From the Inositol phospholipids regulate a variety of
cellular processes including proliferation, survival, vesicular
trafficking, and cytoskeletal organization. Recently, two novel
phosphoinositides, phosphatidylinositol-3,5-bisphosphate
(PtdIns-3,5-P2) and phosphatidylinositol- 5-phosphate
(PtdIns-5-P), have been shown to exist in cells.
PtdIns-3,5-P2, which is regulated by osmotic stress,
appears to be synthesized by phosphorylation of PtdIns-3-P at the D-5
position. No evidence yet exists for how PtdIns-5-P is produced in
cells. Understanding the regulation of synthesis of these molecules
will be important for identifying their function in cellular signaling.
To determine the pathway by which PtdIns-3,5-P2 and
Ptd-Ins-5-P might be synthesized, we tested the ability of the
recently cloned type I PtdIns-4-P 5-kinases (PIP5Ks) Although phosphoinositides represent only a small fraction of the
total cellular lipids, they play critical roles as intracellular signaling molecules. Phosphoinositides regulate a variety of cellular processes including proliferation, survival, vesicular trafficking, and
cytoskeletal organization (1). The levels of phosphoinositides in the
cell are modulated by external stimuli which regulate the activities of
phosphoinositide kinases, phosphatases, and lipases. Several of these
enzymes have recently been identified and cloned (1-5), and this work
has facilitated the understanding of how phosphoinositides are
regulated and what roles they play in cellular signaling. It is now
recognized that
PtdIns-4,5-P21
has intrinsic signaling capabilities, in addition to its role as a
substrate for diacylglycerol, inositol-1,4,5-trisphosphate, and
PtdIns-3,4,5-P3 synthesis (3). PtdIns-4,5-P2
binds to pleckstrin homology domains (6-8) and plays a role in
exocytosis (9-11). It also regulates the activity of several
actin-binding proteins (12). This finding, in combination with the
evidence that Rho family small G proteins associate with type I PIP5Ks
which synthesize PtdIns-4,5-P2 (13-15), suggests that
PtdIns-4,5-P2 may participate in Rho family-mediated actin
cytoskeletal rearrangements. As mentioned, PtdIns-4,5-P2 is
synthesized by the type I PIP5Ks which phosphorylate PtdIns-4-P on the
D-5 position of the inositol ring. Rameh et al. (16)
recently discovered an alternative pathway for synthesis, demonstrating
that type II PIPKs produce PtdIns-4,5-P2 by phosphorylating PtdIns-5-P on the D-4 position of the inositol ring. Although this work
also provided evidence for the existence of PtdIns-5-P in cells, it
remains unclear how this lipid is synthesized.
The D-3 phosphoinositides, PtdIns-3-P, PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 also have distinct roles in cellular
signaling. PtdIns-3-P, which is present constitutively in cells, plays
a role in Golgi to vacuole trafficking in yeast (17).
PtdIns-3,4-P2 and PtdInsP3, which accumulate
rapidly after cell stimulation in contrast to PtdIns-3-P, interact
directly with pleckstrin homology domains (7, 8, 18-20) and Src
homology 2 domains (21). The binding of PtdIns-3,4-P2 and
PtdInsP3 to these domains likely results in the recruitment
to the membrane of multiple signaling proteins which could initiate
secondary signaling cascades. PtdIns-3,4-P2 and
PtdInsP3 have also been shown to activate the
serine/threonine kinase, Akt, its upstream kinase, PDK-1, and some
protein kinase C isoforms (22-24). A number of PI3K family members
have now been identified which can catalyze the in vitro
phosphorylation of PtdIns, PtdIns-4-P, and/or PtdIns-4,5-P2
on the D-3 position of the inositol ring (25). Kinetic studies in
32PO4-labeled cells suggest that
PtdIns-3,4-P2 and PtdInsP3 are synthesized by
the phosphorylation of PtdIns-4,5-P2 on the D-3 position by
PI3K to produce PtdIns-3,4,5-P3, followed by a
dephosphorylation of PtdIns-3,4,5-P3 on the D-5 position by
a phosphatase to form PtdIns-3,4-P2 (26). Studies in
platelets provide evidence for an alternative synthetic pathway for D-3
phosphoinositide in which PtdIns is first phosphorylated on the D-3
position to produce PtdIns-3-P, followed by the phosphorylation of the
D-4 position to form Ptd-Ins-3,4-P2 (27-29).
