Akt phosphorylation and nuclear phosphoinositide
association mediate mRNA export and cell
proliferation activities by ALY
Masashi Okada, Sang-Wuk Jang, and Keqiang Ye*
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322
Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 23, 2008 (received for review
March 14, 2008)
Nuclear PI3K and its downstream effectors play essential roles in a
variety of cellular activities including cell proliferation, survival,
differentiation, and pre-mRNA splicing. Aly is a nuclear speckle
protein implicated in mRNA export. Here we show that Aly is a
physiological target of nuclear PI3K signaling, which regulates its
through nuclear Akt phosphorylation and phosphoinositide asso-
ciation. Nuclear Akt phosphorylates Aly on threonine-219, which is
required for its interaction with Akt. Aly binds phosphoinositides,
and this action is regulated by Akt-mediated phosphorylation.
Phosphoinositide binding but not Akt phosphorylation dictates
Aly’s nuclear speckle residency. Depletion of Aly results in cell
growth suppression and mRNA export reduction. Inhibition of Aly
phosphorylation substantially decreases cell proliferation and
mRNA export. Furthermore, disruption of phosphoinositide asso-
ciation with Aly also significantly reduces these activities. Thus,
nuclear PI3K signaling mediates both cell proliferation and mRNA
export functions of Aly.
nuclear PI 3-kinase ? nuclear speckle ? nuclear Akt ? T219 phosphorylation
stimulation. Most of phosphoinositol lipid metabolic enzymes
and PI3K signaling machinery occur in the nucleus. For
example, PI-PLC (1, 2), phosphatidylinositol phosphate ki-
nases (3, 4), PDK1 (5), PTEN (6–8), and Akt (9, 10). Nuclear
phosphoinositides play critical roles in cell growth and differ-
entiation (11). In addition to nuclear membrane, phosphoin-
ositides also associate with the nuclear speckles, a peculiar
nuclear subcompartment enriched in small ribonucleoprotein
(RNP) particles and various splicing factors (12), where many
elements of nuclear phosphoinositide metabolism, including
PI(4,5)P2, are concentrated (4, 13, 14). Nuclear phosphoin-
ositides may be implicated in pre-mRNA splicing and chro-
matin structure (11). It has been shown before that nuclear
PI(4,5)P2assembles in a mitotically regulated particle involved
in pre-mRNA splicing (14). Elements of the transcriptional
and pre-mRNA processing machinery interact with this pool of
nuclear PI(4,5)P2, and PI(4,5)P2immunoprecipitates contain
intermediates and products of the splicing reaction. Alterna-
tive splicing of pre-mRNAs can be regulated by extracellular
signals such as growth factors, cytokines, hormones, and stress
stimuli (15). For instance, insulin-activated PI3K signaling has
been shown to implicate in mammary epithelial–mesenchymal
interaction, which regulates fibronectin alternative splicing
Splicing of pre-mRNA and export of mRNA are normally
coupled (17–19). Pre-mRNA splicing has been proposed to stim-
ulate mRNA export (17, 20). Based on genetic and biochemical
evidence, Reed et al. (21) proposed a model for mRNA export:
nascent pre-mRNA is first packaged into heterogeneous nuclear
RNP (hnRNP) particles. During spliceosome assembly, exons are
packaged by non-hnRNP spliceosome components such as SR
I3K and Akt predominantly locate in the cytoplasm, but
they also occur in the nucleus or translocate there upon
proteins. The spliced messenger RNP is targeted for export by
factors recruited during the splicing pathway, in particular the
mRNA export factor Aly. Other non-hnRNP factors present in the
spliced messenger RNP, such as SR proteins, may also be involved
in linking splicing and export or may simply serve a packaging
function to prevent binding of nuclear retention factors such as
hnRNP proteins. The non-hnRNP factors form a splicing-
mRNA for export, whereas hnRNP proteins retain introns in the
nucleus (21). In yeast, the RNA-binding proteins Yra1 and Mex67,
also known as Aly and TAP in mammalian cells, are required for
mRNA export (22, 23). Recently, it has been shown that Yra1 is
required for S phase entry and affects Dia2 binding to replication
origins. Thus, it has a role in DNA replication distinct from its role
in mRNA export (24). Although Aly directly binds UAP56 and
couples it to spliced mRNA, REF/Aly is dispensable for mRNA
export in Drosophila (25) and Caenorhabditis elegans (26).
