). PtdIns 3-kinase
activities are themselves implicated in vesicle traffic in non-neuronal
cells. For example by analogy to the Saccharomyces cerevisiae VPS34 gene product, which is a PtdIns-specific
PtdIns 3-kinase, the mammalian homologue is likely to play a role in trans-Golgi to lysosome traffic (19, 20). There is also
circumstantial evidence for PtdIns(4,5)P2 3-kinase
activities controlling endosome fusion (21-23) as well as traffic of
the Glut4 transporter to the cell surface (24, 25). In this context,
functional identity of the synaptosomal protein synaptojanin is a key
issue. However, PtdIns(3,4,5)P3 5-phosphatase activity has
not been found associated with synaptojanin (18); thus it has been
important to establish this identity.
To define the function of synaptojanin, specific antisera were derived.
The antisera to p145Isynj as well as to the C terminus
common to both forms p145Isynj and p145synj were
found to react with the purified rodent brain 5-phosphatase, confirming
the presence of p145Isynj (Fig. 2). It is
noteworthy that the common antiserum did not reveal any additional
protein species.
These synaptojanin-specific sera can be shown to immunodeplete the
p145Isynj protein from crude rodent brain extracts, and
this correlates with depletion of PtdIns(3,4,5)P3
5-phosphatase activity (Fig. 3). Thus,
immunoprecipitation with the insert-specific antiserum depleted >90%
of the PtdIns(3,4,5)P3 5-phosphatase activity from rat
brain extracts. This depletion was specific, since inclusion of the
peptide antigen fully competed the activity depletion. Immunoprecipitates with these antisera demonstrated recovery of 5-phosphatase activity, and this was also specific as demonstrated by
antigen competition (Fig. 3, panel A).
The antiserum to the C terminus common to both p145synj and
p145Isynj also immunoprecipitated 5-phosphatase activity
albeit less efficiently than the insert sera. Western analysis of
extracts subjected to immunoprecipitation confirmed the depletion of
p145Isynj (see Fig. 3B) and also demonstrates
that there is very little p145synj expressed (the
insert-specific antiserum almost fully depletes p145synj
immunoreactivity). Consistent with the incomplete immunoprecipitation of the 5-phosphatase activity, the C-terminal antiserum only partially depletes the antigen.
Fractionation of brain extracts was performed to establish the
distribution of p145synj and p145Isynj. Consistent
with our previous analysis of PtdIns(3,4,5)P3 5-phosphatase activity distribution, on hypotonic extraction, p145Isynj
is found to be predominantly cytosolic with little immunoreactive protein detected on the membrane (see Fig. 3C).
To establish that the synaptojanin gene encodes a protein with
intrinsic PtdIns(3,4,5)P3 5-phosphatase activity, an
N-terminally deleted expression construct was prepared that included
the predicted catalytic domain of the protein (see Fig.
4). Transient expression in COS cells correlates with
the production of a protein that was immunoreactive with
p145Isynj antisera (panel A). Activity
determination demonstrated that this protein had
PtdIns(3,4,5)P3 5-phosphatase activity (panel B). Similar observations have been made for a C-terminal deletion that does not encode the splice insert (data not shown).
The results presented here demonstrate that p145Isynj
has intrinsic PtdIns(3,4,5)P3 5-phosphatase activity. The
immunodepletion of PtdIns(3,4,5)P3 5-phosphatase activity,
alongside that of p145Isynj protein shows that in fact this
protein represents the major constitutive activity expressed in brain
tissue. The expression of recombinant proteins encompassing the
catalytic domain (with or without the splice insert) indicates that all
the known splice variants from this gene would retain
PtdIns(3,4,5)P3 5-phosphatase activity. However, the
p145Isynj variant itself appears to be the major form
present in brain based upon the immunodepletion data. This conclusion
is entirely consistent with the purification of this activity from rat
brain, since only a single peak of constitutive activity was observed (16), and as shown here this activity purified to near homogeneity after SDS-polyacrylamide gel electrophoresis yielding
p145Isynj.
