May 20, 1994 - DAndrea, A. D., Yoshimura, A,, Youssoufian, H., Zon, L. I., Koo, J. W., and .... Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264,.
THE
JOURNAL OF BIOIOGICAL CHEMISTRY
Vol. 269,No. 47,Issue of November 25,pp. 29962-29969, 1994 Printed in U.S.A.
Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the Mitogen-activated Protein Kinase Pathway by the Erythropoietin Receptor* (Received forpublication, May 20, 1994, and in revised form, September 2, 1994)
Yasuko Miura, Osamu MiuraS, James N. Ihle8, and Nobuo Aoki From the First Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45Yushima, Bunkyo-ku, Tokyo 113, Japan and the $Department of BiochemistT, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
The erythropoietin receptor (EpoR) belongsto the cytokine receptor family, members of which lack a tyrosine kinase domain.Recent studies, however, have shown that a cytoplasmic tyrosine kinase, JAK2, interacts with the cytoplasmic domain of the EpoR and becomes activated upon binding of Epo to the receptor. Epo has also been shownto stimulate activation of Ras and Raf-1. The present studies were undertaken to examine the possible involvementof Epo-induced tyrosine phosphorylation in activation of the Radmitogen-activated protein kinase ( M A P kinase) pathway and to determine its significance on the growth signaling from the EpoR. In an interleukin (IL)-3-dependent cell line expressing the transfected wild-type EpoR, Epo,or IL-3 induced tyrosine phosphorylation of Shc and its association with Grb2. These cytokines also induced tyrosine phosphorylation and activation of MAP kinase isoforms ERKl and E m . A mutant EpoR with a carboxyl-terminal deletion of 108 amino acids (H mutant), which is mitogenically functional but lacks tyrosine phosphorylation sites inthe carboxyl-terminal region,showed markedly diminished abilities to induce tyrosine phosphorylation of Shc and to phosphorylate and activate MAP kinases. A mutant receptor (PM4 mutant) inactivated by a point mutation, T r p 2 * 2 to Arg, which abrogates the interaction with JAK2, failed toinduce any effect on Shc or MAP kinases. In cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg12’ to Cys, in the extracellular portion of the receptor, neither tyrosine phosphorylation of Shc nor activation of MAP kinases by phosphorylation was detectable without stimulation with Epo or IL-3. These results suggest that the carboxyl-terminal region of EpoR may play a crucial role in activation of MAP kinases through the Ras signaling pathway whichmay be activated by tyrosine phosphorylation of Shc and its association with Grb2. Theactivation of MAP kinases, however, failedto correlate with the mitogenic activity of mutant EpoRs and thusmay not berequired for growth signaling from the EpoR. Erythropoietin (Epo)’ is a hematopoietic growth factor that regulates the growth and differentiationof erythroid progeni-
* This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan, from the Uehara Memorial Foundation, and from the Yamanouch1FoundationforResearchon Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should beaddressed. Tel.: 81-3-3813-6111 (ext. 3218); Fax: 81-3-3818-0448. The abbreviationsused are: Epo, erythropoietin;EpoR, erythropoietin receptor;IL,interleukin; SH2, Src homology 2; SH3,Src homology
tor cells (1,2). The receptor for Epo (EpoR)(3,4) belongs to the cytokine receptor family which includes receptors for most of the hematopoietic growth factors (5, 6). The members of this family characteristicallyhave 4 positionallyconserved cysteines and a Trp-Ser-X-TlpSer (WSXWS) motif in the extracellular domain. Except for a short region showing a limited sequence similarity (7-101, the intracellular region of these receptors is quite diverse and lacks any motifs that would indicate potential function, such as the tyrosine kinasedomain. Most of the hematopoietic growth factors, however, induce tyrosine phosphorylation of cellular proteins (3,6). Furthermore, introduction of a variety of activated tyrosine kinaseshas been shown t o abrogate dependence of hematopoietic cell lines on these hematopoietic growth factors (11).These observations have led to a hypothesis that tyrosinephosphorylation plays a critical role in the growth signaling from the hematopoietic cytokine receptors. Using Epo-dependent cell lines expressing the endogenous or transfected EpoR, we and others have shown that Epo rapidly induces tyrosine phosphorylation of a series of cellular substrates, some of which are also phosphorylated by stimulation with interleukin (1L)-3 (12, 13). We further revealed that a membrane proximal cytoplasmic region, showinga limited homology with the other cytokine receptors, is critical for induction of tyrosine phosphorylation, the expression of a series of immediate-early genes, including c-myc, c-fos, and egr-1, and mitogenesis (10). Upon Epo binding, the carboxyl-terminal region of the EpoR becomes phosphorylated on tyrosine (13, 14) and physically associates with the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (15) but is not required for growth signaling (13, 16, 17). Very recently, we have revealed that Epo stimulation induces binding of JAK2,a member of the J A K family of cytoplasmic tyrosine kinases, to the functionally critical membrane proximal region of the receptor and activates its kinase activity (18, 19). The JAK2 kinase was thus suggested to couple Epo binding to tyrosine phosphorylation. However, little has been known how the signalis transmitted to the downstream signaling elements. For both receptor and cytoplasmic tyrosine kinases, activation of Ras has been regarded as a critical step in inducing mitogenesis or differentiation (20,211. The proteins Grb2, Shc, and mSosl havebeen implicated in the control of Ras by tyrosine kinases in a number of different biological systems. Grb2 is an adapter proteinthat lacks a catalytic domain and is composed of one Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains (22, 23). Grb2 binds to a subset of 3; MAP kinases, mitogen-activated protein kinases;ERK, extracellular signal-regulatedkinase; MEK, MAP kinase/ERK-activatingkinase; MBP, myelin basic protein; GM-CSF, granulocyte-macrophage colonystimulating factor; G-CSF, granulocyte colony-stimulatingfactor; PAGE, polyacrylamide gel electrophoresis.
