Tài liệu Báo cáo khoa học: Receptor association and tyrosine phosphorylation of S6 kinases pdf

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, f

7 Babraham Institute, Cambridge, UK

8 GlaxoSmithKline, Harlow, UK

Keywords

AGC kinases; platelet-derived growth factor

receptor; receptor tyrosine kinases;

ribosomal protein S6 kinase; src

Correspondence

H Rebholz, Box 296, Rockefeller University,

1230 York Ave, New York, NY 10021, USA

Fax: +1 212 327 7888

Tel: +1 212 327 8486

E-mail: hrebholz@rockefeller.edu

(Received 17 August 2005, revised 6

February 2006, accepted 8 March 2006)

doi:10.1111/j.1742-4658.2006.05219.x

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth The activation process of S6 kinase involves a complex and sequential series of multiple Ser⁄ Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB⁄ Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein–protein interaction domains However, the mechanism of integration of S6K into a multi-enzyme com-plex around activated receptor tyrosine kinases is not clear In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR) The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway Fur-thermore, src kinase phosphorylates and coimmunoprecipitates with S6K

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.

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,

which originate from different translational start sites

Nucleocytoplasmic shuttling has been shown for both

cytoplasmic forms of S6Ks All four isoforms lack

canonical protein–protein interaction domains, such as

Src homology 2 (SH2), phosphotyrosine binding

domain (PTB), Src homology 3 and WW, and have no

pleckstrin homology (PH) domain, which would enable

membrane association via lipid-binding Instead, in

their C-terminal regions, S6K1 and S6K2 possess

either a PDZ domain-binding motif or a proline-rich

region, respectively, through which S6Ks could bind

other signaling molecules

S6 kinases are activated through mitogen- and

nutri-ent-mediated pathways Growth factor-activated

recep-tor tyrosine kinases (RTKs) recruit PI3K which, via its

effectors PKB⁄ Akt and PDK1, mediates S6K

activa-tion [4] Another major player in the activaactiva-tion of S6K

is the mammalian target of rapamycin, mTor (FRAP)

which senses the level of amino acids and possibly

other nutrients within a cell [5] The activation of S6K

is a multistep phosphorylation event, involving several

ser⁄ thr kinases Initially, a series of serines and

threo-nines in the C-terminal autoinhibitory domain become

phosphorylated, followed by two sites within the

hydrophobic linker domain (S371 and T389) [6,7]

Phosphorylation at T389 by mTor or an

mTor-dependent kinase enables PDK1 to bind to S6K via its

PIF binding pocket [8] Finally, PDK1 phosphorylates

T229 in the activation loop and hereby fully activates

S6K [8] Protein phosphatases PP2A and PP1 have

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 vitroand 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

to mitogenic stimuli and that this phosphorylation coincides with receptor recruitment The use of platelet derived growth factor (PDGF) receptor mutants defici-ent in signaling via specific pathways and SU6656, a src-specific inhibitor, indicated that both, RTK and src activities are needed for tyrosine phosphorylation of S6Ks We have mapped the major src-dependent tyro-sine phosphorylation site to a tyrotyro-sine in the N-termi-nus of S6K1 and 2 Tyrosine phosphorylation does not affect the activity or subcellular localization of S6Ks

Results

S6 kinases are tyrosine phosphorylated by various receptor and nonreceptor tyrosine kinases

In the present study, we addressed whether S6K acti-vation involves tyrosine phosphorylation and trans-location to the plasma membrane In recent years, a number of AGC kinases such as PKB⁄ Akt, PDK1, various PKCs and PKD but not S6Ks have been shown to be tyrosine phosphorylated [18–23] Initially,

we used a baculoviral expression system in Sf9 insect cells We infected Sf9 cells with viruses expressing either cytoplasmic EE[Glu-Glu]-tagged S6K1 or S6K2 together with a panel of RTKs or the cytosolic tyro-sine kinase fyn When we immunoprecipitated S6Ks with an anti-EE-tag IgG and probed the membrane with phosphotyrosine antibody (4G10), tyrosine phos-phorylation of S6K1 and 2 was reproducibly observed when HGFR (hepatocyte-growth factor receptor), PDGFR (platelet-derived growth factor receptor) and

