Genomic tagging reveals a random association of endogenous PtdIns5P 4-kinases IIalpha and IIbeta and a partial nuclear localization of the IIalpha isoform.

Abstract

PtdIns5P 4-kinases IIalpha and IIbeta are cytosolic and nuclear respectively when transfected into cells, including DT40 cells [Richardson, Wang, Clarke, Patel and Irvine (2007) Cell. Signalling 19, 1309-1314]. In the present study we have genomically tagged both type II PtdIns5P 4-kinase isoforms in DT40 cells. Immunoprecipitation of either isoform from tagged cells, followed by MS, revealed that they are associated directly with each other, probably by heterodimerization. We quantified the cellular levels of the type II PtdIns5P 4-kinase mRNAs by real-time quantitative PCR and the absolute amount of each isoform in immunoprecipitates by MS using selective reaction monitoring with 14N,13C-labelled internal standard peptides. The results suggest that the dimerization is complete and random, governed solely by the relative concentrations of the two isoforms. Whereas PtdIns5P 4-kinase IIbeta is >95% nuclear, as expected, the distribution of PtdIns4P 4-kinase IIalpha is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIalpha was 2000-fold more active as a PtdIns5P 4-kinase than the IIbeta isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIbeta may be to target the more active IIalpha isoform into the nucleus.

Full text

Page 1

Biochem. J. (2010) 430, 215–221 (Printed in Great Britain)doi:10.1042/BJ20100340
215
Genomic tagging reveals a random association of endogenous PtdIns5P
4-kinases IIα and IIβ and a partial nuclear localization of the IIα isoform
Minchuan WANG*1, Nicholas J. BOND†1, Andrew J. LETCHER*, Jonathan P. RICHARDSON*2, Kathryn S. LILLEY†,
Robin F. IRVINE*3and Jonathan H. CLARKE*3
*Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K., and †Cambridge Centre for Proteomics, Department of Biochemistry, Tennis Court Road, Cambridge
CB2 1GA, U.K.
PtdIns5P 4-kinases IIα and IIβ are cytosolic and nuclear
respectively when transfected into cells, including DT40 cells
[Richardson, Wang, Clarke, Patel and Irvine (2007) Cell.
Signalling 19, 1309–1314]. In the present study we have genomi-
cally tagged both type II PtdIns5P 4-kinase isoforms in DT40
cells. Immunoprecipitation of either isoform from tagged cells,
followed by MS, revealed that they are associated directly with
each other, probably by heterodimerization. We quantified the
cellular levels of the type II PtdIns5P 4-kinase mRNAs by
real-time quantitative PCR and the absolute amount of each
isoform in immunoprecipitates by MS using selective reaction
monitoring with14N,13C-labelled internal standard peptides. The
results suggest that the dimerization is complete and random,
governedsolelybytherelativeconcentrationsofthetwoisoforms.
Whereas PtdIns5P 4-kinase IIβ is >95% nuclear, as expected,
the distribution of PtdIns4P 4-kinase IIα is 60% cytoplasmic
(all bound to membranes) and 40% nuclear. In vitro, PtdIns5P
4-kinase IIα was 2000-fold more active as a PtdIns5P 4-kinase
than the IIβ isoform. Overall the results suggest a function of
PtdIns5P4-kinaseIIβ maybetotargetthemoreactiveIIα isoform
into the nucleus.
Key words: DT40 cell, genomic tagging, nuclear signalling,
phosphatidylinositol, phosphatidylinositol 5-phosphate kinase.
INTRODUCTION
Among the many known intracellular roles for inositol lipids,
the best understood are those in the cytoplasm. However, there
are also a number of less well studied functions for these lipids
withinthenucleus(forreviews,see[1–4]).Thereisclearevidence
in the nucleus for the minor route of PtdIns(4,5)P2 synthesis
[5], the 4-phosphorylation of PtdIns5P by type II PtdIns5P
4-kinases. The probable major route of PtdIns5P synthesis in
animals is by 4-dephosphorylation of PtdIns(4,5)P2[6,7], with
the re-phosphorylation of PtdIns5P by type II PtdIns5P 4-kinases
serving to remove it. Thus the PtdIns(4,5)P24-phosphatases and
the type II PtdIns5P 4-kinases can form a reversible pathway
regulating the levels of PtdIns5P (see Lecompte et al. [8] for
evolutionary arguments for this pathway). PtdIns5P is present in
the nucleus [9] and evidence has been presented that it regulates
the activity of the transcription factor ING-2 (inhibitor of growth
family 2) [10]. Nuclear levels of PtdIns5P increase during the
cell cycle [9] or when cells are stressed [11], and it is generally
accepted that a key regulator of PtdIns5P levels in the nucleus is
PtdIns5P4-kinaseIIβ whoseactivityinstressedcellsisdecreased
as a result of it being phosphorylated, and thus inhibited, by
the p38 MAPK (mitogen-activated protein kinase) [11]. This,
together with an increase in nuclear PtdIns(4,5)P24-phosphatase
[7], causes the increase in nuclear PtdIns5P. Nuclear PtdIns5P
4-kinase IIβ has also been reported to associate with and regulate
the activity of Cul3 (cullin 3)–SPOP (speckle-type POZ protein)
ubiquitin ligase [12].
The nuclear localization of PtdIns5P 4-kinase IIβ is mediated
by a novel nuclear localization sequence [13] consisting of a
17-amino-acidacidicα-helix,numberedα-helix7inthePtdIns5P
4-kinaseIIβ structuredescribedbyRaoetal.[14].Wehaveshown
previouslythatanydisruptionofthisα-helixinPtdIns5P4-kinase
IIβ leads to a cytosolic localization [13,15]. On the other hand,
PtdIns5P 4-kinase IIα, which is cytosolic when transfected, lacks
this α-helix 7 altogether, and merely introducing that helix into
PtdIns5P 4-kinase IIα is sufficient to target the enzyme to the
nucleus[13].However,thereisevidence,thoughnotyetdefinitive,
that some endogenous PtdIns5P 4-kinase IIα may be nuclear
[13,16,17].
We established previously that endogenous PtdIns5P 4-kinase
IIβ is indeed mostly nuclear by genomic tagging of the enzymein
DT40cells[18].WealsoshowedthatwhenPtdIns5P4-kinaseIIα
isacutelytransfectedintoDT40cellsitis,asinothercells,cytoso-
lic [18]. In the present study we have used the specificity of gen-
omic tagging together with quantitative MS with internal peptide
standardstoshowthatendogenousPtdIns5P4-kinasesIIα andIIβ
associateinvivo,probablybyrandomheterodimerization,andthat
approximately 40% of the endogenous IIα isoform is nuclear.
MATERIALS AND METHODS
TheculturingofDT40cells,extractionofgenomicDNA,Western
blotting, protein purification and immunoprecipitation of tagged
proteinswereallperformedasdescribedinRichardsonetal.[18].
Tagging PtdIns5P 4-kinase IIα
This was accomplished using the same strategy as for the
tagging of PtdIns5P 4-kinase IIβ, described previously [18,19].
Abbreviations used: AQUA, absolute quantification; Cul3, cullin 3; DAPI, 4?,6-diamidino-2-phenylindole; qRT-PCR, quantitative real-time PCR; SPOP,
speckle-type POZ protein; SRM, selective reaction monitoring; UPLC, ultra-performance liquid chromatography; UTR, untranslated region; WT, wild-type.
1These authors contributed equally to this work.
2Present address: Ion Channels and Cell Signalling Centre, Division of Basic Sciences, St George’s University of London, London SW17 0RE, U.K.
3Correspondence may be addressed to either of these authors (email rfi20@cam.ac.uk or jhc30@cam.ac.uk).
c ?The Authors Journal compilation c ?2010 Biochemical Society
www.biochemj.org
Biochemical Journal