29962
Activation of MAP Kinases by the Epo Receptor A
29963
B - Ep IL3
-
Ep 113
-
Ep 113
-
-
Ep IL3 - Ep IL3
200 -
200
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97 69 mu.
ZShc
46 -
46" " U
TCL
aShc
aEpoR
dAK2
aJAKl
-
Shc
uShc
FIG. 1. Substrates of tyrosine phosphorylation induced b y Epo or IL-3. A, 32DIEpoR-Wt cells, a clone of an IL-3-dependent cell line expressing the transfectedwild-type murine EpoR cDNA, were washed outof IL-3 for 12 h and left unstimulated (-1 or stimulated withEpo ( E p ) or IL-3 (IL3)for 10 minbefore solubilization. Equivalent amountsof the cell lysates were subjected to immunoprecipitation with anti-Shc (uShc), anti-EpoR (OlEpoR), anti-JAK2 (dm), or anti-JAK1 (aJAK1) antiserum. Aliquots of the cell lysates (TCL), from 5 x 10" cells, and the immunoprecipitates, from 1 x loG cells, were resolved by 6.6% SDS-PAGE and subjected to immunoblotting with an anti-phosphotyrosine monoclonal antibody 4G10. B , the relevant portion of the membrane was stripped and reprobed with anti-Shc to demonstrate equivalent loading of Shc. The molecular mass markers are indicated and given in kilodaltons. The positions which to p150, JAK2, the tyrosine-phosphorylatedform of EpoR (EpoR-PY), and Shc migrated are indicated with arrows. The background band around kDa 50 is the immunoglobulin heavy chain from the immunoprecipitation.
autophosphorylated receptor tyrosine kinases through its SH2 ability to activate MAP kinases. However, activation of MAP domain (22,231 and simultaneously associates through its SH3 kinases failed to correlate with themitogenic activity of mutant domains with mSosl, a guanine nucleotide-releasing protein EpoRs. that activates Ras by inducing exchange of GDP for GTP on Ras (24). Grb2 thus repositions mSosl adjacent to Ras, which is MATERIALS AND METHODS located at the plasma membrane. Theshc gene encodes three Cells and Reagents-A clone of 32D cells, an IL-3-dependent cell line overlapping proteins of 46, 52, and 66 kDa which possess a originally isolatedfrom long term bone marrow cultures, hasbeen precarboxyl-terminal SH2 domain and a glycine/proline-rich re- viously described (37). 32D clones expressing the wild-type or various gion but no obvious catalytic domain (25).Shc proteinsbecome mutant EpoRs and IL-3-dependent DA3 cells expressing thewild-type RPMI tyrosine-phosphorylated upon activation of a variety of recep- EpoR were also describedpreviously (10, 13) and maintained in 10% fetal calf serumand 10% tor tyrosine kinases (25-27) and are phosphorylated constitu- 1640mediumsupplementedwith conditioned medium a s a source of IL-3. tively in cells transformed by the v-Src or v-Fps tyrosine ki- WEHI-3 An expression plasmid for activated mutant EpoR with an ArglZ9to nases (28). Shchas beenimplicated in activation of Ras, Cys mutation was constructedby primer-mediated mutagenesis using because the tyrosine-phosphorylated Shcproteins associate the polymerase chain reaction method a s described previously (10). with Grb2 and mSosl(26,29,30). Moreover, overexpression of Transfection of the plasmid into DA3 cells and isolation of a clone Shc proteins led to Ras-dependent neurite outgrowth inPC12 expressing the mutantEpoR were also carried out as described previously (13). In brief, DA3 cells were transfected with 10 pg of the excells (26). pression plasmid and 1 pg of the pSV2neo plasmid by electroporation Recent studies have revealed that tyrosine kinase pathway and selected in medium containing G418. Six clones were isolated by activates mitogen-activatedprotein kinases ( M A P kinases), limiting dilution and confirmed to grow in medium containing neither also known as extracellular signal-regulated kinases (ERKs), IL-3 nor Epo. These clones were then analyzed by"'I-Epo binding in a Ras-dependent manner (31, 32). MAP kinases are serine/ assays, and theclone that bound the highest radioactivity wasused for threonine kinases that phosphorylate well studied regulatory the subsequent studies. For measurement of the cell number increase, cells were cultured in proteins, including