The properties of the native purified 5-phosphatase protein have been
characterized and provide evidence of a much higher affinity for
PtdIns(3,4,5)P3 over PtdIns(4,5)P2. Indeed the
protein was purified on the basis of activity determined against
32P-labeled PtdIns(3,4,5)P3 in the presence of
an ~10-fold excess of unlabeled PtdIns(4,5)P2 (16). This
relative preference for PtdIns(3,4,5)P3 contrasts with the
related p145SHIP (12-14) which has been reported to be
devoid of 5-phosphatase activity toward PtdIns(4,5)P2 and
the 43-kDa inositol polyphosphate 5-phosphatase which does not
hydrolyze inositol lipids (26, 27). Interestingly p145SHIP
itself has poor activity toward PtdIns(3,4,5)P3 compared
with the 75-kDa type II phosphatase (11, 15). The molecular basis for
these distinctions must await structural analysis.
The PtdIns(3,4,5)P3 5-phosphatase activity of synaptojanin
implicates the complex regulation of
PtdIns(4,5)P2-PtdIns(3,4,5)P3-PtdIns(3,4)P2 inositol lipid metabolism in the directional cycle of events required for neurotransmitter granule recycling in the synapse. However it is
clear that both p145Isynj and p145synj are largely
cytosolic in brain extracts although there is a higher proportion of
synaptojanin in the particulate fraction of synaptosomes (data not
shown) which is in agreement with recent studies (28). Hence, a dynamic
interaction of synaptojanin with the membrane has to be proposed. Such
behavior would be compatible with many other "membrane-interacting"
proteins (e.g. protein kinase C (29)) and
"membrane-metabolizing" proteins (e.g.
PtdIns-phospholipase C (30)) involved in regulatory processes. The
ability of amphiphysin to interact with synaptojanin and so tether it
to neurotransmitter granules may be a crucial element in the protein's
role (18).
The involvement of 3-phosphorylated polyphosphoinositides in vesicle
traffic processes in mammalian cells has been implied through the use
of the fungal metabolite wortmannin (5, 22, 31) which is a potent
inhibitor of a number of the phosphoinositide 3-kinases (32, 33).
However, the specific role of PtdIns(3,4,5)P3 is unknown in
this context. Indeed it is not clear whether
PtdIns(3,4,5)P3 or actually PtdIns(3,4)P2 is
the key element. The identification of p145Isynj as a
PtdIns(3,4,5)P3 5-phosphatase provides evidence that in neuronal cells the metabolism of PtdIns(3,4,5)P3 in
granules or following fusion at synaptic termini is a critical
component in their cycling. Whether this is a part of the cycling
itself, for example by marking the vesicle at one stage in its cycle,
or whether this is simply a negative regulatory device to effectively
remove PtdIns(3,4,5)P3 from the vesicle remains to be
determined.
We thank Drs. N. Q. McDonald and A. E. Stewart for helpful discussions. We are also grateful to Drs. D. Nicholls and J. Ryves for synaptosome preparations.
Protein Phosphorylation Laboratory,
Department of Biochemistry and
Molecular Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Microsequencing of Purified Rat Brain PIP3
5-Phosphatase
oligonucleotide) and XhoI site (3
oligonucleotide). An EcoRI-XhoI-digested PCR
fragment was subcloned into a pCDNA3 derivative which had been
modified to contain a myc epitope (MEQKLISEEDL) followed by an in-frame
EcoRI site. Expression of the myc-tagged protein was driven
from a cytomegalovirus promoter. The correct sequence of the myc-tagged
phosphatase construct was confirmed by DNA sequence analysis. COS7
cells were transfected by electroporation.
Fig. 1.
The purified PtdInsP3
5-phosphatase is identical to synaptojanin. The purified
PtdInsP3 5-phosphatase (16) was subjected to Edman
microsequencing using Asp-N and Lys-C digests. The determined peptide
sequences (light gray and black boxes) are
aligned with the synaptojanin sequence (18) (shown in
white). Synaptojanin consists of at least two forms due to
splicing which inserts a short peptide sequence (GVGAPPSPGVTRREME) in
the C-terminal part of the protein (black box). Sequences
which were used as antigens for antibody production are shown as
black boxes; these had also been identified by Edman
microsequencing.