Page 2

216
M. Wang and others
Primers were used for the PCR amplification of genomic DNA
upstream and downstream of the stop codon of the gene encoding
PtdIns5P 4-kinase IIα, PIP5K2A (5?construct, forward pri-
mer 5?-GCATGGTACCTCAGAGTCATGTTAGCTGTG-3?and
reverse primer 5?-GCATTCTAGACGTCAAGATGTTGGCAAT-
AAAG-3?; 3?construct; forward primer 5?-CATGGATCCTCC-
CCTCATGTCACACCGGACAG-3?and reverse primer 5?-GC-
ATGCGGCCGCGTCTGTCAGTGCTACAGAAGTG-3?).
subsequent insertion of the puromycin selection cassette, plasmid
linearization, transfection and selection of DT40 lines that had
incorporated the insert, and PCR confirmation of the correct
incorporation, were all as described in Richardson et al. [18].
The
Fractionation of DT40 cells
DT40 cells were swollen and disrupted by syringing as
described previously [18], but the subsequent fractionation was
modified as follows. Samples were layered on to 3 ml of 1.8 M
sucrose solution and spun at 35000 rev./min for 1 h on a
SW55Ti rotor using a Beckman Optima L-100 XP ultracentri-
fuge (Supplementary Figure S1 at http://www.BiochemJ.org/
bj/430/bj4300215add.htm). After centrifugation, two separate
volumes were taken from above the buoyant membranes (fraction
C, the top 0.4 ml, and fraction P, the remaining volume). The
cytoplasmic membranes (fraction M) were added to 0.5 ml of
lysis buffer P1 (0.1% Triton X-100 and 0.5% protease inhibitor
cocktail in PBS buffer). The nuclei were collected, and their
purity determined by brightfield microscopic inspection after
staining in Trypan Blue solution or by fluorescent microscopic
inspection using a wheat germ agglutinin (conjugated to Alexa
Fluor®555) plasma membrane stain in combination with DAPI
(4?,6-diamidino-2-phenylindole) nuclear staining. The nuclei
were lysed in 0.8 ml of lysis buffer (50 mM sodium phosphate
buffer, pH 7.4, containing 300 mM NaCl, 0.1% Triton X-100
and 0.5% protease inhibitor cocktail) on ice for 20 min, and
the supernatant (fraction N) was collected after centrifugation.
Identical proportions of each fraction were taken for SDS/PAGE
and immunoblotting with anti-histone or anti-α-actin antibodies,
and the remainder was used for affinity purification of the FLAG–
(His6)2-taggedproteinsusingTALONbeads,followedbyWestern
blotting with anti-FLAG antibodies [18].
Pull-down using anti-FLAG antibody
The anti-FLAG M2 monoclonal antibody (Stratagene) was
coupledtoProteinG–Sepharose
pimelimidate dihyrochloride, and these were incubated for 2 h at
4◦C with 400 μl of DT40 cell lysate [18]. Beads were pelleted by
centrifugationandwashedtwiceinPBSbuffer,andboundprotein
eluted with 3× FLAG peptide buffer [100 μg/ml 3× FLAG
peptide (Sigma–Aldrich) and 0.5% protease inhibitor cocktail
in TBS (Tris-buffered saline)].
beadsusing dimethyl
Lipid kinase assays
PtdIns5P4-kinaseassayswerecarriedoutasdescribedpreviously
[20] with slight adaptations. Substrate (300 pmol of PtdIns5P)
lipid was dried under vacuum and micelles made by sonication
in kinase buffer (50 mM Tris/HCl, pH 7.4, containing 10 mM
MgCl2, 80 mM KCl and 2 mM EGTA). Recombinant human
type II PtdIns5P 4-kinase isoforms, cloned and purified as
described in [21], were added to the reaction mixture with 10
μCi of [γ-32P]ATP for 90 min at 30◦C. Lipids were extracted and
separatedbysilica-gelTLC[20]anddetectedbyautoradiography.
Radioactivity was quantified by scintillation counting of labelled
PtdIns(4,5)P2.
Table 1 Proteotypic peptides selected for MS internal standards
Two peptides were selected that were unique to each chicken PtdIns5P 4-kinase II isoform [IIα;
UniProt accession number Q5F356, IIβ; predicted NCBI (National Center for Biotechnology
Information) accession number XP_418120.2]. Values in square brackets are the molecular
mass (Da) of the unlabelled and labelled peptides respectively; values in parentheses are the
mass/charge ratio of the doubly charged precursor ions (Th). *, has a stable isotope charge.
ProteinAQUA peptide
PtdIns5P 4-kinase IIα
PtdIns5P 4-kinase IIα
PtdIns5P 4-kinase IIβ
PtdIns5P 4-kinase IIβ
SAPLANDSQAR* [1128.6/1138.6*] (565/570*)
FGIDDQDFQNSLTR* [1654.8/1664.8*] (828/833*)
SAPVNSDSQGR* [1116.5/1126.5*] (559/564*)
FGIDDQDYQNSVTR* [1656.7/1666.7*] (829/834*)
qRT-PCR (quantitative real-time PCR)
Total mRNA was extracted from approx. 3×107DT40 cells
from three different cultures of each clone tested, with the SV
Total RNA Isolation System (Promega) and cDNA libraries
were constructed from each preparation with the Sprint RT
(reverse transcriptase) complete kit (Clontech). Primers for
singleplex qRT-PCR were designed using Primer3 [22]. Owing
to the high sequence similarity between PIP5K2A and PIP5K2B,
primer pairs were designed spanning exon 10 and the 3?-UTR
(untranslated region) (PIP5K2A, forward 5?-TCAAAGCGCT-
TCTTGGACTT-3?and reverse 5?-CTACCCTCGTGGTCACTG-
CT-3?; PIP5K2B forward 5?-GCAGTACTCCAAACGCTTCA-3?
and reverse 5?-GGCAAGTAGCCCCTCTTCTC-3?). Primer pairs
specific to the tagged genes were generated from exon 10
and the inserted tag sequence (PIP5K2A-tag forward 5?-CG-
CAGAAATTTCAACCGTTA-3?;
AGAGATCTCCACCGTGAACC-3?; common reverse 5?-CTT-
GTCATCGTCGTCCTTGT-3?). A primer set was also designed
against the chicken β-actin housekeeping gene, spanning exons
1 and 2 (forward 5?-TGGCAATGAGAGGTTCAGGT-3?and
reverse 5?-CGGATATCCACATCACACTTCA-3?). All primers
were obtained from Sigma–Aldrich and produced single PCR
products in conventional reactions. PCR amplification was
performed over 40 cycles (95◦C for 15 s, 60◦C for 30 s) in
a 96-well plate using SYBR Green I master mix (Applied
Biosystems, Warrington, U.K.) and Ct(threshold cycle) values
were collected using a CFX96 qRT-PCR detection system
(Bio-Rad Laboratories). Dissociation curves for each primer
set indicated a single product and no-template controls were
negative after 40 cycles. Control reactions using RNA from
each extraction as a template were negative, showing that the
preparationswerefreefromgenomicDNAcontamination.Primer
and template concentrations were optimized to give Ct values
of 20–27. Validation experiments showed equivalent relative
amplification efficiencies between products from PIP5K2 genes
andthechickenβ-actinreferencegene(SupplementaryFigureS2
at http://www.BiochemJ.org/bj/430/bj4300215add.htm). Nor-
malized expression ratios were calculated by the 2−??Ctmethod
[23].
PIP5K2B-tag
forward5?-
MS
Absolute protein quantification of PtdIns5P 4-kinase II isoforms
was achieved by isotope dilution and SRM (selective reaction
monitoring). Briefly, proteotypic peptides unique to each isoform
were selected as internal standards giving consideration for mass
spectra, size and residue composition (see below and Table 1).
Internal standards were synthesized (Anaspec) incorporating a
single13C6,15N4-labelled arginine residue at the C-terminus and
each was independently quantified by amino acid analysis [24].
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 3

