(Received for publication, October 19, 1995; and in revised form, December 13, 1996)
From the
In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase
(InsP 5-phosphatase) is the major isoenzyme hydrolyzing the
calcium-mobilizing second messenger InsP
. Activity of this
enzyme could be measured in both soluble and particulate fractions of
tissue homogenates. The protein sequence showed a putative C-terminal
isoprenylation site (CVVQ). In this study, two mutants have been
generated. The first mutant (C409S) has a serine replacing a cysteine
at position 409 of the wild-type enzyme. The second mutant (K407D1) is
a deletion mutant that lacks the last five C-terminal amino acids.
These constructs were individually expressed by transfection in COS-7
cells. Western blot analysis of wild-type transfected cells indicated
that both soluble and particulate fractions had a 43-kDa immunoreactive
band, with a higher proportion of the original homogenate associated
with the particulate part. On the contrary, when the two mutated
constructs were transfected in COS-7 cells, the phosphatase was
predominantly soluble. Confocal immunofluorescence studies showed the
wild-type enzyme to be present on the cell surface of transfected COS-7
cells and in subcellular compartments around the nucleus. This was not
observed for the two mutants, where uniform immunofluorescence labeling
was observed throughout the cytosol. Recombinant type I InsP
5-phosphatase expressed in Escherichia coli was a
substrate of purified farnesyltransferase. Altogether, the data
therefore suggest a direct participation of Cys-409 in a C-terminally
anchored InsP
5-phosphatase by farnesylation.
In response to several extracellular signals, two second
messengers, inositol 1,4,5-trisphosphate (InsP) (
)and diacylglycerol, are produced(1) . InsP
mobilizes intracellular Ca
(2) , while
diacylglycerol is the specific activator of C-type protein
kinases(3) . InsP
concentration is dependent upon
the relative activities of phospholipase C, together with several
enzymes that either phosphorylate or dephosphorylate this molecule:
InsP
5-phosphatase is responsible for dephosphorylation
into inositol 1,4-bisphosphate(4) , while InsP
3-kinase leads to the synthesis of inositol
1,3,4,5-tetrakisphosphate (InsP
)(5) . The
InsP
molecule itself has been shown to interact with
several candidate receptors. It is involved, at least in some cells, in
the mechanism of Ca
entry and/or in the regulation of
neurotransmitter
release(6, 7, 8, 9, 10) .
The recent cloning of a cDNA encoding an InsP
-binding
protein in porcine platelets shows that it is a member of the
GTPase-activating protein family (11) .
Three isoenzymes of
InsP 5-phosphatase have been described: (a) a type
I 43-kDa protein initially reported in human erythrocyte membranes (4) and later purified from many sources (12, 13, 14, 15, 16, 17) , (b) a 75-kDa protein originally isolated from human
platelets(18) , and (c) a protein identified due to
its deficiency in the Lowe or oculocerebrorenal syndrome(19) .
A truncated mutant of this protein was recently shown to possess
InsP
, InsP
, and
phosphatidylinositol-4,5-bisphosphate 5-phosphatase activities (20) .
Type I InsP 5-phosphatase hydrolyzes both
InsP
and InsP
, with higher affinity for
InsP
(K
= 1 µMversus 10 µM for InsP
), but
lower velocity (ratio of V
= 11 in favor
of InsP
)(14) . Phosphatidylinositol
4,5-bisphosphate is not a substrate(21) . In many tissues, the
activity appears to be associated with the particulate fraction of cell
homogenates; in rat brain, for example, only 5% of the total activity
of homogenates could be measured in the soluble part of the
tissue(22) . In intestinal epithelial cells, the activity was
much greater in the basolateral region of the cell in comparison with
the brush border, suggesting that the sites of InsP
generation and inactivation are in close proximity(23) .
This is opposite to the polarized distribution of particulate
5-phosphatase in hepatocytes(24) . In these studies, however,
cellular distribution was determined by enzymatic assay following cell
fractionation without any distinction between possible isoenzymes.
cDNAs encoding type I InsP 5-phosphatase have been
isolated(25, 26, 27) . All studies have
demonstrated a putative C-terminal isoprenylation site (CVVQ).
Insertion of type I InsP
5-phosphatase into membranes could
therefore be assured by post-translational modification of this
motif(28) . This hypothesis was tested in the present study by
comparison for the first time of biochemical data and immunofluorescent
localization of the phosphatase in transfected cells. Labeling of COS-7
cells at the cell surface and in subcellular compartments surrounding
the nucleus could be shown for intact InsP
5-phosphatase,
but not for two mutants of the isoprenylation motif.