transcription factors, membrane proteins, 25-cm2culture flasks a t a density of 1 x 105/ml(5 ml of culture) in 10% cytoskeletal elements, and otherprotein kinases (31, 33).MAP fetal calf serum-containing RPMI 1640 mediumsupplemented with kinases are thus thought to be key intermediate regulatory 10% WEHI-3 conditioned medium or with 4 unitdml human recombiproteins functioning in signal transductionnetworks. MAP ki- nant Epo. Culture media were changedevery 2 days.Viable cell counts nases achieve maximum activity when phosphorylated on both were determined by trypan blue staining. The preparation and propertiesof rabbit polyclonal antisera against tyrosine and threonine residues by MAP kinase/ERK-activating kinase (MEK), a dual specificity kinase (31, 32). MEK in the cytoplasmic portion of recombinant murine EpoR (38) or against synthetic peptidesfrom JAKl andJAK2 (39) have been described. Antiturnis phosphorylated andthusactivated by a serine/ phosphotyrosine monoclonal antibody(4G10)andrabbitantisera threonine kinase, Raf-l(31,32). Itknown is that activated Ras against Shc and ERKl (erkl-CT) were purchased from Upstate Biotechphysically associates with Raf-1 and that phosphorylation is nology, Inc. (Lake Placid,NY). A rabbit antiserum againstERK2 and a required for activation of Raf-1 (32). However, the mechanism monoclonal antibody against Grb2 were purchasedfrom Transduction Laboratories(Lexington, KY). Recombinant human Epo was kindly how Ras activates Raf-1 remains to be known. The cascade of intracellular signal transducing events lead- provided by Sankyo Pharmaceutical Co. Ltd. (Tokyo, Japan). Immunoprecipitation and Immunohlotting-For stimulationwith ing to activation of MAP kinases may also be involved in the Epo or IL-3, cells were starved for 12 h without IL-3 in complete meEpoR signaling, because Epo also activates Ras (34, 35) and dium. The cells were then left unstimulated a s a negative control or induces phosphorylation and activation of Raf-1 (36). The pre- stimulated with a saturating concentrationof Epo or IL-3 a t 37 "C for sent studies were undertaken to examine the involvement of 10 min. The cells were lysed in a lysis buffer containing 1%Triton Ras-activating proteins andMAP kinases in the EpoR signal- X-100, 20 mM Tris-HCI (pH 7.5), 150 mhl NaCI, 1 mM EDTA, 100 PM ing. Epo was found to induce tyrosine phosphorylation of Shc sodiumorthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 For immunoprecipitation of MAP pg/ml each of aprotinin and leupeptin. and itsassociation with Grb2 in Epo-stimulated cells. Epo also kinases, 1 x 10' cells were lysed in 100 p1 of boiling lysis buffer coninduced tyrosine phosphorylation and activation of MAP ki- taining 1%SDS and 10mM Tris-HCI (pH 7.4) andboiled for another 5 nases. Among mutant EpoRs, the ability to induce phosphoryl- min. The lysate was then diluted 10-fold with the Triton X-100 lysis ation of Shc and itsassociation with Grb2 correlated with the buffer and denatured MAP kinases were immunoprecipitated with anti-
Activation of MAP Kinases by the Epo Receptor
29964 MAP kinase antibodies.
appropriately and varying amounts of the samples were subjected to For immunoprecipitation, a relevant antibody was added to the ly- immunoblot analysis using antibody against phosphotyrosine,Shc, or sates along with protein A-Sepharose beads and incubated for 4 h a t MAP kinases. Kinase Assays of Anti-MAP Kinase Immunoprecipitates in Myelin 4 "C. The beads were washed extensively, and the proteinsbound to the beads were resolved by SDS-PAGE, transferred to polyvinylidene dif- Basic Protein fMBP)-containingGels after SDS-PAGE-Determination luoride membranes, immunoblotted with the indicated antibody, and of MAP kinase activity in MBP-containing gel was carried out essentially as described (40). In brief, anti-ERK1 immunoprecipitates were developed by the enhanced chemiluminescence (ECL) system (Amersham Corp.). Aliquots of the cell lysates were also subjected to immu- prepared a s described above and electrophoresedon an SDS-12% polynoblotting after