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J Biol Chem, Vol. 273, Issue 15, 8741-8748, April 10, 1998
From the Type I phosphatidylinositol 4-phosphate
(PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of
phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in
various cellular processes. Here, we report the cloning of the third
member (PIP5K Recent advances in cell biology have revealed that
phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at
appropriate times and intracellular sites appear to be required for
complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2),1
is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P2 serves as a substrate for
phosphoinositide-specific phospholipase C (EC 3.1.4.11), generating the
second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate
(1). PtdIns(4,5)P2 can also be phosphorylated by
phosphoinositide 3-kinase (EC 2.7.1.137), generating
phosphatidylinositol 3,4,5-trisphosphate
(PtdIns(3,4,5)P3), the synthesis of which is activated
in signaling pathways of several growth factor receptors (2).
Furthermore, PtdIns(4,5)P2 modulates the activity of
numerous enzymes, including actin-binding proteins (3), binds
pleckstrin homology domains (4-6), and has been suggested to
play a role in exocytosis (7-9). The major pathway of
PtdIns(4,5)P2 synthesis is that mediated by type I
phosphatidylinositol-4-phosphate 5-kinases (PIP5K; EC 2.7.1.68),
phosphorylating the D-5 position of the inositol ring of
phosphatidylinositol 4-phosphate (PtdIns(4)P). Despite these important
functions of PtdIns(4,5)P2, direct investigations of the
intracellular roles and of mechanisms regulating synthesis of this
lipid molecule are limited. Until recently, a major factor hindering
progress in this field was the absence of molecular tools.
Recently, cDNAs encoding two isoforms of type I PIP5K have been
cloned (10, 11). Herein, we report molecular cloning of a third isoform
of type I PIP5K (PIP5K Cloning of Murine Type I PIP5K Northern Blotting--
A murine multiple tissue Northern
blot (CLONTECH) was hybridized according to
the instructions of the manufacturer with an [ Production of Antibody Specific to the Epitope Tagging and Expression of PIP5K Isoforms by Recombinant
Adenoviruses--
Epitope (influenza virus hemagglutinin (HA))-tagged
cDNAs were generated as described previously (10). Recombinant
adenoviruses bearing the cDNA of PIP5K isoforms with or without the
HA tag were constructed as described previously (15-17). COS7 cells
(1.5 × 106 cells) maintained in Dulbecco's modified
Eagle's medium with 10% fetal calf serum were infected with
recombinant adenoviruses (3-5 × 107 plaque-forming
units) as described previously (17). Three days later, cells were lysed
with 1 ml of lysis buffer (50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EGTA, 15 mM
MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride) and used for Western blotting and
immunoprecipitation.
Constructions of Mutant cDNAs--
Substitution mutant
cDNAs were constructed by oligonucleotide-directed mutagenesis. For
example, the PIP5K PtdIns(4)P Kinase Assay--
Immunoprecipitation was performed
using a monoclonal antibody against the HA epitope (12CA5) and protein
G-Sepharose 4 First Flow (Pharmacia Biotech Inc.). The
immunoprecipitates were used for the PtdIns(4)P kinase assay. A
standard assay for phosphorylation of PtdIns(4)P was carried out in an
incubation medium containing a final concentration of 50 mM
Tris/HCl (pH 7.5), 100 mM NaCl, 15 mM
MgCl2, 1 mM EGTA, 100 µM
PtdIns(4)P, and 50 µM [
Type I Phosphatidylinositol-4-phosphate 5-Kinases
CLONING OF THE THIRD ISOFORM AND DELETION/SUBSTITUTION ANALYSIS
OF MEMBERS OF THIS NOVEL LIPID KINASE FAMILY*
§,,,, and Third Department of Internal Medicine,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, and the
¶ Third Department of Internal Medicine, Yamaguchi University
School of Medicine, Kogushi, Ube, Yamaguchi 755, Japan
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
) and the characterization of members of the type I
PIP5K family. Type I PIP5K has two alternative splicing forms,
migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis.
The amino acid sequence of the central portion of this isoform shows
approximately 80% identity with those of the 
and 
isoforms.