Heterodimerization of type II PtdIns5P 4-kinases
217
For quantifying total protein in unfractionated cells, 7 mg of
lysate digest, with 30 pmol of internal standards was separated by
strongcationexchange(0–150 mMKClover90 minona200 mm
NEST column with a 2.1 mm internal diameter). Subsequent
fractions were each dried, resuspended in 0.1% formic acid and
analysed by UPLC (ultra-performance liquid chromatography)–
SRM. To determine the ratio of the PtdIns5P 4-kinase IIα and
IIβ isoforms in pull-downs, protein precipitates were digested
by incubation overnight with 40 ng of trypsin, with 100 or 200
fmol of internal standards, and analysed by nano-UPLC–SRM as
appropriate.
Nano-flow UPLC (300 nl/min) was used for separation of
pull-down digests and normal-flow UPLC (400 μl/min) used for
the second dimension of separation for lysate digests. In both
cases peptides were eluted from preformed 1.7-mm-diameter
BEH packed columns [Waters; nanoUPLC (150 mm column
with 75 mm internal diameter) and UPLC (100 mm column with
2.1 mm internal diameter)] using gradients of linearly increasing
acetonitrileconcentrationfrom0to45%over40 min(nano-flow)
or 10 min (normal-flow).
SRM was conducted using a Quattro Premier XE (Waters).
For each peptide monitored by SRM, up to four transitions
were configured to monitor the four most intense fragment
ions (Supplementary Table S1 at http://www.BiochemJ.org/bj/
430/bj4300215add.htm). Transition dwell times were 5 ms for
two-dimensional UPLC–SRM and 50 ms for nanoUPLC–SRM.
Signals derived from internal standards and analytes of interest
weremeansmoothed(windowof2with1iteration)andintegrated
using QuantLynx (Waters). Ratios of standards and analyte peak
integrals were calculated and analyte concentration determined.
RESULTS AND DISCUSSION
Immunoprecipitation of genomically tagged PtdIns5P 4-kinase IIβ
We planned to use the DT40 cell line JPR3, in which PtdIns5P
4-kinaseIIβ isgenomicallytaggedwithaFLAG–(His6)2tag[18],
to identify protein partners that associate with this enzyme. As a
proof of principle that we could specifically pull-down and detect
endogenous PtdIns5P 4-kinase IIβ by MS, we conducted a series
of pull-downs from 3 l of cells using anti-FLAG antibody beads,
followed by three washes of the beads and elution with 3× FLAG
peptide. We performed this on triplicate samples from WT (wild-
type) and JPR3 cells in parallel, and MS analysis revealed that
PtdIns5P 4-kinase IIβ is the major protein pulled down, and that
it was detectable only in pull-downs from JPR3 and not from WT
cells (results not shown). With one exception (see below), the
next 20 most abundant proteins identified (mostly contaminants)
were present in much lower amounts and were common to WT
and JPR3 pull-downs, establishing that this stringent protocol is
highly specific for the tagged PtdIns5P 4-kinase IIβ.
However,unexpectedlywealsodetectedPtdIns5P4-kinaseIIα,
which was identified as the other major protein present, and was
againfoundonlyinpull-downsfromJPR3cellsandnotWTcells;
quantitative results (Table 2) confirm that this co-precipitation of
PtdIns5P 4-kinase IIα with the IIβ form is highly reproducible.
Given that only the IIβ form is FLAG-tagged in JPR3 cells,
these results unambiguously establish that the two PtdIns5P
4-kinase II isoforms associate with each other and the absence
of any detectable third protein suggests that their association
is direct. As both PtdIns5P 4-kinase IIβ [14] and IIα [25] are
homodimers in solution and the proposed dimerization interface
is conserved between the two isoforms [14], it is likely that this
association reflects a heterodimerization (Supplementary Figure
S3 at http://www.BiochemJ.org/bj/430/bj4300215add.htm), but
Table 2
sets of internal peptides
PtdIns5P 4-kinase II isoform ratios determined using two separate
RatiosofPtdIns5P 4-kinaseIIβ/IIαinanti-FLAGantibodypull-downsfromJPR3cellsmeasured
bytherelevantpeptidesets(559/565and829/828)andquantifiedbyinternallabelledstandards
(see Table 1). ND, not determined.
Ratio IIβ/IIα
Experiment Peptide set 559/565Peptide set 829/828
JPR3 exp. 1
JPR3 exp. 2
JPR3 exp. 3
JPR3 exp. 4
1.39
ND
1.37
1.75
0.92
1.41
ND
1.47
as yet we have no results directly addressing the structure of the
interaction.
Cellular ratio of PtdIns5P 4-kinase IIα and IIβ
There are many examples of different isoforms of the same
enzymic activity heterodimerizing to regulate localization and
function, such as the myotubularins [26]. However, these
phenomena are usually based on associations of transfected
proteins and the actual extent of association of endogenous
species, and how this may be regulated, have not been quantified.
Our genomic tagging approach gave us a unique opportunity
to address these issues, and prompts three questions: (i) Is
this PtdIns5P 4-kinase II association random and complete, i.e.
dictated only by the relative mass levels of the two isoforms? (ii)
Given that PtdIns5P 4-kinase IIβ is mostly nuclear [18], are there
therefore significant levels of endogenous PtdIns5P 4-kinase IIα
in the nucleus? (iii) What is the physiological relevance of these
phenomena?
To answer the first question, we needed to know both the mass
ratio of the enzymes in the JPR3 DT40 line, and their relative
levels in the pull-downs from JPR3 cells. Both PtdIns5P 4-kinase
II isoforms are expressed at very low levels in DT40s, and were at
thelimitofaccuratequantificationbyMS,withPtdIns5P4-kinase
IIα being the predominant isoform (see below). Additionally we
quantified the mRNA levels by qRT-PCR of WT and JPR3 DT40
cells,andtheresultsshowninFigure1andSupplementaryFigure
S2 show that the ratio of PtdIns5P 4-kinase IIβ to IIα mRNA is
approx. 0.23 and is similar in WT and JPR3 cells. Moreover,
the results also suggest that the tagged and untagged PtdIns5P
4-kinase IIβ alleles are expressed in approximately equal
amounts. If these proportions are reflected in the protein level,
and an association between the isoforms were random, then we
can define the cellular PtdIns5P 4-kinase IIβ/IIα ratio as x, and
the proportion of PtdIns5P 4-kinase IIβ that is tagged as t, so the
ratioofPtdIns5P4-kinaseIIβ toIIα inapull-downfromPtdIns5P
4-kinase IIβ-tagged cells will be (2−t)x−1 (see the Sup-
plementary Experimental section at http://www.BiochemJ.org/
bj/430/bj4300215add.htm). Thus if we take x as 0.23 and t as
0.5,randomassociationwouldgiveapredictedPtdIns5P4-kinase
IIβ/IIα ratio in the pull-downs from JPR3 cells of 1.36:1.
Quantification of PtdIns5P 4-kinase IIα and IIβ by quadrupole MS
MS was applied to quantify PtdIns5P 4-kinase II isoforms
at the protein level using an AQUA (absolute quantification)
[27] isotope dilution strategy. Given the high sequence identity,
suitable proteotypic candidate peptides were limited. Unique
candidates were ranked according to their perceived fitness for
quantification by considering the quality of empirical data and
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 4

