From the Departments of In an attempt to further understand how nuclear
events (such as gene expression, nuclear import/export, and cell cycle
checkpoint control) might be subject to regulation by extracellular
stimuli, we sought to identify nuclear activities under growth factor
control. Using a sensitive photoaffinity labeling assay that measured
[ Growth factor binding to cell-surface receptors can initiate
signals that are propagated through the cell by a cascade of protein-protein interactions, ultimately to impact upon specific cellular functions and regulate cell growth. The activities of signaling molecules must be tightly regulated to maintain the integrity
of cellular communication, as loss of regulation in these processes can
give rise to defects in cell growth and metabolism that may lead to
human disease. Given the importance of signaling processes in cell
growth, a great deal of effort has gone into the elucidation of
proteins participating in signaling pathways that start at the level of
receptor activation and culminate in the stimulation of a nuclear
activity. Multiple cascades have now been identified that result in the
activation of different nuclear mitogen-activated protein kinases,
including the extracellular receptor-activated kinases and the
stress-responsive c-Jun N-terminal kinase/stress-activated protein
kinase and p38 (1, 2). Extracellular receptor-activated kinase
activation is the outcome of mitogen-stimulated Ras signaling, whereas
c-Jun N-terminal kinase/stress-activated protein kinase and p38
activities are often stimulated by pathways involving the Cdc42 and Rac
GTP-binding proteins (2-6). Although these different signaling
pathways were originally thought to be independently regulated, later
work showed that cross-talk between the individual mitogen-activated
protein kinase pathways exists. A common functional outcome of the
activation of these signaling pathways is a translocation of the
activated mitogen-activated protein kinase to the nucleus and
subsequent activation of specific transcription factors and gene
expression (2-6).
How other nuclear functions might be influenced in response to
extracellular stimulation is less clear. However, it is attractive to
envision how critical nuclear activities such as RNA metabolism and
export, nuclear protein import, and cell cycle control might be subject
to regulation as downstream targets of extracellular stimuli. With this
in mind, we set out to identify novel nuclear activities that were
growth factor-responsive. Using a photoaffinity labeling approach, we
identified the nuclear cap-binding complex (CBC)1 as such an activity
based on the enhanced ability of its ~20-kDa subunit (CBP20) to
undergo a photocatalyzed incorporation of [ Cell Culture Conditions--
Rat pheochromocytoma (PC12) cells
were maintained in Dulbecco's modified Eagle's medium containing 5%
fetal bovine serum, 10% horse serum, and antibiotic/antimycotic
solution (Sigma). All other cell types, including HeLa, BHK21, and
COS-7 cells, were maintained in Dulbecco's modified Eagle's medium
with the addition of 10% fetal bovine serum and antibiotic/antimycotic solution. Prior to growth factor treatment, cells were switched to
serum-free medium for 40 h. Growth factors (NGF (Life
Technologies, Inc.), heregulin Cell Fractionation and Nuclear Lysis--
Tissue culture cells
were washed twice on the plate with TBS and then lysed in a buffer
containing Hanks' solution (20 mM Hepes, pH 7.4, 5 mM KCl, 137 mM NaCl, 4 mM
NaHCO3, 5.5 mM D-glucose, and 10 µM EDTA), 0.3% (v/v) Nonidet P-40, 1 mM
sodium orthovanadate, 1 mM DTT, 1 mM
phenylmethanesulfonyl fluoride, and 10 µg/ml each leupeptin and
aprotinin. The lysate was then centrifuged at 800 rpm for 15 min at
4 °C. The supernatant was microcentrifuged for 10 min at 4 °C,
and then the resulting supernatant was saved as the cytoplasmic
fraction. The nuclear pellet was washed twice with an equal volume of
Hanks' solution with 0.2% (v/v) Triton X-100 and centrifuged at 800 rpm for 15 min at 4 °C. The resulting pellet was treated as the
purified nuclear fraction. The nuclei were then lysed in a buffer
containing 50 mM Tris, pH 7.4, 1% (v/v) Triton X-100, 400 mM KCl, 1 mM sodium orthovanadate, 1 mM DTT, and protease inhibitors as described above. The
samples were incubated on ice for 30 min and microcentrifuged for 10 min at 4 °C, and the supernatant was used as the whole nuclear
fraction. For nuclear fractionation, nuclei were isolated from tissue
culture cells, and nuclear membranes and nuclear soluble fractions were then prepared as described by Davis and Blobel (17) with some modification. The whole nuclear fraction was resuspended in 50 mM Tris-HCl, pH 7.4, 10% (w/v) sucrose, 1 mM
sodium orthovanadate, 1 mM DTT, 1 mM
MgCl2, and protease inhibitors. DNase-I (5 mg/ml) and RNase
A (1 mg/ml) were added, and the nuclei were then incubated for 15 min
at 37 °C. Following the incubation with DNase-I, the nuclei were
underlaid with 30% sucrose and then subjected to centrifugation in a
swinging bucket rotor for 10 min at 20,000 × g to
generate a soluble nuclear fraction and a nuclear membrane fraction.