In this report we show that Aly is a downstream target of nuclear
PI3K signaling cascade. Nuclear Akt directly binds Aly and phos-
phorylates it on the T219 residue. Interestingly, Aly directly inter-
acts with nuclear PI(4,5)P2and PI(3,4,5)P3, which is essential for its
nuclear speckle residency. Depletion of Aly markedly blocks cell
cycle progression and reduces cell growth and mRNA export, and
these processes are regulated by Akt phosphorylation and
Akt Phosphorylates Aly on T219 Residue. Aly contains an RNA
recognition domain (RRM) and GR (glycine-arginine)-rich do-
we noticed that amino acids 29–34, RGRAGS, and amino acids
214–219, GTRRGT, correspond to a motif that is identified as a
consensus Akt phosphorylation element present in numerous Akt
substrates (Fig. 1A). In vitro Akt kinase revealed that both
N-terminal (amino acids 1–107 and amino acids 1–181) and
C-terminal (amino acids 107–257 and amino acids 181–257) frag-
ments were robustly phosphorylated by active Akt. By contrast, the
middle RRM domain (amino acids 107–181) was not phosphory-
lated (Fig. 1B Upper). Mutation with S34A or T219A in Aly
abolished the phosphorylation of fragments 1–107 and 181–257,
suggesting that both residues can be phosphorylated by Akt in vitro
(Fig. 1C Upper). To explore whether Aly can be phosphorylated by
Akt in intact cells, we performed a metabolic labeling assay with
Author contributions: M.O., S.-W.J., and K.Y. designed research; M.O. and S.-W.J. per-
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
June 24, 2008 ?
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pretreated with PI3K inhibitors, wortmannin and LY294002, re-
spectively, and followed by EGF stimulation. Compared with
control, EGF elicited potent Aly phosphorylation, which was
substantially blocked by PI3K inhibitors (Fig. 1D, first panel).
Immunoblotting with phospho-T219 antibody verified this obser-
vation (Fig. 1D, last panel). Furthermore, metabolic labeling re-
Aly. The phosphorylation was slightly decreased in S34A and
indicating that T219 residue in Aly is a major Akt phosphorylation
site. Interestingly, T219A but not S34A mutation abolished Akt
association by Aly (Fig. 1E, second panel), fitting with the obser-
vation that T219 but not S34 is the predominant phosphorylation
site. The interaction between Akt and Aly was further explored in
supporting information (SI) Fig. S1 and SI Text. Aly phosphoryla-
tion was markedly decreased when Akt1 was depleted by its siRNA
compared with control siRNA. Overexpression of active plasma
membrane myristoylated Akt substantially enhanced Aly phos-
phorylation, underscoring that Akt is the physiological upstream
kinase for Aly (Fig. 1F Left). Aly T219 phosphorylation was
selectively diminished in Akt1 but not Akt2 knockout MEF cells
(Fig. 1F Right), indicating that Akt1 specifically mediates Aly
phosphorylation. Collectively, these data support that Aly is a
physiological substrate of Akt.
Nuclear Phosphoinositides Bind Aly. Nuclear phosphoinositides as-
sociate with pre-mRNA splicing machinery (19). To investigate
whether Aly binds to phosphoinositol lipids, we conducted an in
GFP-Aly. Aly bound avidly to PI(3,4,5)P3, weakly to PI(3,5)P2and
PI(4,5)P2, and not at all to other phosphoinositol lipids (Fig. 2A
Upper). Endogenous Aly robustly interacted with both PI(3,4,5)P3
and PI(4,5)P2, but it failed to bind either PI(3)P or PI(3,4)P2(Fig.
To explore whether Aly/PI(3,4,5)P3association is regulated by
nuclear extracts from PC12 cells, pretreated with NGF for various
time points. Immunoblotting analysis revealed that binding oc-
several Akt phosphorylation motifs. (B) Aly N terminus and C terminus are
termini of Aly were phosphorylated by Akt (Upper). Coomassie brilliant blue
residues in Aly are Akt phosphorylation sites. S34A and T219A mutation
in vivo. The transfected HEK293 cells were serum-starved and fed with32P-
orthophosphate for 4 h. The cells were treated with 100 ng/ml EGF for 20 min
after wortmannin (100 nM) or LY294002 (10 ?M) treatment. GST-Aly was
Western blotting. (E) T219 is the major phosphorylation site by Akt in vivo.
