Localization of phosphatidylinositol phosphate kinase IIgamma in kidney to a membrane trafficking compartment within specialized cells of the nephron.

Abstract

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kgamma. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kalpha and PIP4Kbeta, PIP4Kgamma is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kgamma, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kgamma had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kalpha, bacterially expressed recombinant PIP4Kgamma was completely inactive but did have the ability to associate with active PIP4Kalpha in vitro. Overall our data suggest that PIP4Kgamma may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

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doi:10.1152/ajprenal.90310.2008
295:1422-1430, 2008. First published Aug 27, 2008;
Am J Physiol Renal Physiol
Jonathan H. Clarke, Piers C. Emson and Robin F. Irvine

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Localization of phosphatidylinositol phosphate kinase II? in kidney to
a membrane trafficking compartment within specialized cells of the nephron
Jonathan H. Clarke,1Piers C. Emson,2and Robin F. Irvine1
1Department of Pharmacology, University of Cambridge and2Laboratory of Molecular Neuroscience, Babraham Institute,
Cambridge, United Kingdom
Submitted 15 May 2008; accepted in final form 24 August 2008
Clarke JH, Emson PC, Irvine RF. Localization of phosphatidyl-
inositol phosphate kinase II? in kidney to a membrane trafficking
compartment within specialized cells of the nephron. Am J Physiol
Renal Physiol 295: F1422–F1430, 2008. First published August 27,
2008; doi:10.1152/ajprenal.90310.2008.—PIP4Ks (type II phosphati-
dylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate
(PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P
levels. In this study, we investigated the expression, localization, and
associated biological activity of the least-studied PIP4K isoform,
PIP4K?. Quantitative RT-PCR and in situ hybridization revealed that
compared with PIP4K? and PIP4K?, PIP4K? is expressed at excep-
tionally high levels in the kidney, especially the cortex and outer
medulla. A specific antibody was raised to PIP4K?, and immunohis-
tochemistry with this and with antibodies to specific kidney cell
markers showed a restricted expression, primarily distributed in epi-
thelial cells in the thick ascending limb and in the intercalated cells of
the collecting duct. In these cells, PIP4K? had a vesicular appearance,
and transfection of kidney cell lines revealed a partial Golgi localiza-
tion (primarily the matrix of the cis-Golgi) with an additional presence
in an unidentified vesicular compartment. In contrast to PIP4K?,
bacterially expressed recombinant PIP4K? was completely inactive
but did have the ability to associate with active PIP4K? in vitro.
Overall our data suggest that PIP4K? may have a function in the
regulation of vesicular transport in specialized kidney epithelial cells.
phosphoinositide; phosphatidylinositol 5-phosphate 4-kinase; collect-
ing duct; Golgi apparatus
POLYPHOSPHOINOSITIDES are quantitatively minor lipid compo-
nents of cells and are increasingly being recognized as making
diverse and important contributions to many aspects of cell
physiology, for example, in ion channel regulation, membrane
trafficking, cell proliferation, and cytoskeletal rearrangement
(for reviews see Refs. 10, 18, 32, 40, 46). Phosphatidylinositol
5-phosphate (PtdIns5P) is the most recent polyphosphoinosi-
tide to be found, first identified as the primary substrate for the
type II PtdInsP kinases (33). Here these enzymes are referred
to as PIP4Ks (consistent with their substrate specificity as
PtdIns5P 4-kinases), although it should be noted that gene
nomenclature still refers to them as PIP5K2s. As a minor
component of cell polyphosphoinositides, it is suggested that
PtdIns5P either provides a specific lipid pool for a (minor)
localized production of PtdIns(4,5)P2or that it is a signaling
molecule in its own right. Suggested functions include nuclear
responses (9, 13), insulin signaling, and protein kinase regula-
tion (5, 22, 31, 38) and phosphatase activation (39).
PtdIns5P can be synthesized from PtdIns by a PtdIns 5-kinase
(37) or by dephosphorylation from PtdIns(3,5)P2or PtdIns(4,5)P2
(28, 41–43). The main route of PtdIns5P metabolism is via
PIP4Ks, which are represented in vertebrates by three charac-
terized isoforms, ?, ?, and ?. PIP4K? is found predominantly
in the cytosol and can be recruited to the plasma membrane
(15, 45). PIP4K? is localized to the nucleus (3, 7, 36), where
there is evidence that it regulates PtdIns5P levels (21), proba-
bly in concert with type I PtdIns(4,5)P2 4-phosphatase (47),
although it has also been seen to associate with the cytosolic
TNF receptor (6). As cellular PtdIns5P levels are modified in
response to stress (21, 47), to bacterial infection (28), to
receptor-mediated signaling (26, 45), and during cell cycle
progression (8), the ability of the PIP4Ks to localize to specific
compartments, or to shuttle between them, may be intrinsic to
their function.
