signaling to the dead box—regulation of dead-box p68 rna helicase by protein phosphorylations
TRANSCRIPT
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Cellular Signalling 17 (
Signaling to the DEAD box—Regulation of DEAD-box p68 RNA
helicase by protein phosphorylations
Liuqing Yang, Chunru Lin, Zhi-Ren Liu*
Department of Biology, Georgia State University, University Plaza, Atlanta, GA 30303, USA
Received 22 January 2005; accepted 4 March 2005
Available online 31 May 2005
Abstract
P68 nuclear RNA helicase is essential for normal cell growth. The protein plays a very important role in cell development and
proliferation. However, the molecular mechanism by which the p68 functions in cell developmental program is not clear. We previously
observed that bacterially expressed his-p68 was phosphorylated at multiple sites including serine/threonine and tyrosine [1] [L. Yang, Z.R.
Liu, Protein Expr. Purif. 35 (2004) 327]. Here we report that p68 RNA helicase is phosphorylated at tyrosine residue(s) in HeLa cells.
Phosphorylation of p68 at threonine or tyrosine residues responds differently to tumor necrosis factor alpha (TNF-a) induced cell signal.
Kinase inhibition and in vitro kinase assays demonstrate that p68 RNA helicase is a cellular target of p38 MAP kinase. Phosphorylation of
p68 affects the ATPase and RNA unwinding activities of the protein. In addition, we demonstrate here that phosphorylation of p68 RNA
helicase controls the function of the protein in the pre-mRNA splicing process. Interestingly, phosphorylation at different amino acid residues
exhibits different regulatory effects. The data suggest that function(s) of p68 RNA helicase may be subjected to the regulation of multiple cell
signal pathways.
D 2005 Elsevier Inc. All rights reserved.
Keywords: DEAD box; RNA helicase; TNF-a; Protein phosphorylation; ATPase
1. Introduction
Modulation of dynamic RNA structures and complex
RNA-protein interactions is essential for every biological
process that is involved in RNA metabolism. It is generally
believed that a super-family of so-called DEAD or DExH
box RNA helicases executes this essential role in the cells
[2–5]. RNA helicases use the energy derived from ATP
hydrolysis to dissociate the RNA–RNA and/or RNA–
protein interactions [6–8]. The DEAD or DExH box RNA
helicases are characterized by a core region of 290–360
amino acids (helicase core) that consist of eight conserved
sequence motifs. The proteins also have variable C- and N-
terminal. RNA helicases are involved in a wide spectrum of
biological processes of RNA metabolism. Thus, an individ-
0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2005.03.008
* Corresponding author.
E-mail address: [email protected] (Z.-R. Liu).
ual RNA helicase must be highly substrate specific both in
time and space in cells to perform its own specific task.
The nuclear p68 RNA helicase is a prototypical DEAD
box family of RNA helicase [9,10]. As an early example of
a cellular RNA helicase, the ATPase and the RNA
unwinding activities of p68 RNA helicase were documented
with the protein that was purified from human 293 cells
[11–13]. P68 RNA helicase plays a very important role in
cell proliferation and development [14,15]. The protein is
expressed in all dividing cells of different vertebrates
[10,15]. The protein shows clear cell cycle-related local-
ization in the nucleus. Related to the role of p68 RNA
helicase in cell proliferation, the protein was shown to play a
critical role in the tumorigenesis process [16–18]. Never-
theless, the biological functions of the protein at a molecular
level are not well understood [19]. It has been suggested that
p68 RNA helicase might be involved in transcription
regulation [20–23] and DNA methylation pathways [24].
Recently, the experiments carried out in our laboratory
2005) 1495 – 1504
L. Yang et al. / Cellular Signalling 17 (2005) 1495–15041496
demonstrated that p68 RNA helicase is an essential human
splicing factor in vitro that plays a role in unwinding the
transient U1 :5V splice site duplex [25,26]. Consistently,
other research laboratories also suggested that p68 RNA
helicase has a functional role in the pre-mRNA splicing
process [27–30].
An interesting question is how the biological function(s)
of p68 RNA helicase is linked to cell growth signals. It was
noted early that p68 can be phosphorylated in vitro by
Protein Kinase C (PKC) [31]. Moreover, Akileswaran and
colleagues found that a kinase anchoring protein AKAP95 is
associated with p68 RNA helicase [32]. The AKAP95
functions as an anchor for cAMP-dependent kinase in the
cell nuclear matrix. These observations strongly argued that
p68 RNA helicase might be a downstream target for
multiple protein kinases. Thus, elucidation of the molecular
mechanism by which the function and activities of p68
RNA helicase are regulated by protein phosphorylation will
provide a link for the role(s) of p68 RNA helicase in cell
development programs.
We previously reported that bacterially expressed p68
RNA helicase is phosphorylated at serine/threonine and
tyrosine residues [1]. Here, we report that cellular p68
RNA helicase is phosphorylated at tyrosine residues.
Phosphorylation of p68 is coupled to TNF-induced signal
pathway. In response to TNF stimuli, p68 change its
phosphorylation status, including decrease in tyrosyl
phosphorylation and increase in threonine phosphorylation.
Phosphorylation affects the biochemical activities of the
protein. Our data also demonstrated that phosphorylations
on serine/threonine or tyrosine residues inhibit each other.