218
M. Wang and others
Figure 1Differential expression of PtdIns5P 4-kinase II isoforms from endogenously tagged alleles
Expression from tagged and untagged PIP5K2A and PIP5K2B alleles determined by qRT-PCR using the comparative threshold cycle (Ct) method. Normalized expression is presented as the relative
fold increase above untagged PIP5K2B in WT cells (n=9). PCR was performed on cDNA made from RNA extracts of WT, JPR3 and MW2 cells as described in the Materials and methods section.
residue composition. The two best proteotypic peptides for each
isoform were selected and AQUA peptides for use as internal
standards synthesized (Table 1).
Initially the cellular PtdIns5P 4-kinases IIα and IIβ in
whole cell lysates were analysed; 7 mg of lysate digest from
2×1010cells, with 30 pmol of AQUA peptides, were separated
by two-dimensional LC and peptides were quantified by
selective reaction monitoring using a triple quadrupole mass
spectrometer. Preliminary experiments indicated which of the
first-dimension fractions the AQUA (and analyte) peptides would
elute in. Two transitions were monitored for each peptide
across the selected first-dimension fractions by UPLC–SRM and
quantifiablesignalswithacceptablesignal-to-noiseresponsewere
integrated (Figure 2). Signals were within the linear response
range of the instrument (results not shown) enabling direct
calculation of the analyte from the known amount of internal
standards. Both diagnostic peptides for PtdIns5P 4-kinase IIα
were detected and the protein was quantified at 1.5 pmol/mg
of total protein, but no quantifiable signals were detected for
PtdIns5P 4-kinase IIβ.
We then turned to analysis of the anti-FLAG immunoprecipit-
ations from JPR3 cells, and good quantification of both isoforms
was achieved. Pre-elution wash steps did not affect the PtdIns5P
4-kinase IIβ/IIα ratio observed, but provided a better signal-to-
noise response allowing more reliable quantification; the data
from four independent experiments using two different sets of in-
ternalAQUApeptides(Table2)yieldedamean+−S.E.MPtdIns5P
4-kinase IIβ/IIα ratio in the immunoprecipitates of 1.39+−0.10.
Thisiscloseenoughtotheratioof1.36predictedabovetosuggest
strongly that the association between PtdIns5P 4-kinases IIα and
IIβ is random and complete.
Tagging PtdIns5P 4-kinase IIα
To gain independent data of this association, and to answer
directly the second question posed above (the likelihood of some
endogenous PtdIns5P 4-kinase IIα being nuclear), we knocked
the same FLAG-(His6)2tag into the 3?-end of one allele of the
PIP5K2A gene by a strategy identical with that which we used
previously for the PIP5K2B gene [18]. This proved more difficult
to do than for the PIP5K2B gene, and once we had achieved
it, expression of tagged protein in several DT40 lines, including
the one we studied most (MW2), declined during the first eight
passages (assessed by Western blotting), stabilizing at a level
per cell approx. 5-fold lower than the tagged PtdIns5P 4-kinase
IIβ expression in JPR3 cells (results not shown). Moreover,
PCR analysis of the MW2 cells revealed unequal transcription
of the tagged and untagged alleles (Figure 1), and an increase
in overall PtdIns5P 4-kinase IIα transcription, presumably to
compensate for the low expression of tagged protein. Clearly
there are problems with tagging this isoform that do not apply
for PtdIns5P 4-kinase IIβ in JPR3 cells, for which expression
of tagged protein was always stable, and where no distortion of
transcription is evident compared with WT cells (Figure 1). This
distortion of both transcription and translation is probably due to
a gene-specific effect of the tag, particularly of the puromycin
selection cassette in the 3?-UTR. Overall these factors limit the
quantificationthatcanbeachieved,buttheMW2linenevertheless
yielded quantitative results to complement data from JPR3 cells.
In two independent immunoprecipitation experiments where
quantification was clear the PtdIns5P 4-kinase IIα/IIβ ratios were
11:1 and 8.8:1. As discussed above, our analysis by Western blot-
ting suggested that the level of total tagged PtdIns5P 4-kinase IIα
protein in MW2 cells was approx. 20% that of IIβ in JPR3 cells.
So if we assume that the untagged PtdIns5P 4-kinase IIα allele
was translated into protein with the same efficiency in MW2 cells
asinWTandJPR3cells,thenfromFigure1wecantoafirstdegree
ofapproximationestimatethatx=0.2andt=0.05inMW2cells.
These parameters lead to a predicted PtdIns5P 4-kinase IIα/IIβ
ratioinpull-downsfromMW2cellsof10.3:1,whichisconsistent
with the mean of 9.9:1 from the above experiments.
Nuclear PtdIns5P 4-kinase IIα
Immunocytochemistry in these tagged DT40 cell lines is not
possible with such low expression levels [18], and so we
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 5

