Báo cáo khoa học: Phosphorylation of Hrs downstream of the epidermal growth factor receptor docx

Bache1, Camilla Raiborg1, Anja Mehlum1, Inger Helene Madshus2and Harald Stenmark1 1 Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello,

Phosphorylation of Hrs downstream of the epidermal

growth factor receptor

Kristi G Bache1, Camilla Raiborg1, Anja Mehlum1, Inger Helene Madshus2and Harald Stenmark1

1

Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, Oslo, Norway;

2

Institute of Pathology, the National Hospital, Oslo, Norway

The hepatocyte growth factor-regulated tyrosine kinase

substrate Hrs is an early endosomal protein that is thought

to play a regulatory role in the trafficking of growth factor/

receptor complexes through early endosomes Stimulation

of cells with epidermal growth factor (EGF) rapidly leads to

phosphorylation of Hrs, raising the question whether the

receptor tyrosine kinase phosphorylates Hrs directly Here,

we present evidence that a downstream kinase, rather than

the active receptor kinase is responsible We show that the

nonreceptor tyrosine kinase Src is able to phosphorylate Hrs

in vitroand in vivo, but that Hrs is nevertheless phosphory-lated in Src-, Yes- and Fyn-negative cells Moreover, we show that only 10–20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at mul-tiple tyrosines located in different parts of Hrs These results suggest that Hrs is a substrate for several kinases down-stream of the EGF receptor

Keywords: endosome; membrane trafficking; Src; tyrosine kinase

The growth factor or cytokine stimulated phosphorylation

of macromolecules regulates cellular functions such as

adhesion, cytoskeletal function, membrane trafficking and

gene transcription (reviewed in [1]) The hepatocyte growth

factor regulated tyrosine kinase substrate Hrs is a protein

that becomes rapidly phosphorylated upon growth factor

and cytokine stimulation [2,3] It was first shown to be

phosphorylated in cells treated with HGF as well as

platelet-derived growth factor ( PDGF) and epidermal growth factor

(EGF) [2] Later, Hrs was also identified as a tyrosine

phosphorylated protein in hematopoietic cells treated with

the cytokines interleukin-2 (IL-2) and

granulocyte-macro-phage colony-stimulating factor (GM-CSF) [3] Even though

Hrs was identified as a phosphorylated protein, neither the

responsible kinase(s), the exact phosphorylation site(s) nor

the functional significance of the phosphorylation have been

identified so far Recent results suggest that in order for Hrs

phosphorylation to take place, endosomal localization

of Hrs is required [4] Hrs is localized to early endosomes

via a phosphatidylinositol 3-phosphate (PtdIns3P)-binding

FYVE domain [5] and a coiled-coil domain [6] It has been

shown to bind the signal transducing adaptor molecules

STAM [3] and STAM 2/Hbp [7,8], the SNARE protein

SNAP-25 [9], and the signal transmitter downstream of the

transforming growth factor-b/activin receptors, Smad 2 [10]

In addition, a functional clathrin box motif has recently been found in the C-terminal part of Hrs, and direct binding between Hrs and clathrin was discovered, suggesting that clathrin is recruited to early endosomes via Hrs [11] Putative homologues of Hrs have been identified in various eukaryotes, including the class E vacuolar protein sorting molecule Vps27p in Saccharomyces cerevisiae [12] More than 40 vps mutants have been identified in Saccharomyces cerevisiae [13–15], and a subset of 13 of these are designated as the class E vps mutants These mutants accumulate vacuolar, endocytic and trans-Golgi markers in an aberrant multilamellar structure, the class E compartment [16–18] Mice that lack Hrs die during early embryogenesis, demonstrating that Hrs is an essential protein The embryos show severe defects in ventral folding morphogenesis and contain enlarged transferrin receptor-positive structures [19] Drosophila larvae that lack Hrs likewise contain enlarged endosomes, and electron micro-scopy has revealed that the formation of endosomal invaginations is inhibited in the absence of Hrs [20] Because Hrs is an essential protein with functions in membrane trafficking and signal transduction, it will be important to determine the functional significance of its phosphorylation

In this paper, we have investigated whether Hrs is phosphorylated by the EGF receptor or a downstream kinase We find that Hrs is phosphorylated on multiple tyrosines by kinases downstream of the receptor While the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs both in vitro and in vivo, other tyrosine kinases also phosphorylate Hrs

M A T E R I A L S A N D M E T H O D S

Antibodies and plasmid constructs Anti-myc Ig was from the 9E10 hybridoma [21] The horse-radish peroxidase (HRP)-conjugated anti-phosphotyrosine

Correspondence to H Stenmark, Department of Biochemistry,

The Norwegian Radium Hospital, Montebello N-0310 Oslo, Norway.