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

To test whether tyrosine phosphorylation would also occur in mammalian cells, we transiently transfected Cos7 cells with S6Ks and PDGFR Cells were starved for 24 h and stimulated for 30, 60 or 180 min with PDGF-BB When S6Ks were immunoprecipitated via their EE-tag, we found PDGF-dependent tyrosine phosphorylation of S6Ks Tyrosine phosphorylation reached its maximum at 30 min of stimulation and decreased after 1 h (Fig 2A) Time course experiments using 5 and 10 min of stimulation were also performed and indicated that S6Ks are already tyrosine phos-phorylated within 5 min (data not shown) Further-more, PDGFR was found to coimmunoprecipitate with S6Ks In this system, the association appears to

be constitutive However, one has to take into account that the receptor is strongly overexpressed and there-fore may be partially active even in starved cells The fact that the top band of the coimmunoprecipitated PDGFR (representing the mature receptor) exhibits a slightly weaker pY signal in the starved sample than in the PDGF-treated sample further indicates that the receptor is partially but not fully active when cells are

C

Fig 1 Tyrosine phosphorylation of S6K1 and S6K2 in Sf9 cells and in vitro (A) Sf9 cells were infected with baculoviruses encoding either EE-tagged S6K1 or S6K2 and a receptor tyrosine kinase (EGFR, HGFR, PDGFR) or the cytosolic tyrosine kinase fyn Cells were lyzed 2 days postinfection and S6Ks were immunoprecipitated with anti-EE IgG Samples were resolved by SDS ⁄ PAGE, transferred onto nitrocellulose membrane and analyzed by immunoblotting with monoclonal antibodies against phosphotyrosine (4G10) (B) In vitro tyrosine phosphorylation

of S6K by PDGFR S6Ks were immunoprecipitated from Sf9 cells (using anti-EE IgG) and then subjected to an in vitro tyrosine kinase assay 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.

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 p70 S6K1 Activated S6K usually migrates as four dis-tinct bands on a SDS⁄ PAGE gel due to multiple phos-phorylation The tyrosine phosphorylated bands of S6K overlap with two of the activated and slower migrating bands

We have shown that S6Ks can be detected in a complex with PDGFR when they are transiently expressed We further tested the interaction between endogenous S6Ks and PDGFR (Fig 2C) We found that PDGFR is specifically associated with S6K1 in a serum-inducible manner indicating that, under physio-logical circumstances, S6K is only recruited to acti-vated RTKs

S6K translocates to PDGF-induced membrane ruffles

Immunofluorescence studies in fibroblasts showed that PKB⁄ Akt is recruited to membrane ruffles upon mito-gen treatment [24] We therefore decided to investigate

if S6K is also recruited to the plasma membrane upon mitogenic stimulation We used PDGF as a stimulus

as it is well known to generate ruffling in NIH3T3 cells Serum-starved NIH3T3 cells were stimulated for various times, fixed and stained with an antibody against the C-terminus of S6K1 Using an fluoroscein isothiocyanate (FITC)-labeled secondary anti-rabbit IgG and phalloidin to stain actin, S6K was shown to

be evenly distributed in the cytoplasm of starved cells We could also detect colocalization with stress fibers which has been described previously [25] (data not shown) Platelet-derived growth factor (PDGF)

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.

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

points tested Fig 3A shows a 5-min treatment with

PDGF

In addition, we used v-src transformed Swiss 3T3

cells in this study as they show very strong

PDGF-inducible ruffling Serum-starved Swiss 3T3 cells were

stimulated with PDGF for 5 min, fixed and stained

with antibody against the C-terminus of S6K1

Simi-larly to NIH3T3 cells, PDGF treatment lead to a

redistribution of the bulk of S6K towards the nucleus

or the perinuclear region and of a small fraction of

S6K1 to membrane ruffles (Fig 3B) In a western blot

on total cell lysate from NIH3T3, this S6K-antibody is

very specific and solely recognizes S6K1 (p70 and p85)

We used the same antibody for immunofluorescence

studies These data suggest that S6K may translocate

towards the membrane where it could participate in

multienzyme complexes consisting of RTKs and other

signaling molecules

Tyrosine phosphorylation is dependent on PDGFR-src signaling

Upon stimulation, the PDGF receptor dimerizes and autophosphorylates The generated phosphotyrosine sites constitute binding sites for a variety of down-stream proteins with SH2 domains In order to deter-mine the signaling pathways resulting in S6K tyrosine phosphorylation, we utilized a panel of PDGFR mutants where specific tyrosine sites were mutated to phenylalanines The PDGFRb Y763⁄ 1009F mutant is deficient in signaling via Shp2 phosphatase, while PDGFRb Y579⁄ 581F is unable to bind and activate 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.