directly mixed with equal volumes of 2 x Laemmli's acrylamide gel containing 0.5 mg/ml MBP. After removing SDS with buffer containing 20% 2-propano1, the gel was denatured with 6M guasample buffer and heated a t 100 "C for 5 min. For reprobing with a different antibody, the membranes were treated at 50 "C for 30 min nidine HCI and then renatured in a 0.04% Tween 40-containing buffer. with stripping buffer containing 100 mh1 2-mercaptoethanol, 2% SDS, Phosphorylation of MBP was carried outby incubating thegel a t 22 "C and 62.5 mM Tris-HC1 (pH 6.7). for 1h in 40 mM HEPES (pH 7.51, 0.1 mM EGTA, 20 mM MgCI,, 20 p~ For determination of the stoichiometry of tyrosine phosphorylationof ATP, and 25 pCi of [Y-:'~P]ATP. After incubation, the gel was washed Shc andMAP kinases, 2x 10' cells stimulated with 75 unitdm1Epo for extensively with 5% trichloroacetic acid, 1% pyrophosphate solution, of phospho10 min were lysed in 1ml of the lysis buffer. After clarification, 50pl of dried, and subjected to autoradiography. The radioactivity the lysate was mixed with the equal volume of 2 x SDS buffer and boiled rylated MBP was also quantified by Bio-Imaging Analyzer BAS2000 for 5 min, whereas500 pl of the lysate was immunoprecipitated with 10 (Fuji Film, Tokyo). pg of the anti-phosphotyrosine 4G10 antibody conjugated with agarose beads and subsequently eluted with 50pl of 1 x SDS buffer by boiling RESULTS for 5 min. Thewhole cell lysate and the immunoprecipitate were diluted Epo Induces Tyrosine Phosphorylation of Shc andIts Asso32DIEpoR-Wt
51 04 0
1
20
31
Days
420
32DIEpoR-H
31
42
3
ciation with Grb2-To explore the signal transduction pathways from the EpoR, we first examined whether Epo stimulation induces tyrosine phosphorylation of Shc, which has been implicated in coupling receptor tyrosine kinases to the Ras signaling pathways. As shown in Fig. lA, Epo stimulation induced tyrosine phosphorylation of 150-, 130-, 97-, 92-, 72-, 70-, 52-, and 49-kDa proteins in the32D cells expressing thewildtype EpoR (32DlEpoR-Wt). IL-3 stimulation induced an almost identical pattern of tyrosine phosphorylation except that the 72-kDa protein was notphosphorylated after IL-3 stimulation. In accordance with our previous results (13,18, 19),anti-phosphotyrosine blotting of immunoprecipitates obtained with relevant antisera showed that the 72- and 130-kDa substrates were the EpoR and JAK2, respectively, whereas it was revealed that JAKl was not phosphorylated after stimulation with these cytokines in the 32D cells examined. Anti-phosphotyrosineblotting of anti-Shc immunoprecipitates revealed the presence of tyrosine-phosphorylated 52- and
32DIEwR-PM4
4
Days
Days
FIG.2. Growth of transfected 32D cells in E - 3 and Epo. Viable cell numbers of 32D/EpoR-Wt, 32DiEpoR-H, and 32DiEpoR-PM4 cells (0) or with either10% WEHI-3 condicultivated without growth factor tioned medium (0) or 4 units/ml Epo ( 0 )are shown.
A
WT
H
"-
B
PM4
WT
"
- E p l U - Ep 1 1 3 - Ep
200
200-
FIG.3. Induction of tyrosine phosphorylation of Shc and its association with Grb2 in cells expressing the wild-type or mutant EpoR.32DEpoRWt cells (WT)and 32D transfectants expressing the H or PM4 mutant of EpoR ( H or PM4, respectively) were either left unstimulated (-) or stimulated (+) for 10 min with Epo (Ep) or IL-3 ( I D )and solubilized. Aliquots of the cell lysates (A)or immunoprecipitatesobtainedwithantiShc (B) were subjected to5 2 0 % gradient SDS-PAGE followed by immunoblot analysis with the 4G10 anti-phosphotyrosine monoclonal antibody. The filtersused in B was stripped and reprobed with anti-Shc ( C ) or anti-Grb2 ( D ) todemonstrate equivalent loading of Shc or coimmunoprecipitation of Grb2withShc, respectively. Thepositions of p150, Shc,and Grb2 are indicated.
-
113
Ep 1 1 3
.. .
VI.
"-
-
e
"-
200
Ep 113
H
- Ep 1 1 3 -
"
WT
-
Ep 1 1 3
-
Ep 1 1 3
PM4
-
Ep 1 1 3
97 69 46-
22 aShc aShc
Ep 1 1 3
H
-
30-
-
aShc aPY
"-
D
PM4
fShc
IP : Blot :
4P150
-
200
30 22
-
IP : Blot :
-
97 69 46-
Ep 1 1 3
-t:Shc
Total cel lysate Blot : aPY
WT
-
Ep 1 1 3
30 22
46-
C
-
PM4
97 69 46 -
-
....
