|
 |
|
EMBO reports 4, 8, 793–799 (2003)
doi:10.1038/sj.embor.embor900
AOP Published online: 11 July 2003
Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo
Camilla Burnett & Ken Howard
|
|
|
|
MRC Laboratory for Molecular Cell Biology, and
Department of Physiology, University College London, Gower
Street, London WC1E 6BT, UK
To whom correspondence should be addressed
Ken Howard Tel: +44 20 7679 2248; Fax: +44 20 7679 7805;
ken.howard@ucl.ac.uk
Received 14 March 2003; Accepted 11 June 2003; Published online 11 July 2003.
|
|
|
|
Abstract
Wunen (Wun), a homologue of a lipid phosphate phosphatase (LPP), has a
crucial function in the migration and survival of primordial germ cells (PGCs)
during Drosophila embryogenesis. Past work has indicated that the LPP
isoforms may show functional redundancy in certain systems, and that they have
broad-range lipid phosphatase activities in vitro, with little apparent
specificity between them. We show here that there are marked differences in
biochemical activity between fly Wun and mammalian LPPs, with Wun having a
narrower activity range than has been reported for the mammalian LPPs.
Furthermore, although it is active on a range of substrates in vitro,
mouse Lpp1 has no activity on an endogenous Drosophila
germ-cell-specific factor in vivo. Conversely, human LPP3 is active,
resulting in aberrant migration and PGC death. These results show an absolute
difference in bioactivity among LPP isoforms for the first time in a model
organism and may point towards an underlying signalling system that is
conserved between flies and humans.
EMBO reports 4, 8, 793–799 (2003)
doi:10.1038/sj.embor.embor900
AOP Published online: 11 July 2003
|
|
|
|
Introduction
Primordial germ cells (PGCs) are the stem-cell progenitors of the germ
line. In Drosophila, as in most organisms, the PGCs originate in an area
of the developing embryo that is remote from their target tissue, the somatic
gonad. During their migration through the embryo in search of the somatic
gonad, they are subject to several guidance cues that are essential for a
successful journey (Wylie, 1999;
Starz-Gaiano & Lehmann, 2001).
The process of PGC migration provides a system for studying the
control of cellular migration in general. wunen (wun) and its
closely related counterpart wunen 2 (wun2) encode
Drosophila homologues of mammalian lipid phosphate phosphatases (LPPs).
Wun and Wun2 have dynamic expression patterns, but are most strongly evident in
the hindgut primordium at stage 10, becoming localized to the side of the
midgut from which the PGCs migrate. In embryos that are mutant for both genes,
the PGCs scatter on exiting the midgut at stage 10, and fail to complete
migration to the gonads. Ectopic expression of Wun or Wun2 in the mesoderm at
stage 10 repels PGCs from its permissive environment, and is concomitant with a
marked reduction in the number of PGCs by stage 13. This activity can be
prevented by point mutations in the conserved catalytic residues, indicating
that these proteins function as enzymes to dephosphorylate an unknown
substrate. The observation that misexpression of either protein results in PGC
death shows that this substrate, which is presumably an attractive signal for
the PGCs, also regulates their survival (K.H., unpublished data;
Starz-Gaiano et al., 2001;
Zhang et al., 1996, 1997).
The LPPs are grouped into three isoforms, 1, 2 and 3, and the human
genes also have splice variants (Waggoner et al.,
1999). We analysed sequence alignments of Wun and Wun2 with mammalian
LPPs, and conclude that both proteins have the greatest homology with human
LPP3.
Past work has indicated that the three known LPP isoforms can
dephosphorylate a broad range of lipid phosphates, notably lysophosphatidic
acid (LPA), phosphatidic acid (PA), ceramide-1-phosphate (C(1)P),
diacylglycerol pyrophosphate (DGPP) and sphingosine-1-phosphate (S(1)P), with
relatively little apparent specificity (Dillon et al.,
1997; Jasinska et al., 1999;
Kai et al., 1997; Roberts
et al., 1998; Waggoner et al.,
1996). Apart from Wun and Wun2, little is known about the biological
functions of these proteins. Dri42 (differentially expressed in rat intestine
42), the rat homologue of LPP3, is upregulated during differentiation of the
crypt cells in the small intestine (Barila et al.,
1996), whereas the human splice-variant LPP1- 1 is
downregulated in human colon-tumour tissue (Leung et
al., 1998). In 2000, Zhang et al. reported a homozygous
null mutation in murine Lpp2 that produced viable, fertile mice with no
detectable phenotype. In addition, Lpp2 is expressed at lower levels than the
Lpp1 and Lpp3 isoforms, leading to the proposal that Lpp2 functions redundantly
with them (Zhang et al., 2000).