Northern blot analysis revealed that the isoform is highly
expressed in the brain, lung, and kidneys. Among three isoforms, the
isoform has the greatest Vmax value for the
PtdIns(4)P kinase activity and the isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the
three isoforms, the minimal kinase core sequence of these isoforms were
determined as an approximately 380-amino acid region. In addition,
carboxyl-terminal regions of the and isoforms were found to
confer the greatest Vmax value and the highest
phosphatidic acid sensitivity, respectively. It was also discovered
that lysine 138 in the putative ATP binding motif of the isoform is
essential for the PtdIns(4)P kinase activity. As was the case with the
isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem.
272, 7578-7581), overexpression of either the or the isoform induced an increase in short actin fibers and a decrease in
actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient
substitution mutant also induced an abnormal actin polymerization,
suggesting a role of PIP5Ks via structural interactions with other
molecules.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
) from a cDNA library of the murine
pancreatic -cell line MIN6 (12). This novel isoform has two
alternative splicing forms of 87 and 90 kDa and is the most markedly
stimulated by phosphatidic acid of the three isoforms. These molecular
identifications revealed that PIP5K isoforms constitute a novel lipid
kinase family, distinct from phosphoinositide 3-kinases, phosphatidylinositol 4-kinases, and diacylglycerol kinases.
Demonstration of structural characteristics is essential for
understanding the intracellular roles of these isoforms and the
mechanisms by which they are regulated. Therefore, in this report,
several aspects of the structural characteristics of these isoforms
were also studied in vitro and in vivo. We found
that a central region, consisting of approximately 380 amino acids, is
sufficient for PtdIns(4)P kinase activity and that carboxyl-terminal
regions are important for modulation of the kinase activities of these isoforms. We also found that expression of either the or the isoform leads to actin rearrangement in COS7 cells, as was the case
with the isoform (13), and that the central region is sufficient
for this effect. Furthermore, surprisingly, the expression of a
kinase-deficient substitution mutant generated a similar effect in COS7
cells.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
--
A polymerase chain
reaction (PCR) using degenerate oligonucleotide primers and
screening of a MIN6 cell cDNA library were as described previously
(10). To obtain a 5' sequence of PIP5K, a MarathonTM
cDNA amplification kit (CLONTECH) was
used according to the instructions of the manufacturer. The reverse
transcription was performed using MIN6 cell poly(A)+ RNA
and an antisense primer 5'-GGTGACGTAGAAGACAGAGCC-3'. The first PCR was
performed using adapter primer 1 (CLONTECH) and an
antisense primer, 5'-CTTCACTGGGGAAGAAGA TGC-3'. The second PCR was
performed using adapter primer 2 (CLONTECH) and an
antisense primer, 5'-GTGGCCCAGCTTCTTCCCATG-3'. The first and second PCR reactions were conducted with inclusion of dimethyl sulfoxide (5%),
without which only shorter products were obtained. Individual clones
were sequenced following subcloning into pGEM-T vector (Promega, WI) as
described above. The consensus of three independent clones confirms the
sequence of the 5' region of PIP5K.
-32P]dCTP-labeled 0.4-kb
Aor51HI-PstI fragment from the 3' portion of the
isoform cDNA.
Isoform and Western
Blotting--
An oligopeptide, CASDEEDAPSTDIYF, was custom synthesized
and conjugated to keyhole limpet hemocyanin (Research Genetics, AL) and
injected into female New Zealand rabbits employing standard protocols
(14). The COS7 cell lysates (10 µg/lane) and murine brain lysate (50 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) (7.5%) and then probed with the antisera raised against the
above peptide (1:100 dilution). Blots were developed using ECL reagents
(Amersham, UK).
-K138A mutant cDNA was generated using primers
5'-ATGAATTCATCATCGCAACCGTTCAG-3' (underlined nucleotides
encode a mutated alanine) and 5'-CTCCTGACTGCATGCAATACAGC-3'. The amino-
and carboxyl-terminal deletion mutants were generated by using either
endogenous restriction enzyme sites or a PCR-based strategy.
