From the Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas Texas 75235
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ABSTRACT |
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Phosphatidylinositol phosphates (PIPs) perform
central functions in signal transduction and membrane traffic.
Synaptojanin is a PIP 5-phosphatase that is expressed in a
brain-specific and a ubiquitous splice variants and is thought to
constitute the major PIP 5-phosphatase in mammalian brain (Woscholski,
R., Finan, P. M., Radley, E., Totty, N. F., Sterling, A. E., Hsuan, J. J., Waterfield, M. D., and Parker, P. J. (1997) J. Biol. Chem. 272, 9625-9628). We now
describe synaptojanin 2, a novel isoform of synaptojanin that, similar
to synaptojanin 1, contains an N-terminal SAC1-like sequence and a
central 5-phosphatase domain but a distinct, unique C-terminal
sequence. Transfection studies demonstrated that synaptojanin 2, like
synaptojanin 1, is an active PIP phosphatase. An interesting feature of
synaptojanin 1 is the presence of a long open reading frame in the 3
region of the brain mRNA that in non-brain tissues is joined to the
coding region by alternative splicing, resulting in a shorter
synaptojanin 1 form in brain and a longer form in peripheral tissues
(Ramjaun, A. R., and McPherson, P. S. (1996) J. Biol. Chem. 271, 24856-24861). Although it exhibits no homology
to synaptojanin 1 in this region, synaptojanin 2 also contains an open
reading frame in the 3
region that is subject to alternative splicing.
Similar to synaptojanin 1, alternative splicing of synaptojanin 2 is
tissue-specific and creates a shorter isoform expressed in brain and a
longer form in peripheral tissues. The similar alternative splicing of
two homologous proteins in a region of non-homology raises the
possibility of evolutionary convergence and supports the significance
of the variants. Analysis of mRNAs from three brain regions at
different developmental stages revealed that alternative splicing of
synaptojanin 2 is a developmentally late event, occurring only after
the first postnatal week after the generation of neurons and initial
synaptogenesis.
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INTRODUCTION |
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Phosphatidylinositol phosphates (PIPs)1 are minor components of the cellular phospholipids with major roles in signal transduction and membrane traffic (1, 2). PIPs can carry phosphates at the 3, 4, and 5 positions of the inositol ring in various combinations. The synthesis and hydrolysis of PIPs are mediated by a tightly regulated network of PIP kinases and phosphatases with distinct substrate specificities (1, 2). Studies in permeabilized PC12 cells demonstrated that a PIP 5-kinase is essential for Ca2+-triggered exocytosis (3), suggesting that generation of PIPs with 5-phosphates is involved in Ca2+-regulated exocytosis. PIPs have also been proposed to function in endocytosis because they bind to a number of proteins involved in endocytosis (4-6). Because PIPs have such a central role in membrane traffic and signal transduction, termination of the PIP signal by phosphate hydrolysis must be a key regulatory step. A PIP 5-phosphatase called synaptojanin was recently discovered that is enriched in synaptic nerve terminals (7, 8) and may serve to inactivate the PIP 5-phosphate signal.
Synaptojanin is ubiquitously expressed and hydrolyzes soluble inositol 5-phosphates as well as lipidic PIP2 and PIP3 phosphorylated in the 5-position (8, 9). Synaptojanin is composed of three principal domains: an N-terminal domain homologous to the yeast membrane trafficking protein SAC1, a central domain that carries the phosphatase activity, and a C-terminal domain that is not similar to any currently known protein but highly enriched in proline. Three homologues of synaptojanin have been identified in the yeast genome that also contain an N-terminal SAC1 domain and a middle 5-phosphatase domain but differ from synaptojanin and from each other in their C-terminal sequences (1). Synaptojanin was first identified as a phosphoprotein purified on immobilized Grb2 (7). Later the C-terminal proline-rich domain of synaptojanin was shown to interact with the SH3 domain of amphiphysin, suggesting a function in endocytosis (10). However, the relative significance of the enzymatic activity and protein interactions of synaptojanin have not been elucidated.
In addition to the three principal domains, the synaptojanin mRNA
contains an open reading frame in the 3 region separated from the
proline-rich C-terminal sequence by a stop codon (8). Although
synaptojanin was initially thought to be brain-specific, PCR
experiments revealed that it is ubiquitously expressed and that it is
alternatively spliced (11). In brain, synaptojanin is present in a
splice variant containing a stop codon after the proline-rich domain,
whereas in peripheral tissues the stop codon is spliced out, and a
longer protein is produced in which the open reading frame is fused to
the proline-rich sequence. Thus two different forms of synaptojanin are
produced, a shorter form that is expressed primarily in neurons in
brain where it seems to be concentrated in nerve terminals and a longer
ubiquitous form (11).