GST-Aly wild type, S34A, and T219A construct transfected HEK293 cells were
treated with NLS-Akt adenovirus. After 36 h of infection, cells were metabol-
ically labeled. GST-Aly were pulled down and analyzed by autoradiography
and immunoblotting. S34A mutation weakly decreased Aly phosphorylation,
The T219A mutant did not bind to Akt (second panel). (F) Ablation of Akt
abolishes Aly phosphorylation. Knocking down of Akt eliminated Aly T219
phosphorylation, whereas overexpression of active myristoylated Akt en-
selectively diminished in Akt1 but not Akt2 knockout MEF cells (top panel in
Aly is an Akt substrate in vitro and in vivo. (A) Schematic diagram of
binds to phosphatidylinositol lipids. GFP-Aly transfected HEK293 cells were
harvested, mixed with phosphatidylinositol lipid-conjugated beads, and in-
cubated for 3 h at 4°C. Lipid-bound proteins were analyzed by immunoblot-
ting. Exogenous and endogenous Aly associated with phospholipids, espe-
cially PI(4,5)P2 and PI(3,4,5)P3. (B) NGF mediates the interaction between
PI(3,4,5)P3and Aly. PC12 cells were treated with NGF for various time points.
The cell lysates were incubated with PI(3,4,5)P3-conjugated beads. After ex-
(C) T219 phosphorylation is required for Aly/PI(3,4,5)P3association. (D) The N
terminus of Aly is the PI(3,4,5)P3binding site. (E) Amino acid sequence of
N-terminal Aly. Positive amino acid residues were indicated in gray. (F) R27/
29/31 and R79/K81 are important for Aly’s PI(3,4,5)P3binding activity. R27/29/
31A and R79A/K81A mutants lost its PI(3,4,5)P3interaction activity.
Aly binds to phosphatidylinositol-(3,4,5)-Tris-phosphate. (A) Aly
www.pnas.org?cgi?doi?10.1073?pnas.0802533105Okada et al.
curred at 30 min, which peaked at 60 min and partially decayed at
between Aly/PI(3,4,5)P3. Aly phosphorylation correlated with its
binding activity to PI(3,4,5)P3beads (Fig. 2B Bottom). Preincuba-
tion with PI3K inhibitors substantially decreased the association
between PI(3,4,5)P3and Aly. Consequently, Aly phosphorylation
by Akt was markedly blocked by PI3K inhibitors (Fig. 2C), under-
scoring that Aly phosphorylation by Akt is necessary for the
binding. To map which portion is required for its association with
PI(3,4,5)P3, we prepared various Aly truncates. PI(3,4,5)P3selec-
tively interacted with full-length Aly and the N-terminal fragment
(amino acids 1–107) but not other fragments (Fig. 2D Upper),
indicating that the N terminus of Aly is required for the binding
activity. Aly N terminus contains a GR-rich domain. All of the
positive residues are highlighted in red (Fig. 2E). To determine
which R or K residues are essential for Aly binding to PI(3,4,5)P3,
we prepared numerous point mutants with K or R changed into A.
R27/29/31A and K79A/K81A mutation disrupted the association
between Aly and PI(3,4,5)P3, whereas other mutants exhibited
slightly decreased affinity (Fig. 2F). Taken together, these data
demonstrate that Aly strongly binds nuclear PI(3,4,5)P3and that
Akt phosphorylation mediates its affinity to PI(3,4,5)P3.