[View Larger Version of this Image (9K GIF file)]
Fig. 2.
The purified PtdInsP3
5-phosphatase is recognized by antibodies against synaptojanin.
The purified PtdInsP3 5-phosphatase (16) was subjected to
SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF
membrane (Western blot). The PVDF membrane was then incubated with
insert and C terminus antibodies in the presence (+) or absence () of
the corresponding peptide antigen. The position of the 145-kDa band
(PtdInsP3 5-phosphatase) is indicated by an
arrow.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Antibodies against synaptojanin
immunoprecipitate PtdInsP3 5-phosphatase activity from rat
brain cytosol. Rat brain cytosol and membrane fractions were
prepared as described earlier (16). A, antibodies raised
against the unique insert region (insert) and the C terminus
(C-term.) were bound to protein A beads. For comparison,
protein A beads were loaded with a bovine IgG fraction
(control). Antibody-loaded beads were then incubated for
3 h with rat brain cytosol at 4 °C in the presence (+) or absence () of competing peptide antigen. The supernatants were separated by centrifugation, and the beads were washed and then assayed
for PtdInsP3 5-phosphatase activity as described (16). The
autoradiograph of a thin layer chromatography separation of the lipid
products of the phosphatase assay is shown; the position of the
phosphoinositides are indicated by arrows. B,
aliquots of the supernatants from the immunoprecipitation experiment
(see panel A) were separated by SDS-polyacrylamide gel
electrophoresis and transferred onto PVDF membranes (Western blot). The
membranes were incubated with insert or C terminus antibodies. The
position of the 145-kDa band is indicated. C, the
subcellular fractions, i.e. crude extract (E),
cytosol (C), and membrane (M), were subjected to
SDS-polyacrylamide gel electrophoresis and then transferred onto PVDF
membranes (Western blot). The PVDF membranes were then incubated with
antibodies raised against synaptojanin (insert or C terminus) as
described above (see Fig. 2). The position of the 145-kDa band
(synaptojanin) and the presence (+) of competing antigen peptide are
indicated.
[View Larger Version of this Image (19K GIF file)]
Fig. 4.
A synaptojanin construct expressed in COS
cells has PtdInsP3 5-phosphatase activity.
Cells were harvested 48 h after transfection. The cells were lysed
either in SDS-polyacrylamide gel electrophoresis sample buffer (for
Western blot) or 1% Triton X-100 in Tris-buffered saline (for
immunoprecipitation). A, SDS extracts of COS cells
expressing empty vector (vector), the myc-tagged 5-phosphatase core
domain not containing the splice insert (Syn core), or a myc-tagged
N-terminal truncated form (Syn N-del) were subjected to gel
electrophoresis and transferred onto nitrocellulose. Western blots were
performed as described in the legends to Figs. 2 and 3 except using the
anti-myc monoclonal antibody 9E10 and an antimouse horseradish
peroxidase second antibody. B, Triton extracts of the COS
cells expressing 5-phosphatase constructs (see above) were subjected to
immunoprecipitation using the anti-myc monoclonal antibody 9E10 coupled
to protein A beads, and the washed immunoprecipitates were assayed for
PtdInsP3 5-phosphatase activity as described in Fig.
3.
[View Larger Version of this Image (13K GIF file)]
*
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: Protein Phosphorylation
Laboratory, Imperial Cancer Research Fund, P. O. Box 123, Lincoln's
Inn Fields, London WC2A 3PX, UK. Tel.: 44-171-269-3460; Fax:
44-171-269-3092.
1
The abbreviations used are: PtdIns,
phosphatidylinositol; PIP3, phosphatidylinositol
3,4,5-trisphosphate; PCR, polymerase chain reaction; PVDF,
polyvinylidene difluoride.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.