KD PDGFR However, in the S6K2⁄ KD PDFGR

sample the expression level is comparable to

S6K⁄ wtPDGFR of the starved sample Expression of

the Y763⁄ 1009F mutant when compared to

wtPDGFR did not alter tyrosine phosphorylation of

S6K Interestingly, the phosphotyrosine signal of

S6Ks from cells expressing the Y579⁄ 581F receptor is

strongly reduced This result suggests that src kinase

may be involved in tyrosine phosphorylation of

S6 kinase When the membrane was re-probed with

anti-S6K1 IgG, the migration of multiple bands

representing S6K1 was similar in wtPDGFR and the Y579⁄ 581F mutant hinting that the activation process was probably not altered by the lack of tyrosine phos-phorylation

To investigate the involvement of src and PDGFR

in tyrosine phosphorylation of S6K further, we studied the effect of inhibitors on tyrosine phosphorylation of S6Ks As expected, genistein, a broad-range tyrosine kinase inhibitor, reduced the PDGF-induced phospho-tyrosine signal of both S6Ks Similarly, PP1, an inhib-itor acting on src, but also PDGFR, c-kit and abl [28] reduced tyrosine phosphorylation of S6K very strongly Finally, the src-specific SU6656 also showed an inhibi-tory effect on the phosphotyrosine signal in S6K (Fig 4B) Interestingly, LY294002 and rapamycin, inhibitors of PI3Kand mTor, respectively, while being effective in inhibiting S6K activity (as shown by phos-pho-S6 blot), did not reduce but rather slightly enhanced tyrosine phosphorylation of S6K (supple-mentary Fig S2)

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-stimustimu-lated 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 The reason therefore may be that stimulation leads to S6K translocation towards the plasma membrane where it may interact with src Furthermore, we could

B

A

Fig 4 Tyrosine phosphorylation of S6K is mediated via a

PDGFR-src pathway (A) Cos7 cells transfected with wt or mutant

forms of PDGFRb (KD PDGFRb K634A, PDGFRb579 ⁄ 581F,

PDGFRbY763 ⁄ 1009F) and EE-tagged S6K1 or 2, starved and

stimul-ated with PDGF (40 ngÆmL)1) for 15 min Lysates were incubated

with anti-EE IgG bound to protein A-sepharose followed by western

blot analysis using anti-pY IgG The membrane was stripped and

reprobed with anti-S6K IgG The total lysate (30 lg) was also tested

for PDGFR expression (B) Effect of inhibitors on tyrosine

phos-phorylation of S6Ks Hek293 cells transiently expressing PDGFR

and either S6K1 or S6K2 were starved for 24 h Sixty minutes

before stimulation, cells were incubated with a panel of inhibitors

(genistein 100 l M , PP1 50 l M and SU6656 4 l M ), then stimulated

with PDGF (40 ngÆmL)1) EE-S6Ks were immunoprecipitated with

anti–EE IgG, transferred to nitrocellullose membrane and probed

with antiphosphotyrosine (4G10) followed by anti–S6K IgG Total

lysate (30 lg) was tested for PDGFR expression.

detect endogenous S6K1 and src in a complex in

expo-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-ated and purified recombinant S6K1 N-terminal domain and subjected it to an in vitro kinase assay with several cytoplasmic tyrosine kinases such as src, lyn, syk and btk As a result, all kinases were able to phosphorylate the S6K1 N-terminal domain (Fig 6B)

An almost complete mobility shift of the domain could

be seen in the presence of src Even though N-terminal sequences of S6K1 and S6K2 are only conserved to 38%, both contain a tyrosine residue, S6K1Y39 and S6K2Y45, equally followed by a glutamate at +1 indicative for a src phosphorylation site Using mass spectrometry, we could confirm the S6K1Y39 site as being tyrosine phosphorylated in vitro (supplementary Fig S3) In order to determine if these residues consti-tute major phosphorylation sites in full length S6K we generated EE-tagged phenylalanine mutants When these mutants were subjected to an in vitro tyrosine kinase assay, they were much less tyrosine phosphoryl-ated by src than wt S6K (Fig 6C) The level of S6K autophosphorylation was also assessed and was hardly detectable under the experimental conditions More importantly, overexpression of the mutants together with src (527F) in Hek293 cells led to a strongly

