Receptor association and tyrosine phosphorylation
of S6 kinases
Heike Rebholz
1,2
, Ganna Panasyuk
3
, Timothy Fenton
1,2
, Ivan Nemazanyy
3
, Taras Valovka
4
,
Marc Flajolet
5
, Lars Ronnstrand
6
, Len Stephens
7
, Andrew West
7
and Ivan T. Gout
2,3
1 Ludwig Institute for Cancer Research, London, UK
2 Department of Biochemistry and Molecular Biology, University College London, UK
3 The Institute of Molecular Biology and Genetics, Kyiv, Ukraine
4 Institute of Veterinary Biochemistry and Molecular Biology, University Zurich, Switzerland
5 Rockefeller University, New York, NY, USA
6 Lund University, Department of Experimental Clinical Chemistry, Malmo, Sweden
7 Babraham Institute, Cambridge, UK
8 GlaxoSmithKline, Harlow, UK

in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or
src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction
in tyrosine phosphorylation of S6K. In addition, we mapped the sites of
tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively.
Mutational and immunofluorescent analysis indicated that phosphorylation
of S6Ks at these sites does not affect their activity or subcellular localiza-
tion. Our data indicate that S6 kinase is recruited into a complex with
RTKs and src and becomes phosphorylated on tyrosine ⁄ s in response to
PDGF or serum.
Abbreviations
btk, Bruton’s tyrosine kinase; CSFR, colony stimulating factor receptor; DBS, donor bovine serum; DMEM, Dulbecco’s modified Eagle’s
medium; FBS, fetal bovine serum; FITC, fluoroscein isothiocyanate; HGFR, hepatocyte-growth factor receptor; PDGF, platelet-derived
growth factor; PDGFR, platelet-derived growth factor receptor; PDK1, 3-phosphoinositide-dependent protein kinase-1; PH, pleckstrin
homology; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKB ⁄ Akt, protein kinase B; PKC, protein kinase
C; PTB, phosphotyrosine binding domain; RTK, receptor tyrosine kinase; S6K, ribosomal protein S6 kinase; SH2, Src homology 2.
FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2023
Ribosomal protein S6 kinase (S6K) is a serine ⁄ threon-
ine kinase belonging to the family of AGC kinases,
which includes protein kinase A (PKA), protein kinase
B (PKB ⁄ Akt), protein kinase C (PKCs), p90 ribosomal
S6 kinase and 3-phosphoinositide-dependent protein
kinase-1 (PDK1). AGC kinases share a high homology
in their kinase domains and have a similar mode of
activation [1].
There are two isoforms of S6 kinase, S6K1 and 2.
Both have highly homologous kinase and kinase
extension domains flanked by the less conserved
N- and C-terminal regulatory regions which are
responsible for their differential regulation [2,3]. S6K1
and S6K2 have cytoplasmic and nuclear isoforms,

been found in a complex with S6Ks [9,10]. PP2A has
further been shown to be the major phosphatase
responsible for the dephosphorylation and inactivation
of S6K [11] and its activity is stimulated upon inhibi-
tion of mTor [12].
The main known physiological substrate of S6 kin-
ases is the 40S ribosomal protein S6. Several other
in vitro and in vivo substrates have been recently identi-
fied, including pro-apoptotic protein Bad1 [13], cyto-
skeletal protein neurabin [14] and transcriptional
activator CREM [15].
Knockout studies in mice and Drosophila provided
evidence that S6K is an important regulator of cell size
and growth [16,17]. In S6K2(– ⁄ –) cells S6 phosphoryla-
tion is strongly reduced whereas in S6K1(– ⁄ –) almost
no reduction can be observed. This finding indicates
that S6 protein is not the major substrate for S6K1
in vivo as it cannot compensate for the lack of S6K2.
Hence, it is possible to imagine that S6K1 exerts some
effects via other substrates. It is also plausible that
changes in subcellular localization bring S6K in
contact with different substrates. Indeed, we have
shown that PKC-mediated phosphorylation of S6K2
at S486 leads to a retention of the kinase in the cyto-
plasm [2].
Here we report, for the first time, that both isoforms
of S6 kinase, S6K1 and S6K2, are associated with
RTKs and recruited to membrane ruffles upon growth
factor stimulation. Furthermore, we have shown that
S6Ks become phosphorylated on tyrosine in response