Heterodimerization of type II PtdIns5P 4-kinases
219
Figure 2 Detection of AQUA and analyte peptides by MS
Integrated signals derived from two selected transitions (see Supplementary Table S1 at http://www.BiochemJ.org/bj/430/bj4300215add.htm) for (A) the internal AQUA standard peptide
(570.38>770.87, top panel; 570.38>699.89, lower panel) and (B) the corresponding analyte peptide (565.38>760.87, top panel; 565.38>689.99, lower panel) for quantification of
PtdIns5P 4-kinase IIα in a whole cell lysate by SRM.
previously used cell fractionation to show that most of the
genomically tagged PtdIns5P 4-kinase IIβ is nuclear [18].
In the present study we quantified the cytoplasm-to-nuclear
ratio of the type II PtdIns5P 4-kinase isoforms by another
fractionation protocol (Supplementary Figure S1 and see the
Materials and methods section) that is more quantitative than
that used previously [18] in that we quantified all fractions to
give a complete balance sheet of the distribution of the enzymes
and markers. Figure 3(A) gives representative images of nuclei
obtained from this protocol. The results (Figure 3B) confirm our
previous observation [18] that >95% of PtdIns5P 4-kinase IIβ is
nuclear.Notethatthefractionationprotocolhascausedsignificant
leakage of histones (as exemplified by histone H1) from the
nuclei (Figures 3B–3D), indicating some damage to the nuclear
membrane. The retention of most of the PtdIns5P 4-kinase IIβ
in the nucleus (Figure 3B), in contrast with this histone leakage,
suggeststhattheenzymeisnotfreelydiffusiblewithinthenucleus
but is interacting with nuclear structures.
Examination of the nuclear fraction with DAPI staining
revealed <1% nuclei with any detectable cytoplasmic debris
attached (Figure 3A). We also probed for remaining plasma
membrane using wheat germ agglutinin, and again found <1%
of nuclei had any detectable signal (Figure 3A). Additionally we
quantified cytoplasmic contamination of the nuclei by examining
for actin by Western blot, and found it to be low (Figure 3D);
indeed, the levels of actin (10% of total) that are detected may be
mostly accounted for by bona fide intranuclear actin [28] and/or
F-actin closely associated with the nucleus [29]. This protocol
is therefore apparently effective enough to remove >90% of
the cytoplasm from the nuclei, yet fractionation of MW2 cells
showed that 40% of the tagged PtdIns5P 4-kinase IIα is nuclear
and 60% cytoplasmic (Figures 3C and 3D). This suggests that
in contrast with transfected PtdIns5P 4-kinase IIα [13,18], the
endogenous enzyme is significantly nuclear. It also is important
to emphasize another difference; we found that endogenous
cytoplasmicPtdIns5P4-kinaseIIα isinthemembranousfraction,
not in the cytosol (Figures 3C and 3D). We are not sure to which
membranePtdIns5P4-kinaseIIα isattached,butfromourcurrent
knowledge we suggest that it is most likely to be the plasma
membrane, to which it could be targeted by its interaction with
type I PtdIns4P 5-kinases [30].
Lipid kinase activity of PtdIns5P 4-kinase IIα and IIβ
The simplest interpretation of our results overall is that
the PtdIns5P 4-kinases IIα and IIβ associate randomly, and
one consequence may be that PtdIns5P 4-kinase IIβ therefore
targets the IIα to the nucleus. The third question posed above
then remains: what is the physiological significance of these
events? One possibility is that the two isoforms could be
differentially regulated within the nucleus, e.g. we have shown
that PtdIns5P 4-kinase IIα is phosphorylated by casein kinase II
on a residue that is unique to that isoform [31,32]. However,
another (not mutually exclusive) possibility is that the two
isoforms have very different enzymic activities. This idea is
mademorelikelybyourpreviousdemonstrationofanassociation
between PtdIns5P 4-kinases IIα and IIγ; the latter had negligible
activity when we assayed it in comparison with the former,
and IIγ may target IIα to the vesicular compartment in which
it (IIγ) is localized [21,33]. To address the issue directly we
assayed the PtdIns5P 4-kinase activity of recombinant human
PtdIns5P 4-kinases IIα and IIβ in parallel (Supplementary Figure
S4 at http://www.BiochemJ.org/bj/430/bj4300215add.htm and
Supplementary Table S2). The specific activity in μmol/min/mg
of protein of PtdIns5P 4-kinase IIα is 3.25×10−2(S.E.M.+−1.9
× 10−4, n=4), and that of IIβ is 1.57×10−5(S.E.M.+−7.9 ×
10−7, n=4). Thus strikingly PtdIns5P 4-kinase IIα is 2000-fold
more active than the IIβ isoform, which means that in a PtdIns5P
4-kinaseIIα/IIβ heterodimertheactivityofthePtdIns5P4-kinase
IIα is going to be by far the most significant.
Conclusion
WeshouldnotethatoursuggestionthatPtdIns5P4-kinaseIIβ may
target IIα to the nucleus could throw a new light on experiments
where PtdIns5P 4-kinase IIβ has been increased by transfection
[34] or decreased in a knockout mouse [35], both of which cause
alterations in (cytoplasmic) insulin sensitivity, an effect primarily
evident in muscle and liver. One consequence of manipulating
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 6

220
M. Wang and others
Figure 3Fractionation and Western blotting of DT40 cell lysates
(A) Purity of isolated nuclei were assessed by adhering whole cells or nuclei (prepared as described in the Materials and methods section) to coverslips using cell-tak and decorating with a
combination of wheat germ agglutinin plasma membrane stain (conjugated to Alexa Fluor®555) and DAPI nuclear stain. Scale bar = 5 μm. Representative Western blot for fractionation of (B)
JPR3 or (C) MW2 cell lysates. The same proportion of each cell lysate fraction (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/430/bj4300215add.htm) was either Western blotted for
actin or histone H1, or used for immunoprecipitation and detection of FLAG-tagged protein as described in the Materials and methods section. (D) Quantification of PtdIns5P 4-kinase IIα, actin and
histone H1 levels in Western blots of MW2 cell lysate fractionations [the cytosolic fractions (C+P) were combined]. Results are means+
and histone H1). C+P, cytosol; M, cytoplasmic membranes; S, sucrose cushion; N, extracted nuclei.
−S.E.M. (n=4 for PtdIns5P 4-kinase IIα; n=3 for actin
PtdIns5P 4-kinase IIβ might be to alter significantly the amount
of the IIα isoform present in the cytoplasm, and this would be
especially evident in muscle and liver; of the tissues we analysed
these have the highest PtdIns5P 4-kinase IIβ/IIα ratios [21].
More importantly, the apparently complete and random
association(probablyheterodimerization)ofendogenousproteins
that we have revealed and quantitatively examined, plus the much
greater enzymic activity of PtdIns5P 4-kinase IIα compared with
IIβ, make it reasonable to pose the question: is a (or even the)
major function of PtdIns5P 4-kinase IIβ simply to act as a IIα
nuclear-targetingprotein?Thismaynotbethewholestory,e.g.the
requirement for PtdIns5P 4-kinase IIβ to be catalytically active
to fulfil at least some of its nuclear functions is suggested by
a striking dominant-negative effect of a kinase-dead PtdIns5P
4-kinase IIβ construct on Cul3–SPOP ubiqutin ligase [12]. As
discussed previously, there are precedents for inactive or nearly
inactive isoforms of proteins targeting active isoforms to cellular
locations, an example taken from phosphoinositide metabolism
being the myotubularins [26]. Much of the evidence for this idea
is based on association of transfected proteins, and the degree,
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 7