A newly discovered phosphoinositide in the PI3K pathway has recently
been described. Analysis of phosphoinositides present in smooth muscle
cells revealed an inositol containing lipid with chromatographic
properties similar to PtdIns-3,4-P2, which we proposed to
be PtdIns-3,5-P2 (30). Whiteford et al. (31)
recently verified that PtdIns-3,5-P2 is present in
fibroblasts. Dove et al. (32) also found
PtdIns-3,5-P2 in Saccharomyces cerevisiae and
showed that the level of this lipid is regulated by osmotic strength
changes (32). The synthesis of PtdIns-3,5-P2 is blocked by
the PI3K inhibitor wortmannin (31). Analysis of the specific activity
of the D-3 and D-5 phosphates indicates that PtdIns-3,5-P2 is synthesized primarily by phosphorylation of PtdIns-3-P at the D-5
position (31, 32). The specific activity data and the fact that
PtdInsP3 is absent in cells under conditions where
PtdIns-3,5-P2 is found argue that a phosphatase is unlikely
to be involved in PtdIns-3,5-P2 synthesis.
The only known kinases that phosphorylate the D-5 position of the
inositol ring are the type I PIP5Ks. As mentioned previously, the type
II PIPKs have recently been demonstrated to phosphorylate the D-4
position of the inositol ring (16). While the function of
PtdIns-3,5-P2 in the cell is unknown, we were interested in the pathway by which it is synthesized and tested whether type I PIP5Ks
can phosphorylate the D-5 position of PtdIns-3-P to form PtdIns-3,5-P2. We find that type I PIP5Ks do phosphorylate
PtdIns-3-P at the D-5 position. We also find that these kinases can
produce PtdIns-5-P from PtdIns and can synthesize PtdInsP3
from either PtdIns-3-P or PtdIns-3,4-P2.
Materials--
PtdIns and PtdIns-4-P were obtained from Avanti
Polar Lipids Inc. and Sigma, respectively. Synthetic dipalmitoyl
PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,5-P2
were synthesized as described (34).2
[3H]PtdIns-4-P,
[3H]PtdIns-4,5-P2,
[3H]Ins-1,3,4-P3,
[3H]Ins-1,4,5-P3, and
[ Plasmids--
N-terminal HA-tagged murine cDNAs of type I
PIP5K Expression and Purification of Recombinant PIP5Ks
Isoforms--
Glutathione S-transferase fusion proteins of
the type I PIP5Ks were expressed in Escherichia coli and
purified with glutathione-Sepharose beads as described previously (15).
Proteins were eluted from glutathione-Sepharose beads with 50 mM Tris buffer, pH 8.0, containing 20 mM
reduced glutathione and then concentrated in a Centricon filter after
several washes with buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 5 mM dithiothreitol). Glycerol was added to the proteins
before storing at Cell Culture and Transfections--
293 E1A cells were
maintained in Dulbecco's modified medium containing 10% heat
inactivated fetal calf serum. Cells were transfected with LipofectAMINE
using 4 µg of HA-tagged PIP5Ks per 10-cm plate. Cells were harvested
18 h after transfection.
Immunoprecipitation of PIP5Ks from 293 E1A Cells--
Cells were
rinsed with phosphate-buffered saline and then lysed in 600 µl of
lysis buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 4 µg/ml each leupeptin and pepstatin,
and 200 µM AEBSF). The clarified lysates were incubated
with 1.5 µg of an anti-HA antibody (12CA5 from Boehringer Mannheim)
and protein A-Sepharose beads for 3 h at 4 °C. The beads were
then washed twice with lysis buffer and twice with TNM (50 mM Tris, pH 7.5, 50 mM NaCl, and 5 mM MgCl2).
Kinase Assays--
PIP5Ks were assayed in 50-µl reactions
containing 50 mM Tris, pH 7.5, 30 mM NaCl, 12 mM MgCl2, 67 µM lipid substrate,
133 µM phosphoserine, and 50 µM
[ HPLC Analysis--
Deacylated lipids were mixed with
3H-labeled standards and analyzed by anion-exchange
high-pressure liquid chromatography (HPLC) using a Partisphere SAX
column (Whatman) as described previously (35). To separate PtdIns-4-P
from PtdIns-5-P and PtdIns-3,4-P2 from
PtdIns-3,5-P2, a modified ammonium phosphate gradient was used. The compounds were eluted with 1 M
(NH4)2HPO4, pH 3.8, and water using
the following gradient: 0% 1 M
(NH4)2HPO4 for 5 ml, 0 to 1% 1 M (NH4)2HPO4 over 5 ml,
1 to 3% 1 M (NH4)2HPO4
over 40 ml, 3 to 10% 1 M
(NH4)2HPO4 over 10 ml, 10 to 13% 1 M (NH4)2HPO4 over 25 ml, and 13 to 65% 1 M
(NH4)2HPO4 over 25 ml. Eluate from the HPLC column flowed into an on-line continuous flow scintillation detector (Radiomatic Instruments, FL) for isotope detection.