Starz-Gaiano et al. reported that null mutations in either wun or
wun2 also present no detectable phenotype, with embryonic development
and PGC migration occurring normally. By contrast, removal of both genes
results in highly perturbed PGC migration, with PGCs scattering widely on
exiting the midgut at stage 10. This led to the suggestion that these LPPs
function redundantly (Starz-Gaiano et al.,
2001).
Given the remarkable bioactivity of the potential lipid substrates for
these enzymes, and the specificity of some of their receptors (Takuwa et al., 2002), we found it curious that the
separate isoforms present such broad-range activity in biochemical assays and
seem to be functionally redundant in vivo. We were interested in
examining any potential specificity in substrate recognition among the fly and
mammalian isoforms in vitro for a range of known substrates, and in
vivo by investigating their ability to dephosphorylate the PGC-specific
guidance molecule that functions as a substrate for Wun. We show here that
there are marked differences in relative activity between immunopurified Wun
and the mammalian isoforms in a biochemical phosphate-release assay, with Wun
showing negligible activity for two of the substrates. We created transgenic
flies expressing mouse Lpp1 and human LPP3, and show that, although active on
all of the tested substrates in vitro, mouse Lpp1 is completely inactive
in the germ-cell migration bioassay. Conversely, overexpression of human LPP3
results in aberrant PGC migration and death, with a remarkably similar
phenotype to Wun overexpression. This demonstrates a distinct difference in
bioactivity between the isoforms for the first time, and may point towards an
underlying signalling system that is conserved between flies and humans.
Results
wun and wun2 have the same messenger RNA expression
pattern and the same ectopic expression phenotype, indicating a high level of
conservation between the two genes. To investigate the relationship between
mammalian LPPs and Wun and Wun2, we performed ClustalW alignments
(http://www.ebi.ac.uk/clustalw) of the entire amino-acid sequences
of each protein alongside those of the human LPPs, LPP1, LPP2 and LPP3 (Fig. 1). This showed almost complete conservation of the
phosphatase domains. A phylogenetic tree, as calculated by ClustalW, indicates
that Wun and Wun2 are most homologous to human LPP3 (Fig.
2).
|
|
Figure 1
Sequence alignment of lipid phosphate phosphatase proteins. The
conserved phosphatase domains are shown in light blue, with those residues
thought to be required for catalytic activity shown in dark blue (Brindley & Waggoner, 1998). The position of the
WunD:248>T mutation is shown in white. Six transmembrane domains are
indicated, as predicted by TMPred
(http://www.ch.embnet.org/software/TMPRED_form.html;
Hofmann & Stoffel, 1993), and are shown in
boxes. Amino acids that are identical between all five sequences are indicated
by asterisks, and are shown on a dark pink background or by dark pink letters.
Where conserved substitutions have been identified, the amino acids are
indicated by a colon, and are shown on a purple background or by purple
letters; where semi-conserved substitutions have been identified, amino acids
are indicated by a dot, and are shown on a green background or by green
letters. The GenBank accession numbers for each sequence are as follows: human
lipid phosphate phosphatase 1 (LPP1), AB000888 (Kai et al.,
1997); human LPP2, AF047760
(Roberts et al., 1998); human LPP3,
AB000889 (Kai et
al., 1997); Wunen, U73822
(Zhang et al., 1997); Wunen2,
AF331162 (Starz-Gaiano
et al., 2001).
|
|
|
|
|
|
Figure 2
Phylogenetic tree of Wunen and Wunen 2 with the human lipid
phosphate phosphatase isoforms. The tree was generated using the sequences
shown in Fig. 1. LPP, lipid phosphate phosphatase.
|
|
|
Alongside Wun, we focused on mouse Lpp1 and human LPP3, as they had
been previously cloned and characterized (Kai et al.,
1996, 1997). We tagged each protein at
the carboxyl terminus with green fluorescent protein (GFP). We also tagged
WunD:248>T, which has a mutation in the sixth transmembrane domain that
disrupts the putative catalytic site (Neuwald,
1997) and should result in a catalytic null (Fig.
