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J Biol Chem, Vol. 273, Issue 46, 30061-30064, November 13, 1998
From the A novel ribosomal S6 kinase, termed
p70 S6 kinase p70 S6 kinase was identified as an insulin/mitogen-activated
protein kinase in mammalian cells, whose major known substrate is the
40 S ribosomal subunit protein S6 (1-4). Two isoforms of the enzyme,
denoted p70 In addition to the role of p70 Construction and Screening of a HEK293 Uni-ZAP Library and DNA
Sequencing Analysis--
Total RNA was isolated from HEK293 cells as
described (10), and poly(A)+ mRNA was selected by using
the Dynabeads mRNA purification kit (Dynal). An oligo(dT)-primed
library was constructed in UNI-ZAP XR vector from 5 µg of HEK293
mRNA, using the Uni-ZAP cDNA synthesis kit (Stratagene).
Packing into phages was carried out by using Gigapack III Gold
Packaging extracts (Stratagene). The human EST cDNA clones were
obtained from the UK HGMP Resource Center. cDNA encoding p70 Northern Blot Analysis--
Membranes containing
poly(A)+ RNA samples from various human tissues and human
Construction of Plasmids and Expression of GST Fusion
Protein--
The full-length coding sequence corresponding to p70 Cell Cultures and Transient Expression Analysis--
CHO cells
stably overexpressing human insulin receptors (CHO-IR) and HEK293 cells
were maintained and cultured as described earlier (11) in Ham's F-12
medium or Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, respectively. Cells were transfected with plasmids
containing p70 Antibodies, Immunoprecipitation, Immunoblot, and p70 S6 Kinase
Assay--
The anti-phosphopeptide antibody against proline-directed
site Ser434 of p70
Cell lysis and immunoprecipitation were carried out as described
previously (11). Immunoblotting was performed using the ECL method
according to the manufacturer's protocol (Amersham Pharmacia Biotech).
p70 S6 kinase activity was determined in the immunoprecipitates by
using 40 S ribosomal subunit as substrate as described earlier (11).
To detect possible p70 S6 kinase isoforms, we carried out
immunoblot analysis of chromatographic fractions of lysates of
serum-treated HEK293 cells that had been treated with or without
rapamycin. We employed for immunoblotting the anti-pS434 Ab, because
Ser434 and its surrounding amino acid residues are highly
conserved both in mammalian p70 Peptide sequences surrounding the proline-directed site
Ser434 of human p70 It is known that the two isoforms of the p70 Northern blot analysis of human tissues revealed a single 2.2-kb
transcript for p70
COMMUNICATION
Molecular Cloning and Characterization of a Novel p70 S6
Kinase, p70 S6 Kinase 
Containing a Proline-rich Region*
§¶,
,,,, and
Ludwig Institute for Cancer Research,
London W1P 8BY and the Department of Biochemistry and Molecular
Biology, University College London, London WC1E 6BT, United Kingdom,
the § Institute of Molecular Biology and Genetics, Kyiv 143, Ukraine, and the Biosignal Research Center, Kobe University,
Kobe 657-8501, Japan
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
(p70), which has a highly conserved amino acid
sequence compared with that of p70/p85 S6 kinase (p70
) within the
catalytic, kinase extension, and autoinhibitory pseudosubstrate
domains, was identified. However, the amino acid sequence of p70
differs from that of p70 in the noncatalytic amino-terminal region
and in the carboxyl-terminal tail, which contains a proline-rich
region. The majority of the regulatory phosphorylation sites identified
in p70 are conserved in p70. Two isoforms of p70, referred to
as 1 (495 amino acids) and 2 (482 amino acids), could be
expressed from the single gene either by alternative mRNA splicing
or by the use of alternative start codons. Here we report the
characterization of p702. Similarly to p70, the catalytic
activity of p70 toward ribosomal protein S6 could be rapidly
activated by serum, insulin, and phorbol ester in transiently
transfected cells. The p70 kinase was found to be significantly less
sensitive to wortmannin and rapamycin than p70. These results
indicate that p70 has the potential to participate in the regulation
of protein synthesis and the cell cycle.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
1 and p702 (p70 S6 kinases 1 and 2), are known
to be generated from a single gene by alternative mRNA splicing and
the use of alternative translational start sites (5). The 525-amino
acid p701 isoform differs from the 502-amino acid p702 isoform
only at the amino terminus (5). p701 is known as p85 S6 kinase
because of its reduced mobility when analyzed by
SDS-PAGE.1 The 23-amino acid
extension at the amino terminus in p701 contains a nuclear
localization signal that constitutively targets this isoform to the
nucleus, whereas p702 appears to be expressed exclusively in the
cytoplasm (6, 7).