9769-
H
"
-
IP : Blot :
aShc aGrb2
- 4Grb2
Activation of MAP Kinases by the Epo Receptor 49-kDa proteins incells stimulated withEpo or IL-3 (Fig.lA ). These proteins were identified a s Shc by reprobing with antiShc (Fig. 1B). In addition, tyrosine phosphorylated 210- and 150-kDa proteins were coimmunoprecipitated with Shc after Epo or IL-3 stimulation. To address the functional significance of tyrosine phosphorylation of Shc, we next examined previouslycharacterized 32D clones expressing various mutantEpoRs (10,13).32DEpoR-H cells express the truncated H mutant lacking the carboxylterminal 108 amino acids. Although this mutant lacks the tyrosine phosphorylation sites in the carboxyl-terminal region and failsto associate with PI 3-kinase (151, it retains theabilities to activate JAK2 (18, 19) and to transduce a mitogenic signal (10, 13).The mitogenic response of 32D/EpoR-H, which was selected by the G418 resistance, to Epo was thus comparable with that to IL-3 as determined by [”]thymidine incorporation (10) or by the growth curves (Fig. 2). 32DEpoR-PM4 cells express the PM4 mutant, which contains a mutation, Trp2*’ to Arg (W282R), in a membrane proximal region of the cytoplasmic domain that shows homology with other members of the cytokine receptor superfamily (10). Having lost the ability t o associates with JAK2 to activate its kinaseactivity (19), the PM4 mutant fails to induce tyrosine phosphorylation of cellular substrates, including thereceptor itself, and toelicit a mitogenic response(10,13). 32DEpoR-PM4, thus, failed to grow in response to Epo, a s shown in Fig. 2. The abilitiesof these mutantEpoRs to induce tyrosine phosphorylation of cellular proteins were first examined by antiphosphotyrosine blotting of total cell lysates. As shown in Fig. 3A, Epo induced tyrosine phosphorylation of p130 (JAK2)and p92 in 32DEpoR-Hcells. In most of the repeated experiments, the tyrosine phosphorylation of JAK2 in these cells was enhancedas comparedwith thatinthe32DEpoR-Wt cells, whereas p70 showed variable degreesof tyrosine phosphorylation in response to Epo. On the other hand, thetyrosine phosphorylation of p52 and p49, which correspond to Shc, afterEpo stimulation in 32DEpoR-H was constantly and remarkably diminished as compared with that observed after IL-3 stimulation in this cell line or after Epo stimulation in32DEpoR-Wt. The PM4 mutant failed to induce any detectable tyrosinephosphorylation of cellular substrates in response to Epo (Fig. 3A). The tyrosine phosphorylation of Shc in these cell lines was then directlyexamined by anti-phosphotyrosine blotting of anti-Shc immunoprecipitates. Results shown in Fig. 3B confirmed that the Epo-induced tyrosine phosphorylation of Shc was severely impaired or abolished in 32DEpoR-H or 32D/ EpoR-PM4, respectively. Because tyrosine-phosphorylated Shc hasbeen shown to activate the Ras signaling pathway by physically associating with the Grb2-mSoslcomplex through the SH2domain of Grb2, the anti-Shc immunoprecipitateswere reprobed with anti-Grb2 to examine the association of Grb2 with Shc in Epo-stimulated cells. As shown in Fig. 30, in 32DEpoR-Wt, Grb2was shown to coimmunoprecipitate with Shc after stimulation with Epo or IL-3. 32DEpoR-H cells also showed the Epo-induced association of Grb2 withShc. However, the amountof Grb2 associated with Shc was markedly decreased in accordance with the decrease in tyrosinephosphorylation of Shc. Epo failed to induce the association of Grb2 with Shc in 32DEpoR-PM4 (Fig. 30). nrosine Phosphorylation and Activation of MAP Kinases Induced by Epo Stimulation-Recent studies have revealed that activation of MAP kinases by a variety of receptor tyrosine kinases ismediated by Ras (31,32).Since Epo has been shown to stimulate the activation of Ras (34, 35), we next examined whether Epo stimulation induces tyrosine phosphorylation and activation of MAP kinases, which are activatedby phosphoryl-
46
-
46
-
29965
aPY
aMA PK
IVK
U U WT ti FIG.4. Tyrosine phosphorylation and activation of MAP kinases after stimulation with Epo or IL-3.32D clones expressing the wild-type EpoR (WT) or H mutant (H) were left unstimulatedor stimulated with Epo ( E p )or IL-3 (1.53) for 10 min. The cells were lysed and the lysates were immunoprecipitatedwith anti-ERK1 (erltl-CT).A, the immunoprecipitates wereresolved by 8.4% SDS-PAGE and subjected to immunoblotting with anti-phosphotyrosine (&Y) followed by reprobing with anti-ERK1 (OlMAPK).Aliquots of the immunoprecipitates were also subjected tothe MAP kinase assay inMBP-containing gel ( N K )as described under “Materials and Methods.” The positions of 44-kDa ERKl and 42-kDa ERK2 are indicated. B , the activities of ERKl and ERK2 in 32D/EpoR-Wt (WT) or 32D/EpoR-H (H) cells shown in A were also quantified by Bio-Imaging Analyzer BAS2000. Increase inactivity of ERKl or ERK2 induced by Epo stimulation is expressed a s a percentage of that induced by IL-3 stimulation.