Interestingly, the different phosphorylation exhibits differ-
ent regulatory effects on its function and biochemical
activities. The data suggest that function(s) of p68 RNA
helicase is subject to the regulation of different signal
pathways.
2. Materials and methods
2.1. Recombinant p68 RNA helicase expression
Expression and purification of recombinant p68 and
other recombinant proteins were carried out by the same
experimental procedures as previously described [33,34].
2.2. Dephosphorylation and phosphorylation of p68 RNA
helicase
Dephosphorylation of recombinant p68 RNA helicase or
the immunoprecipitated cellular protein was carried out with
PP2A and/or PTP1B. Approximately 5 Ag of protein was
incubated with 4 U of the phosphatase in manufacture
suggested buffer conditions in total volume of 50 Al at 30 -Cfor 90 min. The reactions were either directly used for
ATPase, RNA unwinding and splicing assays or used for re-
phosphorylation by the procedure described in the next
paragraph.
Protein phosphatase inhibitors, okadaic acid for PP2A
and vanadium for PTP1B, were added to the above
dephosphorylation reactions. PKC or v-Abl kinase, was
added to the reaction mixture. ATP was added to a final
concentration of 100 mM. Additional 10 ACi of [g-32P]ATPwere added for 32P labeling of the proteins as indicated. The
phosphorylation reactions were further incubated at 30 -Cfor 90 min. The re-phosphorylated proteins were directly
used for ATPase, RNA unwinding and splicing assays or
Western blot assays.
2.3. Cell culture and nuclear extracts
HeLa S3 cells were grown in Ham’s F12K medium
supplement with 10% fetal bovine serum, penicillin (100 U/
ml), and streptomycin (100 Ag/ml). The nuclear extracts from
the HeLa cells were made using nuclear extract preparation
kit (Active-motif). In the cases of treatment of cells with
TNF-a, TNF-a (15 ng/ml) were added to cell culture
medium. After the treatment for indicated time, the cells
were immediately harvest for nuclear extracts preparations.
2.4. Immunoprecipitation and Western blot
Immunoprecipitation experiments were performed as
described in previous studies [26,35]. HeLa nuclear extracts
(80 Al) were diluted to 200 Al with NETS buffer (150 mM
NaCl, 50 mM Tris–HCl, pH7.5, 5 mM EDTA, 0.05%
NP40). The antibody PAbp68-rgg was then added to the
mixture. The solution was subsequently incubated at 4 -Cfor 2 h. Protein A agarose beads slurry (40 Al) was then
added and the mixture was rotated at 4 -C for 5 h. The beads
were recovered and washed with 5�600 Al NETS buffer
with 0.08% SDS. Finally, the precipitated proteins were
analyzed by SDS-PAGE followed by Western blot or were
used in a further reaction described in the appropriate text.
The Western-blot analyses were performed with the
commercially available ECL Western-blot and detection kit
(Amersham Biosciences). The supernatant from culture
medium of hybridoma cells P68-rgg were used in the blotting
experiments as 5 : 1 dilution. The polyclonal antibody
PAbp68-rgg was used in 1 :3000 dilution. The antibodies,
PY20, 16B4, and 14B3, were used with 1 :2000 dilution.
2.5. Kinase competition assays
About 5 Ag of recombinant p68 was dephosphorylated
by both PP2A and PTP1B. The dephosphorylated protein
was separated from the added protein phosphatases by Ni-
NTA beads. After extensive washes, the dephosphorylated
protein was eluted with 250 mM imidazole in protein buffer
(50 mM Tris–HCl pH=7.5, 100 mM NaCl, 0.5 mM DTT,
10% glycerol). The protein was then micro-dialysed
against the same protein buffer with 100 mM of imidazole.
L. Yang et al. / Cellular Signalling 17 (2005) 1495–1504 1497
For the kinase competition, 1 /10 of the above dephos-
phorylated p68 was first incubated with one protein
kinase(s) in the presence of 3 mM of non-radioactive
ATP for 60 min at 37 -C. After the pre-incubation, the
reaction mixture underwent micro-dialysis to exchange the
kinase buffer and remove non-radioactive ATP. The second
protein kinase(s) along with 10 ACi of [g-32P]ATP were
added to the reaction mixture. The kinase reaction was
further incubated at 37 -C for 45 min. The reaction
mixture was subsequently analyzed by 10% SDS PAGE
and subjected to auto-radiography. For the kinase com-
petition with the cellular p68 RNA helicase, the cellular
p68 that was immunoprecipitated from 200 Al of HeLa
nuclear extracts was dephosphorylated on the protein A
agarose beads. After extensive washes, the dephosphory-
lated p68 was eluted at 100 mM glycine pH=2.0. The
elution fractions were collected in 500 mM Tris–HCl,
pH=8.5, 100 mM NaCl for immediate neutralization. The
protein was then micro-dialysed against protein buffer, 100
mM Tris–HCl, pH=7.5, 50 mM NaCl, 1 mM DTT.
Subsequent kinase competition assays were essential the
same as that with the recombinant protein.
2.6. ATPase and RNA unwinding assays
ATPase activities were determined by measuring the
released inorganic phosphate during ATP hydrolysis using a
direct colorimetric assay as previously reported [33,36,37].
RNA unwinding activities were determined by the method
similar to that which was described in our previous report
[33].