in protein synthesis, it has been
shown that p70 S6 kinase is required during the G1 phase of
the cell cycle (6, 8). In these experiments, neutralizing antibodies
against p70 were shown to prevent the serum-induced entry of cells
into S phase. However, in a recent report on targeted disruption of the
p70 gene in murine embryonic stem cells, it was demonstrated that
p70
/ cells still proliferate at a rate slower than
the parental cells (9). These results suggest that p70 has a
positive influence on cell proliferation but that the disruption of
this gene is not lethal. In the present study, using immunoblot
analysis with the anti-phosphopeptide antibody against the
(Ser/Thr)-Pro motif in the autoinhibitory pseudosubstrate domain of
p70, several novel immunoreactive bands were found in the fractions
of HEK293 cells separated by an anion exchange column chromatography.
These observations suggested to us that isoforms of p70 S6 kinase,
other than p70, exist and prompted a search of the expressed
sequence tag (EST) data base that revealed potentially novel isoforms
of p70 S6 kinase. Here we report the identification and
characterization of a novel isoform of p70 S6 kinase, designated p70 S6
kinase (p70).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
was
isolated by screening of 1 × 106 primary phages from
the HEK293 Uni-ZAP library with a 32P-labeled 0.65-kb
EcoRI/NotI fragment derived from the human EST clone AA410355. Positive cDNA clones were isolated and rescued as
Bluescript plasmids by in vivo excision (Stratagene).
Sequencing analysis of selected clones were performed on an Applied
Biosystem 373A DNA sequencer. As a result, we obtained a cDNA
clone, designated clone 53, which contains a full-length coding
sequence of human p70.
-actin cDNA probe were purchased from
CLONTECH. The following probes were used for the
detection of p70 and p70 messages: (i) a 476-bp
HindIII fragment spanning 56 bp of the 3' end coding region
and 420 bp of the 3' noncoding region of the human p70 (EST clone,
AA425599) and (ii) a 650-bp fragment spanning 518 bp upstream of the
stop codon and about 130 bp of noncoding region of the human p70
(EST clone, AA410355).
2
(amino acids 14-495 of p701) was amplified by polymerase chain
reaction using the clone 53 as a template and cloned into the
pcDNA1 vector (Invitrogen) in-frame with the amino-terminal FLAG
epitope. The expression vector of rat p701, pcDNA1 FLAG p701,
was constructed previously (11). A DNA fragment encoding the amino
acids 443-495 of p701 was amplified by polymerase chain reaction
and cloned into the pGEX-4T expression vector (Amersham Pharmacia
Biotech). Expression and purification of a GST fusion protein
containing amino acids 443-495 of p701 (GST/p70C fusion protein)
were carried out according to the manufacturer's protocol (Amersham
Pharmacia Biotech).
and p70 inserts using LipofectAMINE under
conditions recommended by the manufacturer (Life Technologies,
Inc.).
1 (anti-pS434 Ab) was purchased from
New England Biolabs. The anti-peptide antibody against the
carboxyl-terminal end of p701 (anti-p70C Ab) was from Santa Cruz.
