From the Department of Pharmacology,
§ Center for Basic Neuroscience, Department of Molecular
Genetics, and Howard Hughes Medical Institute and the
Department
of Biochemistry, University of Texas Southwestern Medical Center,
Dallas, Texas 75390
Received for publication, December 6, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Phosphatidylinositolpolyphosphates (PIPs) are
centrally involved in many biological processes, ranging from cell
growth and organization of the actin cytoskeleton to endo- and
exocytosis. Phosphorylation of phosphatidylinositol at the D-4
position, an essential step in the biosynthesis of PIPs, appears to be
catalyzed by two biochemically distinct enzymes. However, only one of
these two enzymes has been molecularly characterized. We now describe a
novel class of phosphatidylinositol 4-kinases that probably corresponds
to the missing element in phosphatidylinositol metabolism. These
kinases are highly conserved evolutionarily, but unrelated to
previously characterized phosphatidylinositol kinases, and thus
represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells,
only phosphorylate phosphatidylinositol, are potently inhibited by
adenosine, but are insensitive to wortmannin or phenylarsine oxide.
Although they lack an obvious transmembrane domain, they are strongly
attached to membranes by palmitoylation. Our data suggest that
independent pathways for phosphatidylinositol 4-phosphate synthesis
emerged during evolution, possibly to allow tight temporal and spatial
control over the production of this key signaling molecule.
Phosphatidylinositol 4-kinases
(PtdIns1 4-kinases) catalyze
the phosphorylation of phosphatidylinositol (PtdIns) on the D-4 position of the inositol ring. The product of this reaction,
phosphatidylinositol 4-phosphate (PtdIns4P) is a major precursor in the
synthesis of phosphatidylinositolpolyphosphates (PIPs), including
PtdIns3,4P2, PtdIns4,5P2, and
PtdIns3,4,5P3, which participate in signal transduction, membrane trafficking, and cytoskeletal reorganization (1-5). Two
classes of PtdIns 4-kinases, Types II and III, have been identified; the enzyme originally designated Type I was subsequently found to be a
PtdIns 3-kinase (6). Mammalian Type II PtdIns 4-kinase (henceforth
referred to as PI4KII) is a 55-kDa integral membrane protein believed
to account for most of the PtdIns 4-kinase activity in cells (7). It
can be distinguished from the Type III kinases (PI4KIIIs) by virtue of
its lower Km values for ATP and PtdIns, its
insensitivity to inhibition by wortmannin, and its sensitivity to
adenosine and monoclonal antibody 4C5G (8). The two closely related
Type III kinases, III Materials--
Q-Sepharose, SP-Sepharose, and DEAE-dextran were
from Amersham Pharmacia Biotech; protein G-Sepharose was from
Zymed Laboratories Inc.;
L- Purification of PI4KII--
PI4KII was partially purified from
membranes of bovine adrenal chromaffin granules (17), obtained by
floating lysed granules over a 1.2 m sucrose cushion. Membranes
were washed in a solution containing 20 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol, 1 mM EDTA, and a
range of protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride and 10 mg/l each of
Na-p-tosyl-L-lysine
chloromethyl ester,
Na-p-tosyl-L-arginine
methyl ester, Na-p-tosyl-L-lysine
chloromethyl ketone, leupeptin, and pepstatin A (buffer A) plus
0.5 M NaCl to remove loosely bound proteins, then
solubilized in buffer A containing Triton X-100 (1:100
protein/detergent weight ratio). After centrifugation to remove
insoluble material, the extract was diluted 5-fold with buffer
containing 20 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol (buffer B) and
loaded on a Q-Sepharose column equilibrated with buffer B containing
1% Triton X-100 and 20 mM NaCl. The flow-through was directly loaded on an SP-Sepharose column and eluted with a linear 20-250 mM NaCl gradient. Fractions containing highest
PtdIns 4-kinase activity were pooled and electrophoresed.
PtdIns 4-kinase was identified by assaying renatured gel
slices (13). The most active band was submitted for amino acid sequence
analysis (Argo BioAnalytica, Inc.).
Purification of PI4KIII--
PI4KIII was partially purified from
bovine adrenal medulla following the procedure of Downing et
al. (18). Briefly, tissue was extracted with buffer A containing 1 M NaCl, and the fraction of supernatant precipitated at
40% saturation of ammonium sulfate was dialyzed against buffer A
containing 30 mM NaCl. Dialyzed sample was loaded on a
DEAE-cellulose column, and PI4KIII was eluted with a linear 30-500
mM NaCl gradient.