Endonuclease BamHI, EcoRI, or NcoI
digestion, followed by subcloning into the SwaI site of a
cosmid vector (pAdex1CA) (16) generated mutants containing amino acids
1-456 with extra asparagine, 1-392, or 1-308 with extra
lysine-leucine-isoleucine-lysine-leucine-valine, due to a poly-linker
sequence of the vector, respectively. Amino-terminal deletion mutants
were generated using an inner antisense primer, 5'-CTCCTGACTGCATGCAATACAGC-3', and an appropriate sense primer containing a SalI site for connection with a sequence for
the HA epitope. Carboxyl-terminal mutants were generated using an inner
sense primer, 5'-CTCTATTCCACAGCCATGGAATCC-3', and an appropriate antisense primer containing a stop codon and a BamHI site.
The mutant cDNAs, confirmed by DNA sequencing, were subcloned into pBluescript containing a sequence coding the HA-epitope.
-32P]ATP (5 µCi/tube). For determination of the Km and the Vmax for PtdIns(4)P, concentrations of 10, 30, 100, and 200 µM were used with ATP at 25 µM. For determination of the Km for
ATP, concentrations of 5, 10, 30, and 100 µM were used
with PtdIns(4)P at 100 µM. To investigate the effects of
phosphatidic acid, Triton X-100TM was added at a final
concentration of 0.1%. The phosphorylation reaction was stopped by
adding 20 µl of 8 M HCl and 160 µl of chloroform:methanol (1:1). Lipids were separated by developing thin
layer chromatography plates (Silica gel 60, Merck) in
chloroform:methanol:15 M ammonium hydroxide:water
(90:90:7:22).
Immunofluorescence-- COS7 cells were plated on coverlips in Dulbecco's modified Eagle's medium with 10% fetal calf serum and infected with recombinant adenoviruses the next day. After 18 h, cells were fixed with 3% paraformaldehyde and incubated with anti-HA monoclonal antibody (12CA5) in phosphate-buffered saline with 0.2% gelatin at room temperature for 45 min. After washing three times with phosphate-buffered saline-gelatin, cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (DAKO) and rhodamine-conjugated phalloidin (Molecular Probes) for 30 min. Slides were observed under a Bio-Rad confocal microscope system (MRC 1024).
Materials-- PtdIns(4)P from bovine brain and phosphatidic acid were purchased from Sigma. Oligonucleotides were custom synthesized and purchased from either Japan Bio-service Inc. (Saitama, Japan) or Becks Inc. (Itabashi, Tokyo).
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RESULTS |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Cloning of the Third Member of Type I PIP5K Family--
A
polymerase chain reaction (PCR) using degenerated primers and
subsequent screening of a MIN6 cell cDNA library, as described previously (10), identified a class of partial coding sequences with
homology to type I PIP5K and isoforms. The third cDNA sequence, designated PIP5K, contains four overlapping clones, one of
which has an additional 78-base pair sequence at the 3' terminus and
possibly arises by alternative splicing of the PIP5K gene. In-frame
stop codons upstream from the first ATG codon of this cDNA could
not be identified in the initial study. Therefore, to obtain an
additional 5' sequence, an adapter ligation/PCR-based method
(MarathonTM, CLONTECH) was employed.
Although an additional 172 base pairs and another ATG codon were
obtained, there were no in-frame stop codons in a 79-base pair sequence
upstream from this ATG codon. Nonetheless, this ATG codon was concluded
to be the initial translation codon for the following reasons. First,
the ATG codon is in a favorable position for translation according to
Kozak's rules (Fig. 1, Ref. 19). Second,
as shown in Fig. 2A,
recombinant proteins of the isoform with or without the 26 carboxyl-terminal amino acids expressed via adenoviral vectors migrated
almost identically to either of the doublet bands (87 and 90 kDa) from
brain tissue on SDS-PAGE. The isoform has two alternative splicing
forms, consisting of 635 and 661 amino acids with calculated molecular masses of 69,563 and 72,469 Da, and isoelectric points of 5.40 and
5.27, respectively (Fig. 1). Because the 87-kDa protein was predominantly expressed in brain tissue (Fig. 2A) and MIN6
cells (data not shown), the isoform without the 26 carboxyl-terminal amino acids was used in subsequent analyses. As shown
in Fig. 3, the central portions of the
three type I isoforms were found to be very similar (approximately 80%
identity) in amino acid sequence. In addition, the amino-terminal
sequence of the type isoform shows partial homology with that of
the isoform (approximately 40% identity) whereas the
carboxyl-terminal regions differed in length and amino acid sequence
among the three isoforms. An entire coding sequence of murine cDNA
homologous to human PIP5KII (20, 21), which was recently revealed to
be phosphatidylinositol 5-phosphate (PtdIns(5)P) 4-kinase (22), was
also cloned from a MIN6 cell cDNA library (data not shown). The
murine PIP5KII consists of 405 amino acids, one residue less than
its human counterpart, with only seven conserved amino acids differing
between the two (Fig. 3).