2.7. In vitro pre-mRNA splicing
Splicing reactions were carried out with pPIP10A in 40%
HeLa nuclear extracts or p68 depleted HeLa nuclear
extracts. p68 RNA helicase was depleted from the nuclear
extracts by the experimental procedure that was described in
our previous report [25]. To reconstitute the splicing
activity, the phosphorylated/dephosphorylated his-tag pro-
tein was added to the p68-depleted nuclear extracts to a final
concentration of ¨20 ng/Al. The mixture was incubated at
30 -C for 15 min under normal in vitro splicing conditions.
About 25 fmol of pre-mRNA pPIP10A was then added to
the 10 Al of pre-incubated extracts and the splicing reaction
was incubated at 30 -C for an additional 150 min. The
splicing products were analyzed by 12% urea-PAGE.
3. Results
3.1. P68 RNA helicase is phosphorylated at tyrosine
residues in HeLa cells
We previously reported that bacterially expressed p68
RNA helicase was serine/threonine and tyrosine phosphory-
lated [1]. We questioned whether the endogenous p68 in
the human cells was also serine/threonine and tyrosine
phosphorylated. To this end, we used human HeLa cells as
an example. HeLa nuclear extracts were freshly made from
cultured HeLa cells. To prevent the dephosphorylation of
the target proteins by endogenous protein phosphatases,
Okadaic acid, inhibitors to PP2A, or phosphotyrosine
phosphatase inhibitor set (Vanadium, Calbiochem) were
added to the nuclear extracts. The cellular p68 RNA
helicase was immuno-precipitated from the HeLa nuclear
extracts by a polyclonal antibody PAbp68-rgg, raised
against bacterially expressed recombinant C-terminal
domain (aa 437–614) of p68. Our Western blot experi-
ments verified that the cellular p68 is the only antigen in
the HeLa nuclear extracts that is recognized by this
antibody (data not shown). The immuno-precipitates were
subsequently subjected to Western blot analyses using
specific antibodies against the phospho amino acids. The
immuno-precipitates from the HeLa nuclear extracts that
were supplemented with Okadaic acid were probed by the
antibodies 16B4 (anti-phosphoserine) or 14B3 (anti-phos-
phothreonine). The immuno-precipitates from the nuclear
extracts that were supplemented with vanadium were
probed by the antibody PY20 (anti-phosphotyrosine). It
is evident that p68 RNA helicase precipitated from the
HeLa nuclear extracts was not recognized by 14B3 and
16B4 (Fig. 1A, C, lane 2). The precipitated protein,
however, was recognized by the antibody PY20 (Fig. 1B).
To further verify that the protein recognized by the
antibody PY20 was identical to p68 RNA helicase, we
carried out another Western blot experiment using a
monoclonal antibody (p68-rgg) raised against bacterially
expressed C-terminal p68 (Fig. 1D). Overlay of these two
Western blots indicated that the protein recognized by
PY20 was identical to p68 RNA helicase. Our previous
experiments demonstrated that the antibody PY20 specif-
ically recognizes tyrosine phosphorylated protein [1].
Thus, our immunoprecipitation and subsequent Western
blot experiments indicated that the cellular p68 RNA
helicase in HeLa cells was tyrosyl phosphorylated. The
precipitated p68 could not be recognized by 14B3 and
16B4 may suggest that p68 is not phosphorylated at the
serine/threonine residues. However, it is also possible that
the antibodies do not recognize all serine/threonine
phosphorylation sites. Phosphorylation of p68 in HeLa
cells was further confirmed by metabolism 32P labeling
(data will be reported elsewhere).
3.2. Phosphorylation of p68 RNA helicase is controlled by
TNF-induced cell signals
In an effort to identify the cell signal pathway(s) that is
responsible for stimulation of the phosphorylations of p68
RNA helicase, we tested the effects of a few growth
hormones and cytokines on p68 phosphorylations. Tumor
necrosis factor (TNF-a) induces a cell signaling pathway
Fig. 2. TNF-a induced cell signals regulate phosphorylation of p68 RNA
helicase. Western blot of cellular p68 RNA helicase from HeLa nuclear
extracts by antibodies, (A) p68-rgg, (B) PY20, and (C) 14B3. P68 RNA
helicase was immunoprecipitated from HeLa nuclear extracts by the
polyclonal antibody PAbp68-rgg. The HeLa nuclear extracts were made
after the cells were treated with 15 ng/ml of TNF-a for indicated time
points.
Fig. 1. Cellular p68 RNA helicase from HeLa cells is phosphorylated at tyrosine residue. Western-blot analyses of cellular p68 RNA helicase by monoclonal
antibodies 16B4 (A), PY20 (B), and 14B3 (C), and p68-rgg (D). The cellular p68 RNA helicase was immunoprecipitated via the polyclonal antibody PAbp68-
rgg. The Western blots were carried out with; 600 ng of BSA treated with PP2A then PKC and v-Abl kinases (lane 1 in A and C), 600 ng of BSA treated with
PTP1B then PKC and v-Abl kinases (lane 1 in D), and the immunoprecipitated p68 was treated with; no treatment (lane 2 in A, C), PP2A then PKC (lane 3 in
A, C), PP2A then v-Abl (lane 4 in A, C), PTP1B (lane 2 in D), PTP1B then PKC (lane 3 in D), and PTP1B then v-Abl (lane 4 in D). The precipitated cellular
p68 RNA helicase was dephosphorylated and/or phosphorylated via appropriate protein kinases or protein phosphatases as indicated prior to the Western blot
analyses. In (B), the p68 was immunoprecipitated via PAbp68-rgg raised from two separate rabbits (8207 and 8209).