Photoaffinity Labeling with
[ Purification of an 18-kDa Protein from Bovine Retinal Tissue That
Incorporates [ Cloning and Expression of Recombinant CBP20--
CBP20 was
cloned by polymerase chain reaction from HeLa cell cDNA (a generous
gift from Dr. Wannian Yang, Cornell University). 5'- and 3'-primers
were designed using the published sequence for Homo sapiens
CBP20 (GenBankTM accession P52298), and the
CBP20 gene was then amplified from the HeLa cell cDNA
using 40 polymerase chain reaction cycles (1 min at 94 °C, 1 min at
55 °C, and 1 min at 72 °C). The 470-base product was inserted
into a cloning vector (pCR2.1) using a TA cloning kit (Invitrogen) and
then subcloned into the mammalian expression vector pcDNA3
(Invitrogen) and into the Escherichia coli expression vector
pGEX-2TK.
E. coli cells transformed with the pGEX-2TK-CBP20 vector
were grown in a 1-liter culture, and expression of glutathione
S-transferase (GST)-CBP20 protein was induced for 3 h
using isopropyl-
Using the LipofectAMINE protocol (Life Technologies, Inc.), a
hemagglutinin-tagged form of CBP20 (HA-CBP20) was transiently transfected into BHK21 cells according to the manufacturer's
directions. Following a 5-h incubation with serum-free medium
containing the lipid-DNA complex, the medium was removed and replaced
with medium containing 10% fetal bovine serum. Cells were allowed to
grow in the presence of serum for ~20 h and were then switched to
serum-free medium for 40 h prior to stimulation with serum.
Immunoprecipitation and Western Immunoblotting--
A
polyclonal antibody generated against recombinant CBP80 (
For Western blot analysis, proteins were transferred to polyvinylidene
difluoride membranes following SDS-PAGE. The polyvinylidene difluoride
membranes were blocked with 2.5% (w/v) bovine serum albumin in TBS
plus 0.1% Tween 20 for 1 h at room temperature. After blocking,
the membranes were incubated with either 12CA5 or RNA Binding Assays--
UV cross-linking was done essentially as
described by Rozen and Sonenberg (20), except that the RNA probe was
transcribed from BamHI-cleaved pBluescript II KS with T3 RNA
polymerase (Promega).
Pre-mRNA Splicing Reactions--
Splicing extracts were
prepared from HeLa cells (serum-starved for 40 h prior to
stimulation with 100 nM heregulin for 24 h) as
described by Lee and Green (21). pBSAd1 precursor linearized by
Sau3AI was transcribed using T3 RNA polymerase in the
presence of m7GpppG dinucleotide cap. Splicing reactions
were then carried out as described by Izaurralde et al. (7).
In brief, 60 µg of splicing extract were preincubated for 15 min at
30 °C with 1 mM MgCl2. 5 mM
creatine phosphate, 1.5 mM ATP, 2.5 × 104
cpm of labeled precursor mRNA, and an additional 1 mM
MgCl2 were then added in a final volume of 20 µl, and the
reactions were incubated for 2 h at 30 °C. Splice products were
visualized by separation on a 10% denaturing polyacrylamide gel,
followed by autoradiography.
The overall goal of these studies was to identify nuclear
activities that could represent novel downstream targets in
receptor-coupled signaling pathways. One of the assays we used to
identify such activities was the photocatalyzed incorporation of
[ A purification scheme was developed using bovine retinal nuclei, which
were a particularly rich source of this 18-kDa nuclear activity. A
series of three chromatography steps resolved the activity, as assayed
by [ First, we assayed directly the ability of recombinant E. coli-expressed CBP20 to incorporate [ We next examined whether the ability of CBP20 to incorporate
[ Biochemistry,
ABSTRACT
Top
Abstract
Introduction
References
-32P]GTP incorporation into nuclear proteins,
we identified the 20-kDa subunit of the nuclear cap-binding complex
(CBC) as a protein whose binding activity is greatly enhanced by the
extracellular stimulation of serum-arrested cells. The CBC represents a
20- and 80-kDa heterodimer (the subunits independently referred to as
CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on
RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that
the [
-32P]GTP incorporation into CBP20 was correlated
with an increased ability of the CBC to bind capped RNA and have used
the [
-32P]GTP photoaffinity assay to characterize the
activation of the CBC in response to growth factors. We show that the
CBC is activated by heregulin in HeLa cells and by nerve growth factor
in PC12 cells as well as during the G1/S phase of the cell
cycle and when cells are stressed with UV irradiation. Additionally, we
show that cap-dependent splicing of precursor mRNA, a
functional outcome of CBC activation, can be catalyzed by growth factor
addition to serum-arrested cells. Taken together, these data identify
the CBC as a nuclear target for growth factor-coupled signal
transduction and suggest novel mechanisms by which growth factors can
influence gene expression and cell growth.