ation on both tyrosine and serinekhreonine residues (31, 32). To examine tyrosine phosphorylation of MAP kinases, immunoprecipitates obtained with anti-ERK1 (erk 1-CT), which also recognizes ERK2, were examinedby anti-phosphotyrosine blotting. As shown in Fig. 4 A , 42- and 44-kDa species of MAP kinases, which should correspond to ERK2 and ERK1, respectively, were found to be tyrosine-phosphorylated after Epo or IL-3 stimulation in 32DEpoR-Wt.However, tyrosine phosphorylation of MAP kinases induced by Epo stimulation was remarkably reduced in 32DEpoR-H (Fig. 4A). Reprobing of the membrane with anti-ERK1confirmed that the42- and 44-kDa species of tyrosine-phosphorylated proteins are directly recognized by anti-ERK1anddemonstratedequal loading of samples (Fig. 4A, center panel). The anti-ERK1 immunoprecipitates were then subjected to theMAP kinase assay inMBP-
29966
Activation of MAP Kinases by the Epo Receptor
A 200
FIG.5. Stoichiometry of tyrosine phosphorylation of Shcand MAP kinases. Increasing amounts of whole cell lysate from 32DIEpoR-Wt cells stimulated with Epo for 10 min (lanes 1-6) and anti-phosphotyrosineimmunoprecipitate from unstimulated (lane 7 ) or Epo-stimulated (lane 8 ) cell lysate were subjected to anti-phosphotyrosineblotting (leftpanels) or immunoblotting with anti-Shc (A) or anti-MAP kinase( B). Whole cell lysate from 32DlEpoR-Wt cells unstimulated (lane 9 )or stimulated with Epo (lane 1 0 ) wasalsosubjectedtoanti-phosphotyrosine blotting. The amount of sample applied to each lane is represented by the corresponding volume of original cell lysate (2x 10' celldml) and indicated under the panels.
1
1 2 3 4 5 6 7 0 9 1 0
EpoSt,:+
+ + + + + - + - +
2
3
4
5
6
7
0
+ + + + + + - +
-
97 - 1
B
1 2 3 4 5 6 17 20 39 41 50 6 7 0 + + - + - + -b 7 . - 4 RK1 hK2g -. - I
EpoSt.:+ + + + 46
-
Lysate :I32 116
I0 14 I2
1 10 10 I2I2
(X 5Pl)
containing gel. As shown in Fig. 4A (lower panel),Epo stimulation was found to stimulate the kinase activities of both ERKl and ERK2. However, although the effect of Epo on the MAP kinase activity was about half a s much as that of IL-3 in 32DIEpoR-Wt, Epo showed only a marginal effect on the MAP kinase activity in 32DIEpoR-H (Fig. 4, A and B ). In 32DIEpoRPM4 cells, Epo failed to induce any detectable tyrosine phosphorylation or activation of MAP kinases (data notshown). Stoichiometry of the Tyrosine Phosphorylation of Shc and MAP Kinases-We next triedto determine whatfraction of Shc or MAP kinases istyrosine-phosphorylated in response to Epo stimulation. For this purpose, phosphotyrosyl proteins were immunoprecipitated with the 4G10 antibody from Epo-stimulated 32DIEpoR-Wt cell lysateand, along withvarying amounts of whole cell lysate, subjected to immunoblot analyses. First, theefficiency of immunoprecipitation with 4G10 was evaluated by immunoblotting with this antibody. The amount of tyrosine-phosphorylated Shc or MAP kinases in the 4G10 immunoprecipitatewasthenestimated by immunoblotting with anti-Shc or anti-MAP kinase, respectively. As shown in Fig. 5, the efficiency of immunoprecipitation with 4G10 was rather low and varied significantly with each phosphotyrosyl proteins, which may be a t least partly because some phosphorylated tyrosine residues areinvolved in intramolecularor intermolecular interaction with the SH2 domains and thus may not bind with 4G10. Densitometric analysis of theresults shown in Fig. 5, representative of three repeated experiments, revealed that the efficiency of 4G10 immunoprecipitation of tyrosine-phosphorylated Shc orERK-1 was -8 or -5%, respectively, whereas -3 or -0.5% of the totalcellular Shc or ERK-1, respectively, was shown to be present in the4G10 immunoprecipitates. From these results, it was calculated that -38 or -10% of total cellular Shc or ERK-1, respectively, undergoes tyrosine phosphorylation in response to Epo in 32DIEpoR-Wt cells. Thus, Epo induced significant but notnear-stoichiometric tyrosine phosphorylation of Shc and MAP kinases. The rather low stoichiometry of tyrosine phosphorylation, particularly of MAP kinases, is compatible with our results that only a barely detectable activity of MAP kinases wasobserved in Epo-stimulated 32DEpoR-H cells, in which Epo-induced tyrosine phos-
-
"-0-
"
I32 I16 10 1214
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1 20 20
u
U
IP :
Blot :
+ + + + + + - +
aPY
aPY
aPY
aMAPK
0
1
2
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4
0
1
2 3 Days
4
FIG.6. Growth factor-independent growth of DA3 transfectants expressing the constitutively activated mutant EpoR Viable cell numbers of DA3EpoR-Wt and DA3EpoR-Rl29Ccells cultivated without growth factor (0) or with either10% WEHI-3 conditioned medium (0)or 4 unitdml Epo ( 0 )are shown.