Fax: + 47 22508692, Tel.: + 47 22934951,

E-mail: stenmark@ulrik.uio.no

Abbreviations: Hrs, hepatocyte growth factor-regulated tyrosine

kinase substrate; EGF, epidermal growth factor; GM-CSF,

granulo-cyte-macrophage colony-stimulating factor; HGF, hepatocyte growth

factor; PDGF, platelet-derived growth factor; PtdIns3P,

phosphati-dylinositol 3-phosphate; STAM, signal transducing adaptor molecule;

Vps, vacuolar protein sorting; MBP, maltose binding protein;

TGFa, transforming growth factor-a.

(Received 15 March 2002, revised 3 May 2002, accepted 17 June 2002)

Ig (RC20H) was from Transduction Laboratories

Anti-serum against Hrs was raised against the recombinant

protein [11] Human anti-EEA1 serum was a gift from

Ban-Hock Toh Monash University, Melbourne, Australia

Mouse anti-Src Ig and recombinant active Src (p60c–Src)

were from Upstate (Waltham, MA, USA) [c-33P]ATP was

from Amersham HRP-labelled secondary antibodies were

from Jackson Immunoresearch (West Grove, PE, USA)

pGEM-myc-Hrs1–289 and pGEM-myc-Hrs287–775 were

made by PCR with mouse Hrs [2] as the template

pSGT-SrcY527F used for expression of a constitutive active Src

construct was a gift from S Courtneidge (Van Andel

Research Institute, Grand Rapids, MI, USA) Non-fusion

pEGFP-EGFR used for expression of EGF-receptor in

SYF-2 cells was a gift from A Sorkin (University of

Colorado Health Science Center, Denver, CO, USA)

Transient expression in HEp-2 and HeLa cells

The pSGT-SrcY527Fconstruct was expressed in HEp-2 cells

using the Fugene (Roche) system according to the

manufacturer’s instructions, and the cells were analyzed

24 h after transfection pGEM constructs were expressed in

cells using modified Ankara T7 RNA polymerase

recom-binant vaccinia virus and lipofection as described previously

[22]