INTRODUCTION
Top
Abstract
Introduction
References
-32P]GTP in
response to extracellular stimulation. The CBP20 protein and its 80-kDa
binding partner, CBP80, constitute a functional CBC (7-10). This
nuclear complex binds cotranscriptionally to the monomethylated
guanosine cap structure (m7G) of RNA polymerase
II-transcribed RNAs (7, 11, 12) and has been reported to play a role in
diverse aspects of RNA metabolism: it increases the splicing efficiency
of cap proximal introns (7, 13-15), positively affects the efficiency
of 3'-end processing (16), and is required for the efficient transport
of U snRNAs (9). We demonstrate that the incorporation of
[
-32P]GTP by CBP20 reflects the activation of the CBC
and is correlated with its ability to bind capped RNA. A variety of
growth factors and other cellular stimuli can activate the CBC under
conditions that can give rise to a stimulation of the splicing of
precursor mRNAs in an in vitro assay system. The
implications of CBP20 functioning as a novel end point in signal
transduction highlight the importance of RNA metabolism in regulated
cell growth.
EXPERIMENTAL PROCEDURES
1 (residues 177-244; a generous gift
from Dr. Mark Sliwkowski, Genentech), and EGF (Calbiochem) or 25%
fetal bovine serum) were then added to the serum-free medium in the concentrations and for the times indicated under "Results" at 37 °C. Following treatment, the growth factor-containing medium was
removed, and the cells were washed twice with Tris-buffered saline
(TBS; 25 mM Tris-Cl, pH 7.4, 140 mM NaCl, and
1.0 mM EDTA) and then lysed (see below). Cell cycle blocks
were performed in HeLa cells. A G0 block was achieved by
switching to serum-free medium for 22-24 h. For G1/S phase
arrest, 2.5 mM thymidine was added to the growth medium for
22-24 h. 80 ng/ml nocodazole was added to the growth medium for 22-24
h to achieve arrest in M phase. After treatment, cells were collected,
washed twice with TBS, and lysed. To challenge cells with UV
irradiation, the medium was removed from serum-starved cells, and the
cells were then exposed to UV light for 2 min. Following exposure,
cells were replenished with serum-free medium and allowed to recover at
37 °C for the times indicated below.
-32P]GTP--
Photoaffinity labeling of cellular
proteins with [
-32P]GTP was performed as described
previously (18). In brief, the UV cross-linking reaction was carried
out in a buffer containing 50 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM DTT, 20% (v/v) glycerol, 100 mM NaCl, and 500 µM AMP-PNP. Samples (20 µl) prepared from the cell fractionation procedures, described above,
were incubated for 10 min at room temperature with an equal volume of
cross-linking buffer containing [
-32P]GTP (2-3
µCi/sample, 3000 Ci/mmol; NEN Life Science Products ) in a 96-well,
non-tissue culture-treated plate. The samples were then placed in an
ice bath and irradiated with UV light (254 nm) for 15 min. After
irradiation, samples were mixed with 5× Laemmli buffer and boiled.
SDS-PAGE was performed using 15% acrylamide gels. The gels were then
typically silver-stained and dried, and autoradiography was performed
(typically overnight) using Kodak X-Omat XAR-5 film at
80 °C. To
perform competition experiments, competing nucleotides
(m7GpppG and GpppG (New England Biolabs Inc.) and
m7GTP and GTP (Sigma)) were added to the sample prior to
the addition of the [
-32P]GTP-containing cross-linking
buffer. This buffer did not contain AMP-PNP. The samples were then
subjected to UV cross-linking as described above.