We have now identified a second isoform of synaptojanin that is similar in its N-terminal SAC1 and phosphatase domains but differs in the C-terminal domain. Interestingly, this isoform is also subject to alternative splicing at the C terminus despite the lack of homology between synaptojanins in the alternatively spliced sequences. Our data suggest that in brain and other mammalian tissues, PIP signaling may be more complex than previously envisioned and involve multiple differentially regulated PIP 5-phosphatases that are subject to coordinate alternative splicing.
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EXPERIMENTAL PROCEDURES |
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Cloning, Sequencing, and Construction of Expression
Vectors--
GenBankTM was searched using the BLAST programs of the
NCBI. A single candidate synaptojanin homologue was identified, the corresponding clone was obtained from the IMAGE Consortium (clone 360567; Ref. 12), mapped with restriction endonucleases, and sequenced
after subcloning into pBlueScript II using standard procedures. The 5
AvaI fragment of the clone was used as a probe to screen a
mouse brain cDNA library (Stratagene). Pooled
ZAP clones were
plaque-purified and sequenced after in vivo excision using
standard procedures (13, 14). Sequence analysis was performed using the
DNASTAR software package (DNASTAR Inc.). The sequences have been
submitted to GenBankTM (accession number AF026123). A synaptojanin 1 expression vector (pMESJ1) was constructed by amplifying its complete
coding region by PCR using oligonucleotides 1581 and 1578 (sequences
GCGAATTCGGCTGCCTCTGAAGAAAGGAGAATGGCG and
GCGACTAGTTACCCTGTTGGTTGCTCCTGTTGTAGC) and cloning the PCR product into
the EcoRI and SpeI sites of pME18Sf. A
synaptojanin 2 expression vector (pCMVmycSJ2) containing the putative
phosphatase domain and the C-terminal domain (residues 505-1206; see
Fig. 1) N-terminally fused to the myc epitope was created by
subcloning the BamHI-AvrII fragment of the
cDNA into the BglII and XbaI sites of
pCMV5.
RNA Purification and cDNA Synthesis-- Total RNA was purified from 100 mg each of adult mouse brain, postnatal mouse brains (days 1-20), embryonic mouse brain (day 18), adult mouse heart, lung, spleen, liver, kidney, testis, and skeletal muscle tissues using RNASTAT-60 (TEL-TEST Inc). RNA concentration was determined by absorbance at 260 nm. 10 µg of each RNA sample were reverse transcribed using SuperScript System (Life Technologies, Inc.) with oligo(dT)12-18 primer according to the manufacturer's instructions.
PCR Analysis-- Forward and reverse primers GCGGATCCTTACAACGTCAAACAGATCAAAACCAC and GCGAATTCCACTCGGCCTCCTCTCAACACCTCTC (BamHI and EcoRI sites are underlined) were used for PCR amplifications with AmpliTAQ DNA polymerase (Perkin-Elmer). PCRs were performed in 38 cycles, with the first three cycles at 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min followed by 35 cycles at 94 °C for 45 s and 72 °C for 1 min. Products were separated on 2% agarose gels in TAE buffer (40 mM Tris-acetate, pH 8.3, 1 mM EDTA), purified using a Qiaex kit (Qiagen), and cloned into pBlueScript vector after digestion with BamHI and EcoRI for sequencing. The sequences of the PCR products consistently contained multiple substitutions compared with the sequences of the cDNA clones, possibly because they were derived from different mouse strains (see GenBankTM entry).
Measurements of PIP Phosphatase Activities by Synaptojanins-- COS cells transfected with the synaptojanin 1, the synaptojanin 2, or control expression vectors were lysed by sonication in 4 mM CHAPS, 0.1 M NaCl, 50 mM HEPES-NaOH, pH 7.4, 0.5 mM EGTA, 5 mM MgCl2, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/l benzamidine, 10 mg/l leupeptin, and 1 µM pepstatin A. The lysate was incubated on ice for 30 min and cleared by high speed centrifugation (100,000 × g for 1 h). Proteins from the transfected COS cells were immunoprecipitated using a polyclonal rabbit serum raised against bacterially expressed synaptojanin 1 (T694) for synaptojanin 1-transfected COS cells and monoclonal antibody 9E10 against the myc epitope for synaptojanin 2-transfected COS cells because synaptojanin 2 was expressed as a myc epitope fusion protein. All immunoprecipitations were also carried out with control transfected COS cells. Immunoprecipitates immobilized on protein A-Sepharose beads were washed five times at 4 °C in assay buffer (4 mM CHAPS, 0.1 M NaCl, 50 mM HEPES-NaOH, pH 7.4, 0.5 mM EGTA, 5 mM MgCl2). The PIP2 activity of the washed immunoprecipitates was then determined essentially as described (15). For this purpose, a lipid mix containing 0.67 g/liter phosphatidylcholine, 0.33 g/liter phosphatidylserine (from Avanti lipids), and 1.4 µM 3H-phosphatidylinositol-(4,5)-bisphosphate (2.4 Ci/mmol; NEN Life Science Products) was dispersed in assay buffer by sonication. Equal volumes (30 µl) of the lipid mix and the bead suspension were combined and incubated for 30 min at 37 °C. Thereafter 50 µg of total phosphoinositides (Sigma) were added as carrier lipids, and samples were extracted with 225 µl of chloroform:methanol:concentrated HCl (100:200:1) followed by extraction with 75 µl of chloroform and 0.1 N HCl. The chloroform was evaporated under nitrogen, and lipids were redissolved in chloroform and separated by thin layer chromatography on Whatman silica gel 6 nm plates using a n-propanol, NH4OH, and water (65:20:15) as solvent system. The corresponding spots for phosphatidylinositol, PIP, and PIP2 were identified using lipid markers as internal standards visualized with iodine vapors, scraped out, and counted by liquid scintillation counting.