|
|
|
Tissue Distribution of Type I PIP5K--
Northern blotting
analysis was performed using the isoform cDNA probe
corresponding to the sequence close to the termination codon. A
4.8-kilobase mRNA was detected, as a major band, in murine poly(A)+ RNA from different tissues (Fig. 2B).
The tissue distribution of the isoform differed from those of the
and isoforms, being essentially restricted to the brain, lung,
and kidneys.
The Novel cDNA Encodes Type I PIP5K Protein--
To
characterize the enzymatic activity of the third isoform, HA-tagged
proteins of this isoform expressed in COS7 cells were immunoprecipitated using anti-HA-epitope monoclonal antibody 12CA5. The
resulting immunocomplex exhibited PtdIns(4)P kinase activity (Fig.
2C). Although this thin layer chromatography separation did
not provide information about whether the PtdInsP2 produced was PtdIns(4,5)P2 or PtdIns(3,4)P2, the close
sequence similarity with the and isoforms (Fig. 3) strongly
suggests that the isoform is also a 5-kinase. Furthermore, the
PtdIns(4)P kinase activities of the third isoform increased by more
than 10-fold when an equimolar amount of phosphatidic acid was added to
the reaction solutions (Fig. 2D), demonstrating the novel
murine cDNA to encode the type I PtdIns(4)P 5-kinase (23, 24).
Comparison of Kinetic Activities of Type I PIP5K Isoforms--
For
initial characterization of members of the PIP5K family, kinetic
parameters for the PtdIns(4)P kinase activity of these murine isoforms
were studied. For this purpose, recombinant proteins of isoforms with
the HA epitope were expressed and immunoprecipitated with the
anti-HA-epitope monoclonal antibody. One-half of each immunoprecipitate
was used for kinase assay and the other half for Western blotting with
rabbit anti-HA-epitope polyclonal IgG. Lipid kinase activity was
normalized with the protein amount estimated by Western blotting (for
example, see Fig. 5B). Kinetic parameters for these isoforms
are summarized in Table I. While
affinities for PtdIns(4)P and ATP were similar among the three
isoforms, the isoform had the greatest Vmax
value, approximately 3.2-fold and 1.7-fold higher than those of the and isoforms, respectively. Study of phosphatidic acid sensitivity
revealed the isoform to be most sensitive to phosphatidic acid.
|
Deletion Analysis of the Type I PIP5K Defines a Central Kinase
Domain--
Because type I PIP5K isoforms have no sequence homology
with other lipid kinases including phosphoinositide 3-kinases,
phosphatidylinositol 4-kinases and diacylglycerol kinases (25, 26),
it is of great importance to determine their structure and function
relationships. To begin to address this issue, amino- or
carboxyl-terminal deletion mutants of the type I PIP5K isoform were
constructed using endogenous restriction enzyme sites and PCR-based
methods. As summarized in Fig. 4, while
all four mutants with stepwise deletions from the carboxyl terminus to
glutamine residue 400 have activity almost equal to that of the
wild-type isoform, the PIP5K-(1-392)/EcoRI mutant
has little or no PtdIns(4) 5-kinase activity. In contrast to the long
dispensable region in the carboxyl terminus, amino-terminal deletions
had a pronounced effect. Although the first 17-amino acid deletion did
not alter kinase activity, deletion of only 31 amino acids from the
amino-terminal region resulted in significantly reduced kinase activity
(34 ± 9% of the full-length isoform, mean ± S.E.,
n = 3), and a 46-amino acid deletion virtually
abolished kinase activity.