The third isoform, PIP4K? (19), has not yet been associ-
ated with a cellular function, either related to production of
PtdIns(4,5)P2or to the attenuation of PtdIns5P. In the present
study, we have extensively characterized the tissue distribution
of all of the PIP4K isoforms and found an exceptionally high
level of PIP4K? expression in the kidney. We have further
investigated the localization within this organ of PIP4K?,
using a specific antibody to this isoform, and found that it is
remarkably confined to specific cell populations, where it is
localized to vesicular structures. Transfection experiments with
PIP4K? suggest that these may be derived from the Golgi
apparatus. Our results suggest that PIP4K? may be involved in
vesicle trafficking in specific kidney cells and that this would
imply a specialized function for this enzyme.
MATERIALS AND METHODS
PIP4K cloning. PIP5K2 genes were amplified from a whole
human brain marathon-ready cDNA library (Clontech Laborato-
ries, Mountain View, CA) using gene-specific primers (PIP5K2A
forward: 5?-ATGGCGACCCCCGGCAACCTAGGGTC-3? and reverse:
5?-TTACGTCAAGATGTGGCCAATAAAGTC-3?; PIP5K2B forward:
5?-ATGTCGTCCAACTGCACCAGCACCAC-3? and reverse: 5?-
CTACGTCAGGATGTTGGACATAAAC-3?; and PIP5K2C forward:
5?-ATGGCGTCCTCCTCGGTCCCACCAG-3? and reverse: 5?-
TTAGGCAAAGATGTTGGTAATAAAATC-3?). PCR products were
cloned, via incorporated HindIII and BamHI restriction sites, into
appropriate plasmid vectors. Recombinant protein expressed from
Escherichia coli harboring PIP5K2s in pET-32a (Novagen, Madison,
WI) was purified using TALON metal affinity resin (Clontech) and
cleaved with enterokinase (New England Biolabs, Ipswich, MA).
Endotoxin-free plasmid from bacterial clones harboring PIP5K2s in
pEGFP-C1 (Clontech) was prepared (DNA extraction kit; Qiagen,
Huntsville, AL). All constructs were confirmed by sequencing.
Address for reprint requests and other correspondence: J. H. Clarke, Dept. of
Pharmacology, Univ. of Cambridge, Tennis Court Road, Cambridge CB2 1PD,
UK (e-mail: jhc30@cam.ac.uk).
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.
Am J Physiol Renal Physiol 295: F1422–F1430, 2008.
First published August 27, 2008; doi:10.1152/ajprenal.90310.2008.
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Cell culture and transfection. HeLa, HEK 293, COS-7, and NRK
cells were maintained in DMEM (GIBCO, Paisley, UK) supple-
mented with 10% fetal bovine serum, 50 U/ml penicillin, and 50
?g/ml streptomycin. Cells were transiently transfected with plasmid
constructs for 24 h with TransFectin reagent (Bio-Rad, Hercules, CA),
using the manufacturer’s protocol. Cell lysates for immunoprecipita-
tion were made by suspending cells in cold lysis buffer (PBS with 1%
Triton X-100, 5 mM EDTA, 5 mM EGTA, and 100 ?l/ml Sigma
P8340 protease inhibitor cocktail) and centrifuging at 10,000 g for 10
min at 4°C.
Tissue sample preparation. Protein and mRNA samples were
prepared from adult mouse tissues obtained post mortem and imme-
diately frozen on dry ice after collection. Tissues (50 mg) for mRNA
extraction were pulsed in lysing matrix D tubes (Qbiogene) with 1 ml
Tri-Reagent (Sigma-Aldrich, Poole, UK) on a Hybaid Ribolyser.