Fig 5 Tyrosine phosphorylation of S6K is dependent on Src activity (A) Hek293 cells were transfected with S6K1 and either pcDNA3.1 or wild-type src, dominant negative src (DN) or constitutively active src (Y527F) Starved cells were stimulated with 10% FBS (15 min) followed

by a brief treatment with pervanadate (2 min) Phosphotyrosine levels of S6K were assessed by western blot using 4G10 antibody The membrane was stripped and reprobed twice with antibodies against pT389 and S6K1 Total lysates (30 lg) were probed with anti-src IgG (B) Exponentially growing Hek293 cells were lyzed Endogenous S6K1 was immunoprecipitated using an anti-S6K1 IgG, immunocomplexes were separated by SDS ⁄ PAGE and membrane was probed with anti-src IgG As a control, we used ProteinA-sepharose beads to test for the specificity of the coimmunoprecipitation (C) S6K is tyrosine phosphorylated in v-src transformed cells Exponentially growing v-src trans-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.

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 activity to the wild type (data not shown)

Src-induced tyrosine phosphorylation of atypical PKC has been shown to alter its subcellular localization Therefore, we tested the subcellular localization of wild-type S6K and mutants (S6K1Y39F, S6K2Y45F)

by confocal microscopy in NIH3T3 cells but did not observe significant differences In addition, the subcellu-lar localization of S6K1 was simisubcellu-lar in src-deficient (syf)

or syf + src fibroblasts (data not shown) This data

A

D

B

C

E

Fig 6 Determination of a N-terminal tyrosine as src-dependent phosphorylation site (A) Deletion of the N-terminus leads to a loss of phos-photyrosine in S6K Hek293 cells were transiently transfected with WT and truncated mutants of S6K (S6K1, S6K1 DN and S6K1DC) and src 527F Cells were starved for 24 h and stimulated with FBS (15 min) followed by a 2-min treatment with Na 3 VO 4 S6Ks were precipitated, immunocomplexes separated via SDS ⁄ PAGE and blotted with pY antibody Membrane was stripped and reprobed with anti-EE IgG Total lysate (30 lg) was also analyzed for src expression Arrows indicate the truncated S6Ks (B) The N-terminal domain of S6K1 is a substrate for tyrosine kinases One microgram of the purified recombinant N-terminal fragment was used for an in vitro kinase assay using 7 pmol of cytosolic tyrosine kinases src, btk, lyn and syk (C) Tyrosine Y39 ⁄ 45 in S6K1 ⁄ 2 is a substrate for src kinase in vitro S6K1 ⁄ S6K2 and S6K1Y39F ⁄ S6K2Y45F mutants were immunopurified from Hek293 cells and subjected to an in vitro kinase assay using recombinant src kinase Reaction products were analyzed by autoradiography and Coomassie staining as indicated (D) Tyrosine Y39 ⁄ 45 in S6K1 ⁄ 2 is a sub-strate for src kinase in vivo S6K WT and mutants and src kinase were overexpressed in Hek293 cells, which were starved and stimula-ted with 10% FBS for 15 min and for 2 min with Na3VO4 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.

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-pholipids or via binding to phosphotyrosine residues