L. Yang et al. / Cellular Signalling 17 (2005) 1495–15041498
that simultaneously activates a number of downstream
targets, including a number of protein kinases, e.g. JNK,
p38 MAP kinase [38–40]. Thus, we treated HeLa cells
with TNF-a (Sigma-Aldrich). Nuclear extracts were made
from the TNF-a treated cells. The cellular p68 RNA
helicase was immunoprecipitated from the nuclear extracts
via the polyclonal antibody PAbp68-rgg and subsequent
probed by Western blot using antibodies against specific
phospho amino acids. It was evident that the tyrosine
phosphorylation(s) of p68 responded very quickly to the
TNF-a treatment. The phosphorylation at tyrosine became
completely undetectable if the cells were treated with TNF-
a for 30 min (Fig. 2B, lane 3–5). We next asked if the
serine/threonine phosphorylations of p68 were affected by
TNF-a treatment in HeLa cells. Very similar experiments
were performed. P68 RNA helicase was immunoprecipi-
tated from the HeLa nuclear extracts that were made from
TNF-a treated cells. The precipitated cellular p68 was
subsequently probed with monoclonal antibodies against
phospho-serine or phospho-threonine. Unlike in our
observations made with untreated cells, the Western blots
analyses clearly indicated that cellular p68 RNA helicase
was phosphorylated at threonine residue(s) after the cells
were treated with TNF-a (Fig. 2C, lane 2 and 3), but not
at serine residue(s) (data not shown). Interestingly,
phosphorylation of p68 at threonine residue(s) only
occurred in a very narrow time window of TNF-
a treatments. The phosphorylation(s) was detected at 5
min of TNF-a treatment, reached the maxim at about 30
min, and rapidly decreased thereafter (Fig. 2C, lane 2 and
3, and data not shown). Our data indicated that phosphor-
ylations of p68 RNA helicase were controlled by TNF-a
induced cell signal pathway.
3.3. P68 is phosphorylated at threonine residue(s) by p38
MAP kinase
One important downstream target in the TNF-a induced
signal pathway is the activation of p38 MAP kinase [38,41].
Since we observed the threonine phosphorylation of p68 in
response to the TNF-a treatment, we reasoned that p68
L. Yang et al. / Cellular Signalling 17 (2005) 1495–1504 1499
RNA helicase might be targeted by p38 MAP kinase in
cells. To test this possibility, we utilized a commercially
available p38 MAP kinase specific inhibitor, SB203580
(Sigma-Aldrich). HeLa cells were pre-treated with various
concentrations of SB203580. Following the treatments, the
cells were treated by TNF-a for 30 min. Nuclear extracts
were made from the cells. The cellular p68 RNA helicase
was immunoprecipitated from the nuclear extracts and
examined by Western blots using anti-phosphothreonine
antibody. It was very clear that the threonine phosphor-
ylation of p68 is inhibited by this kinase inhibitor (Fig. 3B,
lane 3, 4, and 5). To confirm phosphorylation of p68 RNA
helicase at threonine by p38 MAP kinase, we carried out
phosphorylation reaction with recombinant p68 using the
same HeLa nuclear extracts. Bacterially expressed recombi-
Fig. 3. P68 RNA helicase is a target of p38 MAP kinase. Western blot of cellular p6
RNA helicase was immunoprecipitated from HeLa nuclear extracts by PAbp68-r
different concentrations (indicated) of p38 MAP kinase inhibitor SB205380 follow
RNA helicase (lane 2–4), HCV-NS3 (lane 5), and yeast Saccharomyces cerevisia
untreated (lane 2), treated with TNF-a (15 ng/ml) for 30 min (lane 3–6), and the c
(lane 4). Top panel is Western blot with antibody 14B3 and bottom panel is Cooma
(600 ng) were incubated with corresponding HeLa nuclear extracts (30%) in 30 Aextracts by Ni-NTA beads. The precipitates were separated in 10% SDS-PAGE. T
were indicated on right side. (D) In vitro phosphorylation of; p38 MAP kinase
recombinant p68 (lane 4), recombinant HCV-NS3 (lane 5), and recombinant De
incubated with 0.5 U of p38 MAP kinase in 10 Al reactions in the presence of 4 m
14B3. The middle panel is autoradiography of the SDS-PAGE and bottom panel is
his-p68, his-Ded1p, his-HCV-NS3, and p38 MAP kinase were indicated on right
nant p68 was completely dephosphorylated by PP2A and
PTP1B prior to the addition to the HeLa extracts. It is
evident that the recombinant p68 was phosphorylated at
threonine in the nuclear extracts made from TNF-a treated
cells (Fig. 3C, lane 3). However, under the same conditions,
the recombinant p68 was not phosphorylated if the cells
were pre-treated with p38 MAP kinase inhibitor (Fig. 3C
lane 4). As controls, other recombinant RNA helicases,
HCV-NS3 and yeast Ded1p were not phosphorylated by the
HeLa nuclear extracts made from TNF-a treated HeLa cells
under the same conditions (Fig. 3C, lane 5 and 6). To further
verify phosphorylation of p68 at threonine residue(s) by p38
MAP kinase, we carried out in vitro phosphorylation assay
using a commercially available p38 MAP kinase (Calbio-
chem). Our assays clearly showed that the dephosphorylated
8 RNA helicase from HeLa nuclear extracts by, (A) p68-rgg, (B) 14B3. P68
gg. The HeLa nuclear extracts were made after the cells were treated with
ed by TNF-a treatment for 30 min. (C) Phosphorylation of recombinant; p68
e Ded1p (lane 6) in HeLa nuclear extracts made from HeLa cells that were;
ells were pre-treated with SB203580 (30 AM) prior to the TNF-a treatment
ssie staining of the SDS-PAGE. The dephosphorylated recombinant proteins
l for 60 min. The recombinant proteins were precipitated from the nuclear
he protein bands corresponding to his-p68, his-Ded1p, and his-HCV-NS3
(lane 2), recombinant p68 without addition of p38 MAP kinase (lane 3),
d1p (lane 6) by p38 MAP kinase. Recombinant proteins (¨200 ng) were
M ATP and 3 ACi of [g-32P]ATP. Top panel is Western blot with antibody
Coomassie staining of the SDS-PAGE. The protein bands corresponding to
side.