Samples were purified (RNeasy kit; Qiagen, and Turbo DNase;
Ambion, Austin, TX), and cDNA libraries were constructed by
RT-PCR (Sprint Powerscript kit; Clontech). Protein lysates were
prepared in RIPA buffer (150 mM sodium chloride, 50 mM Tris pH
7.4, 1 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, and 0.1%
SDS) with 10 mM tetrasodium pyrophosphate, 10 mM sodium fluo-
ride, 17.5 mM ?-glycerophosphate, and 100 ?l/ml Sigma P8340
protease inhibitor cocktail, using 10–15 strokes in a 5 ml dounce
homogenizer. Cell debris was cleared by centrifugation at 2,900 g for
20 min at 4°C, and lysates were clarified by further centrifugation at
29,000 g for 45 min at 4°C. Kidney tissue was dissected under a
binocular microscope to produce cortical and medullary samples.
Tissues for immunochemistry from perfused mice were fixed in 4%
paraformaldehyde, sectioned on a Leica cryostat at ?20°C, mounted
on slides, and stored at ?80°C.
Quantitative PCR. Singleplex quantitative PCR was carried out using
specific primers for each PIP5K2 gene, designed to the 3?-untranslated
region (PIP5K2A forward: 5?-AAGAGTCTGATGCCAAGAACCTGT-3?
and reverse: 5?-TGCAGTGCAACTTAAGGATGGTAA-3?; PIP5K2B
forward: 5?-CATCCTCACAGAAGAACATGGC-3? and reverse: 5?-
CCTGGTCATTCACCGTCTCA-3?; and PIP5K2C forward: 5?-CATCT-
TCCACTGCTAATGTGTCTCC-5? and reverse: 5?-TTGAGTTATG-
GCTCTGACTCCTCTCT-3?). Three cDNA libraries were constructed
for each tissue tested and analyzed in triplicate. PCR amplification
was performed in a 96-well plate using SYBR Green I master mix
(Applied Biosystems, Warrington, UK), following the manufacturer’s
protocol. Reactions were heated (50°C for 2 min, 95°C for 10 min)
and cycled 40 times (95°C for 15 sec, 60°C for 1 min) on an ABI
Prism 7700 sequence detection system (Applied Biosystems). Disso-
ciation curves for each primer set indicated a single product, and
no-template controls were negative after 40 cycles. Reactions using
Fig. 1. PIP5K2C is differentially expressed in adult mouse tissues. A: expres-
sion of PIP5K2A, PIP5K2B, and PIP5K2C determined by quantitative PCR
using the comparative threshold cycle (CT) method. Normalized expression is
presented as relative fold increase above PIP5K2A in testis (n ? 9). B: in situ
hybridization using PIP5K2C-specific probes on thin sections of adult mouse
brain (a), liver (b), and kidney (c; not to scale). Control incubations using an
excess of cold probe indicate nonspecific binding (d–f). C: autoradiographic
emulsion staining of mouse kidney cortex tubules, silver grains (arrows)
indicating PIP5K2C mRNA expression (tu, tubule; gl, glomerulus). Scale
bar ? 20 ?m.
Fig. 2. Specificity of anti-PIP4K? antibody. Detection of PIP4K?, but not the
PIP4K? or PIP4K? isoforms, was established in various procedures. A: equivalent
amounts of purified recombinant protein of each isoform were visualized on
SDS-PAGE (i) and detected by Western blotting (ii). B: HeLa cells overexpressing
green fluorescent protein (GFP)-tagged PIP4K? or PIP4K? were fixed and stained
with anti-PIP4K? antibody. Scale bar ? 10 ?m. C: Surface plasmon resonance
analysis using PIP4K?, PIP4K?, and PIP4K? adsorbed to an nitrilotriacetic acid
biosensor. Anti-PIP4K? antibody was passed over the biosensor at 0 s and
exchanged for wash buffer at 45 s. Data were normalized to 6xHis tag control
protein binding.
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RNA as template were negative, showing that the preparations were
free from genomic DNA contamination. Primer concentrations were
chosen to give threshold cycle (CT) values of 20–25. Validation
experiments showed equivalent relative amplification efficiencies be-
tween each PIP5K2 gene and a primer set for mouse ?-actin (forward:
5?-GACGATATCGCTGCGCTGGT-3? and reverse: 5?-CCACGAT-
GGAGGGGAATA-3?), and ?CT values were analyzed using the
comparative CTmethod (23).