Periodate Oxidation--
Periodate oxidation was performed as
described with the following modifications (36). Deacylated lipids were
incubated in the dark at 25 °C with 10 or 100 mM
periodic acid, pH 4.5, for 30 min or 36 h, respectively. The
remaining oxidizing reagent was removed by adding 500 mM
ethylene glycol and incubating the reaction in the dark for 30 min. 2%
1,1-dimethylhydrazine, pH 4.5, was then added to a final concentration
of 1% and the reaction was allowed to proceed for 4 h at
25 °C. The mixture was then purified by ion exchange using a Dowex
50W-X8 cation-exchange resin (20-50 mesh, acidic form), dried, and
applied to an HPLC column. PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 controls used in this experiment were
prepared using recombinant Sf9 cell-expressed PI3K.
PIP5Ks Phosphorylate PtdIns, PtdIns-3-P, and
PtdIns-3,4-P2--
The recent description of the presence
of PtdIns-3,5-P2 in fibroblasts rekindled our interest in
this phosphoinositide and how it is produced. Since it was recently
shown that type II PIPK phosphorylates the D-4 position of PtdIns-5-P
(16), the synthesis of PtdIns-3,5-P2 must either be
catalyzed by a type I PIP5K, a phosphatase, or a new enzyme. To further
define the substrates and products of the type I PIP5Ks, we provided
PtdIns, PtdIns-3-P, PtdIns-4-P, PtdIns-3,4-P2, and
PtdIns-3,5-P2 as substrates and analyzed the products. As
our enzyme source, we used recombinant PIP5Ks I Division of Signal Transduction,
Medicine,
Third Department of Internal Medicine,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, and the
** Department of Medicinal Chemistry, University of Utah,
Salt Lake City, Utah 84112-5820
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and
to
phosphorylate PtdIns-3-P and PtdIns at the D-5 position of the inositol
ring. We found that the type I PIP5Ks phosphorylate PtdIns-3-P to form
PtdIns-3,5-P2. The identity of the
PtdIns-3,5-P2 product was determined by anion exchange high
performance liquid chromatography analysis and periodate treatment.
PtdIns-3,4-P2 and PtdIns-3,4,5-P3 were also
produced from PtdIns-3-P phosphorylation by both isoforms. When
expressed in mammalian cells, PIP5K I
and PIP5K I
differed in
their ability to synthesize PtdIns-3,5-P2 relative to
PtdIns-3,4-P2. We also found that the type I PIP5Ks
phosphorylate PtdIns to produce PtdIns-5-P and phosphorylate
PtdIns-3,4-P2 to produce PtdIns-3,4,5-P3. Our findings suggest that type I PIP5Ks synthesize the novel phospholipids PtdIns-3,5-P2 and PtdIns-5-P. The ability of PIP5Ks to
produce multiple signaling molecules indicates that they may
participate in a variety of cellular processes.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP were purchased from NEN Life Science
Products. LipofectAMINE and Opti-MEM were obtained from Life
Technologies, Inc. All other chemicals were from Sigma.
and
in pBluescript were generated as described previously
(5). The cDNAs of the PIP5Ks lacking the HA-tag were subcloned into
the SalI and NotI sites of the bacterial
expression vector pGEX4T. HA-tagged type I PIP5K cDNAs were also
subcloned into the ClaI and NotI sites of the
mammalian expression vector pEBB (provided by B. J. Mayer). The
mutations PIPKI
D227A and PIPKI
D268A were generated using the
CLONTECH Transformer Site-directed Mutagenesis Kit.
80 °C.