The anti-FLAG monoclonal M2 antibody was from Eastman Kodak. A
polyclonal antibody against p701 carboxyl-terminal end (anti-p70C
Ab) was raised by immunizing rabbits with the GST/p70C fusion
protein as an antigen. Immunoreactive sera were affinity-purified on an
Affi-Gel matrix containing the GST/p70C fusion protein.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
and in Drosophila p70
(12, 13). Among fractions of serum-treated HEK293 cells separated using
an anion exchange column, we detected several immunoreactive bands that were recognized by immunoblotting with the anti-pS434 Ab in a rapamycin-sensitive manner but were not detected with the anti-p70C Ab (data not shown). These data indicated that p70 S6 kinase isoforms may exist in which the Ser434 site is conserved but that do
not have a sequence homologous to the carboxyl-terminal end of p70.
These results prompted us to search for sequences that could encode
mammalian isoforms of p70 S6 kinase in the EST data bases.
1 were used to search the EST data
bases. Extensive analysis of EST clones revealed two nearly identical
human EST cDNA clones (AA284234 and AA410355) that were highly
homologous but not identical to the p70 sequences. Therefore, the
full-length cDNA clone corresponding to the identified EST clones
was isolated from a library of HEK293 cells using the insert of EST
clone AA410355 as a probe. Among 12 isolated overlapping clones, one
clone (clone 53) was found to contain an open reading frame of 495 amino acids. The carboxyl terminus of this clone was identical to the
sequence of EST clone AA410355. The protein encoded by this clone was named p70, because it encodes a protein that is homologous to but
distinct from p70.
, namely p701 and
p702, are expressed from a single gene through alternative mRNA
splicing and the use of alternative translational start sites (5).
Inspection of the sequence of p70 revealed an in-frame AUG codon
close to the 5' end of the cDNA, but this may not be the sole or
preferred translational start site, because the sequence preceding this
AUG lacks a purine at position 
3, which is present just before the
second AUG (starting at amino acid 14) (data not shown). Thus, by
analogy to p70, the novel cDNA may encode two proteins,
designated p701 and p702. If this is the case, the 495-amino acid
p701 would differ from the 482-amino acid p702 only at the amino
terminus (Fig. 1A). A 13-amino
acid extension of the p701 contains a putative nuclear localization
sequence, RGRRAR, which is similar to that found within the 23-amino
acid extension of p701. The overall sequence of p70 is very close to that of p70 with 70% identity and 85% similarity at the protein level and consists of the amino-terminal noncatalytic region, the
catalytic domain, the kinase extension domain, the autoinhibitory pseudosubstrate domain, and the carboxyl-terminal tail. The amino acid
identity to corresponding domains of p70 is 28, 83, 80, 73, and
25%, respectively (Fig. 1B). It has been observed that p70 undergoes a multisite phosphorylation in response to stimulation by insulin or mitogens (3, 4). These multiple phosphorylation sites are
also well conserved in p70 and include: (i) a set of (Ser/Thr)-Pro
motifs clustering in the autoinhibitory pseudosubstrate domain
(Ser423, Ser430, Ser436, and
Ser441 in p701, which correspond to Ser434,
Ser441, Ser447, and Ser452 in
p701) (14, 15); (ii) Ser383 and Thr401
located in the kinase extension domain, which correspond to
Ser394 and Thr412 in p701 (16-18); and
(iii) Thr241 located in the catalytic loop, which
corresponds to Thr252 in p701 (16, 17). The major
differences in the amino acid sequence between p70 and p70 are
located in the amino-terminal noncatalytic region (28% identity and
45% similarity) and in the carboxyl-terminal tail (25% identity and
38% similarity). Within the amino-terminal noncatalytic region,
however, acidic residues are well conserved between amino acids 19-36
of p701 and the corresponding amino acids 29-46 of p701 (hence,
this is called the "acidic region"). A unique feature of the
carboxyl-terminal tail of p70 is the existence of a proline-rich
region, which might be involved in interactions with SH3
domain-containing molecules.
View larger version (42K):
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Fig. 1.
Comparison of p70 1 with p701.
A, the deduced amino acid sequences of human p701
(accession number M60724) (5) and p701 are aligned. Amino acids are
numbered. Conserved residues are boxed. B, the
schematic structures of human p701 and p701 are shown. p702
and p702 start from amino acid 24 of p701 and amino acid 14 of
p701, respectively.