|
when less than
10% (for typical cells, see Fig. isoform (amino acid residues 18 to 399) constitutes the
kinase core domain. Indeed, as described below, this central portion
alone retains kinase activity (see Fig. 6B). This region of
the PIP5K isoform has about 80% amino acid identity with the
corresponding regions of both the and the isoform. The
amino-terminal half of the kinase core domain is especially conserved
among the three isoforms (more than 90%) (Fig. 3). In addition,
sequence alignment between type I PIP5Ks and PIP5KII suggests that
the latter enzyme (PtdIns(5)P 4-kinase) consists essentially of the
kinase region of type I PIP5Ks with approximately 40% identity (Fig.
3).
Mutations in the Putative Nucleotide Binding Region--
In
several protein and lipid kinases, a glycine-rich sequence followed by
a lysine residue 10-30 residues downstream constitutes an important
region for phosphate-transfer reactions (25, 27). Although there is no
typical region for such a glycine-rich sequence, the region of amino
acid residues 121 to 138 in the isoform is similar to the ATP
binding domain of cyclic AMP-dependent protein kinase (PKA,
Fig. 5A). To examine the role
of this region, two isoform mutants were constructed in which
glycine 124 was substituted with valine (PIP5K-G124V mutant) or
lysine 138 with alanine (PIP5K-K138A mutant). A lipid kinase assay
revealed type I PIP5K-G124V to have 67 ± 11% of the
PtdIns(4)P kinase activity of the wild-type protein, while type I
PIP5K-K138A had virtually no kinase activity (Fig.
5B).
|
(Role of Amino- and Carboxyl-terminal Variable Regions of Type I
PIP5Ks--
As indicated above, these type I PIP5K isoforms consist of
a similar central domain and variable amino- and carboxyl-terminal regions. To characterize these domains, amino- and/or carboxyl-terminal deletion mutants of the three isoforms, carrying the HA epitope, were
constructed based on the results of deletion analysis of the isoform (Fig. 6A). As
summarized in Fig. 6B, the central regions of the three
isoforms showed essentially equivalent lipid kinase activities. There
were no marked changes when amino- and/or carboxyl-terminal regions
were deleted from the isoform. As shown in Table I, the isoform
had approximately three-fold higher activity than the isoform. When
the carboxyl-terminal region was deleted from the isoform, the
activity was reduced to a level approaching that of the isoform. In
addition, a deletion of the carboxyl-terminal region of the isoform
also resulted in a reduction in its kinase activity. The phosphatidic
acid sensitivities of these deletion mutants were also examined (Fig.
6C). The central regions alone of the three isoforms can be
stimulated by phosphatidic acid. Although the isoform showed the
highest sensitivity to phosphatidic acid, its carboxyl-terminal
deletion mutant exhibited a magnitude of phosphatidic acid stimulation
similar to that of the isoform.
|
Effects on Actin Polymerization of Overexpressing PIP5K Isoforms
and Their Mutants in COS7 Cells--
As reported previously,
overexpression of type I PIP5K via an adenoviral vector led to a
decrease in typical stress fibers and an increase in disarrayed short
actin fibers (13). In this study, we examined the effects of
overexpressing three type I PIP5K isoforms, type II PIP5K, and their
mutants on actin polymerization (Fig. 7).
Abnormal reorganization of actin fibers was also observed in COS7 cells
overexpressing either the or the isoform (Fig. 7, B
and C). These in vivo analyses using deletion
mutants of the isoform are summarized in Fig. 4. All deletion
mutants of the isoform with the complete kinase core domain induced
abnormal actin reorganization in COS7 cells. In addition, COS7 cells
expressing I-dNdC, I-dNdC, or I-dNdC, mutants in which both
amino- and carboxyl-terminals are deleted from the three isoforms (Fig.