L. Yang et al. / Cellular Signalling 17 (2005) 1495–15041500
recombinant p68 RNA helicase became phosphorylated at
threonine residue(s) by p38 MAP kinase (Fig. 3D, lane 4).
Under the same conditions, the recombinant HCV-NS3 and
yeast Ded1p were not phosphorylated by p38 MAP kinase
(Fig. 3D, lane 5 and 6). The experiments provided evidence
that p68 RNA helicase is phosphorylated at threonine
residue(s) by p38 MAP kinase in response to TNF-a
induced signal.
3.4. The serine/threonine phosphorylation(s) and tyrosine
phosphorylation(s) inhibit each other
The preceding data demonstrated the phosphorylations of
p68 RNA helicase at serine/threonine and tyrosine residues.
The phosphorylations of the protein were coupled to TNF-a
induced cell signal pathway and genotoxic agent mediated
DNA damage signal. Our next question was whether the
serine/threonine phosphorylations and/or tyrosyl phosphor-
ylation affected each other. To this end, we employed
competition kinase assays. The dephosphorylated recombi-
nant p68 RNA helicase was examined in two kinase
reactions. We examined two kinase reactions with
[g-32P]ATP by PKC or v-Abl kinases under the conditions
that the dephosphorylated protein was first phosphorylated
with non-radioactive ATP by PKC or/and v-Abl kinases
prior to [g-32P]ATP phosphorylations by another protein
Fig. 4. (A) PKC and v-Abl kinase competition assays with recombinant p68 RNA
immunoprecipitated from HeLa nuclear extracts. (C) P38 MAP kinase and v-Abl k
is auto-radiography of the SDS-PAGE. The bottom panel is Coomassie staining of
and p38 MAP kinase were indicated on right side. The dephosphorylated p68 was
the presence of 3 mM of non-radioactive ATP for 60 min at 37 -C (competition). A
along with 10 ACi of [g-32P]ATP were added to the competition reaction mixture. T
reaction). The reaction mixture was subsequently analyzed by 10% SDS-PAGE a
kinase. If dephosphorylated p68 was pre-treated with PKC
and non-radioactive ATP, phosphorylation by v-Abl kinase
was then very weak (Fig. 4A, lane 3). Similarly, If
dephosphorylated p68 was pre-treated with v-Abl kinase
and non-radioactive ATP, phosphorylation by PKC was very
weak (Fig. 4A, lane 5). Similar kinase competition experi-
ments were also carried out with p38 MAP kinase and v-Abl
kinase. Our experiments showed that phosphorylation of
p68 by p38 MAP kinase inhibited the phosphorylation by
v-Abl kinase (Fig. 4C, lane 7). The v-Abl phosphorylation
also inhibited p38 MAPK phosphorylation (Fig. 4C, lane 5).
We also tested the kinase competitions with cellular p68 that
was purified from HeLa nuclear extracts. The cellular p68
from HeLa nuclear extracts was first immobilized on protein
A agarose beads by the polyclonal antibody PAbp68-rgg
and dephosphorylated by PTP1B. The proteins were eluted
and further separated by a gel-filtration. The dephosphor-
ylation reaction went to completion as indicated by Western
blot (data not shown). The kinase competitions similar to
that were used to the recombinant protein were applied here
with the dephosphorylated cellular p68. The results obtained
with the cellular p68 were essentially the same as that
obtained with the recombinant protein (Fig. 4B). The kinase
competition results demonstrated that phosphorylations on
serine/threonine residues inhibited the phosphorylations on
tyrosine residues and vice-versa.
helicase. (B) PKC and v-Abl kinase competition assays with cellular p68
inase competition assays with recombinant p68 RNA helicase. The top panel
the SDS-PAGE. The protein bands corresponding to his-p68, v-Abl kinase,
first incubated with appropriate protein kinase(s) indicated in each panel in
fter the pre-incubation, the second protein kinase(s), indicated in each panel
he kinase reaction was further incubated at 37 -C for another 45 min (kinase
nd subjected to auto-radiography.