Molecular Cloning and Expression of Rat Brain cDNA Encoding
PI4KII--
Using a 300-bp (EcoRI/HindIII)
fragment from a mouse EST (GenBankTM accession
number AI385489, obtained from ATCCTM), a rat brain
Analysis of Recombinant PI4KII--
COS cells transfected with
Myc-PI4KII cDNA were washed in PBS; resuspended in solution
containing 0.25 M sucrose, 20 mM Tris-HCl, pH
7.5, 2 mM EDTA, 1 mM dithiothreitol, and
protease inhibitors; and permeabilized by freezing and thawing. The
suspension was centrifuged for 5 min at 1000 × g to
obtain post-nuclear supernatant, which was subsequently centrifuged for
15 min at 200,000 × g to obtain cytosol (supernatant)
and membrane (pellet) fractions. Pellets were extracted for 15 min on
ice with a solution containing 20 mM Tris, pH 7.4, 10%
glycerol, 0.1 M NaCl, 1% Triton X-100, 1 mM
dithiothreitol, and protease inhibitors. Insoluble material was removed
by centrifugation (15 min at 200,000 × g), and the supernatant, representing the membrane-bound pool, was analyzed by
immunoblotting with anti-Myc antibody and assayed for PtdIns 4-kinase
activity. The enzymatic activity was assayed using either total Triton
X-100 extracts of cell membranes (Fig. 2, b and
d-i) or immunoprecipitates obtained with anti-Myc antibody
(Fig. 2c). When membrane extracts were assayed,
the activities of similar extracts from mock-transfected cells were
subtracted from each data point. For immunoprecipitation of recombinant
PI4KII, Triton X-100 extracts of membrane fractions described above
were precleared by 30-min incubation with protein G-Sepharose and then
incubated with anti-Myc antibodies chemically cross-linked to protein
G-Sepharose with DMP.
PtdIns 4-Kinase Assay--
PtdIns 4-kinase activity was measured
by phosphorylation of PtdIns micelles using [ Palmitoylation--
Cells were radiolabeled with
[3H]palmitate (0.3 µCi/ml) for 2 h as described by
Linder et al. (22), washed with cold PBS, then lysed with
buffer containing 50 mM Tris, pH 8.0, 150 mM
NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, and protease
inhibitors for 15 min on ice. Lysates were centrifuged for 15 min at
200,000 × g, and Myc-tagged PI4KII was
immunoprecipitated with anti-Myc antibodies as described above.
Palmitoylation was detected by autoradiography.
Sequence Analysis--
Sequence data base searches against the
nonredundant protein data base maintained at the National Center for
Biotechnology Information (Bethesda, MD) were performed using the
PSI-BLAST program (23) with various parameters run to convergence. The data base hits identified in the initial searches were used as queries
for additional PSI-BLAST searches. The phylogenetic tree was
constructed with the PHYLIP package (24) by the distance method
(protdist program). Neighbor-joining algorithm (neighbor program) was
utilized to construct the phylogenetic tree from the distance matrix.
Other Methods--
COS-7 cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and
antibiotics. Protein concentrations were determined using the modified
Lowry method (25) according to Peterson (26) with bovine serum albumin
as a standard. SDS-polyacrylamide gel electrophoresis was carried out
according to the method of Laemmli (27). Immunoblot analysis was
carried out by the method of Towbin et al. (28) as described
previously (29).
To obtain amino acid sequence information, PI4KII was partially
purified from bovine adrenal chromaffin granule membranes (14) (Fig.
1, a and b). As
reported previously (12-16), a major 55-kDa electrophoretic band was
prominent in SDS gels of active fractions from an SP-Sepharose ion
exchange column. However, a more slowly migrating component, sometimes
poorly resolved, was also evident. The gels were sliced, renatured, and
assayed for PtdIns 4-kinase activity. Only the upper, minor component
of the 55-kDa doublet was active. The lower band was identified as
cytochrome P450 by mass spectrometry.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and III
, belong to the PtdIns 3/4-kinase
superfamily, which also includes the protein kinases TOR/ATM/DNA-PK (9,
10). Although PI4KII was identified more than 30 years ago (11) and
purified to apparent homogeneity more than 10 years ago (12-16), it
had, until now, not been possible to clone it. Here we report the amino
acid sequence of rat PI4KII and show that it represents a large family
of putative lipid kinases highly conserved from yeast to humans, but
bearing little similarity to other known lipid or protein kinases.
Recombinant rat PI4KII has the enzymatic characteristics expected of
the Type II kinases, and, despite lacking an obvious transmembrane
domain, behaves as an integral membrane protein. This tight membrane
association is due to palmitoylation within a cysteine-rich segment in
the center of the protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-phosphatidylinositol was from Avanti Polar-Lipids,
Inc.; diC16PtdIns, diC16PtdIns(3)P,
diC16PtdIns(4)P, and diC16PtdIns (5)P were from
Echelon Research Laboratories Inc.; diethylaminoethylcellulose (DE52)
was from Whatman; [
-32P]ATP and
[9,10-3H]palmitic acid were from PerkinElmer Life
Sciences; cloning reagents (rat cDNA library, reagents for
mutagenesis) were from Stratagene; monoclonal anti-Myc antibody 9E10
was obtained form Cell Culture Center; monoclonal anti-PI4KII antibody
4C5G was from Upstate Biotechnology; dimethyl pimelimidate (DMP) was
from Pierce. All other reagents, including ATP, adenosine, phenylarsine
oxide (PAO), wortmannin, and protease inhibitors were from Sigma.
ZAPII cDNA library was screened using standard procedures
(19), yielding 10 overlapping clones that covered the entire coding
region of PI4KII. A 1.5-kilobase pair cDNA fragment encoding
the full-length sequence was subcloned into
EcoRI/PstI sites of pCMV5Myc vectors. The
mycPI4KII cDNA construct was used as the template for generating
mutants by site-directed mutagenesis using the
QuickChangeTM Site-Directed Mutagenesis Kit, and the
presence of appropriate mutations was confirmed by DNA sequencing.
The deletion mutant
CCPCC was created using the
following primers: 5'-GGCTTCAGAAGCTGTTTGGCCGAGATTGCC-3' and
5'-GGCAATCTCGGCCAAACAGCTTCTGAAGCC-3'. Truncation mutant 92-487 was
made with primers 5'-CCGGAATTCTACACGCCGTTCAGACCCACCGCGAG-3' and
5'-CCCAAGCTTCTACCACCATGAAAAGAAGGGCTTC-3'. COS cells were
transfected with cDNAs of wild type or mutant PI4KII in pCMV5Myc
vectors using DEAE-dextran according to Schwartz and Rosenberg (20).
Typical transfection efficiency was 20%.
-32P]ATP
(10 mCi/mmol) as phosphate donor. Typically, kinase (in 20 mM Tris, pH 7.5, 10% glycerol, 0.1 M NaCl, 1%
Triton X-100, 1 mM dithiothreitol, and protease
inhibitors) was preincubated for 10 min with PtdIns prepared by
sonication with 50 mM Tris, pH 7.5, 1 mM EGTA,
0.4% Triton X-100, and 0.5 mg/ml bovine serum albumin. Reactions were
initiated by adding ATP and MgCl2 at final concentrations
of 0.2 mM and 15 mM, respectively, and carried out at room temperature for 15 min. Phospholipids were extracted according to Bligh and Dyer (21) and separated by thin layer chromatography (TLC) in n-propyl
alcohol/H20/NH4OH (65:20:15) solvent
system. For quantification, a range of [
-32P]ATP
concentrations were also spotted on TLC plates. Radioactive spots were
detected by autoradiography, scraped, and radioactivity measured by
scintillation counting.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Purification and sequence of PI4KII.
a, PtdIns 4-kinase activity of fractions eluted from an
SP-Sepharose column with a 20-250 mM NaCl gradient
(upper panel) and Coomassie Blue-stained SDS gel of the same
fractions (lower panel). b, assay of PtdIns
4-kinase activity of electrophoresed SP-Sepharose fractions. Pooled
fractions from the SP-Sepharose column (cross-hatched in
panel a) were electrophoresed on a 7.5% gel. The gel was
cut into equal slices, which were then renatured and assayed (13).
c, predicted amino acid sequence of rat (Rn),
PI4KII, and human and yeast homologs (Hs,
GenBankTM accession number AL355315; Sc,
GenBankTM accession number CAA89395) identified in the
high-throughput genomic sequence data base at
GenBankTM.
The electrophoretic band containing PtdIns 4-kinase activity was excised for internal amino acid sequence analysis, and the sequences of five peptides were obtained. Although these sequences did not correspond to those of any known proteins, two peptide sequences were found within a 443-bp mouse EST clone. With this information, a 300-bp nucleotide probe was generated and used to screen a rat brain cDNA library. Analysis of the nucleotide sequences of overlapping clones allowed us to determine the complete amino acid sequence of rat PI4KII, corresponding to a protein of Mr 54,305 (Fig. 1c). All five of the original peptide sequences were found in this full-length sequence. Based on the rat sequence we identified predicted homologs in numerous genome data bases, some of which are presented in the phylogenetic tree in Fig. 4. The sequences of putative human and yeast (Saccharomyces cerevisiae) PI4KIIs are aligned with the rat sequence in Fig. 1c.
To confirm that our derived sequence encodes an authentic Type II
PtdIns 4-kinase, the Myc-tagged recombinant protein was expressed in
COS cells, and its properties were characterized (Fig.
2). The expressed protein has an
electrophoretic mobility consistent with its molecular weight of
~55,000, and, like endogenous PI4KII, it is found almost exclusively
in the membrane fraction after high speed centrifugation (Fig.
2a), but can be solubilized by 1% Triton X-100. Homogenates
of cells transfected with rat PI4KII typically have 10-fold higher
specific activities than homogenates from untransfected or
mock-transfected cells. Membrane fractions account for most of this
excess activity (Fig. 2b) correlating well with the
distribution of the overexpressed protein.
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To determine the substrate specificity of PI4KII, the Myc-tagged kinase was immunoprecipitated with anti-Myc antibodies from overexpressing cells. As shown in Fig. 2c, the immunoprecipitated kinase did not phosphorylate PtdIns3P, PtdIns4P, or PtdIns5P to yield PtdInsP2; only PtdInsP was generated, presumably from trace contaminations of PtdIns in some of the PtdInsP preparations. Thus, PI4KII specifically phosphoryltates only PtdIns.
The expressed kinase displays all of the enzymatic characteristics previously reported for the native PI4KII (3, 9, 30, 31): it has relatively low Km values for ATP (~88 µM) (Fig. 2d) and for PtdIns (~45 µM) (Fig. 2e); it is inhibited by low concentrations of adenosine (Fig. 2f), but not by PAO (Fig. 2g) or wortmannin (Fig. 2h); and it is inhibited by monoclonal antibody 4C5G (Fig. 2i), which specifically recognizes the Type II kinase (8). In contrast, the Type III PtdIns 4-kinases have relatively high Km values for ATP (>400 µM) and PtdIns (~120 µM), require millimolar adenosine concentrations for inhibition, and are sensitive to wortmannin (Fig. 2h) and PAO (Fig. 2g).
Although both native and recombinant forms of PI4KII behave as integral
membrane proteins, sequence analysis does not reveal an obvious
transmembrane domain. The only portion of the rat enzyme with a
predominantly hydrophobic character is an alanine/valine-rich segment
(residues 74-95) that is predicted to adopt an -helical conformation. However, a truncation mutant (92) lacking most of this hydrophobic segment still behaves as an integral membrane protein,
i.e. it is soluble only in the presence of detergent (Fig.
3b). An alternative mode of
tight membrane association is through covalently attached lipids. Rat
PI4KII does not contain consensus myristoylation or prenylation motifs,
but a cysteine-rich segment (CCPCC, residues 173-177) is a potential
palmitoylation site. Indeed, wild type PI4KII and truncation mutant
92-487 are palmitoylated in cells, whereas a mutant lacking residues
173-177 (
CCPCC) is not (Fig. 3a). Both
CCPCC and the
92-487,
CCPCC double mutants behave as peripheral, rather than
integral, membrane proteins; they can be solubilized by sodium
carbonate, pH 10, though not by 1 M NaCl (Fig.
3b). The
CCPCC mutant is catalytically inactive, but
there is insufficient evidence to conclude from this that palmitoylation is essential for activity.
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A search of protein sequence data bases using the PSI-BLAST program
(23) identified PI4KII homologs in Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana,
S. cerevisiae, and other organisms (Fig.
4). The only prokaryotic sequences that belong to this family are from Mycobacterium, the organism
in which inositol-containing lipids were first identified (32).
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In addition to the putative orthologs of rat PI4KII, another family of
more distantly related sequences was found using the PSI-BLAST program.
This group is designated FJ-like putative kinases in Fig. 4 for its
best characterized member, Drosophila Four-jointed, a
membrane-bound or secreted protein with no known enzymatic activity, but apparently involved in development of ommatidia (33). Although it
is much too early to assign lipid or protein kinase activity to
Four-jointed protein or its mouse and human orthologs, the homology to
known kinase catalytic domains merits further examination.
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ACKNOWLEDGEMENTS |
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We thank Greg Kutsenok, Vinu Ipe, Sveta Earnst, Ewa Borowicz, and Iza Leznicki for technical assistance; Clark Wells for many helpful discussions; Dr. Melanie H. Cobb for advice and encouragement; and Dr. Clive Slaughter for mass spectrometry.
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FOOTNOTES |
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* This work was supported by Grant GM55562 from the National Institutes of Health.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.
¶ Holds a postdoctoral fellowship from the Deutsche For- schungsgemeinschaft.
** To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-3200; Fax: 214-648-2971; E-mail: jalban@mednet.swmed.edu.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.C000861200
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ABBREVIATIONS |
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The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PIP, phosphatidylinositolpolyphosphate; DMP, dimethyl pimelimidate; PAO, phenylarsine oxide; bp, base pair; EST, expressed sequence tag; PBS, phosphate-buffered saline.
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