3A). Complementary DNAs encoding these proteins were cloned into the
Gal4-regulated transformation vector pUAST, and the sequences of the modified
inserts were confirmed. To verify that they encoded proteins of the predicted
sizes, we transfected them individually into Drosophila S2 cells with
the ubiquitous driver Actin5C–Gal4 and analysed them by western
blotting (Fig. 3B).
|
|
Figure 3
The Wunen protein and confirmation of protein sizes. (A) The
Wunen (Wun) protein, indicating the position of the D:248>T point mutation.
Conserved residues required for catalysis are shown in red (Neuwald, 1997). (B) Western blot confirming that
each protein runs to the correct predicted size. GFP, green fluorescent
protein; LPP, lipid phosphate phosphatase.
|
|
|
We investigated the activity of immunopurified Wun and the two
mammalian LPPs on PA, LPA, S(1)P and C(1)P in a biochemical assay. The results
show that, despite the presence of high levels of D:248>T, it is completely
inactive for any of the substrates. Whereas mouse Lpp1 and Wun showed similar
levels of activity for LPA, human LPP3 was considerably less active (Table
1; Fig. 4). However, human LPP3 showed  1.6
times more activity for PA, than mouse Lpp1 (Table 1),
whereas mouse Lpp1 was 1.7 times more active on C(1)P than was human LPP3
(Table 1). By comparison, Wun showed negligible
activity on C(1)P or PA. We were unable to obtain any results for S(1)P,
possibly due to an inability to present the substrate optimally in this
system.
|
|
Table 1
Relative activities from the phosphate-release assay and
densitometry
|
|
|
|
|
|
Figure 4
PiPer® phosphate-release assay with lysophosphatidic acid.
Fluorescence was measured using a SPECTRAmax™ GEMINI XS Dual Scanning
Microplate Spectrafluorometer. (A) Mean fluorescent readout 
s.d. over time from the three experiments for each protein. The sequential
action of the enzymes involved in the detection system after exposure to
phosphate in solution accounts for the 20-min lag period seen at the start of
the assay. Running phosphate standards alone produces the same lag period (see
supplementary information online).
Plotting phosphate standards against the corresponding fluorescent readout at
the chosen timepoint (t = 65 min) gives a straight line in the presence
of up to 4 nmol, indicating that the detection system shows first-order
kinetics at this timepoint (B). These values are the means s.d.
of the three samples for each standard. LPA, lysophosphatidic acid; LPP, lipid
phosphate phosphatase; RFU, relative fluorescence units; Untrans,
untransfected; Wun, Wunen.
|
|
|
We generated transgenic flies for each construct and crossed them to
the mesodermal driver Twist–Gal4 (Greig &
Akam, 1993). Expression of Wun–GFP disrupted PGC migration and
reduced PGC number, as previously reported for untagged Wun. PGCs are seen in
tissues other than the gonads, having been repelled from the mesoderm and
therefore failing to contact the somatic gonadal precursors (Fig. 5A,F).
|
|
Figure 5
Ectopic expression in the mesoderm. Embryos were immunostained with
anti-Vasa antibody to visualize the primordial germ cells (PGCs; brown) and
with anti-green-fluorescent-protein (GFP) to visualize protein expression
(blue). Embryos in (A–E) are viewed laterally, with the
posterior pole to the right; embryos in (F–J) are viewed
dorsally. Expression of Wunen (Wun)–GFP (A) or human lipid
phosphate phosphatase 3 (LPP3)–GFP (B) at stage 10 results in an
early loss of PGCs compared with the ectopic expression of
WunD:248>T–GFP (C) or mouse Lpp1–GFP (D). These
can be compared with a wild-type embryo (E) with a full complement of
PGCs at the same stage. By the end of embryogenesis, those embryos expressing
Wun–GFP (F) or LPP3–GFP (G) show a marked loss of
PGCs. Embryos expressing WunD:248>T–GFP (H) or mouse
Lpp1–GFP (I), however, show no apparent perturbation or loss of
PGCs, and clearly form two gonads, as seen in a wild-type embryo at the same
stage (J). Wild-type embryos have not been stained with anti-GFP, and
consequently show no blue staining.