on activated RTKs Translocation of PKB⁄ Akt or

PDK1 is mediated through PH domains which

specif-ically recognize the second messenger PIP3 [30,31] A

variety of signaling molecules such as PI3K, IRS1, Src

or GRB2 translocate to the membrane and associate

with activated receptors via their SH2 or PTB

domains PKC translocation is mediated by a variety

of isoform-specific RACKs (receptors for activated

C-kinase) [32] In addition, many AGC kinases have

been shown to be substrates for src kinase which itself

associates with activated RTKs Even though the

phosphorylation events leading to full activation of

S6K have been thoroughly studied, it is not clear if

they involve translocation of S6K to the membrane

However, S6K, in order to be phosphorylated by

PDK1, may be in the vicinity of the membrane

Fur-thermore, Rho family G proteins Rac and Cdc42,

which control cytoskeletal organization, were shown to

associate with and activate S6K [33] As these small

GTPases are most active when they are

membrane-bound, it would be logical for S6K to be colocalized

with its upstream effectors Finally, it was reported

that S6K is complexed with the receptor-associated

p85 subunit of PI3K and that this complex formation

is needed for mTor and PI3K-mediated activation of

S6K [34] We showed that PDGFR can specifically

im-munoprecipitate with S6K which, to our knowledge, is

the first report of coimmunoprecipitation of an RTK

with an AGC kinase We further used

immunofluores-tyrosine kinases and S6K leads to its immunofluores-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 stimuphosphory-lation In contrast to p70 S6K, the p85 isoform is thought to be exclusively localized in the nucleus, and thus, the delayed tyrosine phosphorylation may result from activation and⁄ or translocation of the respective kinase For example, as c-src was shown to be in part localized in the nucleus [35], one possibility could be that src translocates to the nucleus where it can phosphorylate p85 S6K Very poss-ibly both isoforms are part of distinct feedback mecha-nisms via tyrosine phosphatases

For several AGC kinases such as PKB⁄ Akt, PDK1, PKCs and PKD it was shown that tyrosine phosphory-lation results in increased kinase activity [19] [20,23,36] It is known that PI3K activity is involved in v-src transformation and the level of PIP3 is elevated

in v-src transformed cells [37] In v-src transformed cells PKB⁄ Akt activity is enhanced, due to elevated PIP3 levels [38,39] In the case of S6K, there is also evidence pointing towards src-induced S6K activation: Src inhibitor PP1 interferes with S6K activation after insulin, IGF1 and pervanadate stimulation [40] Fur-thermore, S6K activity in v-src transformed cells is higher than in nontransformed cells [29] We could confirm that the level of phospho-S6 is higher in v-src

transformed cells We also found that S6K in v-src

transformed cells but not the parental cells is tyrosine

phosphorylated However, our experimental data

indi-cate that S6K tyrosine phosphorylation does not

corre-late with its activity We propose that in v-src

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

Monoclonal antibody to the EE-tag was a gift from J

Downward, Cancer Research UK The antiphosphotyrosine

4G10 antibody, polyclonal phosphospecific S6 protein

(S235⁄ 236) and anti-src IgG were from Upstate (Lake

Placid, NY, USA) Phosphospecific antibody against

p70S6Kinase (pT389) was purchased from Cell Signaling

(Danvers, MA, USA) Anti-flag (M2) IgG and anti-b-actin

were from Sigma (St Louis, MO, USA) Polyclonal

anti-bodies against the C-terminus of S6K1 and 2 were

des-cribed previously [2] Recombinant human PDGF-BB was

purchased from AutogenBioclear (Calne, UK) LY294002

and rapamycin were from Calbiochem (Nottingham, UK),

genistein from Oxford Biomedical Research (Oxford, MI,

USA), PP1 from Biomol (Exeter, UK), SU6656 and

phal-loidin from Sigma

Construction of expression vectors Baculoviruses containing S6K1 and S6K2, fyn and RTKs have been made as described elsewhere [46] The con-struction of mammalian expression vectors encoding wt S6Ks1⁄ 2, activated and kinase-dead forms of S6K (p70S6K1T389D, p70S6K2T388D and p70S6K1K100R) was previously reported [2] The flag–tagged truncated S6Ks (S6K1DNDC and S6K2DNDC) were from K Yone-zawa (Kobe University, Japan) The mammalian expres-sion constructs for wild-type PDGFRb and kinase dead PDGFR, PDGFR Y579⁄ 581F, PDGFR Y763⁄ 1009F were made as reported [26] [27] Mouse⁄ chicken activated Src (Y527F) and DN src (mouse K296R, Y528F) mam-malian expression constructs were purchased from Upstate

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 mm l-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 with LipofectAMINE (Qiagen, Crawley, UK)

Immunoprecipitation Two days postinfection, Sf9 cells were lyzed in 50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1% Triton X-100, 20 mm NaF, 50 lgÆmL)1 leupeptin, 0.5% aprotinin, 1 mm PMSF, 3 mm benzamidine and