Heterodimerization of type II PtdIns5P 4-kinases
221
regulation and extent of association and targeting in vivo is not
always clear. In the present study we have combined the powers
ofDT40genomictaggingwithMStoplacethistypeoffunctional
relationship, at least for PtdIns5P 4-kinases IIα and IIβ, on a new
level of quantitative clarity.
While the present work was in progress we learned that another
group independently discovered the association between these
two PtdIns5P 4-kinase II isoforms [36].
AUTHOR CONTRIBUTION
MinchuanWangperformedthepull-downs,madetheIIα-taggedcells,didthefractionation
experiments and co-wrote the initial draft of the paper. Nicholas Bond co-designed and
then performed and co-interpreted all the MS experiments, and co-wrote the inital draft.
Andrew Letcher contributed to the pull-downs and fractionation experiments. Jonathan
Richardson made the IIβ-tagged cells. Kathryn Lilley co-designed and co-interpreted
all the MS experiments, and co-wrote the first draft. Robin Irvine conceived the project,
co-designed all the experiments other than the MS and co-wrote all drafts of the paper.
Jonathan Clarke performed the qRT-PCR experiments, made the recombinant enzymes
and assayed their activity, contributed to the fractionation experiments, co-designed all
the experiments other than the MS and co-wrote all drafts of the paper.
ACKNOWLEDGEMENTS
We thank Frederic Langevin, K.J. Patel and members of the Irvine laboratory for helpful
discussions.
FUNDING
This work was supported by a Wellcome Trust programme grant [grant number
079194/Z/06 (to R.F.I.)]. N.J.B. is supported by a Ph.D. studentship from the Engineering
and Physical Sciences Research Council [grant number EP/D013615/1].
REFERENCES
1 Irvine, R. F. (2003) Nuclear lipid signalling. Nat. Rev. Mol. Cell Biol. 4, 349–360
2 Cocco, L., Faenza, I., Fiume, R., Maria Billi, A., Gilmour, R. S. and Manzoli, F. A. (2006)
Phosphoinositide-specific phospholipase C (PI-PLC) β1 and nuclear lipid-dependent
signaling. Biochim. Biophys. Acta 1761, 5095–5021
3 York, J. D. (2006) Regulation of nuclear processes by inositol polyphosphates. Biochim.
Biophys. Acta 1761, 552–559
4 Bunce, M. W., Bergendahl, K. and Anderson, R. A. (2006) Nuclear PI(4,5)P2: a new place
for an old signal. Biochim. Biophys. Acta 1761, 560–569
5 Rameh, L. E., Tolias, K. F., Duckworth, B. C. and Cantley, L. C. (1997) A new pathway for
synthesis of phosphatidylinositol-4,5-bisphosphate. Nature 390, 192–196
6 Ungewickell, A., Hugge, C., Kisseleva, M., Chang, S. C., Zou, J., Feng, Y., Galyov, E. E.,
Wilson, M. and Majerus, P. W. (2005) The identification and characterization of two
phosphatidylinositol-4,5-bisphosphate 4-phosphatases. Proc. Natl. Acad. Sci. U.S.A.
102, 18854–18859
7 Zou, J., Marjanovic, J., Kisseleva, M. V., Wilson, M. and Majerus, P. W. (2007) Type I
phosphatidylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced
apoptosis. Proc. Natl. Acad. Sci. U.S.A. 104, 16834–16839
8 Lecompte, O., Poch, O. and Laporte, J. (2008) PtdIns5P regulation through evolution:
roles in membrane trafficking? Trends Biochem. Sci. 33, 453–460
9 Clarke, J. H., Letcher, A. J., D’santos, C. S, Halstead, J. R., Irvine, R. F. and Divecha, N.
(2001) Inositol lipids are regulated during cell cycle progression in the nuclei of murine
erythroleukaemia cells. Biochem. J. 357, 905–910
10 Gozani, O., Karuman, P., Jones, D. R., Ivanov, D., Cha, J., Lugovskoy, A. A., Baird, C. L.,
Zhu, H., Field, S. J., Lessnick, S. L. et al. (2003) The PHD finger of the
chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell
114, 99–111
11 Jones, D. R., Bultsma, Y., Keune, W. J., Halstead, J. R., Elouarrat, D., Mohammed, S.,
Heck, A. J., D’Santos, C. S. and Divecha, N. (2006) Nuclear PtdIns5P as a transducer of
stress signaling: an in vivo role for PIP4Kβ. Mol. Cell 23, 685–695
12 Bunce, M. W., Boronenkov, I. V. and Anderson, R. A. (2008) Coordinated activation of the
nuclear ubiquitin ligase Cul3–SPOP by the generation of phosphatidylinositol
5-phosphate. J. Biol. Chem. 283, 8678–8686
13 Ciruela, A., Hinchliffe, K. A., Divecha, N. and Irvine, R. F. (2000) Nuclear targeting of the
β isoform of type II phosphatidylinositol phosphate kinase (phosphatidylinositol
5-phosphate 4-kinase) by its α-helix 7. Biochem. J. 364, 587–591
14 Rao, V. D., Misra, S., Boronenkov, I. V., Anderson, R. A. and Hurley, J. H. (1998) Structure
of type IIβ phosphatidylinositol phosphate kinase: a protein kinase fold flattened for
interfacial phosphorylation. Cell 94, 829–839
15 Clarke, J. H., Richardson, J. P., Hinchliffe, K. A. and Irvine, R. F. (2007) Type II PIP
kinases: location, regulation and function. Biochem. Soc. Symp. 74, 149–159
16 Divecha, N., Rhee, S. G., Letcher, A. J. and Irvine, R. F. (1993) Phosphoinositide
signalling enzymes in rat liver nuclei: phosphoinositidase C isoform β1 is specifically,
but not predominantly, located in the nucleus. Biochem. J. 289, 617–620
17 Boronenkov, I. V., Loijens, J. C., Umeda, M. and Anderson, R. A. (1998) Phosphoinositide
signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA
processing factors. Mol. Biol. Cell 9, 3547–3560
18 Richardson, J. P., Wang, M., Clarke, J. H., Patel, K. J. and Irvine, R. F. (2007) Genomic
tagging of endogenous type IIβ phosphatidylinositol 5-phosphate 4-kinase in DT40 cells
reveals a nuclear localisation. Cell. Signalling 19, 1309–1314
19 Mosedale, G., Niedzwiedz, W., Alpi, A., Perrina, F., Pereira-Leal, J. B., Johnson, M.,
Langevin, F., Pace, P. and Patel, K. J. (2005) The vertebrate Hef ortholog is a component
of the Fanconi anemia tumor-suppressor pathway. Nat. Struct. Mol. Biol. 12, 763–771
20 Hinchliffe, K. A., Irvine, R. F. and Divecha, N. (1998) Regulation of PtdIns4P 5-kinase C
by thrombin-stimulated changes in its phosphorylation state in human platelets.
Biochem. J. 329, 115–119
21 Clarke, J. H., Emson, P. C. and Irvine, R. F. (2008) Localization of phosphatidylinositol
phosphate kinase IIγ in kidney to a membrane trafficking compartment within specialized
cells of the nephron. Am. J. Physiol. Renal Physiol. 295, F1422–F1430
22 Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for
biologist programmers. Methods Mol. Biol. 132, 365–386
23 Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression data using
real-time quantitative PCR and the 2−??Ctmethod. Methods 25, 402–408
24 Spackman, D. H., Stein, W. H. and Moore, S. (1958) Automatic recording apparatus for
use in the chromatography of amino acids. Anal. Chem. 30, 1190–1206
25 Hinchliffe, K., Ciruela, A., Morris, J. A., Divecha, N. and Irvine, R. F. (1999) The type II
PIPkins (PtdIns5P 4-kinases): enzymes in search of a function? Biochem. Soc. Trans. 27,
657–661
26 Lorenzo, O., Urbe, S. and Clague, M. J. (2006) Systematic analysis of myotubularins:
heteromeric interactions, subcellular localisation and endosome related functions. J. Cell
Sci. 119, 2953–2959
27 Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W. and Gygi, S. P. (2003) Absolute
quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc.
Natl. Acad. Sci. U.S.A. 100, 6940–6945
28 Rando, O. J., Zhao, K. and Crabtree, G. R. (2000) Searching for a function for nuclear
actin. Trends Cell Biol. 10, 92–97
29 Khatau, S. B., Hale, C. M., Stewart-Hutchinson, P. J., Patel, M. S., Stewart, C. L., Searson,
P. C., Hodzic, D. and Wirtz, D. (2009) A perinuclear actin cap regulates nuclear shape.
Proc. Natl. Acad. Sci. U.S.A. 106, 19017–19022
30 Hinchliffe, K. A., Giudici, M. L., Letcher, A. J. and Irvine, R. F. (2002) Type IIα
phosphatidylinositol phosphate kinase associates with the plasma membrane via
interaction with type I isoforms. Biochem. J. 363, 563–570
31 Hinchliffe, K. A., Ciruela, A., Letcher, A. J., Divecha, N. and Irvine, R. F. (1999) Regulation
of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.
Curr. Biol. 9, 983–986
32 Hinchliffe, K. A. and Irvine, R. F. (2003) CK2 phosphorylation of Type IIa PIPkin. Curr.
Biol. 13, 178
33 Clarke, J. H., Wang, M. and Irvine, R. F. (2010) Phosphatidylinositol 5-phosphate
4-kinases: localization, regulation and function of Type II phosphatidylinositol
5-phosphate 4-kinases. Adv. Enz. Reg. 50, 12–18
34 Carricaburu, V., Lamia, K. A., Lo, E., Favereaux, L., Payrastre, B., Cantley, L. C. and
Rameh, L. E. (2003) The phosphatidylinositol (PI)-5-phosphate 4-kinase type II enzyme
controls insulin signaling by regulating PI-3,4,5-trisphosphate degradation. Proc. Natl.
Acad. Sci. U.S.A. 100, 9867–9872
35 Lamia, K. A., Peroni, O. D., Kim, Y. B., Rameh, L. E., Kahn, B. B. and Cantley, L. C. (2004)
Increased insulin sensitivity and reduced adiposity in phosphatidylinositol 5-phosphate
4-kinase β−/−mice. Mol. Cell. Biol. 24, 5080–5087
36 Bultsma, Y., Keune, W.-J. and Divecha, N. (2010) PIP4Kβ interacts with and modulates
nuclear localization of the high-activity PtdIns5P-4-kinase isoform PIP4Kα. Biochem. J.
430, 223–235
Received 8 March 2010/4 June 2010; accepted 23 June 2010
Published as BJ Immediate Publication 23 June 2010, doi:10.1042/BJ20100340
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 8