Depletion of Aly Suppresses Cell Proliferation and Reduces mRNA
Export. Yra1, a yeast homolog of Aly, is required for S phase entry
and plays a role in DNA replication in yeast (24). To explore
whether Aly mediates cell proliferation, we knocked it down with
its specific siRNA. Ablation of Aly selectively decreased the ex-
but not other G1phase cyclins including cyclin D1 or E1. Cdk4
expression level also remained constant (Fig. 3A). Cyclin A is
during G2/M phases. These results suggest that Aly selectively
further evaluate its role in cell growth, we conducted a BrdU
incorporation assay. Depletion of Aly substantially attenuated
DNA replication (Fig. 3B Left), confirming its cell growth activity
in yeast. Knockdown of Aly reduced BrdU incorporation from
?40% to ?15% (Fig. 3B Right). Colony formation assay demon-
we monitored the cell growth curve. Indeed, ablation of Aly
decreased HeLa cell proliferation (Fig. 3D). Thus, Aly is necessary
export in HeLa cells, we performed an in vitro mRNA export assay
with biotinylated oligo-dT(50) followed by staining with rhoda-
significantly decreased mRNA export (Fig. 3E). Thus, these results
demonstrate that Aly is required for cell proliferation and mRNA
Akt Phosphorylation of Aly Mediates Its mRNA Export and Cell
Proliferation Activities. ToinvestigatewhetherAktphosphorylation
mediates its subnuclear localization, we transfected GFP-Aly wild
type and various mutants into MEF cells. Immunofluorescent
staining showed that all transfected Aly proteins tightly colocalized
with SC-35, a marker for nuclear speckle, demonstrating that Akt
phosphorylation does not influence its subnuclear residency (Fig.
4A). To assess the effect of Akt phosphorylation on Aly’s mRNA
export activity, we transfected HeLa cells with various GFP-Aly
constructs. GFP-Aly T219D displayed comparable activity to GFP
control and wild-type Aly, indicating that Aly phosphorylation by
Akt is sufficient to enhance mRNA export. Noticeably, the un-
phosphorylated T219A mutant markedly decreased mRNA export
Aly’s mRNA export activity.
cell growth effect, we transfected GFP-Aly wild-type and phos-
phorylation mutants into HeLa cells. A BrdU incorporation assay
showed that overexpression of wild-type Aly increased cell prolif-
with a control scrambled RNA or Aly siRNA and incubated for 48 h. The cell lysate was separated by SDS/PAGE and analyzed by immunoblotting. Cyclin A2 and
cyclin B1 protein expression levels were selectively decreased. (B). siAly inhibits S phase progression. HeLa cells were transfected with control or siAly and
incubated for 4 days. Cells were treated with BrdU (20 ?M) for 1 h at 37°C and then fixed by 3% formaldehyde. The fixed cells were treated with 2 M HCl and
are expressed as mean ? SEM from three independent experiments. (C) Aly knockdown prevents cell proliferation. Twenty-four hours after transfection, HeLa
cells were split into six-well dishes (5 ? 104) and incubated for 5 days. After cells were fixed by 3% formaldehyde, cells were stained with crystal violet solution.
(D) Ablation of Aly decreases cell growth. (E) Knockdown of Aly decreases mRNA export. Results are expressed as mean ? SEM from three independent
experiments (P ? 0.005, Student’s t test).
Aly regulates cell cycle progression and cell proliferation. (A) Aly knockdown alters the expression of cell cycle regulators. HeLa cells were transfected
Okada et al.PNAS ?
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eration compared with GFP control. The strongest cell growth
stimulatory effect occurred to the Aly T219D mutant. By contrast,
the unphosphorylated Aly T219A mutant robustly blocked BrdU
incorporation (Fig. 4D Left). Quantitative analysis revealed ?40%
BrdU-positive cells in wild-type Aly transfected cells, which was
increased to ?55% in T219D transfected cells. In contrast, BrdU
incorporation was decreased to ?15% in T219A transfected cells
(Fig. 4D Right). The cell growth curve further supported that Akt
phosphorylation of Aly enhanced its mitogenic activity, whereas
and cell proliferation activities.
Phosphoinositides Binding by Aly Regulates Its Subnuclear Residency,
phosphoinositide interaction regulates Aly’s subnuclear localiza-
tion, we conducted immunofluorescent staining. Whereas wild-
type Aly resided exclusively in the nuclear speckles, Aly mutants
R27/29/31A and R79A/K81A, which failed to associate with
PI(3,4,5)P3, partially colocalized with SC-35. Interestingly, these
(Fig. 5A), indicating that PI(3,4,5)P3binding somehow regulates its
that both Aly mutants R27/29/31A and R79A/K81A evidently
control (Fig. 5B). The cell growth curve further verified this
observation (Fig. 5C), underscoring that nuclear phosphoinositide
association dictates Aly’s cell growth stimulatory effect.