L. Yang et al. / Cellular Signalling 17 (2005) 1495–1504 1501
3.5. Phosphorylation affects the ATPase and RNA unwind-
ing activities of p68 RNA helicase
In our previous study, we showed that the recombinant
p68 is an RNA-dependent ATPase and ATP-dependent
RNA helicase. Therefore, we asked whether or not p68
phosphorylation affects the ATPase and RNA unwinding
activities of the protein. We have observed that phosphor-
ylations on tyrosine or serine/threonine residues have
different effects on the ATPase activity of the protein [1].
We next tested the effects of the phosphorylation of p68 on
the RNA unwinding activity of the protein. Since the
bacterially expressed recombinant p68 was phosphorylated
at serine/threonine and tyrosine residues. Thus, the effects of
dephosphorylation at serine/threonine or tyrosine residue(s)
on the RNA unwinding activity of the protein were
monitored. The unwinding substrate RNA used in our study
contains a short RNA duplex (¨22 bp in length) and long
186 nt and 88 nt 3V overhangs on both sides [33]. It is
evident that the recombinant p68 RNA helicase treated with
PTP1B unwound the substrate RNA (Fig. 5, lane 4). The
proteins that were dephosphorylated by PP2A failed to
unwind the substrate (Fig. 5, lane 5). Thus, our experiments
of ATPase and RNA unwinding assays demonstrated that
phosphorylation on p68 RNA helicase control the ATPase
and RNA unwinding activities of the protein.
3.6. Phosphorylations on tyrosine abolish the function of
p68 RNA helicase in the pre-mRNA splicing process
We have previously showed that p68 RNA helicase is an
essential human splicing factor in vitro [25]. Thus, we asked
whether or not the phosphorylation of p68 RNA helicase
affect the function(s) of the protein in the in vitro pre-
mRNA splicing process. The effects of serine/threonine or
tyrosine phosphorylation on the function of p68 RNA
Fig. 5. RNA unwinding activity of p68 RNA helicase is differently affected by p
RNA helicase. dsRNA, 2.5 fmol, unwinding substrate were incubated with 150 ng
60 min. The his-tag p68 was pretreated with; no treatment (lane 3), 1 Al of PTP1B p
1 Al PP2A (lane 6), 1.5 Al of v-Abl kinase was added to PTP1B and PP2A treated h
PKC was added to PTP1B and PP2A treated his-p68 after addition of protein phosp
for 8 min. The sample was loaded on the gel immediately after heat denature. La
helicase in the spliceosome were tested in previously
established in vitro reconstitution system [25]. To obtain
the recombinant p68 with serine/threonine or tyrosine
phosphorylation(s), the recombinant protein was completely
dephosphorylated by PP2A and PTP1B. The dephosphory-
lated protein was then re-phosphorylated by PKC or v-Abl
kinase. The PKC or v-Abl re-phosphorylated p68 RNA
helicase was added back to the p68 depleted HeLa extracts.
The splicing activity of the reconstituted HeLa extracts was
monitored. The splicing substrate pPIP10A (derivative of
major late transcript of adenovirus) was used in the splicing
assays. Our experiments showed that the splicing activity of
the p68 depleted HeLa nuclear extracts was recovered by
addition of dephosphorylated (Fig. 6, lane 7) or serine/
threonine re-phosphorylated p68 (Fig. 6, lane 8). The
splicing activity, however, could not be restored by addition
of the tyrosyl phosphorylated p68 (Fig. 6, lane 9). The
results suggested that tyrosyl phosphorylation of p68
inhibited pre-mRNA splicing process in HeLa nuclear
extracts. To further confirm the effects of p68 on the
splicing activity were indeed due to tyrosyl phosphorylation,
we re-phosphorylated the dephosphorylated his-p68 via
ATP-g-S by Abl kinase. The tyrosyl thio-phosphorylated
his-p68 could not restore the splicing activities of p68
depleted HeLa extracts (Fig. 6, lane 11).
4. Discussion
In this report, we demonstrated that p68 RNA helicase is
phosphorylated at multiple amino acid residues, including
serine/threonine and tyrosine. We presented evidence to
show that protein phosphorylation play a very important
role in regulating the biochemical activities. Protein
phosphorylation is a major mechanism that links the protein
function with various important cell signal pathways, such
hosphorylations. RNA unwinding activities of the recombinant his-tag p68
of the recombinant p68 RNA helicase at 37 -C in the unwinding buffer for
rotein tyrosine phosphatase (lane 4), 1 Al of PP2A (lane 5), 1 Al PTP1B and
is-p68 after addition of protein phosphatase inhibitors (lane 7), and 1.5 Al ofhatase inhibitors (lane 8). Lane 1 is the dsRNA substrate denatured at 95 -C
ne 2 is the dsRNA alone.
Fig. 6. Tyrosyl phosphorylation inhibits the function of p68 in the in vitro
pre-mRNA splicing. Splicing reactions were carried out with pPIP10A in
40% intact HeLa nuclear extracts (lane 2–4) or p68 depleted HeLa nuclear
extracts (lane 5–11) at 30 -C for 150 min. The HeLa nuclear extracts were
supplemented with; no supplements (lane 2), v-Abl kinase (lane 3), and
protein tyrosine phosphatase inhibitor vanadium (lane 4), and the
recombinant his-tag p68 RNA helicase (¨20 ng/Al). The recombinant
p68 RNA helicase was pre-treated with appropriate protein kinases and/or
protein phosphatases as indicated in each lane (lane 5–11) prior to the
addition of p68 to the p68-depleted extracts. The splicing products were
analyzed by 12% urea-denature PAGE and subjected to the autoradiog-
raphy. Lane 1 is the splicing substrate pPIP10A alone.