|
|
|
The Wun catalytic-null D:248>T had no biological activity. PGC
migration occurred normally, even in the presence of high levels of the mutant
protein in the mesoderm (Fig. 5C,H). Starz-Gaiano et al. (2001) have shown previously
that point mutations in the conserved catalytic domain of Wun2 abolish its
biological activity in vivo. However, this is the first time that loss
of phosphatase activity, as confirmed in a biochemical assay, has also been
shown to abolish biological function, and this indicates that converting a
single aspartic acid to a threonine at residue 248 is sufficient to remove
catalytic activity both in vitro and in vivo in Wun.
Surprisingly, expression of human LPP3–GFP also resulted in
highly perturbed PGC migration and a marked reduction in PGC number. In some
embryos, only one or two PGCs remain at the end of embryogenesis (Fig. 5B,G). Human LPP3 may therefore recognize and
dephosphorylate the same PGC-specific substrate in flies as Drosophila
Wun.
Conversely, expression of biochemically active mouse Lpp1–GFP
gave no phenotype, with no aberrant migration or apparent PGC loss (Fig. 5D,I). Mouse Lpp1 is therefore incapable of
dephosphorylating the same germ-cell-specific factor as Wun in vivo,
demonstrating an absolute difference in functional bioactivity between the LPP1
and LPP3 isoforms.
The LPPs have been shown to have ecto-enzymatic activity (Ishikawa
et al., 2000; Jasinska et al.,
1999; Roberts et al., 1998).
Although we do not know if the germ-cell migration phenotype is caused by an
extracellular activity of Wun or human LPP3, we wanted to exclude the
possibility that mouse Lpp1–GFP fails to be correctly trafficked and to
confirm that all of the GFP-tagged proteins are trafficked to the cell surface.
We examined embryos that express these proteins in the mesoderm by confocal
microscopy. All of the proteins were detected on the mesodermal cell surface
(Fig. 6A). None of the proteins localized to
intracellular membrane structures when transiently expressed in the embryonic
mesoderm by this method. We biochemically analysed the cell surfaces of intact
S2 cells that expressed either human LPP3–GFP or mouse Lpp1–GFP by
incubating them with N-hydroxysuccinimide–biotin in solution and
running the lysates over a monomeric-biotin-binding avidin column. Western blot
analysis also confirmed each protein's presence at the cell surface (Fig. 6B).
|
|
Figure 6
Confirmation of proteins present at the cell surface. (A)
Images obtained by confocal microscopy, showing Wunen
(Wun)–green-fluorescent-protein (GFP), mouse lipid phosphate phosphatase
1 (Lpp1)–GFP and human LPP3–GFP at the surface of mesodermal cells
in Drosophila embryos. Protein expression is driven by
Twist–Gal4. (B) Biotinylation of human LPP3–GFP and
mouse Lpp1–GFP at the surface of S2 cells, detected with anti-GFP
antibody. E, elutions (protein that is biotinylated and has bound to the
column); TL, total lysate before capture on the column; W, washes (protein that
is not biotinylated and has not bound to the column).
|
|
|
Discussion
Although we have not performed detailed kinetic analyses, we show here
that the fly LPP Wun and the mammalian LPP1 and LPP3 isoforms differ in their
relative activities on PA, LPA and C(1)P in the PiPer® phosphate-release
assay. Although Wun can dephosphorylate LPA with a similar efficiency to mouse
Lpp1, it shows negligible activity on both PA and C(1)P. This indicates that
there are distinct differences in substrate preference between the fly and
mammalian enzymes, with Wun showing a narrower activity range in this assay
than was previously reported for the mammalian LPPs. Both mouse Lpp1 and human
LPP3 are active on all three substrates. We found that mouse Lpp1 has a
1.7-fold higher activity on C(1)P than human LPP3 in this assay. Human LPP3,
however, had a 1.6-fold higher activity than mouse Lpp1 on PA. Previous data on
the activity for each isoform on these substrates has tended to vary (Kai et al., 1997; Roberts et
al., 1998). The inconsistencies that have been observed for the
same isoform on the same substrates may be due to the particular assay
conditions or the method of enzyme preparation. This could account for the
variation in relative activities between Wun and human LPP3 in vitro,
despite their close homology. Alternatively, there may be fundamental
differences between the fly and mammalian isoforms that we are unable to
uncover without a more detailed analysis of their structures.