Biochem. J. (2010) 430, 215–221 (Printed in Great Britain)doi:10.1042/BJ20100340
SUPPLEMENTARY ONLINE DATA
Genomic tagging reveals a random association of endogenous PtdIns5P
4-kinases IIα and IIβ and a partial nuclear localization of the IIα isoform
Minchuan WANG*1, Nicholas J. BOND†1, Andrew J. LETCHER*, Jonathan P. RICHARDSON*2, Kathryn S. LILLEY†,
Robin F. IRVINE*3and Jonathan H. CLARKE*3
*Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, U.K., and †Cambridge Centre for Proteomics, Department of Biochemistry, Tennis Court Road, Cambridge
CB2 1GA, U.K.
Figure S1Summary of cell fractionation method
DT40 cell lysate was prepared as described in the Materials and methods section in the main
paper, layered on to 1.8M sucrose and ultracentrifuged. Various fractions were harvested from
the preparation. Fraction C and Fraction P were pooled as the cytosolic component. Fraction
M consisted of the cytoplasmic membranes. Fraction S was composed of the residual sucrose
cushion. The pelleted cell nuclei were further solubilized to make Fraction N. All fractions were
used for direct Western blotting or immunoprecipitation.
EXPERIMENTAL
Calculations of ratios of isoforms expected in pull-downs from
tagged cells and in nuclei
Assuming both alleles are tagged (i.e. all protein is tagged)
If PtdIns5P 4-kinase IIβ/IIα is x, let IIβ be 1, so IIα is 1/x,
and so the total PtdIns5P 4-kinase II content is (1 + 1/x). If
dimerizationisrandom,outofthetotal(=1)IIβ,1/[1+(1/x)]will
homodimerize with IIβ, and (1/x)/(1 + 1/x) will heterodimerize
with IIα, and the homodimerized IIα will be (1/x2)/[1+(1/x)].
In a pull-down from cells with PtdIns5P 4-kinase IIβ tagged,
the number of IIβ molecules pulled down is 1, and the number
of IIα molecules is (1/x)/[1 +(1/x)]. So the IIβ/IIα ratio in that
pull-down is [1 + (1/x)]/(1/x). In a pull-down from cells with
PtdIns5P 4-kinase IIα tagged, the amount of IIα pulled down is
1/x and the amount of IIβ pulled down is (1/x)/[1+(1/x)]. So
IIα/IIβ = [1+(1/x)]
To calculate the proportion of IIα that is cytoplasmic, we
assume that only IIα in homodimers is cytoplasmic. This is
(1/x2)/[1 + (1/x)]. If we divide this by the total IIα (1/x), then
the proportion of total IIα that is cytoplasmic is (1/x)/[1+(1/x)],
or 1/(x+1) (and the ratio of cytoplasmic to nuclear IIα is 1/x).
Figure S2
by qRT-PCR
Relative amplification efficiencies of target and reference genes
Validation experiments to determine equivalent PCR amplification efficiencies of WT or
endogenouslyFLAG-(His6)2-taggedmRNAfromPIP5K2A andPIP5K2B genesrelativetothatof
the reference housekeeping gene (chicken β-actin). Line equations of each plot indicate slope,
error bars represent S.D. (n=9).
Assuming only one allele is tagged
Any dimers containing only untagged protein will be missed as
they will not be pulled down. This will decrease heterodimers
pulled down in direct proportion to the degree of tagging, but it
will decrease homodimers to a varying degree (dictated by the
ratio of isoforms), because only homodimers consisting of two
untagged species will avoid the pull-down. If t is the proportion
of a protein tagged (and other parameters are as above), for a
pull-down from PtdIns5P 4-kinase IIβ-tagged cells, the IIβ/IIα
ratio will be:
1 − (1 − t)2+ (t/x)
t/x
which can be simplified to 2x−tx+1. For a pull-down from a
PtdIns5P 4-kinase IIα-tagged cell, the IIα/IIβ ratio will be
1These authors contributed equally to this work.
2Present address: Ion Channels and Cell Signalling Centre, Division of Basic Sciences, St George’s University of London, London SW17 0RE, U.K.
3Correspondence may be addressed to either of these authors (email rfi20@cam.ac.uk or jhc30@cam.ac.uk).
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 9