L. Yang et al. / Cellular Signalling 17 (2005) 1495–15041502
as many growth hormones and cytokines. The function of
p68 RNA helicase has been shown to be critical for cell
normal growth and regulation. Dysregulating the cellular
function of the p68 leads to tumor progression and cell
proliferation [15,16,18,42]. Thus, regulation of the cellular
function(s) of p68 RNA helicase by protein phosphorylation
provides an excellent explanation for the role of p68 in the
cell development program and proliferation. The p68 RNA
helicase is phosphorylated at multiple sites, including
serine/threonine and tyrosine amino acid residues, which
reflect a complex regulation mechanism. P68 seems to be an
essential multifunctional RNA helicase that is required to
unwind RNA structures or dissociate RNA protein inter-
actions in multiple cellular processes. It is conceivable that
different phosphorylation may be employed to regulate the
function(s) of p68 in response to different external stimuli.
Obviously, the physiological importance of phosphory-
lation on p68 RNA helicase has to be established by
identifying the cell signal pathway(s) and the cellular
protein kinase(s) that are responsible for phosphorylation
of p68. Regulation of phosphorylation/dephosphorylation of
p68 by TNF-a signal pathway certainly established one
connection between cell signal pathways and p68 phosphor-
ylation. TNF-a has been shown in regulating cell apoptosis,
proliferation, and survival [38,43]. Given the role played by
p68 RNA helicase in cell developmental programs, it is not
difficult to imagine that p68 is one cellular target that
execute TNF-a signal downstream. Interestingly, phosphor-
ylation at tyrosine residue(s) or phosphorylation on threo-
nine residue(s) was reciprocally regulated by TNF-a
treatment. This opposite regulatory effects correlated very
well with the different effects of the tyrosine or threonine
phosphorylation on the biochemical activities of the protein,
as well as the function of the protein in the spliceosome.
TNF-a stimulated threonine phosphorylation(s) of p68 was
quickly dephosphorylated upon a longer time of TNF-a
treatment, which suggested that TNF-a also activated a
protein phosphatase to dephosphorylate p68 at threonine
residue(s). Decreases and disappearance of tyrosine phos-
phorylation(s) of p68 upon the TNF-a treatment is intrigu-
ing. It is likely that TNF-a treatment activated a protein
tyrosine phosphatase (PTP) that dephosphorylated the
tyrosyl phosphorylation(s). In supporting this conjecture,
we observed that tyrosyl phosphorylation(s) of recombinant
p68 was dephosphorylated in HeLa nuclear extracts that
were made from TNF-a treated HeLa cells, while the
tyrosyl phosphorylation(s) was not dephosphorylated in
HeLa nuclear extracts that were made from untreated HeLa
cells (data not shown). In addition, it is known that TNF-a
induced cell signal inhibits cell proliferation through
activation of a protein tyrosine phosphatase (SHP-1) [44].
Given that HeLa cell is a human cancer cell line, it is
tempted to speculate that tyrosine dephosphorylation and/or
threonine phosphorylation(s) of p68 may be an apoptotic
response to TNF-a treatment.
Protein tyrosine kinase(s) (PTK) that is responsible for
phosphorylation of p68 on the tyrosine residues is an open
question. Since p68 RNA helicase was shown to predom-
inately localize in the cell nuclear [4], it would be expected
that a nuclear PTK phosphorylates p68 RNA helicase in
cells. The c-Abl kinase is a protein tyrosine kinase that
localizes to both cell nuclear and cytoplasm [45]. The kinase
is a regulator of the DNA damage response system [46,47].
We showed that recombinant p68 could be phosphorylated
by bacterially expressed truncated form of MuLV v-Abl
kinase and c-Abl. Cellular p68 purified by immunoprecipi-
tation from HeLa nuclear extracts could also be phosphory-
lated by the Abl kinases. Moreover, we observed that c-Abl
kinase co-immunoprecipitated with p68 in an immunopre-
cipitation experiment with nuclear extracts made from A549
and Caco-2 cells (data will be reported elsewhere). Thus, c-
Abl kinase is likely a candidate PTK that phosphorylates
p68 RNA helicase on tyrosine residues. It is also possible
that p68 RNA helicase may be a target of protein tyrosine
kinases that are Fmis_-localized to the cell nuclear. It has
L. Yang et al. / Cellular Signalling 17 (2005) 1495–1504 1503
been demonstrated that members of the src family of PTK,
the intracellular tyrosine kinase BRK/Sik, and the receptor
tyrosine kinases (RTK) can change their localization
patterns in response to external stimuli [48–50]. Thus, all
these protein tyrosine kinases are all potential candidates
that phosphorylate p68 RNA helicase.