Confocal immunofluorescence microscopy

For studying localization of Hrs in starved and EGF

stimulated cells, Hep-2 cells were grown on coverslips,

starved for 4 h in serum-free medium, and stimulated (or

not) for 8 min with EGF (100 ngÆmL)1) The cells were then

permeabilized with 0.05% saponin and fixed with 3%

paraformaldehyde before staining with the antibodies

indicated, as described [23] Coverslips were examined using

a Leica TCS NT confocal microscope equipped with a

Kr/Ar laser and a PL Fluotar 100·/1.30 oil immersion

objective

EGF/TGFa stimulation and immunoprecipitation of Hrs

Hep-2 cells were starved for 4 h in serum-free medium and

then stimulated (or not) for 8 min with EGF or TGFa (both

100 ngÆmL)1) for 10 or 20 min at 4C with EGF The cells

were washed three times with ice-cold NaCl/Piand lysed for

15 min in lysis buffer ( 125 mMKAc, 25 mMHepes, 25 mM

MgAc, 5 mM EGTA, 0.5% NP40, 1 mM dithiothreitol,

pH 7.2) at 37C or supplemented with mammalian

phos-phatase inhibitor cocktail II and protease inhibitor cocktail

(Sigma) After preclearance of the lysate by centrifugation, it

was incubated with 25 lL protein A–Sepharose

(Pharma-cia) preincubated with 20 lL anti-Hrs Ig

Immunoprecip-itates were washed three times with washing buffer (125 mM

KAc, 25 mM Hepes, 2.5 mM MgAc, 5 mM EGTA, 1 mM

dithiothreitol, pH 7.2) supplemented with phosphatase

inhibitor cocktail II, and Hrs was eluted in sample buffer

for sodium dodecyl sulfate polyacrylamide gel

electrophor-esis (SDS/PAGE) or two dimensional PAGE (8M urea,

65 mM dithiothreitol, 2% (w/v) Pharmalyte (Pharmacia

AB) and 0.5% (v/v) Triton X-100) (10% gels) For the 2D

PAGE the material was subjected to isoelectric focusing on

linear Immobiline Dry Strips, pH 3–10 (Amersham

Phar-macia Biotech, Uppsala, Sweden), with the PharPhar-macia Multipor II apparatus and procedure After equilibration in 2% (w/v) SDS, the strips were placed on top of a vertical SDS/PAGE gel and subjected to electrophoresis with a Bio-Rad Protean II apparatus Following SDS/PAGE, proteins were transferred to 0.45 lm pore size poly(vinylidene fluoride) membranes (Millipore) and incubated with

prima-ry and secondaprima-ry antibodies before detection with the SuperSignal chemiluminescence kit from Pierce In all experiments, the membranes were stripped and reblotted with anti-Hrs Ig to verify equal loadings of the samples

Immunoprecipitation of Hrs from HeLa cells transfected with a constitutively active Src mutant, or SYF-2 cells transfected with EGF-receptor

HeLa cells transfected or not with the plasmid expressing active Src were starved for 4 h before lysis and immuno-precipitation of Hrs as described above Hrs was eluted

in sample buffer for SDS/PAGE peGFP-EGFR was expressed in SYF-2 cells and and starved for 4 h before stimulating or not with EGF (100 ngÆmL)1) for 8 min The cells were lysed and Hrs immunoprecipitated as described, and eluted in sample buffer for SDS/PAGE Following SDS/PAGE, proteins were transferred to 0.45-lm pore size poly(vinylidene difluoride) membranes (Millipore) and incubated with primary and secondary antibodies before detection with SuperSignal chemiluminescence from Pierce

In vitro phosphorylation of Hrs Mops buffer (10 mMMgCl2, 50 mMMops pH 7.0, 150 mM NaCl) supplemented with 25 lCiÆmL)1 [c-33P]ATP was mixed with 1 lg maltose binding protein (MBP), 1 lg MBP–Hrs or buffer only, and 0.5 U recombinant active Src was added after heating of the samples to 30C After

20 min at 30C, the reactions were stopped by adding 3·SDS-sample buffer Following SDS/PAGE, the gel was fixed and dried and left overnight in a phosphorimager (Molecular Dynamics)

R E S U L T S

Phosphorylation of Hrs at 37 °C and 4 °C Inhibition of endocytosis by dominant-negative dynamin expression and hypertonic media prevents the EGF-induced phosphorylation of Hrs This suggests that the phosphory-lation of Hrs takes place on endosomes [4] As the activation

of the EGF receptor occurs efficiently at 4C [24,25], whereas endocytosis is blocked, we studied the EGF-induced phosphorylation of Hrs at this temperature in order

to further investigate the relationship between endocytosis and Hrs phosphorylation As shown in Fig 1, essentially no Hrs phosphorylation of Hrs was detected in unstimulated cells (lane 1), whereas EGF stimulation for 8 min at 37C caused a strong tyrosine phosphorylation of Hrs (lane 2) When the cells were stimulated with EGF for 10 or 20 min

at 4C (lanes 3–4), the phosphorylation was much weaker than that detected at 37C, supporting the idea that endocytosis of the receptor/ligand complex is important for the phosphorylation of Hrs Nevertheless, some phosphory-lation occurred even at 4C, indicating that endocytosis is

not an absolute requirement for Hrs phosphorylation and

that a small fraction of Hrs may be phosphorylated at the

plasma membrane

Hrs phosphorylation does not require an active

EGF receptor kinase

It has been proposed previously that the EGF receptor

tyrosine kinase may be responsible for the phosphorylation

of Hrs [4,26], and our finding that Hrs is phosphorylated to

some extent at 4C does not exclude this possibility To

investigate if the activated EGF receptor kinase

phosphory-lates Hrs directly, we measured Hrs phosphorylation upon

the stimulation of EGF receptors with two ligands that have

different properties While EGF remains bound to the

receptor at the acidic endosomal pH, thus activating the

receptor kinase, transforming growth factor a (TGFa)

rapidly dissociates and leaves the EGF receptor inactive

after internalization into early endosomes [27,28]