In situ hybridization. Two oligonucleotide probes were designed to the
PIP5K2C sequence, one in the 3?-untranslated region (5?-GACTGGGTG-
GATTGAGTTATGGCTCTGACTCCTCT-3?) and one in the coding se-
quence(5?-ATAGGAGATAAGGAAACGGCCATCACTGCCTTCAG-
3?), which only identified PIP5K2C when BLAT searched against the
European Molecular Biology Laboratory mouse database. Probes
were 3?-tail-labeled with [35S]dATP (NEN, Hounslow, UK), hybrid-
ized with 20-?m mouse tissue sections and autoradiographed for 5
wk, as described previously (12). Slides of interest were dipped in
autoradiographic emulsion and after development (12 wk) were coun-
terstained with hematoxylin and eosin, and images were captured
using an Axioskop II light microscope.
Antibody analysis and Western blotting. A peptide (amino acids
333–352), unique to the variable region of the mouse PIP4K? se-
quence, was used to raise a custom polyclonal antibody (NeoMPS,
San Diego, CA), which was subsequently purified from rabbit serum
by affinity matrix chromatography. Surface plasmon resonance anal-
ysis was carried out on a Biacore 3000 instrument (GE Healthcare
Life Sciences, Bucks, UK) using two optical biosensor chips: car-
boxymethylated dextran preimmobilized with nitrilotriacetic acid
(NTA) or streptavidin (SA). Purified PIP4K recombinant protein and
control 6xHis tag protein were adsorbed to the NTA chip at concen-
trations of 1–9 ng/mm2. Anti-PIP4K? antibody was passed over the
chip at 10 ?g/ml to detect specific interaction, and data were normal-
ized to the nonspecific control. Biotinylated anti-PIP4K? and control
antibodies were bound to the SA chip, to which PIP4K? was then
applied at saturating levels, to show specific antibody-binding inter-
action.
Protein samples (50 ?g) were resolved on 10% polyacrylamide
gels, and Western blots were carried out as described previously (16),
using anti-PIP4K? at 0.5 ?g/ml. Anti-PIP4K? was neutralized by
saturating with excess of antigenic peptide for 30 min. Other antibod-
ies used in this study were diluted to the manufacturers recommen-
dations: goat polyclonal antibodies to aquaporin 1 (AQP1), aquaporin
2 (AQP2) and Tamm-Horsfall protein, and polyclonal rabbit antibod-
ies to lgp110 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit
polyclonal antibodies to TGN38 were a gift from G. Banting; mouse
Mabs to EEA1, BiP, and GM130, and rabbit polyclonal antibodies to
p115 (BD Transduction Laboratories, Oxford, UK); rabbit polyclonal
antibodies to calreticulin (Calbiochem, La Jolla, CA); mouse Mabs to
tubulin (Sigma-Aldrich); and goat polyclonal antibodies to golgin160
and rabbit polyclonal antibodies to catalase, p58K, mannose 6-phos-
phate receptor, and mannosidase II (Abcam, Cambridge, UK). Alexa
Fluor 568 phalloidin and TO-PRO-3 iodide were used to directly stain
actin and DNA (Molecular Probes, Paisley, UK). Horseradish perox-
idase-conjugated secondary antibodies and SuperSignal West Dura
substrate were used for Western blotting (Pierce Protein Research
Products, Rockford, IL), and Alexa dye-conjugated secondary anti-
bodies were used for fluorescence microscopy (Molecular Probes).
Immunoprecipitation with anti-PIP4K? rat Mabs (16) and protein
G-Sepharose (GE Healthcare) was carried out for 16 h at 4°C. Beads
were washed in cold PBS and used directly for Western blotting or
kinase assay.
Immunocytochemistry and immunohistochemistry. Mammalian
cells expressing green fluorescent protein-tagged constructs were
fixed in 4% paraformaldehyde for 30 min on ice. Cells were perme-
abilized with 0.1% Triton X-100 in PBS for 10 min and blocked with
4% fish skin gelatin (Sigma-Aldrich) in PBS for 30 min and then
incubated for 60 min with primary and secondary antibodies (2%
gelatin in PBS) with PBS washes between and after incubation. Slides
were mounted using ProLong Gold antifade reagent (Molecular
Probes), and images were taken on a Leica TCS SP5 laser scanning
confocal microscope running LAS AF software (Leica Microsystems,
Fig. 3. PIP4K? is expressed in adult mouse
tissues. Lysates were probed with PIP4K?-
specific antibody (i) and with neutralized an-
tibody after saturation with immunizing pep-
tide (ii). Whole tissue lysates (A) and protein
preparations from different kidney regions (B)
were probed with the same antibody. Equiva-
lent protein loading was based on total lysate
concentration. Purified recombinant PIP4K?
was used to identify endogenous PIP4K? at
the correct molecular mass (arrow).