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RESULTS |
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Identification of Synaptojanin 2-- The yeast genome contains three genes for synaptojanin-like proteins, but only a single such gene was described in mammals (1). Synaptojanin may be considered as corresponding to two isoforms because it is alternatively spliced in a tissue-specific manner (11). Yet it seemed unlikely that the mammalian genome would not contain additional 5-phosphatases of this family in view of the apparent complexity of synaptojanin-like PIP 5-phosphatases in yeast. Therefore we searched the EST data bank for synaptojanin homologues. We identified a single mouse EST closely related to synaptojanin (clone 370576) and named the corresponding protein synaptojanin 2. Northern blots of synaptojanin 2 showed that it is ubiquitously expressed, with the highest levels in heart and brain (data not shown).
Sequencing of the mouse EST clone suggested that it is nearly full-length. Screening a mouse cDNA library allowed us to isolate more 5
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PIP Phosphatase Activities of Synaptojanins 1 and 2--
Recent
studies demonstrated that synaptojanin 1 is a PIP phosphatase that
hydrolyzes PIP2 and PIP3 (9, 10, 15). The homology of synaptojanin 2 to synaptojanin 1 suggests that synaptojanin 2 may also function as a phosphatase. To directly test this hypothesis, we expressed synaptojanin 1 and 2 by transfection in COS cells. Synaptojanin 1 was expressed as a full-length protein, and synaptojanin 2 was expressed as a truncated protein that was N-terminally fused to
the myc epitope to allow efficient immunoprecipitation. COS cells transfected with the synaptojanin vectors, and control plasmids were subjected to immunoprecipitations using antibodies to synaptojanin 1 in case of the synaptojanin 1 transfections (Fig.
3A) or antibodies to the
myc epitope in case of the synaptojanin 2 transfections (Fig. 3B). The PIP phosphatase activity of the
immunoprecipitates was measured using 3H-labeled
PIP2 and thin layer chromatography for detection of the
hydrolysis products (15). The assays unequivocally demonstrated that
both synaptojanins hydrolyze PIP2 to PIP (Fig. 3). No
hydrolysis to phosphatidylinositol was detected as would be expected
from the predicted specificity of synaptojanins as 5-phosphatases (1,
15). The background activity of the control immunoprecipitates was at
the level of the assay buffer for synaptojanin 2, suggesting that the
entire PIP2 phosphatase activity measured was due to the
transfected protein. In the case of synaptojanin 1, however, the
background activity obtained with the immunoprecipitates with the
synaptojanin 1 antibodies from the control transfected COS cells was
higher than the buffer background, presumably because the synaptojanin
1 antibody reacts with low levels of endogenous synaptojanin 1 present
in the COS cells. Together these data show that synaptojanins 1 and 2 have comparable PIP phosphatase activities and that mammals contain
multiple related PIP 5-phosphatases.
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Alternative Splicing of Synaptojanin 2--
The 3-untranslated
region of the synaptojanin 2 clone we analyzed contains a long open
reading frame (243 residues) that starts 23 bp after the stop codon in
a reading frame different from that of the preceding synaptojanin 2 sequence. The sequence of this open reading frame in the
3
-untranslated region is enriched in proline (15.0% of residues) but
exhibits no other remarkable features. Data base searches failed to
detect any similarity of this sequence with current entries.
Synaptojanin 1 also contains a large open reading frame in the
3
-untranslated region of the brain mRNA that is proline-rich
(14.3% of residues). Alternative splicing joins this open reading
frame to the 5
coding region in peripheral tissues (11). The presence
of a large open reading frame in the 3
region of synaptojanin 2 suggests the possibility that this reading frame may also be joined to
the synaptojanin 2 coding sequence by alternative splicing in some
tissues, similar to the situation with synaptojanin 1. To test this
possibility, we performed PCR experiments on first strand cDNA from
a variety of tissues (Fig. 4).