-32P]ATP (10 µCi/assay). When PtdIns was used as
the lipid substrate, no carrier lipid was used, and when
[32P]PtdIns-3-P was used as substrate, unlabeled ATP was
used in place of [
-32P]ATP. Reactions were stopped
after 10 min by adding 80 µl of 1 N HCl and then 160 µl
of CHCl3:MeOH (1:1). Lipids were separated by thin-layer
chromatography (TLC) using 1-propanol, 2 M acetic acid
(65:35, v/v). Phosphorylated lipids were visualized by autoradiography and quantified using a Bio-Rad Molecular Analyst. The PtdIns used in
all experiments was separated from contaminating phosphoinositides by
TLC purification using
CHCl3:MeOH:H2O:NH4OH
(60:47:11:1.6). After identification using an iodine-stained
Ptd-Ins standard, the PtdIns was scraped from the TLC plate and
eluted in the same solvent solution. Following lyophilization, PtdIns
was resuspended in chloroform and then extracted once with MeOH, 1 N HCl (1:1) and once with MeOH, 0.1 M EDTA
(1:0.9). [32P]PtdIns-3-P and
[32P]PtdInsP3 were generated by incubating
recombinant Sf9 cell-expressed PI3K with 500 µM
[
-32P]ATP (50 µCi/assay), 10 mM
MgCl2, 20 mM Hepes, pH 7.0, and 1 mM PtdIns or PtdIns-4,5-P2, respectively, for
1 h at room temperature. The reaction was stopped with 1 mM EDTA and 50 µl of 3 N HCl, and the lipids
were extracted with 200 µl of CHCl3:MeOH (1:1). Kinetic
analysis of the bacterially expressed PIP5K enzymes was performed using
1-100 µM of the phospholipid substrates and 0.64 µg of
PIP5K I
or 0.08 µg of PIP5K I
in reactions allowed to proceed
for 1 or 4 min, respectively.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and PIP5K I
(according to the nomenclature proposed by Ishihara et al.
(5)) that were produced as glutathione S-transferase fusion
proteins in bacteria. The products of the phosphorylation reactions
were analyzed by TLC using a system that separates PtdInsP, Ptd-InsP2, and PtdInsP3, and visualized
using a Molecular Analyst. As expected, the type I PIP5Ks converted
PtdIns-4-P to PtdInsP2 (Fig.
1) (4, 5). Surprisingly, the enzymes were
also capable of synthesizing other lipid products. Both PIP5K I
and
PIP5K I
phosphorylated PtdIns-3,4-P2 to produce
Ptd-InsP3 and phosphorylated PtdIns-3-P to produce
PtdInsP2 and PtdInsP3 (Fig. 1). PtdIns was also converted to PtdInsP and PtdInsP2 by the type I
PIP5Ks (although bacterially expressed PIP5K I
was more active than
PIP5K I
at phosphorylating PtdIns). In contrast,
PtdIns-3,5-P2 was a poor substrate for both enzymes (1/5 as
good as PtdIns-3,4-P2). The PtdInsP2 produced
in the reactions using PtdIns-3,4-P2 and
PtdIns-3,5-P2 as substrate most likely resulted from a
contaminant in either the synthetic lipid substrates (which were judged
to be greater than 99% pure based on 1H and
31P NMR) or in the carrier lipid phosphoserine. The
kinase-dead PIP5K mutants, PIP5K I
D227A and PIP5K I
D268A, did
not catalyze the phosphorylation of any lipids, as expected (data not
shown).
View larger version (43K):
[in a new window]
Fig. 1.
Recombinant type I PIP5K and
phosphorylate several substrates in addition to PtdIns-4-P. The
activities of the bacterially expressed recombinant PIP5K I
(0.3 µg) and PIP5K I
(0.1 µg) were assayed using PtdIns, PtdIns-3-P,
PtdIns-4-P, PtdIns-3,4-P2, and PtdIns-3,5-P2 as
substrates. The products of the reactions were separated by thin layer
chromatography. Lanes containing the PIP5K I
products were exposed
on the PhosphorImager (Bio-Rad) four times longer than lanes containing
the PIP5K I
products. The PtdIns-3,5-P2 phosphorylation
products of both PIP5Ks were run on a different TLC plate. The data are
representative of five experiments.