Which cellular serine/threonine protein kinase that
directly targets p68 under the treatment of TNF-a is also
an intrigue question. We identified p68 RNA helicase as a
target of p38 MAP kinase in response to the TNF-a
treatment, which further confirm the role of p68 in TNF-a
induced signal pathway. P38 MAP kinase usually target
transcription factors or other protein kinases [51–54]. Thus,
phosphorylation of p68 by p38 MAP kinase seems
consistent with the potential role of p68 RNA helicase in
transcription activation [20–22]. The p68 RNA helicase has
an IQ motif, a potential PKC phosphorylation site, in its C-
terminal domain. Both the protein purified from rat PC12
cells and the recombinant protein expressed in E. coli can be
phosphorylated by PKC in vitro [31,34]. In addition, p68
RNA helicase was shown to associate with PKC in the
nuclear matrix of the neuroblastoma cells [55]. All these
observations seem to argue that p68 is a substrate of PKC.
Nevertheless, solid evidence to demonstrate the phosphor-
ylation of p68 RNA helicase by PKC in cells is not
presented. P68 RNA helicase was also shown to associate
with a cAMP-dependent kinase (PKA) anchoring protein in
HEK293 and COS cells [32]. Thus, it is also possible that
p68 may be phosphorylated by cAMP-dependent protein
kinase. We reason that phosphorylation(s) of p68 by PKC,
PKA, may be cell/tissue type specific. Recently, we indeed
observed that p68 RNA helicase that was immunoprecipi-
tated from nuclear extracts made from normal human lung
tissue and MCF-7 cells was threonine phosphorylated (data
will be reported elsewhere). We noted that, in contrast to our
observation, p68 RNA helicase was observed as an
unphosphorylated form in HeLa cells by metabolic inor-
ganic 32P labeling the cells [56]. We do not know the
experimental conditions for labeling and detection of protein
phosphorylation used by Uhlmann-Schiffler and co-work-
ers. It is, however, possible that not all phospho proteins in
the cells were detected by the metabolic labeling.
P68 RNA helicase was shown to function in the pre-
mRNA splicing process [25]. In this report, we further
demonstrated that the function of p68 in the spliceosome is
regulated by protein tyrosine phosphorylation. The pre-
mRNA splicing process is an essential step in the eukaryotic
gene expression pathway [57–59]. It is likely that this
process is subjected to regulation by a number of cell
growth signals [57]. Tyrosine phosphorylation is involved in
major signal pathways that regulate the cell growth and
differentiation [60,61]. It has been shown that both pre-
mRNA splicing and mRNA transport are regulated by the
tyrosine kinase activity of src [62]. Therefore, controlling
the pre-mRNA splicing by tyrosine phosphorylation of p68
RNA helicase may represent a critical regulatory point for
this important process in the eukaryotic gene expression
pathway. In our previous report, we showed that p68 RNA
helicase functioned at the transient U1:5Vss duplex [25,26].
We suggested that p68 may unwind the U1:5Vss duplex
during the spliceosome assembly process. In this report, we
demonstrated that the tyrosyl phosphorylated p68 RNA
helicase lost its dsRNA-dependent ATPase and RNA
unwinding activities and the tyrosyl phosphorylated p68
do not support splicing. Although, the bacterially expressed
his-p68 without the treatment of protein phosphatases could
not restore the splicing activity of p68 depleted extracts
most of the time (data not shown), curiously, we repeatedly
observed that the his-p68 without dephosphorylation some-
times could partially restore the splicing activity of the p68
depleted extracts (Fig. 5, lane 6). Interestingly, when the
untreated recombinant his-p68 could recover the splicing
activity, the same recombinant protein often also demon-
strated RNA unwinding activity (data not shown). Our
explanation is that a fraction of his-p68 occasionally was not
completely phosphorylated at tyrosine residue(s) by E. coli.
Thus, the tyrosyl unphosphorylated his-p68 unwound RNA
and supported splicing.
Interestingly, the cellular p68 RNA helicase is tyrosine
phosphorylated in HeLa cells. Nevertheless, the pre-mRNA
splicing process is not inhibited in the intact HeLa nuclear
extracts. The tyrosine phosphorylated recombinant protein,
however, did not restore the splicing activity of p68 depleted
extracts. It is possible that only a portion of cellular p68 is
tyrosyl phosphorylated. Another possibility is that the
tyrosine phosphorylation on the endogenous p68 is dephos-
phorylated by a protein tyrosine phosphatase that is
associated with the spliceosome. Alternatively, the tyrosine
phosphorylations on the cellular p68 are dephosphorylated
before the protein is assembled into the spliceosome
complexes. The tyrosine phosphorylation on the recombi-
nant p68 is somehow not dephosphorylated by the protein
tyrosine phosphatase. In this regard, it will be interesting to
probe the phosphorylation status of endogenous p68 in the
spliceosome complexes. In addition, we cannot rule out
another possibility that the tyrosine phosphorylation sites by
v-Abl kinase on the recombinant protein differ from that of
endogenous protein in the HeLa cells. Currently, we are
mapping the tyrosine phosphorylation site(s) of the cellular
p68 from the HeLa cells and the bacterially expressed
recombinant p68 RNA helicase.
Acknowledgments
We thank Roger Bridgeman for antibody p68-rgg
production. We are grateful to Jenny Yang, Phang C. Tai,
April Ellis, Heena Dey, Amit Khanna, and Shubhalaxmi
Kayarthodi for detailed critical comments on the manu-
script. This work is supported in part by research grants
from National Institute of Health (GM063874) and Georgia
Cancer Coalition to ZRL.
L. Yang et al. / Cellular Signalling 17 (2005) 1495–15041504
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