From the Centre for Molecular Cell Biology,
Department of Medicine, Royal Free and University College Medical
School, Rowland Hill Street, London NW3 2PF, United Kingdom, and the
¶ Ludwig Institute for Cancer Research, Courtauld Building, 91 Riding House Street, London W1P 8BT, United Kingdom
Received for publication, December 18, 2000, and in revised form, February 8, 2001
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ABSTRACT |
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Phosphoinositide lipids regulate numerous
cellular processes in all eukaryotes. The versatility of this
phospholipid is provided by combinations of phosphorylation on the 3',
4', and 5' positions of the inositol head group. Two distinct
structural families of phosphoinositide (PI) kinases have so far
been identified and named after their prototypic members, the PI
3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families,
both of which have been found to contain structural homologues
possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns
4-kinase activity in many mammalian cell types is conferred by the
widespread type II PtdIns 4-kinase, which has so far resisted molecular
characterization. We have partially purified the human type II isoform
from plasma membrane rafts of human A431 epidermoid carcinoma
cells and obtained peptide mass and sequence data. The results allowed
the cDNA containing the full open reading frame to be cloned. The
predicted amino acid sequence revealed that the type II enzyme is the
prototypic member of a novel, third family of PI kinases. We have named
the purified protein type II Phosphoinositides have been implicated in a vast range of cellular
functions, including receptor signaling, vesicle trafficking, endocytosis and cytoskeletal rearrangement (1, 2). Several distinct
metabolic pathways of PtdIns1
phosphorylation exist in eukaryotic cells, producing important effectors such as PtdIns 4,5-bisphosphate, PtdIns 3,4-bisphosphate, and PtdIns 3,4,5-trisphosphate (3). The first step in the
phosphorylation of PtdIns in many receptor-dependent
phospholipase C (PLC) and PI 3-kinase (PI3K) signaling pathways
involves the synthesis of PtdIns 4-phosphate (PtdIns4P) by PtdIns
4-kinase (PtdIns4K) activity (4).
The PI kinase sequences that have been determined so far fall into two
families, the PI3K family (5, 6), which includes all the hitherto known
PI3K and PtdIns4K sequences (3), and the PtdIns phosphate kinase
(PtdInsPK) family (3, 7). All PI3Ks and PtdIns4Ks cloned so far display
significant homology within their kinase domains. In contrast, members
of the PtdInsPK family have quite distinct sequences.
However, recent structural studies have shown that whereas the
PtdInsPKs display little primary sequence homology with the
PI3K family, they share a common protein fold that is also conserved in
many protein kinases (8, 9).
Early chromatographic purification of phosphoinositide kinase
activities from bovine brain (4) and cultured rodent fibroblasts (10)
identified three fractions containing PtdIns kinase activity, termed
types I to III. The type II and III fractions contain different PtdIns4K activities, and the type I fraction was subsequently shown to
contain PI3K activity (11). It is now known that the type I PI3K and
type III PtdIns4K enzymes have related sequences and belong to the PI3K
family (3). However, the fraction containing the type II PtdIns4K
(PtdIns4K II) has so far not been characterized at the molecular level
despite the fact that in many mammalian cells, the predominant pathway
of PtdIns phosphorylation is initiated by this strongly
membrane-associated PtdIns4K (3).
Perhaps surprisingly, although members of the PI3K and PtdInsPK
families have been readily purified and cloned, numerous attempts to
purify the PtdIns4K II enzyme (see Ref. 12 and references therein) have
failed to lead to the cDNA being identified. These preparations
were probably often impure as PtdIns4K II is labile, difficult to
solubilize, and tends to aggregate strongly with other proteins.
Indeed, a published cDNA sequence was subsequently found to encode
a long-chain fatty acid-CoA ligase (13).
Despite the massive increase in genome data, approaches based on
homology to known PtdIns4Ks, such as degenerate polymerase chain
reaction (PCR) primers or data base trawling, have also failed to
identify the PtdIns4K II sequence. Although three related PtdIns4Ks
have so far been cloned (14-18), the failure of such approaches with
PtdIns4K II has raised the question of whether the PtdIns4K II enzyme
might be a proteolytic fragment or splice variant of the known isozymes
or belong to a different structural family altogether.