2024 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS
CSFR (colony stimulating factor receptor) were coex-
pressed. The cytoplasmic tyrosine kinase fyn induced
tyrosine phosphorylation of S6K2 but not S6K1
(Fig. 1A).
Next, we investigated tyrosine phosphorylation of
S6Ks in an in vitro kinase assay with a panel of recom-
binant tyrosine kinases. As PDGFRb induced a strong
phosphotyrosine signal for S6K in insect cells, we tes-
ted this receptor for the ability of its kinase domain to
phosphorylate S6Ks in vitro. As shown in Fig. 1B,
recombinant PDGFRb kinase domain phosphorylated
both S6Ks. We further tested a panel of nonreceptor
tyrosine kinases, including src and fyn, Bruton’s tyro-
sine kinase (btk) and syk in an in vitro kinase assay
using S6K1 and 2 as substrates. As shown in Fig. 1C,
all tested tyrosine kinases, in particular src, phosphor-
ylated both isoforms of S6K in vitro. When tyrosine
kinases were not present in the assay, autophosphory-
lation of S6Ks was hardly detectable. Src kinase and
S6K2 both migrate at 60 kDa in a SDS ⁄ PAGE gel.
Therefore, both autoradiography signals are merged in
the S6K2 sample treated with src. However, when
S6K1 is treated with src, the src autophosphorylation
signal is low in our experiment. For this reason, the
autoradiography signal from the S6K2 plus src sample
should stem mainly from S6K2 phosphorylation.
S6Ks are tyrosine phosphorylated and associated
with receptor tyrosine kinases upon growth
factor stimulation

with PDGFR as kinase for 30 min at 30 °C. 100 ng of PDGFR were used per sample. An autoradiograph and the Coomassie-stained gel are
shown. (C) In vitro tyrosine phosphorylation of S6K by cytosolic tyrosine kinases. P70S6Ks were immunoprecipitated from Sf9 cells (using
anti-EE IgG), then subjected to an in vitro tyrosine kinase assay for 30min at 30 °C. Per sample, 7 pmol of the different tyrosine kinases src,
lyn, syk and btk were used. Autoradiograph and the Coomassie-stained gel are shown.
H. Rebholz et al. S6K tyrosine phosphorylation
FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2025
starved. This activation may be sufficient for S6K
recruitment but not for maximal S6K activation and
tyrosine phosphorylation. The anti-S6K western blot
confirms this hypothesis as in this system S6K is parti-
ally active in starved cells as indicated by the partial
bandshift, with most S6K being the bottom inactive
S6K. With PDGF there is a stronger band shift which
is decreased again after 180 min of stimulation. In a
control experiment it was established that PDGFR,
when expressed alone, does not precipitate with Pro-
tein A Sepharose beads coupled with anti-EE IgG
(data not shown, or also see Fig. S1).
To strengthen our observation, we tested whether
endogenous S6K would also become tyrosine phos-
phorylated. Since NIH3T3 cells express high levels of
endogenous S6K we used them in this study. To
achieve maximal stimulation of multiple RTKs, we
stimulated the cells with serum rather than PDGF.
Endogenous S6K1 was immunoprecipitated from cells
after 30, 60 and 180 min of serum-stimulation. As
shown in Fig. 2B, both variants of S6K1 (p70 and
p85) are phosphorylated on tyrosine in an inducible
manner. Interestingly, the phosphorylation of the nuc-
lear isoform, p85 S6K1 appears delayed compared to

Fig. 2. S6Ks are tyrosine phosphorylated and associated with RTKs.
(A) PDGFR and S6K1 or 2 were expressed in Cos7 cells. Twenty-
four hour post-transfection cells were starved for 20 h and stimul-
ated with 40 ngÆmL
)1
PDGF as indicated. Immunoprecipitated
EE-S6Ks and complexed were separated by SDS ⁄ PAGE, trans-
ferred onto nitrocellulose and blotted with phosphotyrosine (4G10)
antibodies. The upper half of the membrane was reprobed with
anti-PDGFR IgG and the lower part with anti-EE IgG. (B) Tyrosine
phosphorylation of endogenous S6K1. NIH 3T3 cells were starved
in 0.3% DBS for 24 h and stimulated with 10% DBS as indicated.
Endogenous S6K1 was immunoprecipitated using an antibody
against its C-terminus. The immunoprecipitates were treated as in
(A) and the membrane reprobed with the C-terminal antibody. In
this experiment we focused on S6K1 as NIH3T3 cells do not
express S6K2. The results of three individual experiments for
p70 S6K1 were quantified and are shown as histogram. (C) Endo-
genous PDGF receptor coimmunoprecipitates with S6K1 in a stim-
ulation-dependent manner. NIH3T3 cells were starved and
stimulated with 10% DBS for the indicated times. Endogenous
S6K1 was immunoprecipitated with antibody against the C-termin-
us of S6K and immunocomplexes were analyzed by immunoblot-
ting using anti-S6K or anti-PDGFR IgG.
S6K tyrosine phosphorylation H. Rebholz et al.
2026 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS
treatment leads to a redistribution of the bulk of
S6K towards the nucleus or the perinuclear region.
In addition, we reproducibly observed a small frac-
tion of S6K1 in membrane ruffles for various time