HPLC Analysis of the Products from the Phosphorylation of PtdIns-3-P and PtdIns-3,4-P2-- To determine the identity of the products, they were deacylated and analyzed by anion exchange chromatography using HPLC. As expected from previous studies (4, 5), the PtdInsP2 produced using PtdIns-4-P as a substrate for either enzyme was exclusively PtdIns-4,5-P2 (not shown). The product of PtdIns-3,4-P2 phosphorylation by either enzyme was confirmed to be PtdIns-3,4,5-P3, in agreement with a recent study by Zhang et al. (Fig. 2B) (33). However, in contrast to the findings of Zhang et al. (33), the deacylated PtdInsP2 produced by phosphorylation of synthetic PtdIns-3-P with either type I isoenzyme was resolved into two peaks by HPLC analysis (75.5 and 77 min, Fig. 2A). The peak at 77 min comigrated with the glycerophosphorylinositol 3,4-bisphosphate (GroPIns-3,4-P2) standard while the peak at 75.5 min migrated at the position expected for GroPIns-3,5-P2 (31, 32). Zhang et al. (33) did not detect a second peak corresponding to GroPIns-3,5-P2 when analyzing the reaction products of the phosphorylation of PtdIns-3-P by type I PIP5Ks. The best explanation for this discrepancy is that the anion exchange HPLC analysis done by Zhang et al. (33) did not separate GroPIns-3,4-P2 from GroPIns-3,5-P2. We also detected two additional peaks in the reaction products of PtdIns-3-P phosphorylation by type I PIP5Ks (Fig. 2A). The peak at 98 min eluted at the known position for GroPIns-3,4,5-P3 (Fig. 2A). The peak at 95 min (×) was not identified.
|
|
Type I PIP5Ks Phosphorylate PtdIns-3-P to Produce PtdIns-3,5-P2-- Although the major PtdInsP2 product from the phosphorylation of PtdIns-3-P by type I PIP5Ks appeared to be PtdIns-3,5-P2 based on its migration position, we further investigated its identity using periodate. Since this lipid was not PtdIns-3,4-P2 or PtdIns-4,5-P2, it could only be PtdIns-3,5-P2, PtdIns-2,3-P2, or PtdIns-3,6-P2. The latter two products have never been described as existing in vivo, but can be distinguished from PtdIns-3,5-P2 by their sensitivity to periodate treatment. Of this group, only PtdIns-3,5-P2 lacks a viscinal diol for periodate attack of the inositol ring and is therefore resistant to cleavage. We treated the products of PtdIns-3-P phosphorylation with periodate. GroPIns-3,4-P2 (which is sensitive to periodate) and GroPIns-3,4,5-P3 (which is resistant to periodate) were also treated as controls. The reaction products were analyzed by anion exchange chromatography using an HPLC.
Short periodate treatment (30 min) resulted in the conversion of the GroPIns-3,4-P2 and GroPIns-3,5-P2 products to InsP3 and the GroPInsP3 product to InsP4 due to cleavage of the glycerol moiety (Fig. 4A). Since PtdIns-3,4,5-P3 lacks a viscinal diol and is therefore resistant to periodate cleavage (Fig. 4, E and F), we used the InsP4 derived from the PtdInsP3 present in our sample as an internal control for yield after prolonged periodate treatment (36 h). Prolonged periodate treatment resulted in the loss of 50% of InsP4 (Fig. 4B). Similar treatment of Ins-1,3,4-P3 resulted in almost complete loss of radiolabeled material (3% remaining) at the position of Ins-1,3,4-P3 (Fig. 4, C and D). However, under the same conditions, approximately 30% of the GroPInsP2 product of the type I PIP5K
|
Type I PIP5Ks Produce PtdIns-5-P--
The product of the reaction
using PtdIns as substrate was also identified by deacylation and HPLC
chromatography. As shown in Fig. 5,
deacylated PtdInsP produced using TLC purified PtdIns as the substrate
for the E. coli-expressed PIP5K I enzyme migrates distinctly from GroPIns-3-P or GroPIns-4-P at the position of GroPIns-5-P (16). Similar results were obtained using bacterially expressed PIP5K I
as the enzyme source (data not shown). PtdIns-5-P has been found in cells (16), and this result suggests that it could be
synthesized by type I PIP5Ks. A small amount of PtdInsP2, which was identified as PtdIns-4,5-P2, was also produced
using PtdIns as substrate (not shown). Since the PtdIns used in this experiment was TLC purified to eliminate the possibility that PtdIns-4-P was present as a contaminant, this result suggests that type
I PIP5Ks can phosphorylate both the D-4 and D-5 positions of the
inositol ring. However, given that PtdIns-5-P is a poor substrate for
type I PIP5Ks (16), it is likely that the type I PIP5Ks only synthesize
PtdIns-4,5-P2 from PtdIns in a concerted reaction at a low
rate.
|
Substrate Preference of the Type I PIP5Ks--
To investigate the
relative substrate preferences, we determined the apparent
Km, Vmax and
Vmax/Km ratio for the various
substrates of the bacterially expressed type I PIP5Ks. As expected,
PtdIns-4-P was the favored substrate of both the - and
-isoforms.