It was verified that labeling was observed
neither on cells transfected with the pcDNA3 vector alone and using
anti-InsP 5-phosphatase antiserum nor on transfected cells
using the preimmune serum. Labeling using the purified antibodies was
qualitatively identical to that obtained with the total antiserum.
Although these controls were all consistent with the detection of the
genuine InsP
5-phosphatase, we cannot exclude
cross-reactions with unknown related proteins, and all results should
therefore be read as InsP
5-phosphatase-like
immunoreactivity.
Cells were observed under a Nikon Optiphot fluorescence microscope, and images were obtained using a laser-scanning confocal microscope (MRC 1000, Bio-Rad) equipped with an argon-krypton laser and COMOS software (Bio-Rad). Images were further analyzed using Imagespace software (Molecular Dynamics, Inc.).
Figure 1:
Western blot analysis
of InsP 5-phosphatase activities in crude lysates from rat
brain and transfected COS-7 and CHO cells. InsP
5-phosphatase in crude homogenates from rat brain (0.5 µg; lane 1), COS-7 cells transfected with pcDNA3 vector alone (1.5
µg; lane 2), COS-7 cells transfected with DNA encoding
wild-type InsP
5-phosphatase (2 µg; lane 3),
untransfected CHO cells (5 µg; lane 4), and CHO cells
transfected with DNA encoding wild-type InsP
5-phosphatase
(10 µg; lane 5) were subjected to SDS-polyacrylamide gel
electrophoresis (10% gels) and electrophoretically transferred to
nitrocellulose. Immunodetection was performed with antibodies to type I
InsP
5-phosphatase diluted
1000-fold.
Figure 2:
Testing of specificity of InsP
5-phosphatase antibodies in rat cerebellum. A,
immunofluorescent localization of InsP
5-phosphatase in the
Purkinje cell layer (fluorescein isothiocyanate-labeled secondary
antibody); B, high magnification revealing both diffuse and
reticular labeling within the cell and in dendritic trees. Nuclei of
Purkinje cells are unlabeled. g, granular layer; m,
molecular layer; p, Purkinje cell
layer.
When COS-7 cells were transfected with increasing amounts of DNA, the activity increased markedly until a plateau was reached at 1 µg of DNA (data not shown). 5-Phosphatase activity was distributed in soluble and particulate fractions of the homogenates in a 1:2 ratio. The addition of Triton X-100 to the incubation mixture was shown to increase activity in the particulate fraction; this effect was not observed in the soluble fraction. A maximal effect was shown at 0.1% (v/v) detergent with either rat brain or transfected COS-7 cells (data not shown). Western blot analysis indicated that both soluble and particulate fractions of transfected COS-7 cells had a 43-kDa immunoreactive band, with a higher proportion associated with the particulate fraction (Fig. 3A).
Figure 3:
Distribution of InsP
5-phosphatase activity between soluble and particulate fractions of
transfected COS-7 cells with wild-type and C-terminal mutants. A, homogenates of COS-7 cells transfected with pcDNA3 vector
alone (lane 1), soluble and particulate fractions of COS-7
cells transfected with DNA encoding wild-type InsP
5-phosphatase (lanes 2 and 3, respectively),
soluble and particulate fractions of cells transfected with C-terminal
deletion mutant K407D1 (lanes 4 and 5, respectively),
and soluble and particulate fractions of cells transfected with the
C409S point-mutated enzyme (lanes 6 and 7,
respectively). A 35-µl aliquot of protein was applied to each lane
(
0.5-1.5 µg of protein). Western blot analysis was
carried out as described in the legend of Fig. 1. B,
COS-7 cells transfected with pcDNA3 vector alone (a), DNA
encoding wild-type InsP
5-phosphatase (b), DNA
encoding C-terminal deletion mutant K407D1 (c), and DNA
encoding mutant C409S (d). The total homogenate (H),
soluble fraction (S), and particulate fraction (P)
were obtained as described in the legend of Fig. 1and assayed
for InsP
5-phosphatase activity at 10 µM InsP
in the presence of 0.03% Triton X-100. These
results are representative of three different
transfections.
Using antibodies
able to interact with both the soluble and particulate InsP 5-phosphatases, we were able to determine the relative amounts of
these enzymes in transfected COS cells. This was done on Western blots
by measuring the radioactivity of
I-labeled protein A
associated with the 43-kDa protein bands. Taking into account the
InsP
5-phosphatase activity applied to the gel, we have
estimated the specific activities of the two fractions of transfected
cells. The specific activity of the particulate enzyme was 6-fold lower
than that of the soluble enzyme (Table 1). Similar results have
been obtained by comparing soluble and particulate fractions of rat
cerebellum (data not shown).
Figure 4:
Immunofluorescent localization of
wild-type InsP 5-phosphatase in transfected COS-7 cells.
Cells were grown on coverslips and processed for immunofluorescent
staining with anti-InsP
5-phosphatase antibodies and
fluorescein isothiocyanate-labeled secondary antibody.
Immunofluorescent staining of serial confocal sections is shown (A). Optical sections (0.5- or 1-µm intervals) were from
the basal adherent pole (top left) toward the superior pole (bottom right). Immunofluorescence of wild-type InsP
5-phosphatase revealed several spots like reticulum/vesicle
structures that are present throughout the cell (B). A and B point out clear staining at the plasma membrane. In
some cells, immunofluorescence of the plasmalemma membrane was more
discrete (C). The presence of both transfected stained cells
and untransfected unstained cells can be seen at low magnification (D).
Both
InsP 5-phosphatase mutants showed distinct distributions in
transfected COS-7 cells: uniform and diffuse labeling was localized
throughout the cytosol (Fig. 5). The plasma membrane appeared
unlabeled, and no reticular, granular, vesicle-like structures or
perinuclear labeling reminiscent of the Golgi apparatus could be
recognized.
Figure 5: Immunofluorescence pattern of transfected COS-7 cells with the two C-terminal mutants. A and C show immunofluorescent staining of serial confocal sections. Optical sections (0.5- or 1-µm intervals) were from the basal adherent pole (top left) toward the superior pole (bottom right). A and B, C409S mutant; C and D, K407D1 mutant. Both transfected cells show the same diffused cytoplasmic staining.
The immunofluorescence pattern given by anti-5-phosphatase antibodies in COS-7 cells was not confined to this cell line. A similar staining pattern was also seen in transfected CHO cells: a clear and strong staining at the cell plasma membrane, which in most cells appeared as a continuous line with few membrane vesicle-like patches (data not shown).
Figure 6:
Kinetics of protein farnesyltransferase
modification of recombinant InsP 5-phosphatase. Assays
contained 50 ng of purified FTase and the indicated amounts of
recombinant InsP
5-phosphatase (
), Ha-Ras-CVLS
(
), or Ha-Ras-CVLL (
). The substrate was either 2
µM [
H]FPP (A) or 2
µM [
H]GGPP (B). Assays were
conducted as described under ``Experimental Procedures.''
Reactions were stopped by acid precipitation, and prenylated proteins
were determined by filter binding assay.
Figure 7:
Kinetics of protein
geranylgeranyltransferase modification of recombinant InsP 5-phosphatase. Assays contained 50 ng of purified GGTase-1 and
the indicated amounts of recombinant InsP
5-phosphatase
(
), Ha-Ras-CVLS (
), or Ha-Ras-CVLL (
). The substrate
was either 2 µM [
H]FPP (A)
or 2 µM [
H]GGPP (B).
The cloning of cDNAs encoding type I InsP 5-phosphatase has provided molecular tools to study the levels of
enzyme expression in various cells and its subcellular distribution.
Protein kinase C might activate the enzyme, a feedback mechanism that
had been suggested in human platelets(38, 39) . Data
obtained with human recombinant type I InsP
5-phosphatase
(the isoform suspected to be phosphorylated in platelets) have shown
that the purified enzyme was not a substrate of protein kinase
C(36) . Another mechanism was suggested from cDNA analysis: the
C-terminal end of type I InsP
5-phosphatase shows an
isoprenylation site (CVVQ) that may be critically important for
subcellular localization and biological activity as well as for
interaction with regulatory and effector proteins. The two valine
residues are also present in the C-terminal end of one of the Ras
proteins (Ni-Ras), and as for Ras (40) , positively charged
residues are also adjacent to the modified cysteine.
Previous
studies in other cells/systems have suggested that InsP 5-phosphatase (presumably type I) was preferentially associated
with plasma membranes(23, 24) . The data have been
obtained by the use of differential centrifugation of cell homogenates.