Nuclear phosphoinositides influence pre-mRNA splicing and
chromatin structure (11). To investigate whether PI(3,4,5)P3asso-
ciation is required for Aly’s mRNA export activity, we transfected
Aly R27/29/31A and R79A/K81A mutants lacking PI(3,4,5)P3
binding affinity into HeLa cells and performed an mRNA export
assay. Aly mutants displayed a decreased activity in mRNA export
our data suggest that the N terminus of Aly is critical for nuclear
PI(3,4,5)P3binding and that disruption of this interaction impairs
Aly’s cell proliferation and mRNA export activities.
In the present study we demonstrate that Aly is a physiological
T219 phosphorylation in Aly by Akt is critical for the complex
formation. Noticeably, Aly potently interacts with phosphoinositi-
des, for which its N terminus is necessary. In addition to mediating
mRNA export, we surprisingly uncovered that Aly played a critical
role in cell proliferation. Blocking T219 phosphorylation or crip-
pling PI(3,4,5)P3 binding strongly suppresses cell proliferation.
Moreover, T219 phosphorylation is indispensable for its mRNA
export activity. On the other hand, Aly R79A/K81A and R27/29/
31/A mutants, which fail to bind PI(3,4,5)P3, display a marked
decrease in mRNA export activity. Thus, nuclear PI3K signaling
regulates Aly’s cell proliferation and mRNA export activities.
PI3K is important for both transition from G1to S phase and
increase of PI(3)P during the G2/M phase of the cell cycle occur in
with the current paradigm that the nuclear and cytoplasmic phos-
phoinositide cycles function independent of each other. Depletion
cyclin expression. In addition, BrdU incorporation is significantly
decreased in HeLa cells when Aly is inactivated. These results
support that Aly is required for cell cycle progression. This finding
corroborates with the recent report that Yra1, a yeast homolog of
Aly, is required for S phase entry and affects Dia2 binding to
in DNA replication. To explore whether nuclear Akt phosphory-
lation of Aly plays any role in mediating Aly’s cell cycle activity, we
found that T219 phosphorylation strongly enhanced cell prolifer-
ation, and blockade of T219 phosphorylation profoundly blocked
BrdU incorporation and cell proliferation (Fig. 4). In addition, the
cell growth activity of Aly is also regulated by phosphoinositides.
Overexpression of Aly mutants deficient of binding to nuclear
phosphorylation on Aly T219 is required for mRNA export activity (B). Quantitative analysis of mRNA export is shown in C. (D) Akt phosphorylation regulates
Aly’s cell proliferation activity. T219 phosphorylation mimetic mutants enhanced BrdU incorporation, whereas unphosphorylated mutant T219A displayed
t test). (E) Cell proliferation assay. HeLa cells, transfected with various Aly phosphorylation mutants, grew in six-well plates. The cell numbers were counted at
the indicated time points.
Akt phosphorylation mediates Aly’s effect in cell proliferation. (A) Aly phosphorylation does not alter Aly nuclear speckle residency. (B and C) Akt
www.pnas.org?cgi?doi?10.1073?pnas.0802533105Okada et al.
PI(3,4,5)P3 substantially suppresses cell proliferation. Concomi-
tantly, these mutants aggregated in the nucleoplasm outside the
nuclear speckles (Fig. 5). Conceivably, Aly nuclear speckle resi-
dency is necessary for its cell growth activity. Accumulative evi-
dence supports that cell cycle progression and nuclear phospho-
phosphoinositides remain constant throughout the cell cycle, nu-
clear phosphoinositides fluctuate significantly in a cell-cycle-
dependent manner (30, 31). Our finding that Aly is a downstream
association with nuclear phosphoinositol lipids, provides a molec-
ular mechanism for how nuclear PI3K mediates cell cycle progres-
sion and cell proliferation.
In our previous effort to search for nuclear receptors for
PI(3,4,5)P3we identified nucleophasmin/B23 as one of the binding
targets (32). Aly was also one of the binding proteins identified. In
current study we provided further evidence demonstrating that Aly
the N terminus of Aly is involved in this activity (Fig. 2). Protein
sequence analysis reveals that this region is composed of numerous
basic residues. Mutation of R27/29/31 or R79/K81 into alanine
abolishes Aly’s binding affinity to PI(3,4,5)P3, suggesting that these
residues play an essential role in binding to the heavily negatively
charged phosphoinositide head groups. Presumably, the clusters of
79/lysine-81 residues might form a pocket that harbors the phos-
phorylated inositol head group from phosphoinositol lipids.