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Developmental Expression of Alternative Splicing of Synaptojanin 2-- To characterize the developmental time course of alternative splicing of synaptojanin 2, we also used PCR (Fig. 6). Alternative splicing of synaptojanin 2 was not detectable during early development or even in the first postnatal week in mice. In the three brain regions examined, alternative splicing appeared with similar developmental profiles that differed only slightly. It was first observed between postnatal days 9 and 13 in cortex and cerebellum, and a few days later in the olfactory bulb. The appearance of alternative splicing is significantly later than synaptogenesis, which starts in the first week of life, suggesting that alternative splicing may be triggered by synaptogenesis instead of being involved in synaptogenesis.
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DISCUSSION |
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Synaptojanin 1 is a PIP 5-phosphatase that is enriched in nerve terminals and forms a complex with proteins involved in endocytosis (8, 10). Based on its catalytic activity and protein interactions, synaptojanin 1 may function in terminating a PIP signal for exocytosis or in triggering endocytosis, but its precise function is unclear. In view of the importance of PIP 5-phosphates in signal transduction and membrane traffic (1, 2), PIP 5-phosphatases are likely to be key regulatory enzymes. Although synaptojanin 1 constitutes the major PIP 5-phosphatase in mammalian brain (9), the presence of multiple related genes in yeast suggests that PIP 5-phosphatase signaling probably is more complex than could be performed by a single enzyme. In the current study, we have identified a second isoform of synaptojanin 1 named synaptojanin 2 that also exhibits PIP phosphatase activity and is also alternatively spliced. These data suggest that in mammals, multiple synaptojanin PIP 5-phosphatases are active.
A unitary domain map emerges for synaptojanins from the analysis of the two mammalian and three yeast proteins (Fig. 1). All of these proteins consist of an N-terminal SAC1-homology domain, a central catalytic PIP 5-phosphatase domain, and a nondescript C-terminal domain. The SAC1-homology and PIP 5-phosphatase domains are well conserved, whereas the C-terminal regions exhibit no sequence homologies. Thus these proteins represent a family of enzymes with divergent C termini that are evolutionarily conserved from yeast to man. In the three domains of these proteins, only the central domain has a well defined function as a phosphatase, although the biological significance of the phosphatase activity is unclear. The role of the N-terminal SAC1-homology domain is unknown; however, its homology to SAC1, a protein involved in membrane traffic and the cytoskeleton possibly via interaction with inositol derivatives (16, 17), suggests a similar function. The C-terminal domain in synaptojanin 1 functions as a binding domain for SH3 domains in adaptor proteins such as amphiphysin (10). Because it is not conserved in synaptojanin 2, this is unlikely to be a central activity of synaptojanin 2.
The most unexpected feature of the synaptojanins may consist in their coordinate C-terminal alternative splicing. Alternative splicing of synaptojanins 1 and 2 is expressed in a tissue-specific manner. Furthermore, it is developmentally regulated in synaptojanin 2. Alternative splicing appears developmentally very late in brain, after synaptogenesis has started. Because protein production lags behind the mRNA synthesis monitored in the PCR experiments, it seems likely that the production of the short form of synaptojanin 2 either accompanies or follows synaptogenesis.
Synaptojanins 1 and 2 are very similar in that both genes produce splicing variants in which the longer variant encoding a shorter protein is primarily expressed in brain and the shorter variant produces a longer protein in peripheral tissues (Fig. 4 and Ref. 11). However, this similar alternative splicing is unexpected because the C-terminal domains of synaptojanins and their alternatively spliced sequences exhibit no homology. Furthermore, the alternatively spliced sequences are of different sizes. The lack of homology in this region and the differences in size suggest that the coordinate alternative splicing of synaptojanins is not the result of evolutionary conservation but of evolutionary convergence, i.e. that it arose independently in evolution in the two different isoforms. If this is correct, there must have been considerable evolutionary pressure in producing the splice variants, even if they are not homologous to each other. Identification of the functions of synaptojanins and of the roles of their C-terminal domains will be instrumental in clarifying this issue.
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ACKNOWLEDGEMENTS |
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We thank T. Nguyen for the gift of cDNAs and Drs. M. S. Brown and J. L. Goldstein for advice.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1-MH52804 and by funds from the Perot Family Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF026123.
To whom correspondence should be addressed. Tel.: 214-648-5022;
Fax: 214-648-6426.
1 The abbreviations used are: PIP, phosphatidylinositol phosphate; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EST, expressed sequence tag; PIP2, phosphatidylinositol diphosphate; PIP3, phosphatidylinositol trisphosphate; bp, base pair(s).
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REFERENCES |
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