Bacterially expressed PIP5K I
, which was significantly less active
than PIP5K I
, did not efficiently phosphorylate the other
phosphoinositides tested. In contrast, PIP5K I
phosphorylated PtdIns-3-P about one-sixth as well as PtdIns-4,5-P2
(Vmax/Km ratio of 92 pmol/min/mg compared with 564 pmol/min/mg). Given the high ratio of
PtdIns-4-P to PtdIns-3-P in mammalian cells (approximately 20:1), it is
likely that PtdIns-4-P is the major in vivo substrate.
However, it is certainly possible that the type I PIP5Ks account for
the small amount of PtdIns-3,5-P2 observed in
vivo (approximately 1% of PtdIns-4,5-P2) via
phosphorylation of PtdIns-3-P at the D-5 position.
Type I PIP5Ks Expressed in Mammalian Cells Phosphorylate
PtdIns-3-P, PtdIns-3,4-P2, and PtdIns--
The experiments
presented above were performed using enzymes that were expressed in
bacteria. To determine whether type I PIP5Ks expressed in mammalian
cells have the same properties, we repeated experiments with HA-tagged
PIP5K I and PIP5K I
produced in 293 E1A cells. Proteins were
immunoprecipitated and then assayed for their ability to phosphorylate
PtdIns-3-P, PtdIns-3,4-P2, and PtdIns. The products of the
reactions were deacylated and analyzed by HPLC. The PIP5Ks expressed in
293 cells were both quite active, in contrast to the bacterially
produced enzymes where PIP5K I
was significantly more active than
PIP5K I
. Both mammalian expressed type I PIP5K isoforms were able to
phosphorylate PtdIns-3-P to produce PtdIns-3,5-P2,
PtdIns-3,4-P2, and PtdIns-3,4,5-P3 (Fig.
6, A and D). They
were also able to phosphorylate PtdIns-3,4-P2 to produce
PtdIns-3,4,5-P3 (Fig. 6, B and E),
and PtdIns to produce PtdIns-5-P (Fig. 6, C and
F). In contrast to the bacterially expressed enzymes,
however, the mammalian expressed PIP5Ks differed in their ability to
synthesize PtdIns-3,5-P2 relative to
PtdIns-3,4-P2. When PIP5K I
phosphorylated
Ptd-Ins-3-P, it produced more PtdIns-3,5-P2 than
PtdIns-3,4-P2 (Fig. 6B), whereas PIP5K I
produced equal or more PtdIns-3,4-P2 than
PtdIns-3,5-P2 (Fig. 6E). It is unclear whether
this difference between PIP5K I
and PIP5K I
is the result of
PIP5K I
being more efficient at synthesizing
PtdIns-3,5-P2 or less efficient at producing
PtdIns-3,4-P2.
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DISCUSSION |
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We have found that the type I PIP5Ks can synthesize the novel phospholipids PtdIns-3,5-P2 and PtdIns-5-P by phosphorylating PtdIns-3-P and PtdIns, respectively. Our results suggest a potential pathway for PtdIns-3,5-P2 and PtdIns-5-P synthesis in vivo. We also find, in agreement with a recent report from Zhang et al. (33), that the type I PIP5Ks can phosphorylate PtdIns-3-P and PtdIns-3,4-P2 to produce the important signaling molecules PtdIns-3,4-P2 and PtdIns-3,4,5-P3. Taken together, our results demonstrate that in addition to their well established role of synthesizing PtdIns-4,5-P2, the type I PIP5Ks have the in vivo potential to synthesize the following four phosphoinositides: PtdIns-3,5-P2, PtdIns-3,4-P2, PtdIns-3,4,5-P3, and PtdIns-5-P. Since each of these phospholipids is likely to have distinct roles in signaling, the type I PIP5Ks may participate in a variety of cellular processes.