, whereas p70 probe specifically hybridized to
two transcripts of 3.4 and 7.4 kb (Fig.
2). The expression patterns of p70 and
p70 transcripts are remarkably similar, showing ubiquitous
expression in all tissues examined. We have made a mammalian expression
construct (FLAG-tag/p702) that allows us to examine the expression
and subsequent characterization of p70 kinase in vivo and
in vitro. As shown in the left panel of Fig.
3A, FLAG-tagged constructs of
p702 (lane 3) and p701 (lane 2) were
expressed in HEK293 cells as a 60- and a 85-kDa protein, respectively.
Both polypeptides were effectively immunoprecipitated with the
anti-FLAG antibody (data not shown). To facilitate the characterization
of the p70, two types of polyclonal antibodies against the
carboxyl-terminal peptide and the GST/p70C fusion protein were
generated. As shown in the right panel of Fig.
3A, immunoprecipitation with the p70C Ab revealed
specific recognition of p702 but not p701. The polyclonal
antibody against the carboxyl-terminal peptide recognized p702 but
did not exhibit cross-reactivity toward p701, either (data not
shown).
View larger version (51K):
[in a new window]
Fig. 2.
Northern blot analysis in human tissues.
Membranes containing poly(A)+ RNA samples from human
tissues were hybridized with cDNA fragments of p70 (upper
panel), p70 (middle panel), and -actin
(lower panel) labeled by random priming, respectively.
View larger version (24K):
[in a new window]
Fig. 3.
Expression of p70 and stimulation of S6
kinase activity by agonists. A, HEK293 cells were
transfected with mock (c, lane 1), pcDNA1
FLAG p701 (, lane 2), or pcDNA1 FLAG
p702 (, lane 3). After 48 h,
transfected cells were frozen in liquid nitrogen and lysed. Aliquots of
the lysates were subjected to SDS-PAGE and immunoblot with the
anti-FLAG antibody as the first antibody (left panel).
Aliquots of the lysates were also subjected to immunoprecipitation with
p70C Ab, SDS-PAGE, and immunoblot with the anti-FLAG antibody as the
first antibody (right panel). Positions of p701
(p70 ) and p702 (p70 ) are shown by
arrows. B, CHO-IR cells were transfected with
mock (lane 1), pcDNA1 FLAG p701 (lanes
2 and 3), or pcDNA1 FLAG p702 (lanes
4-7). After serum starvation for 16 h, cells were treated
with vehicle (lanes 1, 2, and 4),
107 M insulin for 10 min (lanes 3 and 5), 15% serum for 10 min (lane 6), or 500 nM 12-O-tetradecanoylphorbol-13-acetate for 30 min (lane 7) at 37 °C. After cell lysis and subsequent
immunoprecipitation with the anti-FLAG antibody, immunoprecipitates
were subjected to p70 S6 kinase assay using 40S subunit as substrate.
The samples were separated by SDS-PAGE and transferred onto a membrane.
The membrane was analyzed by autoradiography (upper panel)
and then immunoblotted with the anti-FLAG antibody as the first
antibody (lower panel). 32P incorporation into
S6 quantified by BAS2000 in arbitrary units (phosphoimager units × 103): lane 1, 1.70; lane 2,
21.6; lane 3, 72.7; lane 4, 21.3; lane
5, 58.2; lane 6, 69.8; lane 7, 40.7. The
positions of S6 (S6-P), p701 (p70 ), and
p702 (p70) are shown by arrows.
To study the S6 kinase activity of p702 and p701 in response to
various stimuli, both proteins were transiently expressed in CHO-IR
cells. After various treatments of the transfected cells, p702 and
p701 were immunoprecipitated with the anti-FLAG antibody, and
in vitro kinase activities toward S6 protein of 40S subunit were measured. Almost equal amounts of p701 and p702 were found to be expressed in transfected cells (Fig. 3B, lower
panel). Results shown in Fig. 3B (upper
panel) demonstrated that p701 kinase activity is activated
3.5-fold by 107 M insulin for 10 min
(lane 3), whereas the kinase activity of p702 is
activated 2.8-fold by the same insulin treatment (lane 5).