6A), had enormous amounts of short actin fibers and
relatively few stress fibers (data not shown). In contrast,
PIP5K-(1-392)/EcoRI and PIP5K-(47-539), mutants with
small deletions at the carboxyl- and amino-terminals, respectively, of
the kinase core domain failed to induce abnormal actin rearrangement
(Fig. 7E). All other deletion mutants with the incomplete
kinase core domain (Fig. 4) and type II PIP5K (data not shown)
exhibited behaviors similar to those of
PIP5K-(1-392)/EcoRI and PIP5K-(47-539).
|
-K138A,
also led to a decrease in typical stress fibers and an increase in
short actin fibers (Fig. 7F). In addition, as was the case
with the wild-type isoform (13), COS7 cells expressing the kinase
defective substitution mutant also exhibited decreased adhesion
activity. They became rounded and readily detached from the bottoms of
culture dishes (data not shown).
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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A novel isoform of PIP5K was identified in this study. This novel
isoform (PIP5K) migrated at 90/87 kDa on SDS-PAGE, suggesting this
isoform to be identical or closely related to the type Ib isoform (90 kDa) previously purified (23). The co-existence of three isoforms of
the type I PIP5K in insulin-secreting clonal cells suggests that these
isoforms have specific functions in vivo. Since the 90-kDa
isoform has been reported to have higher activity than the 68-kDa
isoform in restoring Ca2+-regulated catecholamine release
from cytosol-depleted neuroendocrine cells (7), it appears likely that
the isoform plays an important role in regulated secretion. Type I
PIP5K and phospholipase D are postulated to be involved in exocytotic
processes (8). Stimulation of PtdIns(4,5)P2 synthesis by
phosphatidic acid, which may be generated by phospholipase D, was
considered to be important in this process (8). The highest sensitivity
to phosphatidic acid of the PIP5K isoform might be a reason for the
90-kDa isoform playing a more active role in Ca2+-regulated
secretion (7). More specific expression of the isoform in the brain
may reflect an important role of this isoform in neurotransmitter
release.
Molecular cloning of type I PIP5K isoforms allows study of the
structure-function relationships of these important enzymes. Recent
studies revealed that type I PIP5K isoforms can phosphorylate several
lipid substrates other than PtdIns(4)P (28). In this study, we studied
the structure-function relationships with regard to PtdIns(4)P 5-kinase
activity since PtdIns(4)P is the preferred substrate of type I PIP5K
isoforms (28). The minimal kinase core domain of the type I PIP5K
isoforms was determined to be an approximately 380-amino acid region.
The amino acid sequence in this region does not, however, contain
typical sequences homologous to known protein or lipid kinase domains,
the exception being one which exhibits weak homology with a phosphate
binding loop of PKA. Almost complete loss of PtdIns(4)P kinase activity
by substitution of lysine 138 with alanine suggests that this lysine residue plays a role similar to lysine 72 in PKA, which was proposed to
interact with the - and -phosphate groups of ATP (29). It was
also found that the amino-terminal half of the kinase core domain is
highly conserved among type I PIP5K isoforms. Especially, in the region
spanning residue 80 to 161 of the isoform, 98% of 82 residues are
identical or conserved among three isoforms. In addition to the
putative nucleotide binding domain, there may be domains essential for
lipid kinase activities in this region.
Among the three type I isoforms, the isoform has the greatest
Vmax for PtdIns(4)P kinase activity while the
isoform is most markedly stimulated by phosphatidic acid. Our
results using deletion mutants indicate an important role of the
carboxyl-terminal regions for these characteristics. Since the type I
PIP5K isoforms have recently been reported to phosphorylate PtdIns(3)P
and PtdIns(3,4)P2 (28), it would be intriguing to examine
whether amino- and/or carboxyl-terminal sequences are involved in
recognition of these different substrates. In addition, these regions
might be important for possible associations with other unknown
molecules. Further studies are needed to elucidate roles of these
amino- and carboxyl-terminal variable regions.
We also found that overexpression of any one of the three isoforms led
to the production of massive amounts of short actin fibers while
disrupting actin stress fibers in COS7 cells. A surprising result was
that a kinase defective mutant, PIP5K-K138A, induces similar
effects. The mechanism by which PIP5K isoforms and the kinase-deficient
substitution mutant induce such effects remains to be determined. The
causal relationship between short actin fiber formation and disruption
of actin stress fibers is also unclear. It has been reported that
expression of PtdIns(4,5)P2 5-phosphatases in COS7 cells
decreased the number of actin stress fibers via the hydrolysis of
PtdIns(4,5)P2 bound to actin regulatory proteins (13, 30).