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Wetzlar, Germany). Golgi dissociation was achieved by treatment
with Brefeldin A (10 ?g/ml in DMEM) for 30 min at 37°C.
Sagittal and horizontal tissue sections for fluorescent labeling were
pretreated to reduce autofluorescence (20 min in 1 mg/ml sodium
borohydride), blocked for 2 h (1% fish skin gelatin, 0.3% Triton
X-100 in PBS), exposed to primary antibody (24 h at 4°C), and
fluorophore-labeled secondary antibody (4 h) before being mounted as
described previously. Confocal images were spectrally separated by
LAS AF software using reference spectra obtained from unstained
tissue and cells overexpressing green fluorescent protein. Tissue
sections for chemical staining were pretreated for 20 min (20%
methanol and 5% hydrogen peroxide), blocked, and incubated with
primary antibody as described, and then they were incubated with
biotinylated anti-rabbit IgG antibodies before visualization using
Vectastain ABC and DAB substrate kits (Vector Laboratories, Bur-
lingame, CA). Slides were treated with Histoclear and mounted with
DPX reagent (Sigma-Aldrich) before light microscopy.
Lipid kinase assay. PIP4K assays were carried out as described
previously (16) with slight adaptation. Substrate lipid (6 ?M
PtdIns5P) was dried under vacuum, and micelles were made by
sonication in kinase buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 80
mM KCl, and 2 mM EGTA). Recombinant PIP4K was added to the
reaction mixture with 10?Ci [?-32P]ATP for 90 min at 30°C. Lipids
were extracted and separated by silica-gel thin layer chromatography
(16), and results were obtained by autoradiography.
RESULTS
PIP5K2 isoforms have different expression profiles. Expres-
sion studies using RT-PCR have previously suggested that
levels of PIP5K2 transcription are high in different tissues (1,
19). Using a series of isoform-specific PCR primer pairs for
PIP5K2A, PIP5K2B, and PIP5K2C, we quantitatively assessed
the expressed levels of these transcripts in mRNA isolated
from a range of mouse tissues (Fig. 1). Automated quantitative
PCR of synthesized cDNA libraries was completed using three
biological replicates for each tissue. Pooled data were analyzed
by the Livak method (23), normalizing PIP5K2 to the house-
keeping gene ?-actin, and values were calculated as the rela-
tive fold increase above the lowest observed tissue expression
(Fig. 1A). All three isoforms were expressed at higher levels in
the brain, with PIP5K2A expression increased in spleen and
PIP5K2B in muscle. PIP5K2C was especially high in kidney,
as originally reported by Itoh et al. (19). Tissue analysis by in
situ hybridization with a range of PIP5K2C probes confirmed
that transcription of this isoform was upregulated in brain and
kidney, compared with a control tissue, and that the expression
was localized to discrete regions of these organs (Fig. 1B).
Silver grain labeling of PIP5K2C mRNA in the kidney sug-
gested that expression was confined to segments of the nephron
within the cortical and medullary regions and was not present
in the kidney vasculature (Fig. 1C).
Endogenous PIP4K? is differentially expressed in mouse
tissues. A polyclonal peptide antibody to the variable region of
PIP4K? was raised and purified. Due to the similarity of the
sequence of the PIP4K isoforms at the protein level, the
specificity of the antibody for PIP4K? was determined (Fig. 2).
The anti-PIP4K? antibody has no cross-reactivity with PIP4K?
or PIP4K? by Western blot (Fig. 2A) or by immunocytochem-
istry (Fig. 2B). Purified recombinant PIP4Ks were bound to an
NTA sensor chip by 6xHis tag, and surface plasmon resonance
analysis of the binding of anti-PIP4K? antibody indicated
specificity for PIP4K? compared with PIP4K? and PIP4K?
(Fig. 2C). This was confirmed by binding biotinylated anti-
PIP4K? antibody to an SA sensor chip and detecting specific
binding to PIP4K? (data not shown). Direct Western blotting
of a bank of tissue lysates confirmed the presence of significant
endogenous levels of PIP4K? in brain, kidney, ovary, and
testis (Fig. 3A, i). Other tissues had little or no detectable levels
of PIP4K?, assuming representative expression based on
equivalent loading of total lysate protein. Endogenous PIP4K?
was detected as two bands of very similar molecular mass of
?47 kDa, the larger band presumably representing the phos-
phorylated form of mature PIP4K? (19). Crude kidney frac-
tionation and subsequent Western blotting of protein lysates
indicated that PIP4K? was present throughout this organ, but
comparatively higher levels were seen in the medulla (Fig. 3B,
i). Nonspecific bands were visualized using neutralized anti-
body as control, and lower molecular mass immunoreactive
bands were presumed to be products of proteolytic cleavage
(Fig. 3, ii). Interestingly, PIP4K? in heart ran predominantly as
a 40-kDa band, suggesting that processing of this isoform may
be occurring (Fig. 3A).