src [26,27]. PDGFRb K634A is kinase dead. We
transfected Cos7 cells with S6K1 ⁄ 2 and various
PDGFRb mutants. After serum starvation, cells were
stimulated with PDGF and both S6Ks were immuno-
precipitated and analyzed by western blotting. In the
control experiment with kinase dead receptor
(PDGFRb K634A), there was no detectable S6K tyro-
sine phosphorylation (Fig. 4A). Notably, S6K expres-
sion was always reduced when expressed together with
Fig. 3. S6K1 is localized in membrane ruf-
fles upon PDGF stimulation in NIH3T3 cells.
NIH3T3 cells were starved for 24 h,
followed by stimulation with PDGF
(10 ngÆmL
)1
) for 5 min. Cells were fixed,
permeabilized, blocked and probed with
anti-C-terminal S6K1 IgG and secondary
FITC-anti-rabbit IgG. Actin was visualized by
phalloidin staining which was added during
the last 10 min of incubation with FITC-anti-
rabbit IgG. Arrows indicate membrane ruf-
fles in which S6K is present. We also used
v-src transformed Swiss3T3 cells as they
generate very strong PDGF-induced ruffles.
Cells were grown at 35 °C and treated simi-
larly to NIH3T3 cells.
H. Rebholz et al. S6K tyrosine phosphorylation
FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2027
KD PDGFR. However, in the S6K2 ⁄ KD PDFGR

To further investigate if tyrosine phosphorylation
was mediated by the action of src in vivo, we transi-
ently expressed various mutants of src together with
S6K. Expression of wild-type src leads to weak basal
tyrosine phosphorylation which could be enhanced by
serum ⁄ vanadate stimulation. A constitutively active src
(Y527F) induced a much stronger tyrosine phosphory-
lation of S6K1 (Fig. 5A). Dominant-negative src lead
to a complete loss of the phosphotyrosine signal in
immunoprecipitated S6Ks. Interestingly, in starved
cells we could observe that overexpression of a consti-
tutively active version of src (527F) led to a band shift
of S6K1 that was similar to the shift in stimulated
cells. Furthermore the pT389 signal, a marker of S6K
activity, in these starved cells was equal to the signal
from the stimulated cells. This activation was not
reflected by the state of tyrosine phosphorylation,
which was significantly lower in starved than in stimu-
lated cells. In serum-stimulated cells we did not see a
significant effect of src 527F on the activity of S6K
even though src 527F led to its strong tyrosine phos-
phorylation. Phospho-T389 levels and the band shift
of S6K were similar and independent of the src variant
(DN, WT, 527F) in stimulated cells. The most highly
tyrosine phosphorylated S6K from src 527F expres-
sing, stimulated cells was no more active than the
non-tyrosine phosphorylated S6K derived from cells
overexpressing DN src. Interestingly, this constitutively
active src variant still needed stimulation in order to
generate a maximal phosphotyrosine signal on S6K1.