We also show, however, that human LPP3 is highly active when
ectopically expressed in Drosophila embryos, as assayed by the
disruption of PGC migration and survival, which results in a phenotype similar
to the ectopic expression of Wun or Wun2. That human LPP3 shows the same
phenotype in a bioassay as Drosophila Wun suggests a conserved
signalling pathway that regulates germ-cell migration and survival from flies
to humans. Conversely, although active in the biochemical assay, mouse Lpp1 is
completely inactive in vivo, and has no apparent effect on PGC migration
or survival. This shows an absolute difference in functional bioactivity
between the mammalian LPP isoforms. That the LPP isoforms present different
functional outputs when assayed in vivo has a number of implications. It
may be that the Lpp1 isoform simply cannot recognize or catalyse the
dephosphorylation of the specific factor acted on by Wun and LPP3. This would
demonstrate specificity in substrate choice between the isoforms, and may
indicate that the germ-cell-specific factor is not PA, LPA or C(1)P, on which
mouse Lpp1 is active in vitro, particularly as Wun shows negligible
activity on PA and C(1)P in the same assay. Alternatively, there may be
specific components of the pathway that are required for selection and
recognition of the factor. It is possible that as yet unidentified
conformational or structural differences in mouse Lpp1 preclude its association
with these factors, thus inhibiting its enzymatic function in this system.
Thus, LPA could be the factor, and the unnatural presentation and high
concentration of LPA in the biochemical assay may override the specific
selection mechanisms used to regulate activity in vivo, allowing mouse
Lpp1 access to this otherwise inaccessible substrate. Primary sequence analyses
have not identified any immediate candidates for residues conferring such a
difference. We speculate that the observed differences in substrate preference
may be related to the proteins' structure, which are as yet unsolved. We note
that Barila et al. have already studied the properties of the internal
sequences of Dri42 in its trafficking to the cell surface (Barila et al., 1996). The contributions of the
termini to biological and biochemical properties are yet to be reported in any
detail.
In conclusion, we present evidence of differences in relative activity
between the mammalian and fly LPP isoforms on LPA, PA and C(1)P in a
biochemical assay. Our results indicate that the fly LPP Wun has a narrower
activity range than the mammalian LPPs in this assay. We also present evidence
to show that, although active in vitro, mouse Lpp1 cannot
dephosphorylate the same endogenous germ-cell-specific factor as Wun in
vivo, whereas human LPP3 seems to do so. This demonstrates that despite
broad-range activity in Triton-micelle assays, the mammalian LPP isoforms do
show distinct differences in bioactivity when assayed in a physiological
context. We expect that a combination of biochemical and biological data, such
as those presented here, will in time help to identify the physiological
substrate for Wun, which is, presumably, a stem-cell control factor.
Methods
Cloning and expression in S2 cells.
Human LPP3–GFP cloning was performed using the construct made
in EGFPN3 by I. Wada. The Wun–GFP construct was also made in pEGFPN3 by
I. Wada. We transferred sequences to pUAST using conventional restriction
enzyme digestion techniques. The mouse Lpp1 and mutant wun
constructs were made in our laboratory. Where we used PCR, oligonucleotide
mutagenesis or linker insertion, the resulting clones were sequenced through
the entire coding region of the modified protein and the sequences were
deposited in GenBank. All tags were added to C termini.
Drosophila S2 cells were maintained in HyQCCM3 media (Perbio
Science). We used the Effectene Transfection Reagent (Qiagen). Forty-eight
hours post-transfection, cells were washed in PBS, spun at 3,000 r.p.m. and
lysed on ice in lysis buffer (0.5 M Hepes, 5 M sodium chloride, 1 M sodium
fluoride, 0.5 M EDTA, 0.5 M sodium orthovanadate, 0.1% triton X-100, 2 mM
N-ethylmaleimide, and Complete protease inhibitors (Roche)). Western blots were
performed using standard techniques. We used a mouse monoclonal antibody to GFP
(Roche) followed by a horseradish peroxidase (HRP)-conjugated anti-mouse
antibody (Jackson ImmunoResearch Laboratories).