In addition, serum, 12-O-tetradecanoylphorbol-13-acetate
(Fig. 3B, lanes 6 and 7), and
platelet-derived growth factor (data not shown) were all able to
activate p70 kinase activities toward S6 protein.
Because the kinase activity of p70 has been shown to be sensitive to
wortmannin (16, 19) and rapamycin (20, 21) in vivo, the
effects of those inhibitors on the p70 kinase activity were
examined. p701 and p702 were expressed transiently in HEK293 cells, which were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and then treated with various concentrations of rapamycin and wortmannin. Both serum-activated p701 and p702 kinase activities were inhibited by rapamycin and
wortmannin in a dose-dependent manner (Fig.
4, A and B,
upper panel). However, it appears that p702 activity is
less sensitive to rapamycin and wortmannin than p701. The extent of
inhibition of p701 compared with that of p702 were as follows:
92% versus 46% by 20 nM rapamycin; 98%
versus 62% by 200 nM rapamycin; 86% versus 62% by 100 nM wortmannin; and 97%
versus 75% by 1000 nM wortmannin.
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The present results suggest that p70 kinase may be activated in
response to mitogens in vivo through a multisite
phosphorylation mechanism similar to that of p70, which is regulated
by upstream signals dependent on PI 3-kinase and mTOR (mammalian target
of rapamycin). However, unexpectedly, the potency of inhibition by wortmannin and rapamycin for p70 kinase was significantly lower than
that for p70 kinase. This suggests that some mechanisms other than
PI 3-kinase- and mTOR-dependent inputs may regulate p70
activation. For example, there may be unique phosphorylation sites on
p70, which are regulated by unknown mechanisms. Alternatively, other
mechanisms independent of phosphorylation, such as those based on
protein-protein interactions, may exist. One possible mechanism is the
regulation of p70 kinase by the protein-protein interaction via its
proline-rich region in the carboxyl-terminal tail, which may interact
with SH3 domain(s). Thus, we constructed and expressed a p70 mutant
that lacks the carboxyl-terminal tail (amino acids 442-495 of p70)
containing the proline-rich region. However, the potency of inhibition
by wortmannin and rapamycin for the mutant p70 kinase activity was
almost equal to that for the wild-type p70 kinase activity (data not
shown). These results indicated that the proline-rich region is not
sufficient for the difference in the drug sensitivities between p70
and p70. Despite these results, the binding of proteins containing
SH3 domain(s) to the proline-rich region of p70 may play a role in
transmitting signals in the p70-mediated signaling pathway.
Experiments to determine whether SH3 domain-containing proteins bind to
p70 are currently in progress.
In conclusion, we have identified a novel isoform of p70 S6 kinase,
named p70. Further studies are necessary to clarify the regulation
and the role of p70 in the control of protein synthesis and the cell cycle.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Yasutomi Nishizuka for encouragement. We thank Dr. Ushio Kikkawa and Dr. Khatereh Ahmadi for critical reading of the manuscript and Mika Kusu for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan, the Juvenile Diabetes Foundation International, the Sankyo Foundation of Life Science, the Kato Memorial Bioscience Foundation, and the Japan Foundation for Applied Enzymology.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016869.
¶ Visiting professor of the Biosignal Research Center, Kobe University, supported by the Ministry of Education, Science, Sports and Culture of Japan. To whom correspondence may be addressed: Ludwig Inst. for Cancer Research, 91 Riding Horse St., London, W1P 8BY UK. E-mail: ivan{at}ludwig.ucl.ac.uk.
** Supported by a fellowship from the International Union Against Cancer.
To whom correspondence may be addressed: Biosignal Research
Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. E-mail: yonezawa{at}kobe-u.ac.jp.
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; GST, glutathione S-transferase; Ab, antibody; PI, phosphoinositide; kb, kilobase pair(s); bp, base pair(s); CHO, Chinese hamster ovary.
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REFERENCES |
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References
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