nentially growing Hek293 cells (Fig. 5B), strengthening
the hypothesis that src kinase, which localizes to an
activated receptor tyrosine kinase, is a major kinase
responsible for tyrosine phosphorylation of S6K
in vivo.
We also found endogenous S6K1 to be tyrosine
phosphorylated in v-src transformed Swiss3T3 cells
but not in the parental cell line. The src-specific inhib-
itor SU6656 could inhibit this phosphorylation
(Fig. 5C). This is another indication that the phos-
phorylation of native S6K occurs in cells in a src-
dependent manner. It is possible to imagine that S6K
tyrosine phosphorylation occurs during the process of
oncogenic transformation. In these Swiss3T3 cells we
could also observe higher levels of phospho-S6 than in
parental cells confirming earlier reports of elevated
S6K activity [29] (data not shown).
Src kinase phosphorylates S6K in the N-terminus
In order to determine the sites of tyrosine phosphory-
lation, we used N- and C-terminally truncated S6K1.
When these mutants were immunoprecipitated from
Hek293 cells that also transiently expressed activated
src (Y527F), S6K1DC was tyrosine phosphorylated but
not the S6K1DN mutant (Fig. 6A). This indicated that
the major tyrosine phosphorylation site ⁄ s may be
located at the N-terminus of S6K1. To verify our
hypothesis and to exclude that the lack of tyrosine
phosphorylation in the S6KDN mutant might be due
to a conformational change that hinders the access of
tyrosine kinases to their substrate residues, we gener-

formed Swiss 3T3 and parental cells were treated with 4 l
M SU6656 for 16 h before lysis. S6K1 was immunoprecipitated and blotted with
4G10 antibody. The membrane was reprobed with anti-S6K1 IgG.
H. Rebholz et al. S6K tyrosine phosphorylation
FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2029
reduced phosphotyrosine signal (by 88 and 95% for
S6K1 and S6K2, respectively) (Fig. 6D) indicating that
the N-terminal site is the major phosphorylation site
in vivo. However, the possibility that another minor
site exists cannot be excluded.
As tyrosine phosphorylation was detectable upon
growth factor stimulation and therefore paralleled the
activation by S ⁄ T phosphorylation, it was logical to
hypothesize that tyrosine phosphorylation may be
involved in the regulation of S6K activity. As previ-
ously shown, tyrosine phosphorylation is strongly
reduced when the src signaling-deficient PDGFRY579 ⁄
581F mutant is expressed (Fig. 4A). We assayed the
in vitro activity of S6K coexpressed with wild-type
PDGFR or Y579 ⁄ 581F in starved or PDGF-stimula-
ted cells. S6K activity was not altered in the presence
of the src signaling deficient mutant when compared
with wild type (supplementary Fig. S4). Next, we tes-
ted if mutation of Y39 ⁄ Y45 to phenylalanine would
affect the activity of S6Ks. No difference between
wild-type and mutant activities could be observed in
stimulated or starved cells in an in vitro kinase assay,
indicating that tyrosine phosphorylation of this site
does not modulate kinase activity (Fig. 6E). The
S6K1Y39D mutant was also tested and had similar

VO
4
. Immunoprecipitated S6Ks were tested with anti-pY IgG and membrane was
reprobed with S6K antibody. Total lysate was also analyzed for src expression. (E) The activity of S6K1 ⁄ 2 Y39F ⁄ Y45F mutants is not altered.
Hek293 cells were transfected with S6K1 ⁄ 2 or Y39F ⁄ 45F. Cells were starved and stimulated as indicated (15 min FBS). S6K was immuno-
precipitated from these cells, subjected to an in vitro kinase assay using S6 as a substrate. The expression of S6Ks was assessed by
western blotting.
S6K tyrosine phosphorylation H. Rebholz et al.
2030 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS
indicate that src-mediated tyrosine phosphorylation of
S6K does not affect its subcellular localization.
Taken together, we found that S6K becomes tyro-
sine phosphorylated in a PDGFR-src mediated path-
way which involves membrane recruitment of S6K. We
have shown that a subpopulation of S6K is present at
the membrane upon PDGF stimulation and thus in
the vicinity of PKB and PDK1 which are the major
activators of S6K.
Discussion
In this study, we have shown for the first time that
S6Ks become tyrosine phosphorylated and associated
with PDGFR in a ligand-induced manner. In mamma-
lian cells, both events, receptor association and tyro-
sine phosphorylation occur simultaneously and peak
within the first 30 min after stimulation.
Membrane translocation in response to mitogenic
stimuli has been shown for a variety of AGC kinases,
including PKB ⁄ Akt, PDK1, PKD and various iso-
zymes of the PKC family. This is mainly thought to
occur via binding to second messengers such as phos-