PIP4K? expression in kidney is localized to specific cells.
Identification of PIP4K? at a cellular level was shown to be
specific by the use of peptide-saturated primary antibody con-
trols, which demonstrated an absence of signal due to nonspe-
cific binding of primary or secondary antibodies in tissue
immunohistochemistry. Spectral separation was also utilized to
Fig. 4. PIP4K? expression in the adult mouse kidney. Tissue immunohisto-
chemistry using antibody specific to PIP4K? identified the expression of
endogenous protein in kidney sections. A: positive PIP4K? signal detected by
reporter enzyme (i: scale bar ? 10 ?m) or epifluorescence (ii; scale bar ? 40
?m). Negative control reactions are also shown (primary antibody preincu-
bated with antigenic peptide). B: high resolution imaging indicated that
PIP4K? (green) was differentially distributed in the representative kidney
regions indicated after removal of autofluorescence. Nuclei (red) are counter-
stained as contrast. Scale bar ? 40 ?m.
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remove significant autofluorescence from the kidney tissue in
immunofluorescence experiments (Fig. 4A). Analysis of whole
kidney sections confirmed that PIP4K? was expressed through-
out this organ (data not shown). Detailed examination of
representative regions of the kidney at high resolution indi-
cated that the positive signal was seen to be concentrated in the
outer medulla (Fig. 4B), confirming the results obtained for
transcript expression (Fig. 1B), with much less signal being
observed in the cortical labyrinth. This signal was restricted to
whole regions of specific tubules, which could be visualized
using sequential confocal imaging at 2-?m intervals through-
out a 20-?m cryosectioned tissue slice (data not shown). The
PIP4K?-expressing tubules were present in both the cortical
medullary rays and the inner and outer stripe of the outer
medulla but were absent from the tubules of the inner medulla
(Fig. 4B). However, it was possible to observe single PIP4K?-
positive cells within tubules in the cortex and inner medulla
(Fig. 4B).
Costaining sections with selective markers for different
nephron regions allowed accurate determination of the tubules
and cells that expressed PIP4K? (Fig. 5). The absence of
PIP4K? in cortical tubules with a significant brush-border
lumen lining (Fig. 5, A and B) or coincident with the water
channel AQP1 (Fig. 5C), a marker for the proximal convoluted
tubule and thin descending limb of the loop of Henle (29),
suggested that expression was restricted to the distal part of the
nephron. PIP4K?-positive tubules also expressed Tamm-Hors-
fall protein in the outer medulla (Fig. 5, D–F), which suggested
Fig. 5. Endogenous PIP4K? is differentially
expressed in the adult mouse nephron. Actin
(red) staining of cortical regions (A) identified
luminal brush borders (arrow) in proximal tu-
bules (d; distal tubule, p; proximal tubule), and
localization of PIP4K? (green) in distal tu-
bules (B). PIP4K? was not coincident with
AQP1 (red) in the cortex (C) but was present in
Tamm-Horsfall protein (THP; red) positive
tubules in the outer medulla (D–F). PIP4K?
was also seen in THP-negative tubules in this
region (F; arrows). Double staining for
PIP4K? (green) and AQP2 (red) in different
kidney regions (G–L) revealed tubules posi-
tive for PIP4K? but negative for AQP2 in
cortex and outer medulla (G, H; arrows).
Isolated cells in AQP2-positive tubules also
expressed PIP4K? (I–L; arrows). A, B, D–F,
and K: images from horizontal sections. C,
G–J, and L: images from sagittal sections.
Scale bars ? 20 ?m.
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that PIP4K? is localized to cells in the thick ascending limb
(TAL) of the loop of Henle (2). The presence of PIP4K?-
positive tubules in the cortex and outer medulla (Fig. 5, G and
H), but not in the inner medulla (Fig. 5I), and distinct from
tubules expressing the collecting duct marker AQP2 (24),
confirmed that PIP4K? was mainly expressed in TAL. Inter-
estingly, isolated PIP4K?-positive cells were also localized to
the collecting duct but were spatially differentiated from
AQP2, which selectively stained principal cells (24). This
suggested that PIP4K? was also localized to intercalated cells
in cortical and medullary collecting ducts (Fig. 5, J–L).