An opposite mechanism (i.e. via an increase in
PtdIns(4,5)P2) is unlikely to lead an increase in short
actin fibers in cells overexpressing 5-kinases since the
kinase-deficient mutant induced a similar effect, although the
possibility of endogenous 5-kinase activity playing some part in the
effect cannot be ruled out. Indeed, it was reported that overexpression
of type I PIP5K isoforms in COS7 cells did not increase cellular levels
of PtdInsP2 (31), providing evidence that effects on actin
reorganization of overexpressing PIP5Ks were not mediated by the kinase
activity of overexpressed proteins. The fact that the kinase inactive
mutant induces actin reorganization similar to that seen with the
wild-type enzyme suggests that structural interactions with other as
yet unknown molecules mediate this effect. Small GTP binding proteins, Rac and Rho, are possible candidates (32-34). In this regard, it should be noted that the structure of the kinase core domain was found
to be sufficient for inducing abnormal actin polymerization. There may
be a binding site for such an interacting molecule within the kinase
core domain. Future studies should be designed to identify the
molecules interacting with PIP5K isoforms.
Recent findings suggest that PIP5Ks play various roles in signaling pathways, by participating in the synthesis of a number of phosphoinositides (28). The present results suggest that structural interactions are also important in PIP5Ks functions. Much research remains to be done in order to elucidate the complex signaling pathways in which these lipid kinases are involved.
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FOOTNOTES |
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* This work was supported in part by Grant-in Aid for Scientific Research (A) 09357009 (to Y. O.) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from Uehara Memorial Foundation (to Y. O.).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 DDBJ, EBI, and GenBankTM Data Bank with accession number(s) AB006916 and AB009615.
§ To whom correspondence should be addressed: Third Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3815-5411 (ext. 3121); Fax: 81-3-5803-1874; E-mail: ishihara-tky{at}umin.u-tokyo.ac.jp.
1 The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HA, hemagglutinin; PKA, cyclic AMP-dependent protein kinase.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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A. Shisheva, D. Sbrissa, and O. Ikonomov Cloning, Characterization, and Expression of a Novel Zn2+-Binding FYVE Finger-Containing Phosphoinositide Kinase in Insulin-Sensitive Cells Mol. Cell. Biol., January 1, 1999; 19(1): 623 - 634. [Abstract] [Full Text] [PDF]
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I. V. Boronenkov, J. C. Loijens, M. Umeda, and R. A. Anderson Phosphoinositide Signaling Pathways in Nuclei Are Associated with Nuclear Speckles Containing Pre-mRNA Processing Factors Mol. Biol. Cell, December 1, 1998; 9(12): 3547 - 3560. [Abstract] [Full Text]
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T. Itoh, H. Ishihara, Y. Shibasaki, Y. Oka, and T. Takenawa Autophosphorylation of Type I Phosphatidylinositol Phosphate Kinase Regulates Its Lipid Kinase Activity J. Biol. Chem., June 16, 2000; 275(25): 19389 - 19394. [Abstract] [Full Text] [PDF]
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M. Mejillano, M. Yamamoto, A. L. Rozelle, H.-Q. Sun, X. Wang, and H. L. Yin Regulation of Apoptosis by Phosphatidylinositol 4,5-Bisphosphate Inhibition of Caspases, and Caspase Inactivation of Phosphatidylinositol Phosphate 5-Kinases J. Biol. Chem., January 12, 2001; 276(3): 1865 - 1872. [Abstract] [Full Text] [PDF]
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E. B. Rodriguez de Turco, W. Tang, M. K. Topham, F. Sakane, V. L. Marcheselli, C. Chen, A. Taketomi, S. M. Prescott, and N. G. Bazan Diacylglycerol kinase varepsilon regulates seizure susceptibility and long-term potentiation through arachidonoyl- inositol lipid signaling PNAS, April 10, 2001; 98(8): 4740 - 4745. [Abstract] [Full Text] [PDF]
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