There-fore, if Hrs is phosphorylated by the receptor kinase, we

would expect that only EGF, and not TGFa, causes its

phosphorylation

HEp-2 cells, which are well characterized with respect

to EGF and TGFa signalling [27], were serum-starved

and stimulated with EGF or TGFa before the cells were

washed and Hrs was immunoprecipitated from the lysate

We chose to stimulate the cells with the growth factors

for 8 min, as the growth factor-stimulated

phosphoryla-tion of Hrs is maximal at this time point [2–4] The

tyrosine phosphorylation was analyzed by

immunoblot-ting using an antibody reacimmunoblot-ting specifically to

phospho-tyrosine (Fig 2) The membranes were stripped and

reblotted with anti-Hrs Ig to verify equal loadings of the samples We found that both EGF and TGFa had the same ability to stimulate Hrs phosphorylation This indicates that Hrs is not phosphorylated by the EGF receptor kinase and implies the involvement of a downstream kinase

Hrs is a substrate for Src phosphorylationin vitro andin vivo

When considering tyrosine kinases that are activated downstream of the EGF receptor, we regarded Src family kinases as possible candidates, as these kinases are activated downstream of all known receptors that mediate Hrs phosphorylation [29–34] To study whether Src can phos-phorylate Hrs in vitro, active Src was mixed with recom-binant Hrs fused to maltose-binding protein (MBP) As a negative control we used MBP only The phosphoryla-tion was measured by the incorporaphosphoryla-tion of radioactive phosphate, and we found that MBP-Hrs, but not MBP alone, was phosphorylated by Src (Fig 3A) In addition we observed a strong phosphorylation product of about

60 kDa in all lanes, corresponding to autophosphorylated

Fig 3 Src phosphorylates Hrs in vitro and in vivo (A) Radioactive ATP was mixed with Hrs fused to maltose binding protein (MBP) (lane 3) or MBP alone (lane 2) An additional control containing buffer only was also included (lane 1) Recombinant active Src was added, and the samples were incubated at 30 C for 20 min before the reac-tions were stopped by adding 3· sample buffer for SDS/PAGE The gel was fixed and dried and exposed in a phosphoimager over night (B) HeLa cells were transfected or not with an active Src construct and serum-starved for 4 h before Hrs was immunoprecipitated and phos-phorylation analyzed by immunoblotting with anti-phosphotyrosine

Ig (upper panel) or anti-Hrs Ig (lower panel).

Fig 1 Phosphorylation of Hrs is reduced at 4 °C HEp-2 cells were

serum-starved for 4 h before stimulation with EGF for 8 min at 37 C

(lane 2, upper panel) or 10 and 20 min at 4 C ( lanes 3 and 4, upper

panel) Hrs was immunoprecipitated and phosphorylation analyzed by

immunoblotting with phosphotyrosine Ig (upper panel) or

anti-Hrs Ig ( lower panel).

Fig 2 Hrs phosphorylation is caused by both EGF and TGFa Hep-2

cells were serum-starved for 4 h and stimulated with EGF or TGFa as

indicated Hrs was immunoprecipitated and phosphorylation analyzed

by immunoblotting with anti-phosphotyrosine Ig (upper panel) or

anti-Hrs Ig (lower panel).

Src These results show that Hrs is a substrate for Src

phosphorylation in vitro

In order to study if Hrs is a substrate for Src

phosphorylation in vivo, we investigated whether a

consti-tutively active Src construct was able to phosphorylate Hrs

in serum-starved cells HeLa cells were transfected with the

active Src construct and serum-starved for 4 h before lysing

the cells and immunoprecipitating Hrs Phosphorylation

was examined by immunoblotting As expected, no

phos-phorylated Hrs was detected in the serum-starved cells

without the active Src construct However, in the

trans-fected cells we could observe a strong phosphorylation of

Hrs (Fig 3B) These results indicate that Src is able to

phosphorylate Hrs in vivo

After transfection of HeLa cells with the active Src construct and analysis by confocal fluorescence

microsco-py, we observed a number of large vesicular structures that were positively stained for Src and partly colocalized with Hrs and EEA1 (Fig 4) These enlarged structures might represent enlarged endosomes resulting from the hyper-phosphorylation of Hrs or other endosomal pro-teins However, it has been shown that v-Src induces constitutive macropinocytosis in rat fibroblasts resulting in large pinocytic vesicles [35], and the enlarged structures we observed might also be related to this phenomenon In any case, the colocalization between Src and Hrs is consistent with the idea that Hrs is a substrate for Src phosphorylation