Protein immunoprecipitation.
Transfected S2 cells were lysed as before and an equal volume of
lysate was added to an aliquot of Fusion Aid GFP resin (Vector laboratories).
After incubation for 2 h at 4 °C, the resin was washed extensively and
resuspended in a 5 volume of cell lysis buffer plus protease inhibitors
before western blot analysis. Lysate from untransfected S2 cells at the same
density was also immunocaptured as a control.
Phosphate-release assay.
We used the Molecular Probes PiPer® phosphate-release assay
(Cambridge Bioscience). In the presence of inorganic phosphate, maltose
phosphorylase converts maltose to glucose-1-phosphate and glucose.
Gluconolactone and H2O2 are then formed by the action of
glucose oxidase. Using HRP as a catalyst, the H2O2 reacts
with the Amplex™ Red reagent to produce the fluorescent product
resorufin. The increase in detectable fluorescence/absorbance is therefore
proportional to the amount of phosphate present.
Each reaction was performed in triplicate, using 50  l of the
anti-GFP resin containing the immunocaptured proteins. Assays were performed at
37 °C and the resulting fluorescence was measured in a standard
plate-reader in a final volume of 100 l at up to seven timepoints during
the assay period. Each substrate control gave a small but constant value, which
was subtracted from each of the samples. LPA (Sigma) in 50% ethanol and C(1)P
(Affiniti) in 100% ethanol were used at a final concentration of 500 M. PA
(Sigma) in 100% ethanol was used at a final concentration of 70 M in the
supplied buffer with 0.01% phosphate-free triton X-100 (Sigma) and 1 mg
ml-1 fatty-acid-free BSA (Sigma). To test the linearity of
the detection system, a range of phosphate standards were run simultaneously in
triplicate. We examined activity at an arbitrary timepoint that seemed to fall
within the linear range for the enzymes for each assay. The PO4
standards were converted to nanomoles of PO4 at this time, and were
used to produce a standard curve. This was linear in each case, indicating that
the detection system shows first-order kinetics in the presence of up to 4 nmol
of PO4. A line of best fit was used to calculate the amount of
PO4 released from each protein sample. Each sample was western
blotted, and densitometry was performed using BioRad Quantity One software to
compare relative protein amounts in each sample on the same blot.
Ectopic expression.
The constructs were microinjected into
white118 embryos and transformants were recovered using
standard techniques. To visualize the construct in the mesoderm and the PGCs,
we used a mouse monoclonal antibody to GFP (Roche) followed by an
alkaline-phosphatase-conjugated anti-mouse antibody (Jackson ImmunoResearch
Laboratories), and chicken anti-Vasa (K.H.) followed by biotin-conjugated
anti-chicken (Jackson ImmunoResearch Laboratories).
Cell-surface biotinylation.
We used Pierce No-Weigh™ Premeasured NHS-PE04 Biotin (Perbio
Science). The reaction was quenched with 100 mM ammonium chloride. Cells were
washed to remove extraneous unbound biotin, lysed on ice in 1.5 ml cell lysis
buffer (1% Triton X-100) and spun for 5 min at 3,000 r.p.m. at 4 °C. The
supernatant was applied to a Pierce Immunopure Monomeric Avidin column (Perbio
Science) in accordance with the manufacturer's instructions. Fractions were
analysed by western blotting.
Supplementary information
is available at EMBO reports online
(http://www.emboreports.org).
|
|
Acknowledgements
We thank A. Harwood, J. Ryves, P. Makridou, A. Morris, D. Cutler, R.
Lehmann and M. Raff for help and advice, and T. Kornberg for the
Actin5C–Gal4 driver. We also thank H. Kanoh, I. Wada and M. Kai
for sharing materials and for help with cloning. This work was supported by the
Wellcome Trust, grant number 054375.
|
|
References
Barila, D. et al. ( 1996) The Dri 42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein. J. Biol. Chem., 271, 29928–29936. | Article | PubMed | ChemPort |
Brindley, D. & Waggoner, W. ( 1998) Mammalian lipid phosphate phosphohydrolases. J. Biol. Chem., 273, 24281–24284. | Article | PubMed | ChemPort |
Dillon, D.A. et al. ( 1997) Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity. J. Biol. Chem., 272, 10361–10366. | Article | PubMed | ChemPort |
Greig, S. & Akam, M. ( 1993) Homeotic genes autonomously specify one aspect of pattern in the Drosophila mesoderm. Nature, 362, 630–632. | Article | PubMed | ChemPort |
Hofmann, K. & Stoffel, W. ( 1993) TMbase—a database of membrane spanning proteins segments. Biol. Chem. Hoppe Seyler, 374, 166.