The existence of PtdIns-3,5-P2 has only recently been verified in yeast, mammalian, and plant cells (31, 32). Although present in resting cells, the level of this lipid appears to be regulated by osmotic strength changes (32). Since PtdIns-3,4,5-P3 is absent in the cells in which PtdIns-3,5-P2 is found, PtdIns-3,5-P2 is unlikely to be produced by a phosphatase. Analysis of the specific activity of the D-3 and D-5 phosphates indicates that PtdIns-3,5-P2 is synthesized primarily by phosphorylation of PtdIns-3-P at the D-5 position (31, 32). We propose that the type I PIP5Ks are the kinases responsible for catalyzing this reaction in vivo. Given that the level of PtdIns-3,5-P2 is regulated by osmotic strength, it will be interesting to determine whether the activity of the type I PIP5Ks is altered upon changes in osmotic pressure.
PtdIns-3,4,5-P3 is primarily thought to be synthesized by the phosphorylation of PtdIns-4,5-P2 on the D-3 position of the inositol ring by PI3K. Our results suggest an alternative synthetic pathway in which PI3K provides the initial substrate PtdIns-3-P by phosphorylating PtdIns on the D-3 position. Ptd-Ins-3-P is then phosphorylated by the type I PIP5Ks on the D-4 position to produce PtdIns-3,4-P2 and then the D-5 position to produce PtdIns-3,4,5-P3. PtdIns-3,5-P2 is less likely to be an intermediate in the synthesis of PtdIns-3,4,5-P3 since it is a rather poor substrate for type I PIP5Ks. Evidence for this pathway is provided by studies in platelets which demonstrated that PtdIns is first phosphorylated on the D-3 position to produce PtdIns-3-P, followed by the phosphorylation of the D-4 position to form PtdIns-3,4-P2 (27-29).
The type I PIP5Ks appear to be dual specificity kinases since they phosphorylate both the D-4 and D-5 positions of the inositol ring. However, while PtdIns-5-P is produced when PtdIns is used as a substrate for type I PIP5Ks, no PtdIns-4-P is detected. Similarly, PtdIns-3,5-P2 is a poor substrate compared with PtdIns-3,4-P2 for type I PIP5Ks. Therefore, the ability of type I PIP5Ks to phosphorylate the D-4 position of the inositol ring is primarily restricted to the substrate PtdIns-3-P. In general, the type I PIP5Ks favor phosphorylating the D-5 position of the inositol ring, whereas the type II PIPKs favor the D-4 position (16).
The proof of the existence of PtdIns-3,5-P2 and PtdIns-5-P in cells and the finding that the type I PIP5Ks can synthesize these products in vitro increases the complexity of phosphoinositide signaling. The challenge remains to understand the function of the various phosphoinositides and the regulation of their synthesis and degradation. It will be interesting to determine whether extracellular signals that activate growth factor receptors and/or proteins known to interact with type I PIP5Ks such as Rho family small GTP-binding proteins (13-15) alter the specificity of the type I PIP5Ks for their various substrates.
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ACKNOWLEDGEMENTS |
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We thank A. Couvillon and C. Yballe for preparing recombinant PI3K and members of the Carpenter and Cantley laboratories for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM54389 (to C. L. C.), GM36624 (to L. C. C.), and NS 29632 (to G. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Div. of Signal Transduction, Harvard Institute of Medicine, 1007, 330 Brookline Ave., Boston, MA 02115. Tel.: 617-667-0941; Fax: 617-667-0957; E-mail: ktolias{at}bidmc.harvard.edu.
1 The abbreviations used are: PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PtdIns, phosphatidylinositol; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-5-P, phosphatidylinositol 5-phosphate; PtdIns-3,4-P2, phosphatidylinositol 3,4-bisphosphate; PtdIns-3,5-P2, phosphatidylinositol 3,5-bisphosphate; PtdIns-3,4,5-P3 and PtdInsP3, phosphatidylinositol 3,4,5-triphosphate; Ins-1,3,4-P3, inositol 1,3,4-triphosphate; Ins-1,3,5-P3, inositol 1,3,5-triphosphate; Ins-1,4,5-P3, inositol 1,4,5-triphosphate; GroPIns, glycerophosphorylinositol; type I PIP5K, type I phosphatidylinositol-4-phosphate 5-kinase; type II PIPK, type II phosphatidylinositol phosphate kinase; PI3K, phosphoinositide 3-kinase; HPLC, high performance liquid chromatography.
2 J. Chen, L. Feng, and G. D. Prestwich, manuscript in preparation.
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REFERENCES |
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