Fig 4 Hrs colocalizes with constitutively active Src on large endocytic structures Hep-2 cells were transfected with the pSGT-Src Y527F construct that expresses constitutively active Src, and permeabilized with 0.05% saponin before fixation with 3% paraformaldehyde Src was stained with anti-Src Ig (rhodamine) (a), Hrs with anti-Hrs Ig (FITC) (b) and early endosomes with anti-EEA1 Ig (Cy5) (c) Merged images are shown to illustrate colo-calization between active Src and Hrs (yellow) ( d), Hrs and EEA1 ( turquoise) ( e) and active Src and EEA1 (purple) (f) Examples of enlarged structures are pointed out (arrows) Size bar ¼ 5 lm.

Hrs is phosphorylated in cells that lack

Src, Yes and Fyn

The previous results led us to suspect that Src plays an

important role in the phosphorylation of Hrs Therefore, we

wanted to examine whether EGF-induced phosphorylation

of Hrs can take place in cells lacking Src The mouse

fibroblast cell line SYF-2, which is negative for Src and the

related kinases Yes and Fyn, was used for this experiment

The EGF receptor is not endogenously expressed in this cell

line, and therefore had to be transfected in before

serum-starvation and EGF stimulation As expected, no tyrosine

phosphorylation of Hrs was detected in starved SYF-2 cells

(Fig 5, lane 1) However, when the cells were stimulated

with EGF (lane 2), we observed a significant

phosphoryla-tion of Hrs This result demonstrates that kinases other than

Src, Yes and Fyn are able to phosphorylate Hrs

Hrs is phosphorylated on multiple tyrosines,

and only a minor fraction is phosphorylated

Even though the ligand-induced phosphorylation of Hrs has

been documented in many studies, the fraction of Hrs that is

phosphorylated has never been determined Furthermore, it

is not known if Hrs is phosphorylated on single or multiple

tyrosine residues In order to investigate this, we stimulated

Hep-2 cells for 8 min with EGF and analyzed the

phosphorylation of Hrs by 2D PAGE, which should enable

us to separate phosphorylated Hrs from nonphosphorylated

Hrs based on differences in the pI We expected

phosphory-lated Hrs to have a slightly lower pI than

nonphosphory-lated Hrs, and to observe multiple spots if there are multiple

phosphorylation sites In unstimulated cells, Hrs migrated

as a strong spot at pI 6, with a minor spot at slightly lower

pI (Fig 6A) The nature of this minor spot is not known

Significantly, in cells that were stimulated with EGF, we

observed at least three additional spots towards the acidic

side of the gel (Fig 6B) When reprobing the blots with

anti-phosphotyrosine Ig, we confirmed that these three

addi-tional spots corresponded to phosphorylated Hrs (Fig 6D),

whereas we observed no phosphorylation in the

unstimu-lated cells (Fig 6C) To study if the phosphorylation occurs

in the N- or C-terminal regions of Hrs, we transfected an

N-terminal (Hrs1–289) and a C-terminal (Hrs287–775) Hrs

construct into HeLa cells and studied their phosphorylation

(Fig 7) in the presence of EGF Interestingly, both of these

constructs were phosphorylated, although Hrs287–775 showed the relatively strongest phosphorylation We con-clude from these experiments that Hrs is phosphorylated on

Fig 5 Hrs is phosphorylated in cells lacking Src, Yes and Fyn SYF-2

cells, which are negative for the Src family kinases Src, Yes and Fyn,

were transfected with EGF receptor, serum-starved for 4 h and

sti-mulated or not for 8 min with EGF Hrs was immunoprecipitated

and phosphorylation analyzed by immunoblotting with

anti-phospho-tyrosine Ig (upper panel) or anti-Hrs Ig (lower panel).

Fig 6 2D PAGE reveals that only a minor fraction of Hrs is phos-phorylated upon EGF stimulation Hep-2 cells were serum-starved for

4 h and then stimulated (b and d) or not (a and c) for 8 min with EGF Hrs was then immunoprecipitated and analyzed by 2D gel electro-phoresis and immunoblotting with Hrs Ig (a and b) or anti-phosphotyrosine Ig (c and d) Arrowheads indicate additional spots appearing after stimulation, corresponding to phosphorylated Hrs.