Ishikawa, T., Kai, M., Wada, I. & Kanoh, H. ( 2000) Cell surface activities of the human type 2b phosphatidic acid phosphatase. J. Biochem., 127, 645–651. | PubMed | ChemPort |
Jasinska, R. et al. ( 1999) Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters. Biochem. J., 340, 677–686. | Article | PubMed | ChemPort |
Kai, M., Wada, I., Imai, S., Sakane, F. & Kanoh, H. ( 1996) Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase. J. Biol. Chem., 271, 18931–18938. | Article | PubMed | ChemPort |
Kai, M., Wada, I., Imai, S., Sakane, F. & Kanoh, H. ( 1997) Cloning and characterization of two human isozymes of Mg2+-independent phosphatidic acid phosphatase. J. Biol. Chem., 272, 24572–24578. | Article | PubMed | ChemPort |
Leung, D.W., Tomkins, C.K. & White, T. ( 1998) Molecular cloning of two alternatively spliced forms of human phosphatidic acid phosphatase cDNAs that are differentially expressed in normal and tumor cells. DNA Cell Biol., 17, 377–385. | PubMed | ChemPort |
Neuwald, A.F. ( 1997) An unexpected structural relationship between integral membrane phosphatases and soluble haloperoxidases. Protein Sci., 6, 1764–1767. | PubMed | ChemPort |
Roberts, R., Sciorra, V.A. & Morris, A.J. ( 1998) Human type 2 phosphatidic acid phosphohydrolases. Substrate specificity of the type 2a, 2b, and 2c enzymes and cell surface activity of the 2a isoform. J. Biol. Chem., 273, 22059–22067. | Article | PubMed | ChemPort |
Starz-Gaiano, M. & Lehmann, R. ( 2001) Moving towards the next generation. Mech. Dev., 105, 5–18. | Article | PubMed | ChemPort |
Starz-Gaiano, M., Cho, N.K., Forbes, A. & Lehmann, R. ( 2001) Spatially restricted activity of a Drosophila lipid phosphatase guides migrating germ cells. Development, 128, 983–991. | PubMed | ChemPort |
Takuwa, Y., Takuwa, N. & Sugimoto, N. ( 2002) The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities. J. Biochem. (Tokyo), 131, 767–771. | PubMed | ChemPort |
Waggoner, D.W., Gomez-Munoz, A., Dewald, J. & Brindley, D.N. ( 1996) Phosphatidate phosphohydrolase catalyzes the hydrolysis of ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. J. Biol. Chem., 271, 16506–16509. | Article | PubMed | ChemPort |
Waggoner, D.W., Xu, J., Singh, I., Jasinska, R., Zhang, Q.X. & Brindley, D.N. ( 1999) Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction. Biochim. Biophys. Acta, 1439, 299–316. | Article | PubMed | ChemPort |
Wylie, C. ( 1999) Germ cells. Cell, 96, 165–174. | PubMed | ChemPort |
Zhang, N., Zhang, J., Cheng, Y. & Howard, K. ( 1996) Identification and genetic analysis of wunen, a gene guiding Drosophila melanogaster germ cell migration. Genetics, 143, 1231–1241. | PubMed | ChemPort |
Zhang, N., Zhang, J., Purcell, K.J., Cheng, Y. & Howard, K. ( 1997) The Drosophila protein Wunen repels migrating germ cells. Nature, 385, 64–67. | Article | PubMed | ChemPort |
Zhang, N., Sundberg, J.P. & Gridley, T. ( 2000) Mice mutant for Ppap2c, a homolog of the germ cell migration regulator wunen, are viable and fertile. Genesis, 27, 137–140. | Article | PubMed | ChemPort |
|
|
|
|
|
top  |
|
|
|