phorylation of Shc andMAP kinases ismarkedly diminishedas compared with that in 32DIEpoR-Wt. Cells Expressing a Constitutively Activated EpoRMutant Show Epo-independent Growth without Tyrosine Phosphorylation of Shc or Activation of MAP Kinases-Previously, a single point mutation, resulting in anArg to Cys change a t residue 129 of the extracellular domain of EpoR, has been shown to activate thereceptor independent of Epo binding (41,421. The R129C mutant was confirmed to abrogate the factor-dependence of DA3 cells, because six subclones of transfectants, selected by the G418 resistance due to the cotransfected pSV2neo plasmid, grew in the absence of IL-3 or Epo; the clone used in this study grew comparably in medium with or without Epo (Fig. 6). We examined the tyrosine phosphorylation states of Shc andMAP kinases as well as theactivity of MAP kinases in a DA3 clone that expresses this mutant receptor and thus grows without added growth factors. As shown in Fig. 7, antiphosphotyrosine and anti-Grb2 blotting of anti-Shc immunoprecipitates showed that tyrosine phosphorylation of Shc and
Activation of MAP Kinases by the Epo Receptor
A
WT
R129C
B
WT
200
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Ep IL3 - Ep IL3
Ep IL3
-
Ep IL3
-
97 69 46 30
R129C
”
”
-
29967
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30
-
-
--”
4Gtb2
IP : aShc IP : aShc Blot : aGrb2 Blot : aPY FIG. 7. Tyrosine phosphorylation of Shc and its association with Grb2 in cells expressing the constitutively activated mutant EpoR DA3 cells expressing thewild-type EpoR ( W T )or the constitutively activated mutant (RI29C)were cultured in the absence of IL-3 or Epo for 12 h and either left unstimulated (-) or stimulated (+I for 10 min with Epo ( E p ) or IL-3 (IL3). The cells were lysed and subjected to
--
immunoprecipitation with anti-Shc. The immunoprecipitates were resolved by 5-20% SDS-PAGE followed by immunoblotting with anti-phosphotyrosine (A) and reprobing with anti-Grb2( B ) .The positions of p150, Shc, Grb2, and the heavy chainof immunoglobulin (ZgH)are indicated.
its association with Grb2 was dependent on Epo stimulation in these cellsshowing the Epo-independentgrowth. Tyrosine phosphorylation and activation of MAP kinases were also dependent on stimulation with Epo or IL-3 (Fig. 8). These cells were thus found to grow without showing tyrosine phosphorylation of Shc or activation of MAP kinases in growth factordeficient medium.
WT R129C
- 46 aPY
-
Ep IL3
--
I-V
3:;:;
DISCUSSION
The present studies demonstrated that Shc MAP andkinases 46 IVK are among the substratesof tyrosine phosphorylation induced by Epo stimulation. In addition,Epo induced physical association of Shc with Grb2 and activated the catalytic activity of FIG.8. Tyrosinephosphorylation and activation of MAP MAP kinases. However, Epo did not have any effect on these kinases in cells expressing the constitutively activated mutant signaling molecules in cells expressing thePM4 mutant EpoR, EpoR DA3 cells expressing thewild-type EpoR ( W T ) or the activated mutant (RI29C)were left unstimulatedor stimulated withEpo ( E p )or which has a point mutation, Trp282to Arg, abolishing the ability IL-3 (IL3)for 10 mina s indicated. The cells were lysed and the lysates of EpoR to couple with JAK2 to transduce a mitogenic signal. were immunoprecipitated with anti-ERK1 (erhl-CT). The immunopreThe effects of Epo on Shc and MAP kinases were markedly cipitates were resolved by 8.4% SDS-PAGE and subjected to immunodiminished in cells expressing the mitogenically functional, blotting with anti-phosphotyrosine (&Y) followed by reprobing with anti-ERK1 (aA4A.M). Aliquots of the immunoprecipitates were also truncated H mutant, which has lost tyrosine phosphorylation subjected to the MAP kinase assay in MBP-containing gel ( N K ) as sites in the carboxyl-terminal region. In cells expressing the described under “Materials and Methods.” The positions of 44-kDa mutant EpoR that isconstitutively activated by a point muta- ERKl and 42-kDa ERK2 are indicated. tion, Arg’” to Cys, tyrosine phosphorylation of Shc and its association with Grb2 as well a s tyrosine phosphorylation and examined. A tyrosine-phosphorylated protein of 145 kDa (43, activation of MAP kinases were not observed without stimula- 44) or 140 kDa (45), which should correspond to the 15O-kDa tion with Epo or IL-3. Thus, in cells expressing these mutant protein in the present studies,were also found to be associated with Shc in cells stimulated with Epo or IL-3. Although the EpoRs, activation of MAP kinases correlated withtyrosine phosphorylation of Shc and its association with Grb2. Taken identity of this species remains unknown, we found in a previtogether with previous reports showing that Epo activates Ras ous study that this 150-kDa protein coimmunoprecipitated and Raf-1 (34-361, the present results suggest that Shc and with the EpoR or JAK2 in digitonin lysates and underwent Grb2 may mediate activation of the Ras/MAP kinases pathway tyrosine phosphorylation in vitro (19).It is thus possible that from the EpoR and that the