However, such strategies are subjected to potential problems arising
from cellular disruption and subcellular fractionation, where
redistribution of proteins may occur. In the present study, the
subcellular distribution of type I InsP
5-phosphatase
enzyme was performed by indirect immunofluorescence. In both
transfected COS-7 and CHO cells, anti-InsP
5-phosphatase
immunofluorescence displayed a reticular pattern of staining throughout
the whole cell. Furthermore, a signal at the cell membrane was clearly
observed. A crescent-shaped staining around the nucleus suggestive of
the Golgi apparatus was also observed. All these subcellular
localizations (membrane, Golgi apparatus, endoplasmic reticulum, and
vesicle-like structures) were clearly superimposable to previously
reported localizations of other membrane proteins such as opiate and
glutamate metabotropic receptors expressed in COS-7 cells and analyzed
using similar techniques(41, 42) .
Data obtained in
the present studies support the post-translational modification of type
I InsP 5-phosphatase by farnesylation. First, most of the
activity present in crude homogenates of rat cerebellum or transfected
COS-7 or CHO cells was found in the particulate fraction (illustrated
by Western blotting). Deletion of the CAAX motif or the
construction of a 5-phosphatase mutant in which the cysteine has been
replaced by serine resulted in proteins that were localized largely in
the cytosol. This was visualized by activity assay, by Western blotting
after disrupting transfected COS-7 cells, or directly by
immunofluorescence (where uniform labeling was observed throughout the
cytosol). Finally, our data indicated that recombinant type I
InsP
5-phosphatase was a substrate of purified FTase.
A
C-terminal CCVVQ sequence of the 5-phosphatase would be predicted to be
perhaps dually prenylated, as in the case of RhoB that ended with CCKVL
and was shown to contain both farnesyl as well as geranylgeranyl
groups(43) . RhoB was farnesylated as well as
geranylgeranylated by GGTase-1(44) . Our data indicated that
InsP 5-phosphatase was not a substrate of GGTase-1, but was
farnesylated by FTase. Consistent with this, the carboxyl-terminal
residue of the CAAX motif in general determines which
isoprenoid will be added. When X is serine, methionine, or
glutamine (as in the case of type I InsP
5-phosphatase),
proteins are recognized by FTase(45) . The hepatitis D virus
large antigen is a virally encoded protein also containing a C-terminal
CAAX motif where X is glutamine. Recent data
indicated that this protein is farnesylated as well by the same FTase. (
)
The post-translational modification of proteins by
isoprenoids facilitates protein-membrane and protein-protein
interactions. This was shown, for example, for the -subunit of
transducin(46) . It may also affect the enzymatic activity by a
conformational change of the protein. This hypothesis has now been
tested for type I InsP
5-phosphatase. Using anti-43-kDa
protein antisera to titrate the relative amounts of enzyme, we could
demonstrate that particulate InsP
5-phosphatase had a lower
specific activity compared with the soluble enzyme.
Recent data
indicated that type II InsP 5-phosphatase also shares an
isoprenylation motif (CNPL) at the C-terminal end of the protein,
although a proline residue at this position is unusual in mammalian
systems(47) . Immunofluorescent localization of this enzyme in
transfected COS-7 cells was not reported. Furthermore, Jefferson and
Majerus (47) reported that the cysteine C-terminal mutant was
less active relatively than the wild-type enzyme toward the lipid
phosphatidylinositol 4,5-bisphosphate as substrate. No effect on
activity could be seen when InsP
was used as substrate. The
data indicated that membrane association was not required for activity.
Whether isoprenylation could modify the specific activity of this
phosphatase when it is anchored as compared with the soluble enzyme is
not known.
Isoprenylation could target the
InsP-metabolizing enzyme to specific intracellular
distributions, and further studies should establish possible
colocalization with the InsP
receptor and InsP
3-kinase. In the soma of Purkinje cells, the reticular-like
pattern is reminiscent of both InsP
and ryanodine receptors
that have been localized to the endoplasmic reticulum using electron
microscopy(48) .
Perhaps inhibition of the specific activity
of particulate type I InsP 5-phosphatase (compared with the
soluble enzyme) may be regarded as a mechanism of regulation of
InsP
5-phosphatase activity. In this context, the
myristoylated alanine-rich protein kinase C substrate and Ki-Ras
interact with the cytoplasmic surface of the plasma membrane by both
hydrophobic and electrostatic interactions(49) .
Phosphorylation of the myristoylated alanine-rich protein kinase C
substrate reduces the electrostatic interaction, and the protein is
released to the cytosol. It is not yet known whether membrane binding
of Ki-Ras, and perhaps InsP
5-phosphatase, is regulated.