Alteration of one cluster of positively charged residues results in
deformation of the delicate three-dimensional conformation, lead-
ing to loss of binding affinity by Aly to phosphoinositides.
phoinositol lipid binding activity, indicating that nuclear Akt me-
diates Aly’s affinity to PI(3,4,5)P3.
Nuclear speckles are highly dynamic, and their morphology is
tightly linked to the state of mRNA transcription. Inhibition of
mRNA transcription induces these structures to become larger and
fewer in number; the phosphatidylinositol phosphate kinases and
PI(4,5)P2reorganize identically (33). The presence of PI(4,5)P2in
the speckle domains of the nucleus has been related to its involve-
ment in pre-mRNA splicing (14). In fact, when PI(4,5)P2 was
immunoprecipitated from HeLa cell nuclear extracts, some pro-
teins were pulled down with it, resulting in a specific inhibition of
pre-mRNA splicing in the extracts (14). Here we show that
PI(4,5)P2and PI(3,4,5)P3robustly bind to Aly in the speckles and
thus the involvement by nuclear phosphoinositides in pre-mRNA
possibility that PI(4,5)P2or PI(3,4,5)P3binds nuclear matrix pro-
teins and serves as a structural interface between the enzymatic
core of the spliceosome and the matrix itself cannot be excluded
either. Identification of Aly, a nuclear speckle protein implicated in
mRNA export, as one of nuclear phosphoinositol lipid binding
targets is certainly of great help in understanding the exact and
multifaceted functions of these nuclear lipids. Collectively, our
study establishes that Aly is a physiological downstream target of
nuclear PI3K signaling and that nuclear PI(3,4,5)P3 and Akt
coordinately mediate the nuclear effector through the concerted
interaction and phosphorylation. This finding provides a molecular
mechanism of how the cell cycle progression, cell proliferation, and
mRNA export activities of Aly are regulated by nuclear PI3K.
Materials and Methods
Cells and Reagents. HEK293, HeLa, MEF, Akt1, Akt2, and Akt1/2 null cells were
PC12 cells were maintained in DMEM including 10% horse serum, 5% FBS, and
100 units of penicillin-streptomycin. All cells were maintained at 37°C with 5%
CO2atmosphere in a humidified incubator. EGF, NGF, avidin-rhodamine, and
biotin-16–2?-deoxy-uridine-5?-triphosphate were from Roche. Anti-cdk4 and
rosporin, etoposide, BrdU, anti-tubulin, SC-35, GST-HRP, and HA-HRP antibodies
were from Sigma. Terminal transferase was from New England Biolabs. Anti-
phospho-Akt S473 antibody was from Cell Signaling Technology. Anti-ALY
(NB100-670) was from NOVUS. Anti-cyclin A2, cyclin B1, cyclin D1, Akt, HA, and
Upstate Biotechnology. Phosphatidylinositol lipid-conjugated beads were from
Echeron Research Laboratory. siRNA of ALY (1757609) was from Qiagen. The
target sequence is TGGGAAACTGCTGGTGTCCAA.
In Vitro Akt Kinase Assay. Purified Aly fragments or its mutants (0.5 ?g) were
incubated with active Akt in kinase reaction buffer (20 mM Tris, pH 7.5/10 mM
MgCl2) containing 25 ?M ATP and 2.5 ?Ci of [?-32P]ATP for 30 min at 30°C.
Reactions were terminated by addition of Laemmli’s sample buffer and boiling
for 10 min. A portion of the sample (20 ?l) was separated by SDS/PAGE and
analyzed by autoradiography.