Fig 7 Both N- and C-terminal parts of Hrs are phosphorylated Myc-tagged wild-type Hrs or the deletion constructs Hrs1–289 and Hrs287–

775 were transfected into HeLa cells, which were stimulated for 8 min with EGF The expressed constructs were immunoprecipitated from cell lysates using an antibody against the myc epitope, and analyzed by immunoblotting with phosphotyrosine Ig (upper panel) or anti-myc Ig (lower panel) Arrows indicate the position of the Ig heavy chain.

multiple tyrosine residues When comparing the intensity of

the spots corresponding to phosphorylated forms with that

corresponding to nonphosphorylated Hrs, it is striking that

the former is much weaker than the latter one (10–20% as

measured by quantitation of the gel spots) This indicates

that only a fraction of Hrs becomes phosphorylated upon

EGF stimulation of cells

D I S C U S S I O N

In the present work, we show evidence that the

phos-phorylation of Hrs occurs downstream of the EGF

receptor kinase Our findings that Src colocalizes with Hrs

and can phosphorylate Hrs in vitro and in vivo suggest

that this kinase may be involved in the phosphorylation of

Hrs On the other hand, as EGF-dependent

phosphory-lation of Hrs occurs in cells that lack Src, Yes and Fyn,

Hrs must also be a substrate for other kinases Our

2D-gel analysis, which indicates that Hrs is

phosphoryl-ated on multiple tyrosine residues upon EGF stimulation,

is consistent with this view

There are 29 tyrosines in Hrs, and four of these are

conserved in Hrs from nematode, fly, mouse, rat and

human Recent work has shown that tyrosine 334 (and

possibly tyrosine 329) of Hrs is phosphorylated [36] We

found that the N-terminal part (residues 1–289) and the

C-terminal part (residues 287–775) were both

phosphory-lated, with the latter showing the relatively highest

phos-phorylation Together with the fact that multiple spots

representing phosphorylated Hrs could be observed by 2D

PAGE, these findings lead us to conclude that multiple

tyrosines in Hrs are phosphorylated upon EGF stimulation

By transfecting cells with a constitutively active mutant of

Src, we found that active Src has a strong ability to

phosphorylate Hrs in the absence of growth factor/cytokine

stimuli In addition we observed phosphorylation of Hrs

in vitrowhen incubating MBP fusion of Hrs with

recom-binant active Src Src is activated in several signalling

pathways, including those known to lead to Hrs

phos-phorylation, and it localizes to the plasma membrane and

endosomes [37–41] The facts that Hrs has functions in

membrane trafficking and the signalling pathway of growth

factors and cytokines [2,19], that it is localized to the limiting

membrane of early endosomes [26], and that it partially

colocalizes with Src (this study), are consistent with our

finding that Hrs is a substrate for Src phosphorylation It is

evident, however, that kinases other than Src, Yes and Fyn

may also have Hrs as a substrate, because Hrs is still

phosphorylated in Src/Yes/Fyn negative cells upon EGF

stimulation It is possible that different kinases

phosphory-late different tyrosine residues in Hrs, or that alternative

kinases can phosphorylate Hrs in the absence of Src The

functional relevance of the Src-induced phosphorylation of

Hrs thus needs to be investigated further

Previous work has shown that phosphorylated Hrs is

mainly present in cytosol, suggesting that phosphorylation

of Hrs may cause it to dissociate from endosome

mem-branes [4] On the other hand, EGF stimulation does not

cause any detectable redistribution of Hrs from membranes

to cytosol [4] This apparent paradox may be explained by

our present results, which show that only a minor fraction of

Hrs is phosphorylated upon EGF stimulation of HEp-2

cells It will take further work to establish the exact

mechanism behind the cycling of Hrs between the cytosol and endosomal membranes, and the functional consequen-ces of Hrs phosphorylation

A C K N O W L E D G E M E N T S

We thank Sara Courtneidge for pSGT-Src Y527F This work was supported by the Top Research Programme, the Research Council of Norway, the Norwegian Cancer Society, the Novo-Nordisk Founda-tion and the Anders Jahre FoundaFounda-tion.

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