Although there is indirect evidence for the regulation of the PtdIns4K
II activity by serine and tyrosine residue phosphorylation (19, 20),
receptor association (21, 22), heterotrimeric G-proteins (23), and
substrate presentation by the PtdIns transfer protein (22), the
inability to purify or immunoprecipitate the enzyme has precluded
definitive experiments to evaluate its function and regulation.
PtdIns4K II has been identified in the plasma membrane, lysosomal,
microsomal, transport vesicle, and nuclear compartments (4). More
recently this isozyme has been shown to exist in subdomains of the
plasma membrane termed non-caveolar membrane rafts (24), in which
receptor-dependent PLC signaling also appears to be
localized (25). Rafts are typically small, cholesterol-rich membrane
domains of low buoyant density, which are generally insoluble in 1%
Triton X-100 at 4 °C (reviewed in Ref. 26).
To address questions regarding the pivotal role of this enzyme in many
signaling pathways, we set out to purify sufficient human PtdIns4K II
to allow the cDNA to be identified.
Type II PtdIns 4-Kinase Purification--
Plasma membrane rafts
containing PtdIns4K II were isolated from human A431 epidermoid
carcinoma cells, employing similar methods to those described
previously (24). Two chromatographic purification steps were selected
from the work of Deuel and co-workers (27), adapted to microscale
purification and modified using detergents appropriate for
cholesterol-rich rafts. A431 cells were cultured to confluence in six
24 × 24-cm dishes in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum.
Monolayers were washed in phosphate-buffered saline and then scraped
into 12 ml of buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 10 mM EGTA, 0.25 M
sucrose, and protease inhibitors (CompleteTM Mini, EDTA-free; Roche
Molecular Biochemicals) prior to Dounce homogenization. The homogenate
was centrifuged at 4000 × g for 5 min. Membranes were
pelleted from the post-nuclear supernatant by centrifugation at
190,000 × g for 1 h at 4 °C.
Low density membrane rafts were prepared using a modification of the
method of Song et al. (28). Briefly, the 190,000 × g membrane pellet was resuspended in 2 ml of 100 mM Na2CO3, pH 11.0, 10 mM EGTA, 10 mM EDTA, 10 mM
Purified rafts were suspended in 3 ml of base buffer (20 mM
Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing CompleteTM
protease inhibitors and sonicated (10 × 5-s bursts at 40%
maximum power) using a VC130PB sonicator. The dispersed rafts were
solubilized by the addition of Type II PtdIns 4-Kinase Assays--
In-gel assays and product
analysis using TLC were performed as described previously (12, 19).
Solution assays containing 5 µl of MonoS column fractions or 10 ng of
GST-PtdIns4K II Mass Spectrometry--
Proteins were separated by SDS-PAGE and
superficially stained with silver (29). The stained material
comigrating with renatured activity was excised and subjected to
reduction and alkylation using DTT and iodoacetic acid followed by
in-gel digestion using modified trypsin (Promega, Southampton, UK).
Matrix-assisted laser desorption (MALDI) mass spectrometry (MS) was
performed on a Reflex III (Bruker Daltonik, Bremen, Germany) reflector
time of flight mass spectrometer in the reflector mode with delayed
extraction and using a 2,5-dihydroxybenzoic acid matrix. Spectra were
internally calibrated using tryptic autolysis product ions of
monoisotopic m/z 842.5100 and 2211.1046. The
MS-Fit program (ProteinProspector; University of California at San
Francisco, San Francisco, CA) was employed for peptide mass mapping.
For electrospray ionization (ESI) liquid chromatography MS/MS, samples
were loaded on an Ultimate nano-HPLC (LCPackings, Amsterdam, The
Netherlands) via a Famos autosampler (LCPackings). All solvents were
HPLC grade (Rathburn, Walkerburn, Scotland, UK). Separation was
performed on a 75 µm × 150-mm silica C18 (5-µm particle size) PepMapTM column (LCPackings) with 0.1% formic acid in water as solvent
A and 0.08% formic acid in 80% acetonitrile/20% water as solvent B. After equilibration with 5% solvent B, a linear gradient was developed
to 40% solvent B in 32 min using a flow rate of 200 nl/min. The
nano-HPLC was directly coupled to a Q-TOF ESI mass spectrometer
(MicroMass, Manchester, UK) set up for automated liquid
chromatography-MS/MS data acquisition using alternating collision energies.
Isolation of the Type II PtdIns 4-Kinase cDNA--
An
antisense oligonucleotide primer (5'-TGCCTCTGGAGCTACCACCATG) designed
to anneal 12 bases 3' to the predicted stop codon of the putative
PtdIns4K II
A single PCR product conforming to the predicted size of the putative
PtdIns4K II Expression and Purification of Recombinant Protein--
A
restriction fragment containing the ORF was excised from pGEM T-easy,
cloned into the NcoI and SacI sites of pGEX-KG
(31), and expressed in the Escherichia coli XL1 strain
(Stratagene, Amsterdam, The Netherlands) as a GST fusion
protein. Expression of recombinant protein was induced by the addition
of isopropyl-1-thio- Northern Blot Analysis--
A cDNA probe representing the
carboxyl-terminal 300 amino acids of PtdIns4K II Type II PtdIns 4-Kinase Purification--
The vast majority of the
PtdIns4K II in A431 cells is localized within buoyant, non-caveolar
membrane rafts (24). Consequently, purified rafts containing PtdIns4K
II were solubilized in detergents and further fractionated using
sequential anion and cation exchange chromatography. A single peak of
activity was obtained at each stage (data not shown). Individual
fractions from the MiniS column were analyzed by SDS-PAGE using silver
staining (Fig. 1A) and PtdIns4K assays (Fig. 1B). In-gel assays of fraction 3 showed that a band of 52-kDa apparent molecular mass coeluted and
comigrated with the bulk of the activity (Fig. 1C). A much
smaller amount of activity was recovered at lower apparent molecular
mass as reported previously (12). No other protein appeared to elute with the same profile as the 52-kDa protein or the kinase activity, suggesting the absence of a stoichiometrically associated subunit.
Mass Spectrometry--
Peptide mass mapping (32) of the 52-kDa
protein produced 18 peptide masses. When the mass data were used to
search the NCBI non-redundant data base using the MS-fit program they
were found to match most closely a partial cDNA encoding a 30.2-kDa
fragment of a human protein (EMBL accession number AL353952).
This partial ORF lacked the initiating methionine. Searches of the NCBI
human expressed sequence tag data base were performed using the 5'
region of this cDNA clone to complete the ORF. This revealed an
overlapping clone (GenBankTM accession number AW246119),
and further searches using AW246119 revealed two more overlapping
clones (GenBankTM accession number BE302827 and EMBL
accession number AL041898). A clear 1437-bp ORF downstream of an
in-frame stop codon and a Kozak consensus sequence (33) was identified
from these clones. Furthermore, 14 additional overlapping sequences
(GenBankTM accession numbers AA256505, AA495772, AA773590,
BE170428, AW770113, AI674489, AI971186, BE300517, AA234669, AI492121,
AA495828, and AI332390 and DDBJ accession numbers AK024317 and
AK023236) were identified when the NCBI data bases were searched using
the sequence assembled from the four clones (AL353952, AW246119,
BE302827, and AL041898).
The 1437-bp ORF could account for all 18 tryptic peptide ion masses;
these 18 peptides accounted for 37% of the total amino acid sequence
(sequence coverage). To confirm this assignment, the 52-kDa protein
band was prepared as before, but the gel piece was washed with 80%
acetone at
As a separate confirmation of these results, we employed ESI liquid
chromatography-MS/MS to analyze the tryptic digest of the first
preparation of the 52-kDa protein. This produced sequence information
for four peptides, all of which confirmed the sequence assignment. The
inset in Fig. 2 shows the MS/MS spectrum of the tryptic
peptide Ser460-Arg472, in which virtually the
entire series of y-type fragment ions is evident.
The 1437-bp ORF sequence obtained by data base searching was used to
design complimentary oligonucleotide primers. These primers were used
to isolate the cDNA by PCR with a proof-reading polymerase. Extensive DNA sequencing of independently cloned PCR products further
verified the accuracy of the ORF.
Sequence Analysis--
The 1437-bp ORF sequence encoded a
polypeptide containing 479 amino acid residues and a molecular mass of
~54.0 kDa. One motif was found using MacVector, version 7.0 (Oxford
Molecular), a leucine zipper between residues 184 and 205 (Fig.
3A), which may mediate homo- or heterotypic protein-protein
interactions. Although the mammalian enzyme is strongly bound to
membranes, no transmembrane sequence or any established consensus for
acylation was detected. The extended region amino-terminal to the
kinase domain showed no homology with any other known protein. Although
the enzyme is sensitive to inhibition by low micromolar concentrations
of Ca2+ (12), no Ca2+ binding motif was apparent.
Searches of NCBI data bases for homologous sequences revealed a set of
additional sequences from different species including a second human
sequence (NCBI accession number 8922869; see Fig. 3B) and a
single homologue in Saccharomyces cerevisiae (NCBI accession number 6322361; see Fig. 3B) located on chromosome X. Alignment of over 10 different PtdIns4K II
homologues2 revealed several
candidate kinase motifs (34), including a candidate subdomain I P-loop
sequence GSSGSY139, subdomain II ATP-binding
K153, subdomain VIb catalytic residues
DYIIRN307 or DRGNDN314, and subdomain VII
Mg2+ binding motif DNG349. No obvious homology
was apparent between the region amino-terminal to each putative kinase
domain and the corresponding region of the PtdIns4K II Northern Blot Analysis--
The PtdIns4K II Expression and Characterization of Recombinant Protein--
The
GST fusion protein expressed in bacteria had an apparent molecular mass
of ~80 kDa, and removal of the GST polypeptide using thrombin reduced
the mass to ~54 kDa (Fig.
5A). The fusion protein
possessed PtdIns4K activity, which was undetectable in control
preparations containing GST only, and the lipid product comigrated with
a PtdIns4P standard (Fig. 5B). When the fusion protein was
tested against synthetic phosphoinositide substrates it was able to
phosphorylate PtdIns but not PtdIns3P, PtdIns4P, or PtdIns5P
(results not shown). Kinetic analysis of the GST fusion protein using
the Lineweaver-Burk method gave Km values of 28 and
54 µM for ATP and PtdIns, respectively, which fall within the range of previously published values (see Ref. 12 and references therein). The specific activity of the GST fusion protein was ~3.3 nmol/min/mg; this is low compared with the enzyme purified from
various primary tissues (see Ref. 12 and references therein) and may
reflect differences in post-translational modification or incorrect
folding. The recombinant enzyme was activated 32-fold by 0.1% Triton
X-100 (Fig. 5C) and inhibited by adenosine with an
IC50 of 22 µM (Fig. 5D).
Activation by 0.1% Triton X-100 and inhibition by adenosine in the
micromolar range are characteristic properties of PtdIns4K II purified
from mammalian tissues (4). The GST fusion protein was also inhibited
in a dose-dependent manner by the monoclonal antibody 4C5G
(Fig. 5E), which is another characteristic property of
PtdIns4K II that distinguishes it from the type III PtdIns4K isoforms
(35).
The successful purification of PtdIns4K II in sufficient quantity
for sequencing depended on an understanding of the membrane rafting of
this enzyme (24). The purification of these rafts and their efficient
solubilization allowed the development of a rapid, small-scale
purification of the PtdIns4K II enzyme from cultured cells. The
mechanism of PtdIns4K II and PI rafting remains unclear, but it appears
to be required for receptor-dependent signal transduction
(25).
Because of a lack of specific diagnostic reagents, PtdIns4K II activity
is typically characterized by its activation by non-ionic detergents
and inhibition by adenosine and the monoclonal antibody 4C5G (4, 10,
12, 35). The recombinant enzyme displayed identical properties to those
reported for the purified enzyme; GST-PtdInsPK II As the PtdIns4K II The existence of a single PtdInsPK II homologue in S. cerevisiae is worth noting, as the type III homologues, PIK1 and
STT4, have been proposed to account for all of the PtdIns4K activity in
these cells (36). Although a type II-like PtdIns4K had previously been
partially purified from S. cerevisiae (37), the implication was that this was an alternatively spliced or degraded product of the
type III genes. It remains to be shown whether the yeast and other
PtdIns4K II homologues possess PtdIns4K activity.
In summary, the identification of the PtdIns4K II and a second human isoform, type II
. The type II
mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-octylglucoside, and 4 mM deoxycholate, containing
CompleteTM protease inhibitors. The membrane suspension was sonicated
on ice (10 × 5-s bursts at 40% maximum power using a VC130PB
sonicator (Sonics and Materials Inc.), and an equal volume of
80% sucrose in 25 mM MES, pH 6.5, 150 mM NaCl, 10 mM EGTA, and 10 mM EDTA was added. This
solution was transferred to a 12.5-ml ultracentrifuge tube and overlaid
with 4 ml of 35% sucrose and 4 ml of 5% sucrose both in 25 mM MES, pH 6.5, 150 mM NaCl, 50 mM
Na2CO3, 10 mM EGTA and 10 mM EDTA. The discontinuous sucrose gradient was centrifuged
at 4 °C for 16 h at 190,000 × g. Rafts were
then collected at the 5 and 35% sucrose interface, washed with ~12
ml of 10 mM Tris-HCl, pH 8.0, 10 mM NaCl and
repelleted by centrifugation at 4 °C for 1 h at 190,000 × g.
-octylglucoside and deoxycholate to
100 and 40 mM, respectively, diluted 8-fold with base
buffer, and then applied to a MonoQ column (PC 1.6/5; Amersham Pharmacia Biotech) equilibrated in buffer QA (20 mM
Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM DTT, 0.1%
reduced Triton X-100) on a SMART chromatography system (Amersham
Pharmacia Biotech). The column was washed in buffer QA and developed
with a discontinuous salt gradient in buffer QA (20-189 mM
NaCl at 0-19 min, 189-360 mM NaCl at 19-21 min, and
360-900 mM NaCl at 21-24 min). PtdIns4K activity was
collected at ~19-19.2 min, diluted 15-fold with buffer SA (20 mM bis-Tris-HCl, pH 6.0, 50 mM NaCl, 1 mM DTT, 0.1% reduced Triton X-100), and applied to a MiniS
column (PC 3.2/3; Amersham Pharmacia Biotech) equilibrated in buffer
SA. After washing in buffer SA, the column was developed with a
continuous salt gradient in buffer SA (50-900 mM NaCl in 5 min). The peak of PtdIns4K activity was collected at ~2 min.
were performed in 100-µl volumes containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.1% Triton X-100, 1 mM
2-mercaptoethanol, 0.5 mM PtdIns, 0.1 mM EGTA,
and 200 µM [
32P]ATP (1 µCi/assay).
Assays were started by the addition of ATP and incubated for 15 min at
37 °C before being terminated by the addition of HCl to 0.5 M. Under these conditions the addition of radioactive
phosphate to PtdIns was linear (data not shown). The organic phase was
extracted (12), and the lipid products were analyzed by TLC separation
on Silica 60 plates (Whatman Inc.) in propan-1-ol/1
M acetic acid (65:35) containing 1% 5 M
phosphoric acid followed by autoradiography. Spots containing PtdIns4P
were quantitated by scraping and counting in a Beckman LS 6500 liquid scintillation counter.
open reading frame (ORF) was used to prime first strand
cDNA synthesis from 0.5 µg of total RNA isolated from A431 cells
(30). Reverse transcription was performed in 20 µl using the
SuperScriptTM Pre-amplification system (Life Technologies, Inc.). A
2-µl aliquot of this reaction was subsequently used to amplify an
~1.4-kilobase band by PCR using the sense primer 5'-AATTCCATGGACGAGACGAGCCCACTAG (which incorporates an NcoI
restriction site overlapping the predicted initiating methionine codon)
and the antisense primer 5'-TGCCTCTGGAGCTACCACCATG encompassing the predicted termination codon. PCR was performed using Pfu DNA
polymerase (Promega). Cycling conditions were 96 °C for 30 s,
66 °C for 30 s, and 72 °C for 3 min for 40 cycles.
ORF was gel purified and 3'A-tailed with dATP and
Taq DNA polymerase (Promega) before cloning into the pGEM
T-easy vector (Promega) as described by the manufacturer. The identity
of the PCR product was confirmed by sequencing.
-D-galactopyranoside to 0.1 mM, and cells were harvested after 3 h of growth at
30 °C. Bacterial pellets were sonicated in RIPA buffer containing 1 mM EDTA, 1 mM
-mercaptoethanol, and
CompleteTM protease inhibitors. Lysates were cleared by centrifugation
at 20,000 × g for 20 min, after which the recombinant
protein was bound to glutathione-Sepharose (Amersham Pharmacia Biotech)
as described by the manufacturer.
was generated by
PCR using the sense primer 5'-TTTGGCCGTGGACTGCCTTGCC and the antisense
primer 5'-TGCCTCTGGAGCTACCACCATG. The ~900-base pair (bp) PCR product
was gel-purified and labeled with [
-32P]dCTP by random
priming as described previously (30). This was then used to probe a
human multiple-tissue Northern blot (CLONTECH). The
probe was hybridized under high stringency conditions according to the
manufacturer's instructions and then further washed in diethyl
pyrocarbonate-treated 0.1 × SSPE (3 M NaCl, 0.2 M NaH2PO4, 0.2 M EDTA),
0.1% SDS for 1 h at 73 °C. Following autoradiography, the blot
was stripped and reprobed with a human
-actin control probe
(CLONTECH) as directed by the manufacturer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of the human type II PtdIns4K
preparation. A, silver-stained, SDS-PAGE analysis of
MiniS column fractions containing the peak of PtdIns4K activity.
B, the corresponding PtdIns4K assays of column fractions
from panel A. C, renaturation of PtdIns4K
activity from the peak activity fraction corresponding to lane
3 in panel A. The region of the SDS-PAGE gel containing
renatured activity is shown and aligned to scale. The position of the
candidate 52-kDa PtdIns4K II enzyme is indicated by an
arrow.
20 °C, and the peptide digest was desalted with a
ZipTipTM (Millipore) to improve the quality of the MS analysis.
Analysis of this sample by MALDI-MS (Fig. 2) gave 24 peptides matching the
predicted ORF (Fig. 3A) with 44% sequence coverage and a mass accuracy of 100 ppm.
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Fig. 2.
Mass spectrometric analysis. MALDI mass
spectrum of the tryptic peptides from the 52-kDa protein is shown.
Circles indicate ion signals matching tryptic peptide masses
derived from the predicted amino acid sequence. Ion signals marked
K and T can be attributed to human keratin 1 and
tryptic autolysis peptides, respectively. The arrow marks
the ion signal matching the peptide mass of
Ser460-Arg472, for which the inset
shows the MS/MS spectrum with y ions labeled.
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Fig. 3.
Amino acid sequences of human and yeast type
II PtdIns4K homologues. A, the predicted amino acid
sequence of PtdIns4K II showing tryptic peptides identified by MALDI
MS (underlined) and ESI MS/MS (bold). The
putative leucine zipper sequence is boxed. B,
kinase domain alignment for the human PtdIns4K II
, human PtdIns4K
II
, and the S. cerevisiae type II homologue showing
conserved residues. The alignment begins ~30 residues amino-terminal
of each putative P-loop and ends at each predicted carboxyl terminus.
In this alignment PtdIns4K II
and S. cerevisiae kinase
domains show 68 and 30% residue identity, respectively, with the
PtdIns4K II
kinase domain.
enzyme. The
lack of any close sequence similarity with either the PI3K or PtdInsPK
family demonstrated that PtdIns4K II
and its sequence homologues
define a novel, third PI kinase family.
mRNA has an
apparent size of 6.6 kilobases and appears to be ubiquitously expressed
in human tissues (Fig. 4), which is
consistent with the purification of this enzyme from numerous different
primary tissues (see Ref. 12 and references therein). Expression
appeared highest in kidney, brain, heart, skeletal muscle, and placenta
and lowest in colon, thymus, and small intestine.
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Fig. 4.
Distribution of PtdIns4K
II mRNA in human tissues. The
expression of PtdIns4K II
mRNA was analyzed using a human
multiple-tissue Northern blot (CLONTECH) hybridized
with a 32P-labeled fragment of either the PtdIns4K II
(top) or the ubiquitously expressed human
-actin
(bottom) cDNA.
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Fig. 5.
Expression and activity of recombinant
PtdIns4K II . A, the PtdIns4K
II
cDNA was expressed as a GST fusion protein in E. coli. An SDS-PAGE separation stained with Coomassie Brilliant Blue
is shown containing vector only (GST) and the GST-PtdIns4K
II
fusion protein before (GST-II
) and after digestion
with thrombin (II
). The undigested (arrowhead)
and digested (arrow) proteins migrated at their expected
sizes of ~80 and 54 kDa, respectively. B, PtdIns4K assay
of the recombinant GST-PtdIns4K II
fusion protein
(GST-II
) and GST control (GST). The single
product comigrated with an unlabeled iodine-stained PtdIns4P standard
(Std) and no other products were detected. The position of
the origin is indicated by an arrow. C, the
PtdIns4K activity of the recombinant GST-PtdIns4K II
fusion protein
was assayed in the absence (
) and presence (+) of 0.1% Triton X-100.
D, sensitivity of the recombinant protein to adenosine.
GST-PtdIns4K II
was assayed in the presence of a range of adenosine
concentrations as indicated. E, inhibition of GST-PtdIns4K
II
by monoclonal antibody 4C5G. Recombinant fusion protein was
assayed in the presence of the indicated amounts of
4C5G.-32767.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
was activated by
Triton X-100 and inhibited by low concentrations of adenosine and by 4C5G.
enzyme defines a novel sequence family, it should
be clearly distinguished from the previously cloned PtdIns4K
(14),
PtdIns4K
(15-17), and type III (18) isozymes, as well as the PI3K
and PtdInsPK families. PtdIns4K
, PtdIns4K
, and the type III
PtdIns4K belong in the PI3K family. We propose to maintain the type II
assignation for PtdIns4K II, at least until the structural and
functional range of homologous enzymes is clearer, and to name the
human isoform purified in this report and the second isoform identified
by data base searches PtdIns4K II
and PtdIns4K II
, respectively.
The lack of sequence similarity in the amino-terminal region of the
PtdIns4K II
and PtdIns4K II
proteins suggests this region may
confer functional specificity, for example by mediating differential
localization, allostery, or regulation via post-translational modification.
cDNA sequence
has defined a novel family of PI kinases. It is now possible to use
recombinant approaches to provide specific reagents able to
address numerous questions regarding the function of PtdIns4K II,
including the possible functional degeneracy between the different PtdIns4K enzymes. Furthermore an important step has been taken toward
obtaining a molecular definition of the reported rafting, regulation,
complex formation, and biological function of this enzyme in health and disease.
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ACKNOWLEDGEMENTS |
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We thank The Wellcome Trust for financial support (for S. M., M. W., and J. J. H.), Professor Reinhard Wetzker (Jena, Germany) for initiating this project, Dr. Christina Panaretou (Imperial Cancer Research Fund, London, UK) for 4C5G, Professor Richard Bruckdorfer for the generous provision of the swing-out rotor, and Dr. Zac Varghese for use of an ultracentrifuge.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ303098.
§ Supported by The Wellcome Trust.
Wellcome Trust senior fellow in basic biomedical science. To
whom correspondence should be addressed. Tel: 44-20-7433-2821; Fax:
44-20-7433-2817; E-mail: j.hsuan@rfc.ucl.ac.uk.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100982200
2 M. dos Santos and J. J. Hsuan, submitted.
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ABBREVIATIONS |
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The abbreviations used are: PtdIns, phosphatidylinositol; bp, base pair; ESI, electrospray ionization; GST, glutathione S-transferase; MALDI, matrix-assisted laser desorption; MS, mass spectrometry; ORF, open reading frame; PCR, polymerase chain reaction; PI, phosphoinositide; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PtdIns4K, phosphatidylinositol 4-kinase; PtdIns4P, phosphatidylinositol 4-phosphate; PtdInsPK, PtdIns phosphate kinase; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | de Camilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996) Science 271, 1533-1539[Abstract] |
2. | Hsuan, J. J., Minogue, S., and dos Santos, M. (1998) Adv. Cancer Res. 74, 167-216[Medline] [Order article via Infotrieve] |
3. | Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998) Annu. Rev. Biochem. 67, 481-507[CrossRef][Medline] [Order article via Infotrieve] |
4. | Pike, L. J. (1992) Endocr. Rev. 13, 692-706[Abstract] |
5. | Keith, C. T., and Schreiber, S. L. (1995) Science 270, 50-51[Medline] [Order article via Infotrieve] |
6. | Zvelebil, M. J., MacDougall, L., Leevers, S., Volinia, S., Vanhaesebroeck, B., Gout, I., Panayotou, G., Domin, J., Stein, R., Pagès, F., and Waterfield, M. D. (1996) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 351, 217-223[Medline] [Order article via Infotrieve] |
7. | Loijens, J. C., Boronenkov, I. V., Parker, G. J., and Anderson, R. A. (1996) Adv. Enzyme Regul. 36, 115-140[CrossRef][Medline] [Order article via Infotrieve] |
8. | Rao, V. D., Misra, S., Boronenkov, I. V., Anderson, R. A., and Hurley, J. H. (1998) Cell 94, 829-839[Medline] [Order article via Infotrieve] |
9. | Walker, E. H., Perisic, O., Ried, C., Stephens, L., and Williams, R. L. (1999) Nature 402, 313-320[CrossRef][Medline] [Order article via Infotrieve] |
10. | Whitman, M., Kaplan, D., Roberts, T., and Cantley, L. (1987) Biochem. J. 247, 165-174[Medline] [Order article via Infotrieve] |
11. | Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332, 644-646[CrossRef][Medline] [Order article via Infotrieve] |
12. | Wetzker, R., Klinger, R., Hsuan, J., Fry, M. J., Kauffmann, Z. A., Muller, E., Frunder, H., and Waterfield, M. (1991) Eur. J. Biochem. 200, 179-185[Abstract] |
13. |
Yamakawa, A.,
Nishizawa, M.,
Fujiwara, K. T.,
Kawai, S.,
Kawasaki, H.,
Suzuki, K.,
and Takenawa, T.
(1991)
J. Biol. Chem.
266,
17580-17583 |
14. |
Wong, K.,
and Cantley, L. C.
(1994)
J. Biol. Chem.
269,
28878-28884 |
15. | Nakagawa, T., Goto, K., and Kondo, H. (1996) Biochem. J. 320, 643-649[Medline] [Order article via Infotrieve] |
16. |
Meyers, R.,
and Cantley, L. C.
(1997)
J. Biol. Chem.
272,
4384-4390 |
17. |
Balla, T.,
Downing, G. J.,
Jaffe, H.,
Kim, S.,
Zolyomi, A.,
and Catt, K. J.
(1997)
J. Biol. Chem.
272,
18358-18366 |
18. |
Nakagawa, T.,
Goto, K.,
and Kondo, H.
(1996)
J. Biol. Chem.
271,
12088-12094 |
19. |
Kauffmann, Z. A.,
Klinger, R.,
Endemann, G.,
Waterfield, M. D.,
Wetzker, R.,
and Hsuan, J. J.
(1994)
J. Biol. Chem.
269,
31243-31251 |
20. | de Neef, R.-S., Hardy-Dessources, M.-D., and Giraud, F. (1996) Eur. J. Biochem. 235, 549-556[Abstract] |
21. |
Cochet, C.,
Filhol, O.,
Payrastre, B.,
Hunter, T.,
and Gill, G. N.
(1991)
J. Biol. Chem.
266,
637-644 |
22. | Kauffmann, Z. A., Thomas, G. M., Ball, A., Prosser, S., Cunningham, E., Cockcroft, S., and Hsuan, J. J. (1995) Science 268, 1188-1190[Medline] [Order article via Infotrieve] |
23. |
Pike, L. J.,
and Eakes, A. T.
(1987)
J. Biol. Chem.
262,
1644-1651 |
24. |
Waugh, M. G.,
Lawson, D.,
Tan, S. K.,
and Hsuan, J. J.
(1998)
J. Biol. Chem.
273,
17115-17121 |
25. |
Pike, L. J.,
and Miller, J. M.
(1998)
J. Biol. Chem.
273,
22298-22304 |
26. | Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Porter, F. D.,
Li, Y. S.,
and Deuel, T. F.
(1988)
J. Biol. Chem.
263,
8989-8995 |
28. |
Song, K. S.,
Li, S.,
Okamoto, T.,
Quilliam, L. A.,
Sargiacomo, M.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
9690-9697 |
29. | Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858[CrossRef][Medline] [Order article via Infotrieve] |
30. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
31. | Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267[Medline] [Order article via Infotrieve] |
32. | Pappin, D. J. C., Horjup, P., and Bleasby, A. J. (1993) Curr. Biol. 3, 327-332 |
33. | Kozak, M. (1991) J. Cell Biol. 115, 887-903[Abstract] |
34. | Hanks, S. K., and Quinn, A. M. (1991) Methods Enzymol. 200, 38-62[Medline] [Order article via Infotrieve] |
35. | Endemann, G. C., Graziani, A. & Cantley, L. C. (1991) Biochem. J |
36. |
Audhya, A.,
Foti, M.,
and Emr, S. D.
(2000)
Mol. Biol. Cell
11,
2673-2689 |
37. |
Nickels, J. J.,
Buxeda, R. J.,
and Carman, G. M.
(1992)
J. Biol. Chem.
267,
16297-16304 |