PIP4K? has a distinct subcellular compartmentalization.
Tubule sections of medullary TAL showed a distinct concen-
tration of PIP4K? around the lumen (Fig. 6, A–D). Analysis of
a pool of cross-sectioned tubules with a diameter of 23–25 ?m
gave an average fluorescence profile that indicated a sixfold
increase of signal within 3 ?m of the apical membrane of
tubule cells, compared with the basolateral membrane (Fig.
6E). Higher magnification of these cells suggested that PIP4K?
might be present in a vesicular compartment (Fig. 6, C and D).
To further investigate this compartmentalization, PIP4K? ex-
pression was studied in kidney-derived cell lines, but because
endogenous levels of enzyme were not visible by immunocy-
tochemistry in these cells, overexpressed protein levels were
required for colocalization experiments. Cells expressing green
fluorescent protein-tagged PIP4K? were stained with antibod-
ies against different cellular markers (Fig. 7). PIP4K? was not
seen to associate with markers for defined vesicular compart-
ments such as peroxisomes (catalase), endosomes (EEA1,
mannose 6-phosphate receptor), or lysosomes (lgp110) or with
markers for structural components such as tubulin or actin
(data not shown). In our experiments, PIP4K? was also not
seen to associate with endoplasmic reticulum markers [calre-
ticulin (Fig. 7A) and BiP] in contrast with the suggested
endoplasmic reticulum localization seen by Itoh et al. (19).
PIP4K? did, however, show a partial colocalization with a
number of Golgi apparatus markers (golgin160, TGN38, p115,
and p58K), most notably the cis-Golgi marker GM130 (Fig.
7B). Golgi dispersal by treatment of cells with Brefeldin A still
retained partial colocalization of GM130 with the PIP4K?
signal (Fig. 7C) but not with mannosidase II, a Golgi lumen
protein, suggesting that the association was with the matrix
component of this organelle (data not shown).
Lipid kinase activity of PIP4K?. PIP4K, overexpressed in E.
coli cells and purified by metal-affinity resin chromatography,
was used as a source of enzyme for in vitro kinase assays.
PIP4K activity was only observed using the PIP4K? isoform as
the recombinant PIP4K? was inactive (Fig. 8A). Using immu-
noprecipitation experiments with recombinant protein, we
were able to show that PIP4K? can associate with PIP4K?
strongly enough to be selectively purified with a PIP4K?-
specific antibody in vitro (Fig. 8B).
DICUSSION
In this study, we investigated the expression, localization
and associated biological activity of PIP4K?. We discovered a
unique and restricted localization for this PIP4K in kidney
tissue and suggest that this has implications for the physiolog-
ical function of this isoform.
We have shown that the comparative mRNA expression
levels of all three isoforms are significant in brain, where they
have a different spatial distribution (1). PIP4K? is the most
active of the three isoforms in vitro but has a comparably low
mRNA expression in most of the tissues that we tested and is
the most abundant isoform in the spleen, probably reflecting its
role in hematocytes (17, 26). The PIP4K? isoform mRNA is
highly expressed in heart and skeletal muscle cells, consistent
with initial observations (6) and providing a link to insulin
resistance (22). We have confirmed the original observation
that PIP4K? is highly expressed in kidney (19) and also
observe high mRNA expression levels in brain, heart, and testis
compared with other tissues. Our detection of endogenous
PIP4K? protein in tissues is consistent with these transcription
levels, also suggesting that PIP4K? is abundant in the ovary
and may be processed in heart tissue. The distribution of the
PIP4Ks across a range of different tissues, and the observed
differences in subcellular localization and intrinsic activity (36,
45), would suggest that specialized functions could be attrib-
Fig. 6. Distribution of PIP4K? in kidney thick ascending limb (TAL) cells in
situ. Endogenous PIP4K? was detected in thin sections of the outer medulla
region of adult mouse kidney. Saggital kidney section shows longitudinal TAL
tubule sections (A, C), and horizontal kidney section shows TAL tubule
cross-sections (B and D). Scale bars ? 10 ?m. Statistical analysis of PIP4K?
distribution across TAL tubule cross-sections (such as in B), by fluorescence
intensity, indicated peaks in the luminal region (E; n ? 25).