M. Wang and others
Figure S3
heterodimers
Molecular modelling of putative PtdIns5P4-kinase II
(A) Comparison of human and chicken orthologues of PtdIns5P 4-kinases IIα and IIβ by
FUGUE [1] showed protein structure to be identical within 99% confidence limits. Homology
modelling of the three-dimensional structure of the IIα/IIβ heterodimers was performed using
MODELLER[2].ThepeptidesequencesofchickenPtdIns5P 4-kinasesIIα andIIβ wereinitially
aligned with the chain A and chain B sequences of the homodimer structure (PDB code 1BO1)
of human PtdIns5P 4-kinase IIβ [3] respectively by ClustalW [4,5]. The resulting alignments
were manually adjusted using the BioEdit programme (http://www.mbio.ncsu.edu). The model
illustratesthepossiblethree-dimensionalstructureofthePtdIns5P 4-kinaseIIα/β heterodimer
with the blue subunit representing the structure of the PtdIns5P 4-kinase IIα subunit (chain A)
and the silver representing the PtdIns5P 4-kinase IIβ subunit (chain B). The two subunits
are dimerized by forming β-sheet hydrogen bonds between the two β1 strands, which are
highlighted in yellow. (B) A model illustrating the predicted hydrogen bonds formed between
the two β1 strands of the PtdIns5P 4-kinase IIα and IIβ subunits. The magenta arrow
shows the β1 strand of the PtdIns5P 4-kinase IIα subunit and the green the β1 strand of
the PtdIns5P 4-kinase IIβ subunit. Red, oxygen; yellow, carbon; blue, nitrogen.
1 − (1 − t)2+ t
t
which can be simplified to (2−t+x)/x.
As we quantify the cytoplasmic and nuclear proportions of
tagged PtdIns5P 4-kinase IIα in a IIα-tagged cell by Western
blotting (Figure 3 in the main paper), which only sees tagged
protein,thecalculationsforexpectedcytoplasmicIIα areidentical
with those above under conditions where both alleles are tagged,
where t=1.
Table S1 Transitions used to detect PtdIns5P 4-kinase IIα and IIβ
Four transitions were configured for each PtdIns5P 4-kinase II-specific peptide, with either the
unlabelled or stable isotope labelled (*).
Peptide sequenceTransitionCollision energy (eV)
PtdIns5P 4-kinase IIα
SAPLANDSQAR
SAPLANDSQAR
SAPLANDSQAR
SAPLANDSQAR
SAPLANDSQAR*
SAPLANDSQAR*
SAPLANDSQAR*
SAPLANDSQAR*
FGIDDQDFQNSLTR
FGIDDQDFQNSLTR
FGIDDQDFQNSLTR
FGIDDQDFQNSLTR
FGIDDQDFQNSLTR*
FGIDDQDFQNSLTR*
FGIDDQDFQNSLTR*
FGIDDQDFQNSLTR*
SAPVNSDSQGR
SAPVNSDSQGR
SAPVNSDSQGR
SAPVNSDSQGR
SAPVNSDSQGR*
SAPVNSDSQGR*
SAPVNSDSQGR*
SAPVNSDSQGR*
FGIDDQDYQNSVTR
FGIDDQDYQNSVTR
FGIDDQDYQNSVTR
FGIDDQDYQNSVTR
FGIDDQDYQNSVTR*
FGIDDQDYQNSVTR*
FGIDDQDYQNSVTR*
FGIDDQDYQNSVTR*
1: 565.38>689.89
2: 565.38>760.87
3: 565.38>873.89
4: 565.38>970.88
5: 570.38>699.89
6: 570.38>770.87
7: 570.38>883.89
8: 570.38>980.88
1: 828.44>864.91
2: 828.44>979.86
3: 828.44>1222.80
4: 828.44>1337.77
5: 833.44>874.91
6: 833.44>989.86
7: 833.44>1232.80
8: 833.44>1347.77
1: 559.29>648.88
2: 559.29>762.87
3: 559.29>861.86
4: 559.29>958.85
5: 564.29>658.88
6: 564.29>772.87
7: 564.29>871.86
8: 564.29>968.85
1: 829.37>866.90
2: 829.37>981.80
3: 829.37>1109.82
4: 829.37>1339.74
5: 834.37>876.90
6: 834.37>991.80
7: 834.37>1119.82
8: 834.37>1349.74
25
25
25
25
25
25
25
25
30
30
30
30
30
30
30
30
25
25
25
25
25
25
25
25
30
30
30
30
30
30
30
30
PtdIns5P 4-kinase IIβ
Figure S4Specific activity of PtdIns5P 4-kinase II isoforms
Representativethin-layerchromatogramshowinglabelledPtdIns(4,5)P2productfromPtdIns5P
lipid assays. Recombinant human PtdIns5P 4-kinase IIα (0.05μg), PtdIns5P 4-kinase IIβ
(12.5μg) and a control kinase-dead PtdIns5P 4-kinase IIα (KD; 12.5μg) were used in each
reaction as described in the Materials and methods section of the main paper.
c ?The Authors Journal compilation c ?2010 Biochemical Society

Page 10

Heterodimerization of type II PtdIns5P 4-kinases
Table S2Results used to calculate the specific enzyme activity
Raw scintillation results used to calculate specific kinase activities of PtdIns5P 4-kinase IIs.
Assaysusing12.5μgofrecombinantPtdIns5P 4-kinaseIIβ or50ngofrecombinantPtdIns5P
4-kinase IIα were completed as detailed in the Materials and methods section of the main
paper. Counts collected for 5min in the32P channel were corrected for background and used to
calculate mean specific activity.
ProteinRadioactivity (c.p.m)
PtdIns5P 4-kinase IIα
32073.47
26319.75
28414.37
24523.23
3114.77
3611.66
3436.27
2879.39
PtdIns5P 4-kinase IIβ
REFERENCES
1 Shi, J., Blundell, T. L. and Mizuguchi, K. (2001) FUGUE: sequence–structure homology
recognition using environment-specific substitution tables and structure-dependent gap
penalties. J. Mol. Biol. 310, 243–257
2 Sanchez, R. and Sali, A. (2000) Comparative protein structure modeling. Introduction and
practical examples with modeller. Methods Mol. Biol. 143, 97–129
3 Rao, V. D., Misra, S., Boronenkov, I. V., Anderson, R. A. and Hurley, J. H. (1998) Structure
of type IIβ phosphatidylinositol phosphate kinase: a protein kinase fold flattened for
interfacial phosphorylation. Cell 94, 829–839
4 Fukami-Kobayashi, K. and Saito, N. (2002) How to make good use of CLUSTALW.
Tanpakushitsu Kakusan Koso 47, 1237–1239
5 Thompson, J. D., Gibson, T. J. and Higgins, D. G. (2002) Multiple sequence alignment
using ClustalW and ClustalX. Curr. Protoc. Bioinformatics 2, Unit 2.3
Received 8 March 2010/4 June 2010; accepted 23 June 2010
Published as BJ Immediate Publication 23 June 2010, doi:10.1042/BJ20100340
c ?The Authors Journal compilation c ?2010 Biochemical Society