-32P]GTP--
Bovine retinas were
obtained frozen from J. A. & W. L. Lawson Co. (Lincoln, NE). The
retinas (typically 200/batch) were thawed in a buffer containing 50 mM Tris, pH 8.0, 25 mM KCl, 5 mM
MgCl2, and protease inhibitors as described for cell lysate
preparations and then homogenized with a motor-driven Dounce
homogenizer. The homogenate was centrifuged at 2500 rpm in a swinging
bucket rotor to yield a crude nuclear pellet. The nuclei were purified
from this crude preparation using the method described by Blobel and Potter (19), and the soluble nuclear contents were then extracted as
described above. The 18-kDa activity was precipitated using 40-75%
ammonium sulfate, resuspended in 3-5 ml of Buffer A (50 mM
Tris, pH 8.0, 100 mM NaCl, 10 mM MgCl2,
1 mM EDTA, 1 mM DTT, 20 mM KCl),
and loaded onto a fast protein liquid chromatography Superdex-200
Highload 16/60 column as described above. The purification of this
activity was monitored by both silver staining and UV cross-linking to
[
-32P]GTP. The fractions eluted from the Superdex-200
column were assayed for [
-32P]GTP incorporation into
the 18-kDa protein, and six peak fractions (eluting with molecular
masses of ~100-150 kDa) were pooled in a final volume of 12 ml and
loaded directly onto a fast protein liquid chromatography ion-exchange
Mono Q 5/5 column (Amersham Pharmacia Biotech) equilibrated in Buffer A
minus KCl. Bound proteins were eluted from the Mono Q 5/5 column with a
28-ml linear gradient of 100-500 mM NaCl.
[
-32P]GTP-incorporating activity eluted from the Mono
Q 5/5 column with ~300 mM NaCl in a volume of 5 ml. Peak
activity as assayed by [
-32P]GTP incorporation was
eluted from the Mono Q column and applied directly to a Bio-Gel HPHT
hydroxylapatite column (Bio-Rad) equilibrated in 10 mM
potassium phosphate, pH 6.8, 2.5 mM MgCl2, 0.01 mM CaCl2, and 1 mM DTT. Bound
proteins were then eluted, first by stepping the potassium phosphate to
100 mM and then by a 20-ml linear gradient of 100-300
mM potassium phosphate. Peak activity as assayed by the
light-catalyzed incorporation of [
-32P]GTP was found
to elute with ~250 mM phosphate.
-D-thiogalactopyranoside. Following
induction, the cells were pelleted by centrifugation (5000 rpm for 10 min in a JA-10 rotor). The harvested cells were resuspended in 15 ml of
50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 1 mM DTT, and protease inhibitors (as described above) and
then lysed using 15 mg of lysozyme, followed by the addition of 200 mM MgCl2 and 1 mg of DNase-I. Following
centrifugation (100,00 × g for 30 min at 4 °C), the
supernatant was incubated with glutathione-agarose beads for 1 h
at 4 °C to bind the GST-CBP20 protein. Glutathione-agarose-bound CBP20 was washed with 50 mM Tris-HCl, pH 8.0, 0.5% (v/v)
Triton X-100, 200 mM KCl, and 1 mM DTT and then
stored in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 10 µM GTP, and
protease inhibitors. GST-CBP20 was eluted from the glutathione-agarose beads using 10 mM glutathione, pH 8.0, and the GST moiety
was cleaved from CBP20 by the addition of 500 units of thrombin for 30 min at room temperature.
CBP80) was
prepared as described previously (7). Cytosolic and nuclear lysates
were prepared as described above. Prior to immunoprecipitation, the
cytosolic lysate was adjusted to 100 mM NaCl, and the
nuclear lysate was diluted 3-fold with 50 mM Tris-HCl, pH
8.0, 1 mM DTT, and 1 mM sodium orthovanadate.
The lysates were then allowed to incubate at 4 °C for 1 h, with
or without the addition of 5 µl of 12CA5 monoclonal antibody or
CBP80 polyclonal antibody. Following the first incubation, 40 µl
of protein A-Sepharose beads were added to each sample, and the samples were incubated for another hour at 4 °C. The samples were then centrifuged, and the immunoprecipitated pellets were washed four times
with 50 mM Tris-HCl, pH 8.0, 133 mM KCl, 0.33%
Triton X-100, 1 mM DTT, and 1 mM sodium
orthovanadate. The resulting immunoprecipitated pellets were
resuspended in 20 µl of UV cross-linking buffer and were incubated
with [
-32P]GTP and UV cross-linked as described above.
CBP80 antibody for
1 h at room temperature, washed with several changes of TBS and
0.1% Tween 20, and incubated for 30 min at room temperature with sheep
anti-rabbit or sheep anti-mouse horseradish peroxidase-conjugated
antibody (Amersham Pharmacia Biotech) as appropriate. Immunolabeling
was detected by enhanced chemiluminescence (ECL, Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
RESULTS
-32P]GTP into nuclear proteins. The rationale for
this approach was that it would provide a very sensitive assay for
identifying guanine nucleotide-binding activities in the nucleus, in a
manner analogous to the use of phosphorylation assays to identify
growth factor-sensitive phosphosubstrates. Using this assay, we
identified an 18-kDa protein that strongly incorporated
[
-32P]GTP in serum-treated but not serum-starved cells
(see below). We found this activity to be exclusively nuclear and
present in every cell line we examined, including HeLa, PC12, COS-7,
and BHK21 cells, as well as in various mammary epithelial cells. A similar activity was also observed in the yeast Saccharomyces cerevisiae.