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uted to each and hence to the role of PtdIns(5)P in these
locations.
Peptide analysis of the sequence of PIP4K? predicts that this
protein would not be targeted to the endoplasmic reticulum or
plasma membrane due to the absence of a recognized signal
peptide, which is consistent with our observation that PIP4K?,
when overexpressed in cells, is partially colocalized to the
structural component of the Golgi apparatus. Roles for
PtdIns3P and PtdIns4P in membrane trafficking are established
(10, 32), but the recent study (25) of a Golgi-localized phos-
pholipid-inositol phosphatase with a substrate preference for
PtdIns5P presents the intriguing possibility that this phospho-
inositide is also present in cellular vesicles. PIP4K? also has a
role in actin remodeling during endocytic transport (30), and
PtdIns5P levels have been associated with this and with vesicle
translocation to the plasma membrane (38). PIP4K? could be
recruited to the external surface of a specific microsomal
compartment to modify the PtdIns5P signal or to synthesize
PtdIns(4,5)P2.
The restricted expression of PIP4K? within the kidney may
be significant within the context of the specialized function of
different regions of the nephron. Our experiments indicate that
PIP4K? is present in cells constituting the TAL and is also
restricted to intercalated cells in the collecting duct and appears
Fig. 7. Overexpressed PIP4K? localizes to a specific
cellular compartment. Transiently transfected cells, ex-
pressing GFP-tagged PIP4K? (green), were costained
with antibodies to specific markers for different cellular
compartments (red). No specific colocalization was ob-
served with the endoplasmic reticulum marker calreti-
culin (A). Partial colocalization was observed with the
cis-Golgi marker GM130 (B),and this juxtaposed label-
ing was maintained during Golgi dissociation by treat-
ment with Brefeldin A (C). Scale bars ? 10 ?m.
Fig. 8. Activity and binding characteristics of
recombinant PIP4Ks. PIP4K? and PIP4K?
recombinant protein was purified from a bac-
terial source. A: PIP4K assay using equivalent
amounts of recombinant protein from each
PIP4K, showing generation of PtdIns(4,5)P2.
B: immunoprecipitation (IP) of recombinant
PIP4K protein using PIP4K?-specific anti-
body. IPs are from equivalent amounts of
PIP4K?, PIP4K?, or a mixture of the 2 iso-
forms, in buffer. Control IP contains no re-
combinant protein. Western blots (WB) were
probed with either anti-PIP4K? (i) or anti-
PIP4K? (ii). Recombinant protein markers
(rec) indicate PIP4K-positive bands (65 kDa).
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to have a similar subcellular localization in these cell types as
that recently observed for members of the Arf GTPase family
(11), which have known roles in membrane trafficking (34).
These regions are predominantly concerned with homeostasis
by active ion transport and pH regulation and contain a large
number of channels and transporters specific to these tasks (for
reviews see Refs. 20, 27). Phosphoinositide regulation of both
the trafficking to the plasma membrane and the activity of
various collecting duct-localized channels has been reported
(14, 44), but a specific role for PIP4K? in this process has yet
to be established.
PIP4K? has been shown to have PtdIns5P 4-kinase activity
when immunoprecipitated from mammalian cells (19), and the
recombinant protein, purified from E. coli, is inactive, suggest-
ing that a eukaryotic modification to PIP4K? is required for
kinase activation. However, due to the ability of the PIP4Ks to
dimerize (4, 16, 35) in vivo, we also cannot rule out the
possibility that the observed activity associated to PIP4K? is
attributable to PIP4K heterodimers. This raises the possibility
that PIP4K? is able to recruit PtdIns5P 4-kinase activity (in the
form of PIP4K?) to specific compartments, based on the
potential ability to act as a scaffolding protein.
The roles of PtdIns5P, PtdIns(4,5)P2, and hence the PIP4Ks,
in kidney function are unknown. We have shown that PIP4K?
is the predominant PIP4K in the kidney, and we suggest that its
role may be related to its specific localization in this organ.
ACKNOWLEDGMENTS
We thank Dr. Patrick Lynch for practical advice, Søren Nielsen for helpful
suggestions, and Dr. Mihriban Tuna for technical expertise and analysis of
biacore experiments.
GRANTS
This study was supported by a Programme Grant (WT063581) from the
Wellcome Trust and the Biotechnology and Biological Sciences Research
Council.
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