carboxyl-terminal region of the the EpoR-JAK2 complex physically associateswith the 140receptor may play a major role in activation of this pathway. kDa protein, Shc, and Grb2, although the association may be Activation of MAP kinases, however, may not be required for transient or unstable. Although the mechanism of how the EpoR activates Ras has the transduction of growth signal from the EpoR, because the activity of MAP kinases did not correlate with proliferation of remained elusive, inhibition of the Epo-induced activation of Ras by tyrosine kinase inhibitors suggested that it may be cells expressing the mutantEpoRs. Recent studies also demonstrated that Epo or IL-3 induces mediated through tyrosine phosphorylation (34). In a previous tyrosine phosphorylation of Shc and itsassociation with Grb2 study (341, GTPase-activating protein was implicated in the (43-45). Damenet al. (43) alsoobserved that tyrosine-phospho- Epo-induced activation of Ras, because Epo induced tyrosine rylated Shc associates with the EpoR in a human M07cell line phosphorylation of Ras GTPase-activating protein in a human expressing transfected murine EpoR. However, association of erythroleukemia cell line (HEL). On the other hand, it was Shc with the EpoR was not observed in DA3 transfectants in demonstrated in the present study and also in previous studies their studies (43). We also failed to observe any significant (43-45) thatEpo stimulation induces tyrosine phosphorylation association of the EpoR with Shc in the 32D or DA3 clones of Shc and its association with Grb2. Since Shc is strongly
8
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Activation of MAP Kinases by the Epo Receptor
implicated in the activation of Ras through its interaction withthat recognize the sis-inducible element is also utilized by reGrb2 and indirectly with mSos1 (24, 26), Epo-induced activa- ceptors for various growth factors, cytokines, and interferons tion of Ras maybe mediated through tyrosinephosphorylation (55,56). Importantly, the J A K family of tyrosine kinases,which of Shc. In accordance with this idea, in cells expressing the includes JAK2, is stronglyimplicated in this pathway(55,561. mutant EpoRs, tyrosine phosphorylation of Shc and its asso- It is thus tempting to speculate that JAK2, which is activated ciation with Grb2 correlated with activation of MAP kinases, by the H mutant, may activatea latent transcription factor in which are major targets of the Ras signaling pathway (31, 32). the cytoplasm through tyrosinephosphorylation and thus leads Very recently, we found that Epo also induces tyrosine phos- to activationof the promotersof the c-fos gene and other genes phorylation of Vav (46), which has also been implicated in the involved in cellular proliferation. Ras signaling pathway(47). However, the tyrosinephosphorylAcknowledgment-We thank Kaon Okada for expert technical ation of Vav induced by Epo did not show correlation with the assistance. activation of MAP kinases incells expressing the various muREFERENCES tant EpoRs (46). Therefore, a role, if any, of Vav in the Epo1. Koury, M. J., and Bondurant, M. C. (1992) Eul: J. Biochem. 210,649-663 induced activation of Ras remains to be known. 2. Krantz, S. B. (1991) Blood 77,419-434 Recent studies have demonstrated that activation of MAP 3. Ihle, J. N., Quelle, F. W., and Miura, 0. (1993) Semin. Immunol. 5, 375-389 4. Youssoufian, H., Longmore, G., Neumann, D.,Yoshimura, A,, and Lodish, H. F, kinases is involved in signaling from most of the members of (1993) Blood 81,2223-2236 cytokine receptor family. However, the functional significance 5. Bazan, J. F. (1989) Biochem. Biophys. Res. Commun. 164, 788-795 of MAP kinase activation in regulation of growth anddifferen6. Miyajima, A., Kitamura, T., Harada, N., Yokota, T., and Arai, K.(1992)Annu. Reu. Immunol. 10, 295-331 tiation of hematopoietic cells by these cytokines remains elu7. DAndrea, A. D., Fasman, G. D., and Lodish, H. F. (1989) Cell 58, 1023-1024 sive. 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(1993) Cell 74,227-236 kinases also failed to correlate with mitogenesis, since the trun- 19. Miura, O., Nakamura, N., Quelle, F. W., Witthuhn, B. A,, Ihle,J. N., and Aoki, N. (1994) Blood 84, 1501-1507 cated H mutant, whose mitogenic function is intact, showed a 20. Bollag, G., and McCormick, E (1991)Annu. Reu. Cell Biol. 7,601-32 significant impairment in activationof MAP kinases, whereas 21. Satoh, T., Uehara, Y,and Kaziro, Y. (1992)J.Biol. Chem. 267,2537-2541 cells expressing the constitutively activated EpoR mutant grew 22. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li,W., Margolis, B., Lammers, R., Ullrich, A,, Skolnik, E.Y., Bar-Sagi, D., and Schlessinger,J. (1992) Cell 70, in growthfactor-deficient medium withoutshowing any detect431442 23. Matuoka, K., Shibata, M., Yamakawa, A,, and Takenawa, T. (1992) Proc. Nutl. able MAP kinase activation. These observations raise a possiAcud. Scz. U. S. A. 89,9015-9019 bility that activationof MAP kinases may playa role in trans- 24. 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