cence microscopy to show that S6K1 can be localized
at the plasma membrane. In starved cells, S6K1 is
evenly distributed within the cytoplasm and can also
be detected along stress fibers. Upon stimulation, the
majority of S6K1 molecules translocate to the nucleus,
whereas a subpopulation is reproducibly found in
membrane ruffles.
The association between receptor and nonreceptor
tyrosine kinases and S6K leads to its tyrosine phos-
phorylation in vitro and in vivo. The recombinant
kinase domain of PDGFR, as well as cytoplasmic
tyrosine kinases such as src, is able to phosphorylate
S6Ks on tyrosine. In vivo, using PDGFR mutants that
are deficient in signaling via src kinase, we found that
both PDGFR and src kinase activities are needed for
maximal tyrosine phosphorylation of S6Ks. Studies
employing tyrosine kinase inhibitors such as PP1 and
SU6656 validated this finding. PI3K and mTor do not
influence tyrosine phosphorylation of S6K as demon-
strated by the use of the inhibitors LY294002 or rapa-
mycin. This finding is in congruence with the finding
that PDK1 tyrosine phosphorylation is independent of
PI3K activity [20]. The major src-dependent phos-
phorylation sites, S6K1 Y39 and S6K2 Y45 are located
at the N-terminus of S6K.
We observed a difference in phosphorylation kinetics
of the p70 and p85 isoforms of endogenous S6K1
in NIH3T3 cells: Whereas P70 was already phosphoryl-
ated after 30 min, we could only detect p85 phosphory-
lation after 60 min of stimulation. In contrast to

transformed cells S6K could be activated indirectly via
the enhanced action of upstream kinases PKB ⁄ Akt,
PDK1 or PI3K or via the inhibition of ser ⁄ thr phos-
phatases [41,42].
It was shown that some PKCs act in a negative feed-
back loop which controls kit tyrosine kinase activity by
directly phosphorylating two serine residues in the kin-
ase insert of the receptor in a stem cell factor-dependent
manner [43]. Similarly, it was recently published that
S6K activity is required in a negative feedback loop
which down-regulates insulin receptor signaling via
phosphorylation of IRS1 [44,45]. In order to achieve
this, S6K must be recruited to IRS1 and therefore be in
membrane vicinity. It is plausible to speculate that S6K
might not only receive signaling information from acti-
vated PDGF receptors or associated second messengers,
but could regulate their function by phosphorylation.
Bioinformatic analysis of PDGFR kinase domain does
not show the presence of S6K phosphorylation motifs.
An in vitro kinase assay indicated no obvious phos-
phorylation of recombinant PDGFR kinase domain by
S6K. One could speculate that tyrosine phosphorylation
may create an SH2 recognition site and thus may alter
the binding affinities of S6K.
In this study, and for the first time, we demonstrate
receptor association and tyrosine phosphorylation of
S6Ks. Both events occur simultaneously and can be
induced by growth factor stimulation.
Experimental procedures
Materials

Expression of recombinant proteins in bacteria
and Sf9 cells
EE-tagged S6Ks were expressed in Sf9 cells, affinity purified
using monoclonal EE-antibody and eluted with EE-peptide.
PDGFRb cytoplasmic domain recombinant protein was
purchased from Upstate. Tyrosine kinases src, fyn, btk and
syk were purified as described [47]. The N-terminal domain
of S6K1 was subcloned into pET42a (Novagen, Notting-
ham, UK) in frame with a C-terminal His-tag, expressed
in BLR21 DE3 cells, induced and purified with NiNTA
agarose and eluted with 400 mm imidazole.
Cell culture and transfection
Sf9 cells were maintained at 27 °C in IPL41 insect medium
(Invitrogen, Paisley, UK) with yeastolate ultrafiltrate (Gib-
co ⁄ Invitrogen), lipid concentrate and gentamycin (Invitro-
gen). NIH3T3 cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% donor
bovine serum (DBS, Invitrogen), 50 lgÆmL
)1
streptomycin,
50 UÆmL
)1
penicillin and 2 mml-glutamine. Cos7 and
Hek293 cells were cultured in the same conditions than
NIH3T3, but 10% fetal bovine serum (FBS, Invitrogen)
was added instead of DBS. Swiss 3T3 parental and tem-
perature-sensitive v-src transformed cells (F29) were a gift
from M. Frame (Beatson Institute, Glasgow, UK) and were
grown at 35 °C. Cos7 cells were electroporated as described
previously [31]. Hek293 cells were transiently transfected