-32P]GTP incorporation, from the majority of
contaminating low molecular mass proteins (see "Experimental
Procedures"). These steps also resolved an 80-kDa protein (designated
p80), detected by silver staining, which co-purified with the 18-kDa
activity. This putative protein complex was reminiscent of the nuclear
CBC, as the CBC comprises an 18-kDa nuclear protein, CBP20 (for
cap-binding protein 20), stably complexed with an 80-kDa protein, designated
CBP80. The formation of the CBP20-CBP80 heterodimer enables the CBC to bind a guanine derivative, the 7-methylguanosine cap structure (m7GpppN), on RNAs transcribed by RNA polymerase II
(7-10). The similarities between the 18-kDa nuclear activity and CBP20
(both in complex formation and substrate binding) led us to investigate
whether the CBC was a nuclear target for extracellular signals.
-32P]GTP.
Fig. 1A shows GST-CBP20,
thrombin-cleaved CBP20, and the complexed CBC proteins (His-tagged
CBP20 (9) and CBP80 (7)) as visualized by staining with Coomassie Blue.
Fig. 1B shows that the recombinant CBP20 proteins were all
capable of incorporating [
-32P]GTP in a photoaffinity
labeling assay. This activity was greatly enhanced by the presence of
CBP80 (see lane 5), consistent with previous studies that
have demonstrated that complex formation between CBP20 and CBP80 is
necessary for capped RNA binding. The GST control did not show any
cross-linking to [
-32P]GTP.
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Fig. 1.
Recombinant E. coli-expressed CBP20 incorporates
[ -32P]GTP in a photoaffinity
labeling assay. CBP20 was expressed and purified from E. coli as a GST fusion protein. GST-CBP20 (lanes
1 and 2), free CBP20 (the GST was cleaved with
thrombin) (lanes 3 and 4), and
E. coli-expressed CBC (complex proteins designated as
CBP80* and CBP20*) (lane 5) were then
assayed for their ability to incorporate [
-32P]GTP.
Following UV cross-linking, proteins were separated by 15% SDS-PAGE
and visualized by Coomassie Blue staining (A) and
autoradiography (B).
-32P]GTP could be regulated in response to serum.
BHK21 cells were transiently transfected with a HA-tagged CBP20
construct. Following 40 h of serum starvation, the cells were
stimulated with 25% fetal bovine serum for 1.5 h, and HA-CBP20
was immunoprecipitated from cytosolic and nuclear lysates prepared from
either serum-starved or stimulated cells. The immunoprecipitates were
then assayed for the photocatalyzed incorporation of
[
-32P]GTP into CBP20. HA-CBP20 was present in both the
cytosolic and nuclear fractions (Fig.
2B), and CBP80
co-immunoprecipitated with nuclear localized HA-CBP20 equally well
under conditions of either serum starvation or stimulation (Fig.
2A). The large percentage of HA-CBP20 localized to the
cytosol is presumably the result of its overexpression. Nuclear CBP20
demonstrated a marked serum-dependent incorporation of
[
-32P]GTP (Fig. 2C).
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Fig. 2.
Serum-dependent incorporation of
[ -32P]GTP into recombinant
HA-CBP20 expressed in BHK21 cells. Human CBP20 was cloned by
polymerase chain reaction from HeLa cell cDNA and then subcloned
into the mammalian expression vector pcDNA3 to express a HA-tagged
form of the protein. BHK21 cells were transiently transfected with
HA-CBP20 (using 8 µg of DNA/100-mm plate of BHK21 cells). The
transfected cells were serum-starved for 40 h (
) and then
stimulated with 25% fetal bovine serum (+) for 1.5 h. HA-CBP20
was immunoprecipitated (IP) from cytosolic or nuclear
lysates using 12CA5 monoclonal antibody. Immunoprecipitates were then
assayed for [
-32P]GTP incorporation. Proteins were
separated by 15% SDS-PAGE and transferred to Immobilon for Western
blot analysis and autoradiography. A shows the CBP80 protein
co-immunoprecipitating with HA-CBP20 from the nuclear lysates as
detected by Western blotting using CBP80 antiserum. B is a
Western blot using 12CA5 antibody to detect immunoprecipitated HA-CBP20
from cytosolic and nuclear lysates. The [
-32P]GTP
incorporation corresponding to immunoprecipitated HA-CBP20 is shown in
C.
Given that the m7GpppN RNA cap structure is a known
substrate for the CBC, the stimulated incorporation of
[-32P]GTP into CBP20 may reflect an enhanced ability
of the CBC to bind the cap structure on RNA. To address this issue, we
first examined the relative binding affinities of the CBC for different cap analogs by testing their ability to inhibit the incorporation of
[
-32P]GTP into CBP20. PC12 cell nuclear lysates were
immunoprecipitated with antibodies generated against CBP80
(i.e. the binding partner of CBP20) (7), and the
immunoprecipitates were then assayed for photocatalyzed incorporation
of [
-32P]GTP into CBP20 in the absence and presence of
RNA cap analogs or GTP. CBP20 proteins that co-immunoprecipitated with
CBP80 could be efficiently labeled with [
-32P]GTP.
This activity was strongly inhibited by the addition of low
concentrations of cap analogs to the [
-32P]GTP
cross-linking assay and yielded the following binding specificity: m7GpppG > m7GTP > GpppG > GTP
(Fig. 3A). Indeed, the
m7GpppG analog competed with [
-32P]GTP for
binding to CBP20 ~1000 times more effectively than GTP, suggesting
that the CBC most likely binds RNA, rather than GTP, in cells.
|
We further examined whether the CBC shows a regulated binding to capped
RNAs using a PC12 cell line that stably expresses HA-tagged CBP20.
Following starvation, these cells were stimulated with NGF. HA-CBP20
was then immunoprecipitated from the cytoplasmic and nuclear lysates
and assayed for the incorporation of either [-32P]GTP
(Fig. 3B, upper panel) or
m7G32pppN-capped RNA (lower panel).
Both substrates were incorporated into nuclear HA-CBP20 strictly in a
growth factor-dependent manner. Thus, these findings
indicate that the growth factor-stimulated incorporation of
[
-32P]GTP into CBP20 accurately reflects the
activation of the CBC, such that it is induced to bind
m7GpppN-capped RNAs.
We took further advantage of the high sensitivity of the
[-32P]GTP incorporation assay to examine the abilities
of different growth factors to activate the endogenous CBC. Fig.
4A (left panel) shows the results obtained when HeLa cells were first serum-starved and
then treated with EGF and heregulin (the ligand for the Neu-ErbB2/ErbB3 and Neu-ErbB2/ErbB4 heterodimers (22, 23)). Endogenous CBP20 present in
nuclear lysates from HeLa cells was strongly stimulated to incorporate
[
-32P]GTP by heregulin as well as, to a lesser extent,
by EGF. Similarly, in PC12 cells, endogenous CBP20 present in nuclear
lysates was activated by growth factors (Fig. 4A,
right panel). In this case, the incorporation of
[
-32P]GTP into CBP20 was most strongly stimulated by
NGF (as observed in Fig. 3B), followed by heregulin and then
EGF. Fig. 4B shows that in all cases, the growth
factor-stimulated activation of CBP20 was
dose-dependent.
|
In our initial experiments, the incorporation of
[-32P]GTP into CBP20 was assayed after relatively
short periods of growth factor treatment (~15 min). Although this was
sufficient to detect incorporation of the radiolabeled GTP, more
complete time course experiments indicated that near maximal
incorporation occurred following treatment with growth factors for
1 h. An example for PC12 cells is shown in Fig. 4C. In
this experiment, serum-starved PC12 cells were challenged with 100 ng/ml NGF for increasing time periods, up to 24 h. The results
show that near maximal incorporation of [
-32P]GTP into
CBP20 was observed after ~1 h of growth factor addition and that this
level of incorporation was maintained through 24 h. A similar time
course was obtained when PC12 cells were treated with heregulin (data
not shown).
Nuclear lysates from asynchronously growing cells also contain
activated CBP20, suggesting that the growth factor regulation of the
CBC activity may be associated with a particular phase of the cell
cycle. This is illustrated in Fig.
5A. HeLa cells were arrested
in G0 phase by serum starvation, in G1/S phase
by thymidine addition, and in M phase by nocodazole treatment.
Cytoplasmic and nuclear fractions were then prepared (or a mitotic
pellet was prepared in the case of M phase-arrested cells), and the
resulting lysates were assayed for the ability of CBP20 to incorporate
radiolabeled GTP. We found that CBP20 did not incorporate
[-32P]GTP in cells arrested in either G0
or M phase of the cell cycle. However, CBP20 strongly incorporated
[
-32P]GTP in HeLa cells arrested in G1/S
phase. Thus, the activation of the CBC appears to be sensitive to cell
cycle-dependent as well as growth
factor-dependent regulation.
|
To determine whether the CBC might respond to a broader range of
stimuli, we assayed the ability of CBP20 to incorporate radiolabeled GTP under conditions of cellular stress. PC12 cells were first serum-starved and then exposed to UV radiation for 2 min. Following this exposure, the cells were allowed to recover for 30 min or 1 h, and then endogenous CBP20 was assayed for its ability to incorporate
[-32P]GTP. Fig. 5B shows that CBP20 was
strongly stimulated to incorporate radiolabeled GTP in cells that had
been UV-irradiated. We found a similar stress activation of endogenous
CBP20 in COS-7 and HEK-293 cells (data not shown).
Stress response pathways have been shown to be mediated by the low
molecular mass GTP-binding proteins Cdc42 and Rac and to culminate in
transcriptional activation through the stimulation of the nuclear
mitogen-activated protein kinases JNK1 and p38/HOG1 (3-5, 24). Thus,
we examined whether the transient expression of activated Cdc42 would
result in a growth factor-independent activation of the CBC. The
results in Fig. 5C indicate that this is the case. We found
that the transient expression of either a GTPase-defective Cdc42 mutant
(Cdc42 Q61L) or a transforming Cdc42 mutant that is capable of
undergoing the spontaneous exchange of GTP for GDP (Cdc42F28L) strongly
activated CBP20, whereas expression of wild-type Cdc42 showed no
activation. We also have found that expression of V12-Ras stimulates
the incorporation of [-32P]GTP into CBP20 as well as
activated Rac and RhoA (data not shown), although thus far, Cdc42
appears to be the most effective activator.
Taken together, these data suggest that the ability of the CBC to bind RNA cap structures is a tightly regulated process. Previous work by others has defined a role for CBC binding to capped RNAs in important RNA metabolic processes, including pre-mRNA splicing (7, 13-15), U snRNA export (9), and 3'-end processing (16). The ability of growth factors to stimulate the capped RNA-binding activity of the CBC suggests that those metabolic processes that benefit from the recognition of the RNA cap by the CBC (such as pre-mRNA splicing) will also be subject to extracellular regulation. To test this prediction, splicing extracts were prepared from HeLa cells that were either serum-starved or starved and then stimulated with heregulin for 24 h (i.e. conditions that lead to maximal stimulation of CBP20 activity in nuclear lysates (see Fig. 4B)). Creatine phosphate, ATP, and m7GpppG-capped precursor adenovirus mRNA were added to initiate splicing (see "Experimental Procedures"). Extracts prepared from quiescent cells were not competent to splice the m7GpppG-capped precursor RNA (Fig. 6). However, splicing of the m7GpppG-capped RNA was markedly stimulated in extracts prepared from heregulin-treated cells and was ~5-fold higher than the splicing of a nonspecific ApppG-capped RNA probe by the same extract (data not shown). These results indicate that under conditions where growth factor signaling activates CBC, there is a corresponding stimulation in capped precursor mRNA splicing. Because we also observed some increase in ApppG-capped RNA splicing, the possibility exists that other targets, perhaps acting in conjunction with the CBC, may be important in mediating the observed growth factor effect in cap-dependent RNA splicing. Thus, cap-dependent RNA splicing, in addition to CBC-capped RNA binding, is a functional end point for growth factor-coupled signaling pathways leading to the nucleus.
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DISCUSSION |
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The original goal of these studies was to identify novel nuclear
activities that were susceptible to growth factor regulation to further
our understanding of how growth factors exert their effects in the
nucleus. Using a photoaffinity labeling approach to detect nuclear
proteins that specifically incorporate [-32P]GTP, we
detected an 18-kDa nuclear activity that was highly sensitive to the
addition of growth factors to G0 phase-arrested cells. The
fundamental role of this activity in cell growth regulation is
underscored by its response to growth factors, its specific association
with the G1/S phase of the cell cycle, its activation under
conditions of cell stress, and the fact that we have found this
activity in every cell and tissue type examined thus far. It was
therefore interesting to find that the 18-kDa activity corresponds to
the RNA cap-binding protein CBP20, suggesting a necessity for a
regulated nuclear cap binding event in cell growth control.
The m7G(5')ppp(5')N cap structure on RNAs transcribed by RNA polymerase II has been known for some time to be important for the stability of these RNAs (25, 26) and to facilitate different aspects of RNA metabolism, including translation initiation, pre-mRNA splicing, and nuclear transport. In recent years, CBP20 and its 80-kDa binding partner, CBP80 (collectively termed CBC), have been identified as the protein complex that binds to the cap structure in the nucleus and mediates the cap-dependent enhancement of pre-mRNA splicing and export of U snRNAs (7, 9). To our knowledge, this is the first report describing a regulated binding activity by the CBC and thus implies that RNA metabolic processes ascribed to the CBC will be regulated as well. This is supported by our finding that growth factors regulate the in vitro splicing of precursor mRNA in nuclear lysates from HeLa cells.
An understanding of the signaling processes that lead to CBC activation could shed light on how mitogens influence gene expression by modulating RNA metabolism. All indications are that the CBC may receive inputs from multiple pathways. The Ras-Raf-MEK-extracellular receptor-activated kinase signaling cascade is one pathway that is central to mediating growth factor effects in the nucleus, and we have observed that expression of oncogenic Ras G12V in cells results in an activation of the CBC. Stress-activated signaling pathways also induce CBC activation. There are a number of lines of evidence that indicate that signaling pathways stimulated by Rho-like GTP-binding proteins (e.g. Cdc42 and Rac) both participate in cellular stress responses (4-7, 27) and are under growth factor control (25-27). In fact, we have found that activated forms of Cdc42 give rise to an effective activation of the CBC. Given that Cdc42 has been suggested to input into rapamycin-sensitive pathways involving FRAP (FKB12/rapamycin-associated protein) by activating the p70 S6 kinase (27), it is interesting to consider whether the regulation of the CBC is linked to translational control. The cytosolic mRNA cap-binding protein eIF-4E, which plays a critical role in a number of mRNA translational events (29), is also susceptible to growth factor regulation. The phosphorylation of eIF-4E occurs in response to multiple growth factors (including NGF in PC12 cells) and cell cycle arrest (28, 30) and appears to occur downstream of multiple signaling pathways, including the extracellular receptor-activated kinase, c-Jun N-terminal kinase/stress-activated protein kinase, and p38 kinase pathways (29). In addition to its direct phosphorylation, the activity of eIF-4E is also regulated by two other growth factor-responsive factors, the eIF-4E-binding proteins 4E-BP1 and 4E-BP2 (30), and recently, 4E-BP1 has shown to be phosphorylated by the phosphatidylinositol 3-kinase-related kinase FRAP (31). Thus, it will be interesting to see if the cytosolic cap-binding protein eIF-4E and the CBC are similarly or even coordinately regulated through growth factor-initiated signals.
A growth factor-dependent phosphorylation of CBP20 could have a direct effect on its RNA cap-binding activity (similar to eIF-4E), although thus far, we have not been able to detect a growth factor-stimulated phosphorylation of CBP20 in vivo. The cellular levels of CBP20, its ability to bind CBP80, and its nuclear localization are not affected by growth factor stimulation (see Fig. 2). We are currently examining whether growth factors influence the interactions between the CBC and specific regulatory proteins to stimulate the binding of the CBC to capped RNA in a manner analogous to the growth factor-regulated interaction between eIF-4E and the 4E-BP proteins.
Our demonstration that the CBC is susceptible to extracellular
regulation, in conjunction with the previously defined role for the CBC
in RNA processing, makes the CBC an attractive candidate for
translating growth factor signals into altered gene expression by
affecting the metabolism of specific subsets of RNAs. However, given
that the CBC affects both the processing and transport of RNAs
transcribed by RNA polymerase II, the growth
factor-dependent binding of the CBC to capped RNA may
result in a general regulation of gene expression. The reduced ability
of the CBC to bind capped RNAs in the absence of a growth factor signal
could serve as a checkpoint for cell growth by guarding against the
further processing of inappropriate or "leaky" transcripts. This
suggests that altered levels and/or mutations of the CBC might be
capable of deregulating cell growth. Future studies will be directed
toward determining how growth factors influence different aspects of
RNA processing (including precursor mRNA splicing and RNA export)
through the CBC and how overexpression and/or mutation of the CBC
impacts upon normal cell growth.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM40654 (to R. A. C.) and by Department of Defense Grant DAMD17-97-1-7308 (to K. F. W.).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.
To whom correspondence should be addressed. Tel.:
607-253-3650; Fax: 607-253-3659; E-mail: rac1{at}cornell.edu.
The abbreviations used are:
CBC, cap-binding
complex; NGF, nerve growth factor; EGF, epidermal growth factor; TBS, Tris-buffered saline; DTT, dithiothreitol; AMP-PNP, adenosine
5'-(,
-iminotriphosphate); GMP-PNP, guanosine
5'-(
,
-iminotriphosphate); PAGE, polyacrylamide gel
electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin; eIF, eukaryotic initiation factor; snRNA, small
nucleotide RNA.
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REFERENCES |
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