In Vivo ALY Phosphorylation Assay and Metabolic Labeling. TransfectedHEK293
adenovirus and incubated for 24 h. Cells were washed with phosphate-free
mRNA export, and cell proliferation. (A) PI(3,4,5)P3binding mutants localize
outside of nuclear speckles. Wild-type Aly distributed in the nuclear speckles,
colocalizing with SC35. R27/29/31A and R79A/K81A partially localized in the
nuclear speckles and aggregated in nucleoplasm. (B) BrdU incorporation
assay. Aly mutants lacking PI(3,4,5)P3binding affinity suppressed BrdU incor-
poration. Results are expressed as mean ? SEM from three independent
experiments.*, P ? 0.05 (Student’s t test). (C) Aly/PI(3,4,5)P3 interaction is
implicate in mediating cell proliferation. (D) Aly/PI(3,4,5)P3 interaction is
essential for its mRNA export activity. GFP, GFP-Aly wild type, and mutants
deficient of PI(3,4,5)P3binding were transfected into HeLa cells, respectively,
and then incubated for 48 h, followed by mRNA export assay. R79/K81A
R27/29/31A mutant. Results are expressed as mean ? SEM from three
independent experiments.*, P ? 0.001;**, P ? 0.01 (Student’s t test).
Phosphoinositide binding of Aly regulates its subnuclear residency,
Okada et al. PNAS ?
June 24, 2008 ?
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mediumandincubatedfor90mininphosphate-freemedium.Cellsweretreated Download full-text
with 250 ?Ci/ml32P-orthophosphate for 4 h. The labeled cells were treated with
washed with ice-cold PBS three times and lysed and then mixed with GSH beads
for overnight at 4°C. After washing, protein complexes were separated by SDS/
PAGE and analyzed by autoradiography.
Coimmunoprecipitation and in Vitro Binding Assay. A10-cmplateoftransfected
7.4/40 mM NaCl/1 mM EDTA/0.5% Triton X-100/1.5 mM Na3VO4/50 mM NaF/10
mM sodium pyrophosphate/10 mM sodium ?-glycerophosphate/protease inhib-
itor mixture), and centrifuged for 10 min at 16,000 ? g at 4°C. The supernatant
or phosphatidylinositol lipid-conjugated beads. After SDS/PAGE the samples
were transferred to a nitrocellulose membrane. Western blotting analysis was
performed with a variety of antibodies.
Cell Cycle Analysis. For cell counting analysis, transfected HeLa cells were incu-
trypsinized cells were counted by hemacytometer. For crystal violet analysis, 5 ?
were fixed by 3% formaldehyde in PBS and stained by crystal violet solution. In
the BrdU incorporation assay, transfected cells were grown in 20 ?M BrdU
including medium for 2 h and fixed by 3% formaldehyde in PBS. After washing
with PBS, cells were treated with prewarmed 2 M HCl for 15 min at 37°C and
blocked by 2% FBS/PBS and stained by anti-BrdU antibody. Finally, cells were
counterstained with DAPI to visualize the nuclei. For cell cycle regulator protein
expression check, cells were transfected and incubated for 24 h and split into
was adjusted, proteins were loaded onto SDS/PAGE and analyzed by immuno-
Fluorescent in Situ Hybridization for mRNA Export. HeLa cells were transfected
with various GFP-tagged Aly constructs or siRNA and incubated for 24 h. Cells
were fixed by 3% formaldehyde in PBS/diethyl pyrocarbonate (DEPC) for 10 min
at room temperature. After washing with ice-cold PBS/DEPC, cells were perme-
abilized by 0.5% Triton X-100 in PBS/DEPC for 5 min on ice. After cells were
washed with ice-cold PBS/DEPC three times, coverslips were transferred into 2?
SSC and incubated at room temperature for 10 min. Cells were blocked by
blocking mixture (2? SSC, 1 mg/ml tRNA, 10% dextran sulfate, and 25% form-
amide) at room temperature for 30 min and then hybridized in hybridization
sulfate, and 25% formamide] at 37°C overnight. Cells were washed with 2? SSC
for 10 min three times and then washed with 0.5? SSC for 10 min once. After
blocking with 2% FBS in PBS for 30 min at room temperature, cells were stained
by rhodamine-streptavidin for 20 min at room temperature. After cells were
washed with 0.2% Triton X-100/PBS for 10 min three times, cells were washed
with PBS twice. Fluorescent images were taken by using a confocal fluorescence
ACKNOWLEDGMENTS. We are thankful to Dr. Rozanne M. Sandri-Goldin
Michael Green from (University of Massachusetts, Worcester, MA) for the pR-
Grant RO1 NS045627 (to K.Y.).
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