4
. Immunoprecipitation was performed as described
for Sf9 cells, using either anti-EE IgG or polyclonal anti-
body raised against the C-terminus of S6K1 ⁄ 2. NIH3T3
cells were lyzed and subjected to immunoprecipitation in
low salt association buffer (100 mm NaCl, 100 mm
Tris ⁄ HCl pH 8.0, 1% NP40 plus above mentioned inhibi-
tors).
Immunoblot analysis
Proteins were subjected to SDS ⁄ PAGE gel electrophoresis
and transferred onto nitrocellulose membrane. For phos-
photyrosine immunoblots membranes were blocked in 2%
bovine serum albumine (Fraction 5, Sigma) in TBS contain-
ing 0.05% Tween-20, then probed with anti-phosphotyro-
sine IgG (4G10), washed extensively and incubated with
peroxidase-conjugated secondary antibodies (Promega,
Southampton, UK). The antigen–antibody complexes were
detected using enhanced chemiluminescence (ECL, Amer-
sham Pharmacia Biotech).
In vitro S6 kinase assay and tyrosine kinase
assay
The in vitro kinase assay was performed with immunopuri-
fied S6Ks and 40S ribosomes as substrate, which we des-
cribed previously [2]. To test for tyrosine kinase activity
towards S6 kinase, EE-tagged S6Ks were immunoprecipitat-
ed from Sf9 cells with anti-EE IgG immobilized on protein
A-Sepharose. Immunocomplexes bound to beads were
washed twice in lysis buffer and onc0.5 mm EGTA, 10 mm
MnCl
2

4
cells per well and
cultured overnight. The cells were starved in 0.3%
DBS ⁄ DMEM for 24 h and then stimulated with 10 ngÆmL
)1
PDGF for the indicated times. Cells were fixed with 4% for-
maldehyde and permeabilized with 0.2% TritonX-100 in
NaCl ⁄ P
i
. The coverslips were blocked by incubation with
0.5% bovine serum albumin in NaCl ⁄ P
i
, incubated with
rabbit polyclonal antibody against the S6K1, washed and
incubated with goat FITC anti-rabbit IgG. Rhodamin-
phalloidin (Sigma) was added for 10 min. After washing,
the slips were mounted onto microscope slides using moviol
(Sigma). Immunofluorescent staining was analyzed with a
Laser Scanning Microscope LSM510 (Zeiss, Oberkochen,
Germany), using 40 ·⁄1.30 oil Plan-Neofluar immersion
objective (Zeiss, Germany).
Acknowledgements
The authors would like to thank Mike Waterfield for
his critical comments and suggestions, Richard Foxon
for excellent technical assistance, Margaret Frame for
the gift of the v-src transformed cell line and Claus
Spitzfaden for mass spectrometry work. H.R. was sup-
ported by GlaxoSmithKline, G.P. by FEBS Collabor-
ative Experimental Scholarships for Central & Eastern
Europe and I.N. by EMBO Short-term fellowship.

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Supplementary material
The following supplementary material is available
online:
Fig. S1. The kinase or kinase extension domain of S6K
mediates the interaction with PDGFR. To determine
which domain of S6K interacts with the cytoplasmic
domain of PDGFR, we transfected Hek293 cells with
PDGFR and either full-length S6K1 ⁄ 2 (EE-S6K1 ⁄ 2
WT) or deletion mutants which lack both N- and C-ter-
minal regulatory domains (flag-p70S6K1 ⁄ 2DNDC).
S6Ks were immunoprecipitated via their tags and west-
ern blotting was performed with an EE ⁄ anti-flag anti-
body and anti-PDGFR antibody. Both full-length S6Ks
and mutants associated with PDGFR. This suggests

ed S6K fragment. Phosphotyrosine remained stable
and could be detected within the series as a 243 m ⁄ z)1
fragment (163 + 80). The black arrow indicates the
+16 shift caused by oxidized methionine.
Fig. S4. Tyrosine phosphorylation mediated by
PDGFR does not affect S6K activity. Hek293 cells
were transfected with WT or mutant PDGFR and
either isoform of S6K1 or 2, starved and stimulated
with PDGF (40 ngÆmL)1) for 15 min (and Na
3
VO
4
for 2 min). S6Ks were immunoprecipitated with the
EE-antibody, then used for an in vitro kinase assay
with ribosomal protein S6 as a substrate. The total ly-
sate (30 lg) was tested for S6K and PDGFR expres-
sion and b -actin levels.
This material is available as part of the online article
from
S6K tyrosine phosphorylation H. Rebholz et al.
2036 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS