thrombopoietin induces tyrosine phosphorylation and activation

RAPID COMMUNICATION

Thrombopoietin Induces Tyrosine Phosphorylation and Activation of the Janus Kinase, JAK2

By P. Justin Tortolani, James A. Johnston, Chris M. Bacon, Daniel W. McVicar, Akihiro Shimosaka, Diana Linnekin, Dan L. Longo, and John J. O'Shea

Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets that exerts its effects via the receptor, c-Mpl. This receptor is a member of the hematopoietin receptor superfamily and is essential for megakaryocyte maturation; however, the molecular mechanisms of TPO and c-Mpl action have not been elucidated. Recently, the Janus kinases have emerged as important elements in signaling via this family of receptors. In this report, we show that, in the M07e megakaryocytic cell line, which expresses c-Mpl and proliferates in response to TPO, TPO induces phosphorylation of a number

T HROMBOPOIETIN (TPO) is a novel cytokine whose biologic effects implicate it as a major mediator of

megakaryocyte growth and platelet production.'.' TPO sup- ports early megakaryocyte-progenitor colony formation and induces expression of megakaryocyte differentiation markers, polyploidization, and maturation into platelets.' In vivo, TPO significantly expands bone marrow and splenic megakaryocytes and their CD34+ precursors resulting in increased platelet prod~ction."~ Produced by the liver, TPO is a soluble, humoral factor sharing 50% amino acid similarity with erythropoietin (EPO).'

The cell surface receptor for TPO is encoded by the protooncogene c-mpl and has homology to the EPO and interleukin-3 (IL-3) receptors.6-8 Interestingly, within conserved signal transducing domains of these receptors, the so-called Box1 and Box2 regions, c-Mpl exhibits similarity to the IL- 2RP chain and IL-7R,' both of which form heterodimers with the IL-2 receptor common y chain ( y c ) upon ligand binding.'""' Functional studies have indicated that soluble

From the Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesdu, MD; the Laboratory of Experimental Immunology, Laboratory of Leukocyte Biology, Biological Response Modifiers Program, NCI-FCRDC, Frederick, MD; the Institute for Cancer Studies, University of Shef- field Medical School, Shefield, South Yorkshire, UK; and the Kirin Brewing, Ltd, Tokyo, Japan.

Submitted February 14, 1995; accepted April 3, 1995. P.J.T. is a Howard Hughes Medical Institute-NIH Research

Scholar. C.M.B. is supported by grants from the University of Shef- field and the Fulbright Commission.

Address reprint requests to P. Justin Tortolani, Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Insti- tute of Arthritis and Musculoskeletal and Skin Diseuses, National Institutes of Health, Bldg 10, Room 9N-262, 10 Center Dr, MSC 1820, Bethesda, MD 20892-1820.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

This is a US government work. There are no restrictions on its use. 0006-4971/95/8512-0055$0.00/0

of substrates between 80 and 140 kD. Specifically, we show that stimulation with TPO induces the rapid tyrosine phosphorylation of a 130-kD protein that we identify as the Janus kinase, JAK2. However, no detectable tyrosine phosphorylation of JAK1, JAK3, or TYK2 was observed. TPO also induced activation of JAW phosphotransferase activity in vitro. Taken together, these data indicate that JAK2 likely plays a key role in TPO-mediated signal transduction. This is a US government work. There are no restrictions on its use.

c-Mpl can inhibit the effects of TPO on megakaryocyte differentiation,','" and treatment of CD34' progenitors with c-mpl antisense oligodeoxynucleotides greatly inhibits megakaryocyte colony formation without affecting the colony-forming ability of erythroid or myeloid precursors." In addition, mice lacking c-mpl have increased levels of circu- lating TPO as well as a dramatic reduction in both megakaryocytes and platelets, although maintaining normal quantities of other hematopoietic cell types.I4

Hematopoietin receptors, such as c-Mpl, lack intrinsic kinase domains; however, ligand binding characteristically induces tyrosine phosphorylation of multiple intracellular pro- teins, a requisite event for the initiation of signal tran~duction.'~ Recently, members of the Janus family of nonreceptor protein tyrosine kinases (JAKs) have emerged as key mediators of hematopoietin receptor signalling."." Genetic complementation studies have indicated that JAKl and TYK2 are required for interferon (IFN) alp signaling"," and that JAKl and JAK2 are necessary components of the IFN y pathway.I8.'" We and others have shown that JAK3 is functionally coupled to IL-2R yc and that each known cognate ligand (IL-2, IL-4, IL-7, IL-9, and IL- 15) can induce the rapid activation of JAK3 and JAKl (manuscript submitted).1""2,2' In addition, biochemical approaches have shown JAKI, JAK2, and TYK2 to be involved in the signaling cascades of many other hematopoietin receptors including EPO, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-12.1".17.'2~2' Current evidence suggests a model in which ligand-induced receptor homo- or hetero-dimerization leads to activation of receptor-associated JAK kinases, resulting in rapid tyrosine phosphorylation of intracellular substrates.

In the present study, we used the human megakaryoblastic leukemia-derived line, M07e,26 to investigate the role of the JAK kinases in the TPO/c-Mpl signaling pathway. We show that TPO induces the specific and rapid tyrosine phosphorylation and activation of JAK2. These results suggest an important role for JAK2 in TPO signal transduction.

MATERIALS AND METHODS

Reagents und cell culture. Recombinant human TPO was gener- ously provided by Kirin Brewing Ltd (Tokyo, Japan). Recombinant

3444 Blood, Vol85, No 12 (June 15), 1995: pp 3444-3451

For personal use only.on November 17, 2018. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

TPO AND JAK2 ACTIVATION 3445

human IL-2 was kindly provided by Cetus Oncology Corp (Em- eryville, CA). Recombinant human IL-4 was purchased from Pepro- tech (Rocky Hill, NJ). Recombinant human GM-CSF, stem cell factor (SCF), and recombinant human IL-3 were obtained through Dr Craig Reynolds (Biological Response Modifiers Program, NCI- FCRDC, Frederick, MD). Recombinant human IFN-a was obtained from Hoffmann-La Roche (Nutley, NJ). Anti-JAK3 polyclonal rabbit antiserum has been described p rev io~s ly .~~ Anti-JAK1, JAK2, and TYK2 polyclonal rabbit antisera; monoclonal antiphosphotyrosine antibody (4G10); and JAK2 immunizing (cognate) peptide were purchased from Upstate Biotechnology, Inc (Lake Placid, NY). M07e (Genetics Institute, Cambridge, MA), a GM-CSF- and SCF- dependent subline of the human megakaryoblastic leukemia line M07,26 was maintained in RPMI-1640 (Biofluids Inc, Rockville, MD) supplemented with gentamicin (0.1 mg/mL), 1% L-glutamine (Life Technologies), 10% fetal calf serum (FCS; Sigma, St Louis, MO), 100 ng/mL SCF, and 10 ng/mL GM-CSF. TF-l, a GM-CSF- dependent human pluripotent cell was maintained in RPMI- 1640 (Biofluids Inc) supplemented with gentamicin (0.1 mg/mL), 1% glutamine (Life Technologies), 10% FCS, and 10 ng/mL GM- CSF. Human fetal liver cells were procured through Advanced Bio- science Resources Inc (Alameda, CA).

Cell proliferation assay. Cells were washed in serum-free RPMI-1640, resuspended at a concentration of lo5 cells/mL, and then plated in a 96-well microtiter dish (Costar, Cambridge, MA) and incubated at 37°C after adding a 100 pL aliquot of growth factor or media (RPMI-1640, 10% FCS). All assays were performed in triplicate. After 4 days of culture, each well was pulsed with 1 pCi of 3H-thymidine (6.7 Ci/mM; NEN, Boston, MA) for 4 hours and then harvested onto glass fiber filter paper (Filtermat, Skatron, Inc. Sterling, VA). Filter strips were dried and quantitated in a liquid scintillation counter (Model 1216; LKB, Piscataway, NJ).

Immunoprecipitation and irnmunoblotting. For analysis of JAK phosphorylation, 2 X IO’ cells/point were washed free of GM-CSF and SCF and cultured overnight in factor-free medium (RPMI 1640 supplemented as above) before stimulation with TPO (1,000 ng/mL unless otherwise indicated), IL-2 (1,OOO U/mL), IL-4 (1,000 U/mL), GM-CSF (10 ng/mL), or IFNa (1,000 U/mL). After stimulation, the cells were washed in ice-cold phosphate-buffered saline containing 1 mmoVL EDTA and 0.4 mmol/L sodium orthovanadate and lysed in buffer containing 1% Triton X-100 before centrifugation to remove insoluble material. Cleared lysates were immunoprecipitated with polyclonal rabbit antisera precoupled to protein-A Sepharose or monoclonal 4G10 antibody precoupled to protein-G Sepharose and washed in buffer containing 0.1% Triton X-100. Immunoprecipitates were eluted by boiling in 2X sodium dodecyl sulfate (SDS) sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to Immobilon (Millipore, Bedford, MA). The filters were blocked with 1% fish gelatin, 2% goat serum, and 0.1 % bovine serum albumin (BSA) in TBS-Tween (20 mmol/L Tris, 150 mmoVL NaCI, 0.5% Tween) and sequentially incubated with antiphosphotyrosine antibody, biotinylated goat anti- mouse IgG (Oncogene Science, Inc, Cambridge, MA), and horserad- ish peroxidase (HRP)-conjugated streptavidin (Oncogene Science, Inc). Detection was performed using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IN). For reblotting, filters were first treated with 15% hydrogen peroxide and then blocked in tris- buffered saline containing 5% milk and 0.1% Tween. The mem- branes were then incubated with the indicated polyclonal rabbit antisera, washed, incubated with HRP-conjugated goat antirabbit IgG (Boerhinger Mannheim, Indianapolis, IN), washed again, and developed by ECL (Amersham).

Kinase assays were performed as described.28 Briefly, cells were lysed in buffer containing 1 % Triton X-100 and immunoprecipitated with JAK3 antiserum (preclearing) followed by immunoprecipitation

with the JAK2 antiserum. For peptide competition studies, JAK2 cognate peptide (20 pg) was incubated with cell lysates during anti- JAK2 immunoprecipitation reactions. The washed immunoprecipitates were incubated for 5 minutes on ice in 50 pL of buffer containing 20 mmoVL Tris, 5 mmoVL MgCl,, 5 mmoVL MnCI,, and [y3’P] ATP (Amersham) at 200 pCimL. The reaction was termi- nated by the addition of ice-cold wash buffer. The immunoprecipitates were then washed again, eluted, and subjected to SDS-PAGE. Gels were dried and exposed to Kodak X-AR5 film overnight (East- man Kodak, Rochester, NY).

RESULTS

TPO induces proliferative responses in human fetal liver cells and the M07e cell line. TPO has previously been shown to induce the in vitro growth and development of megakaryocytes from hematopoietic stem cell population^.^^ Fetal liver cells are a major source of hematopoietic precursors; therefore, we examined the effects of TPO in this cell population. In accordance with previous finding^:^ TPO induced a large mitogenic response in human fetal liver cells (24-fold increase relative to media alone) as measured by 3H-thymidine incorporation (Fig 1A). To determine if TPO induced an analogous response in a cell line known to ex- press the megakaryocytic and platelet markers gpI, and gpIId III,,3° respectively, as well as the TPO receptor, c-MpZ,I3 we stimulated M07e cells with TPO. Figure 1B shows that TPO- induced proliferation was comparable to that induced by IL- 3, a well-known growth factor for M07e cells.26 In addition, IL-2 and E-4 stimulated moderate proliferation in these cells (4-fold and 8-fold increases relative to medium alone, respectively), and GM-CSF was a potent mitogen. These results suggested that M07e cells proliferate in response to TPO in a manner similar to human hematopoietic progenitor populations and, therefore, would provide a useful in vitro model for the study of the signaling pathways by which TPO might function.

Protein tyrosine phosphorylation induced by TPO. Al- though TPO induces proliferation of human fetal liver cells and M07e cells, the molecular mechanisms of this action are unclear. The receptor for TPO, c-Mpl, is a member of the hematopoietin receptor superfamily, suggesting that tyrosine phosphorylation might be an early event after TPO binding. To examine this possibility, we immunoprecipitated lysates of M07e with antiphosphotyrosine antibodies, followed by immunoblotting with the same antiphosphotyrosine antibody. As shown in Fig 2, TPO induced tyrosine phosphorylation of a number of substrates (80 to 150 kD). In particular, TPO strongly induced tyrosine phosphorylation of a protein of approximately 130 kD (Fig 2, lanes 3 and 4) that was not detectable by antiphosphotyrosine immunoblotting in unstimulated cells (lane 1). The molecular weight of this protein was consistent with it being a Janus kinase family member. Indeed, a phosphoprotein migrating at the same weight is also detectable in the GM-CSF-treated cells (Fig 2, lane 5). It has previously been shown that GM-CSF stimulation causes the tyrosine phosphorylation of the JAK family kinase JAK2,23 suggesting that the 130-kD TPO-induced substrate might also be JAK2.

TPO induces tyrosine phosphorylation and activation of JAK2. To confirm the identity of the 130-kD protein as




3446 TORTOLANI ET AL

B

/ o _ I W media 60000 -

50000- U_ 8 .- 5 40000- * a : 5 30000

4 E

U

C .-

,x20000 c

10000

0

media

0 TPO

IL-2

61 IL-4

IL-3

0 GM-CSF

Fig 1. TPO induces proliferative responses in fetal liver cells and M07e cells. (A) Fetal liver cells were incubated in medium alone or stimulated with TPO (100 ng/mL). (B) M07e cells were incubated in medium alone or stimulated with TPO (100 ng/mL), IL-2 (250 UlmLI, IL-4 (100 UlmL), IL-3 (10 ng/mL), or GM-CSF (10 ng/mL) as indicated. Cell proliferation was measured by 3H-thymidine incorporation and reported in counts per minute (CPM).

JAK2, we stripped the filter in Fig 2 and reprobed with antiserum to JAK2. No observable JAK2 was detected from cells stimulated with medium alone or with the T-cell growth factor, IL-2, which induces tyrosine phosphorylation of JAK3 and JAKl (Fig 3A).” However, a single band corre- sponding to JAK2 was observed in immunoprecipitates from M07e cells stimulated with either of the growth factors, TPO and GM-CSF (Fig 3A, lane 3 through S).

To more firmly demonstrate TPO-dependent phosphorylation of JAK2, we next directly immunoprecipitated JAK2 from stimulated M07e cell lysates followed by antiphosphotyrosine immunoblotting. As shown in Fig 3B, no detectable tyrosine phosphorylation of JAK2 was observed in cells treated with medium alone. In contrast, GM-CSF induced readily detectable tyrosine phosphorylation of JAK2 relative to controls (Fig 3B, lane S). Similarly, tyrosine phosphorylation of JAK2 was observed in TPO-treated cells (Fig 3B, lanes 3 and 4). However, IL-2 stimulation resulted in the tyrosine phosphorylation of the 125-kD JAK3 protein (observed in JAK2 immunoprecipitates as a result of the previously described cross-reactivity of the JAK2 antiserum“’).

The kinetics of tyrosine phosphorylation of JAK2 are shown in Fig 3C. Tyrosine phosphorylation after TPO treatment was maximal at S minutes and was maintained for 60 minutes. This prolonged response contrasts with the decline at 30 minutes previously observed for JAKs analyzed from cells stimulated with other cytokines.”.” In addition, we observed similar levels of JAK2 tyrosine phosphorylation in cells treated with GM-CSF (Fig 3C. lane 6). To ascertain that equal levels of JAK2 were immunoprecipitated for each condition. the filter was stripped and reprobed with antisera to JAK2 (Fig 3R and C, lower panels).

Tyrosine phosphorylation of the JAKs has been shown to

be associated with activation of their kinase activity.”.” We therefore investigated the in vitro kinase activity of JAK2 immunoprecipitates (Fig 3D) before and after TPO stimulation. Cells were stimulated with TPO for 20 minutes and immunoprecipitated with either normal rabbit serum or anti-JAK2 antiserum as described in the Materials and Methods. Low levels of specific kinase activity were observed in JAK2 immunoprecipitates from unstimulated cells (Fig 3D, lane 3) . In contrast. we observed markedly increased kinase activity in JAK2 immunoprecipitates from cells stimulated with TPO. with the major product of phosphorylation being a 130-kD protein, which is consistent with autophosphorylation of JAK2 (Fig 3D. lane 4). Of note, the dose of TPO ( I O 0 ng/mL) reported here to activate JAK2 phosphotransferase activity was sufficient to induce large proliferative effects (Fig IB). To determine if this effect wi~s specific for JAK2, we immunoprecipitated JAK2 in the presence of the peptide to which the antiserum was generated. As shown (Fig 3D. lanes S and 6). this peptide specifically blocked the immunoprecipitation of JAK2 kinase activity observed in response to TPO. Additionally. minimal kinase activity was observed in cell extracts immunoprecipitated with normal rabbit serum (Fig 3D. lanes 1 and 2).

T P O does not induce tyrosine phosphonlntiorl of JA K3. JAKI. or TYKZ. Some cytokines. such as IL-12. 1L-2. and the interferons, induce tyrosine phosphorylation of more than one JAK family kinase.“’.” Therefore, we examined the tyrosine phosphorylation of JAKl. JAK3. and TYK2 upon stimulation of M07e cells by TPO. As indicated in Fig 4, tyrosine phosphorylation of JAK (Fig 4A. lanes 4 through 6). JAKl (Fig 4B, lane 4). and TYK2 (Fig 4C, lane 3) was not induc- ible in response to TPO. Consistent with previous findings,“’.’* IL-2 and IL-4 stimulation induced tyrosine phosphorylation of JAK3 (Fig 4A. lanes 2 and 3. respectively) and JAKl (Fig 4B. lane 2). and IFNa stimulation induced




TPO AND JAK2 ACTIVATION

0 CL l-

0 a l-

220 -

97 -

66 -

1 2 3 4 5 Fig 2. TPO induces protein tyrosine phosphorylation of cellular

substrates. M07e cells were incubated for 15 minutes in medium alone (lane l), with IL-2 (lane 2). with 100 ng/rnL TPO (lane 3). with 1,000 ng/rnL TPO (lane 4), or with GM-CSF (lane 5). and lysates were immunoprecipitated with antiphosphotyrosine monoclonal antibody. The immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine irnrnunoblotting.

tyrosine phosphorylation of both TYK2 (Fig 4C, lane 3) and JAKl (Fig 4B. lane 3). Again, stripping and reprobing with antisera to JAK3, JAKI, and TYK2 (Fig 4, lower panels) indicated that similar levels of protein were immunoprecipitated for each condition.

To confirm that these effects were not restricted to a single cell line, we examined the tyrosine phosphorylation of JAK2 and JAKl in the pluripotent leukemic cell line, T F - I , that expresses c-Mpl.'' As shown in Fig 4D, TPO stimulation induced the tyrosine phosphorylation of JAK2 (lane 2) but not of JAKl (lane S). Consistent with the findings above, GM-CSF induced tyrosine phosphorylation of JAK2 (lane 3). whereas IL-4 stimulation resulted in JAKl tyrosine phosphorylation. Stripping and reprobing with antisera to JAK2 and JAKl indicated that equal levels of protein were immunoprecipitated for each condition. Taken together, these results indicate that, similar to many other cytokines that bind hematopoietin receptors, TPO induces tyrosine phosphorylation and activation of a Janus kinase and that this effect is specific for JAK2.

DISCUSSION

TPO has recently been described as an important mediator of both megakaryocytopoiesis and thrombopoiesis through

3447

its binding and activation of the receptor c-Mpl. In this report, we show that TPO potently induces the proliferation of M07e cells and human fetal liver cells, as well as the rapid tyrosine phosphorylation of several intracellular substrates. In particular, TPO stimulates tyrosine phosphorylation and activation of the JAK family kinase, JAK2. We show that this response is specific for JAK2 because the other known JAK kinases (JAKI, JAK3, and TYK2) are not tyrosine phosphorylated in response to TPO in M07e cells.

TPO exhibits SO% amino acid homology with EPO,' and the receptor, c-Mpl, shares significant homology with the extracellular domain of the EPO receptor.' These structural similarities suggested that TPO and EPO might signal similarly in their respective cell lineages. Indeed, it has previously been shown that EPO stimulates tyrosine phosphorylation and activation of JAK2, which correlates with the induction of mitogenesis in erythroid cells.** In this report we show that JAK2 is similarly tyrosine phosphorylated and activated on TPO stimulation and that TPO induces potent proliferative responses in the M07e megakaryocytic cell line. It is plausible that certain other downstream pathways will also be similar, including activation of the signal transducers and activators of transcription (STATs). This hypothesis is currently under investigation in our laboratory.

The current model of cytokine signaling suggests that ligand binding induces oligomerization of receptor subunits, which brings receptor-associated JAK molecules into sufficient proximity for intermolecular tyrosine phosphorylation. Membrane proximal cytoplasmic domains of many of the hematopoietin receptors, containing conserved Boxl and Box2 regions, are thought to mediate some of these interac- tions and to be essential for proliferation. For both EPOR and the common GM-CSFR subfamily f l chain (pc), JAK2 associates with the Boxl and Box2 membrane proximal regions, and mutations that inhibit the JAK2 association can be correlated with a loss of proliferative responses.**.*'." There is also evidence that the membrane proximal region of c-Mpl is important for mitogenesis? Transfection of wild- type and mutant forms of the oncogene env-mpl (a fusion protein containing the extracellular portion of the virally encoded envelope gene, env, and the cytoplasmic v-mpl se- quence) into a growth-factor-dependent cell line, identified a 69 amino acid, membrane proximal region necessary for factor-independent proliferation.' This information suggested that the pathogenicity of v-mpl is dependent on its ability to deliver a constitutive proliferative signal through this membrane proximal region. To date, a physical association of JAK2 with this region of v-Mpl has not been estab- lished; however, mutations that delete either the Boxl or the Box2 subdomain abolish this factor-independent cell growth: suggesting that this is a likely region for JAK2 association.

c-Mpl also exhibits amino acid similarity with the IL-2Rfl and IL-7R subunits." These subunits form heterodimers with the common IL-2R y chain ( y c ) on ligand binding. However, because all previously characterized yc users (IL-2, -4, -7, -9, and -15) induce tyrosine phosphorylation of JAKl and JAK3 (manuscript submitted),"""." but not JAK2, and because mice lacking yc have no reported platelet defects;*.'' it seems unlikely that TPO uses yc.




3448

C

TORTOIANI ET AL

220 - - ""

97 -

66 -

B

1 2 3 4 5 LL

Time (mins) o G 30 60' 15

220 - - JAK2 + JAK2

97.4 -

66 - """

Time (mins) 220 -

97 -

66 -

5 '5 20' 5

JAK2 JAK3

D

1 2 3 4 5 6

Q 2

,P er

+JAK2 1 2 3 4 5

+ JAK2

97- 1 2 3 4 5 6

Fig 3. TPO induces rapid tyrosine phosphorylation and activation of JAK2. (A) AntiJAK2 immunoblotting. The antiphosphotyrosine immunoblot from Fig 2 above was reprobed with antiserum to JAK2. (B) Tyrosine phosphorylation of JAK2 in response to TPO. M07e cells were incubated for the indicated times in medium alone (lane l ) , with 11-2 (lane 2). with TPO (lanes 3 and 4). or with GM-CSF (lane 5). and lysates were immunoprecipitated with antiJAK2 antiserum. The immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and antiJAK2 immunoblotting (lower panel). (C) Time course of JAKZ tyrosine phosphorylation. M07e cells were incubated for the indicated times in medium alone (lane l) , with TPO (lanes 2 through 51, or with GM-CSF (lane 6). and lysates were immunoprecipitated with antiJAK2 antiserum. Immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and anti-JAK2 immunoblotting (lower panel). (D) In vitro kinase activity of JAK2 immunoprecipitates. M07e cells were incubated for 20 minutes in medium alone (-1 or with 100 ng/mL TPO (+ l , and lysates were immunoprecipitated with either normal rabbit serum (NRS) or anti-JAK2 antiserum in the absence (aJAK21 or presence of cognate peptide laJAK2 + JAKZ peptide). Reaction products were resolved by SDS-PAGE and analyzed by autoradiography.

Because we show that TPO, like GM-CSF, induces tyro- In summary, TPO, in conjunction with its cognate hemato- sine phosphorylation and activation of JAK2, the possibility poietin receptor c-Mpl, regulates the growth and differentia- that c-Mpl forms heterodimers with PE should also be ex- tion of megakaryocytes and platelets. We have shown that plored. Alternatively, TPO signaling may parallel the EPO the JAK family kinase, JAKZ, appears to be uniquely in- model in which only single receptor chains are thought to volved in the early signaling events after TPO stimulation be involved. These questions and others clearly define areas of M07e cells. These findings contribute to the understanding for further investigation. of TPO/c-Mpl signal transduction. Indeed, it is tempting to




A Time (mins)

220 -

0 B l-

' l 5 20'

200 -

97.4 -

66 -

C

e- 1- JAK3

1" 97 -

1 2 3 4 5 6

0 tl

o l " n E

220-

97-

66-

1 2 3

+ JAKl

f JAKl

wm.m 1 2 3 4

220 -

97 - - TYK2 1 2 3 4 5 6

JAK2 JAKI

Fig 4. TPO does not induce tyrosine phosphorylation of JAK1, JAK3, or TYK2. (A) M07e cells were incubated for the indicated times in medium alone (lane l), with IL-2 (lane 2). with IL4 (lane 31, or TPO (lanes 4 through 6). and lysates were immunoprecipitated with anti-JAK3 antiserum. Immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and anti-JAK3 (lower panel) immunoblotting. (6) M07e cells were incubated for 15 minutes in medium alone (lane l), with IL-4 (lane 2). with IFN-a (lane 31, or with TPO (lane 41, and lysates were immunoprecipitated with with anti-JAK1 antiserum. Immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and anti-JAK1 (lower panel) immunoblotting. (C) M07e cells were incubated for l 0 minutes in medium alone (lane l), with TPO (lane 21, or with IFNa (lane 3). and lysates were immunoprecipitated with anti-TYK2 antiserum. Immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and anti-TYKZ (lower panel) immunoblotting. (D) TF-1 cells were incubated for 15 minutes in medium alone (lanes 1 and 4). with TPO (lanes 2 and 5). with GM-CSF (lane 3). or with IL-4 (lane 6). and lysates were immunoprecipitated with antiJAK2 or anti-JAK1 antiserum. Immunoprecipitates were resolved by SDS-PAGE and analyzed by antiphosphotyrosine and anti-JAK2 or anti-JAK1 (lower panel) immunoblotting.




3450 TORTOLANI ET AL

speculate that ultimately pharmacologic activators of JAK2 may be developed that are useful in treating leukopenic and thrombocytopenic patients.

ACKNOWLEDGMENT

We thank Drs M.C. Riedy, Juan Rivera, Andrew Lamer, and Warren J. Leonard for their critical review of this manuscript.

REFERENCES 1. de Sauvage FJ, Hass PE, Spencer SD, Darbonne W, Malloy

BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang W-J, Oles KJ, Hultgren B, Solberg LA Jr, Goeddel DV, Eaton DL: Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature 369:533, 1994

2. Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Sprechter CA, Blumberg H, Johnson R, Prunkard D, Ching AFT, Mathewes SL, Bailey MC, Forstrom JW, Buddle MM, Osbom SG, Evans SJ, Sheppard PO, Presnell SR, O’Hara PJ, Hagen FS, Roth GJ, Foster DC: Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369:565, 1994

3. Kaushansky D, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, Roth GJ, Papa- yannopoulou T, Foster DC: Promotion of megakaryocyte progenitor expansion and differentiation by the c-mpl ligand thrombopoietin. Nature 369:568, 1994

4. Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W: c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 369:571, 1994

5. Bartley TD, Bogenberger J, Hunt P, Li Y-S, Lu HS, Martin F, Chang M-S, Samal B, Nichol JL, Swift S, Johnson MJ, Hsu R-Y, Parker P, Suggss S, Skrine JD, Merewether LA, Clogston C, Hsu E, Hokom MM, Homkohl A, Choi E, Pangelinan M, Sun Y, Mar V, McNinch J, Simonet L, Jacobsen F, Xie C, Shutter J, Chute H, Basu R, Selander L, Trollinger D, Sieu L, Padilla, Trail G, Elliot G, Izumi R, Covey T, Crouse J, Garcia A, Xu W, Del Castillo J, Biron J, Cole S, Hu MC-T, Pacifici R, Ponting I, Saris C, Wen D, Yung YP, Lin H, Bosselman RA: Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77:1117, 1994

6. Vigon I, Mornon J-P, Cocault L, Mitjavila M-T, Tambourin P, Gisselbrecht S, Souryi M: Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: Identification of a member of the hematopoietic growth factor receptor superfamily. Proc Natl Acad Sci USA 89:5640, 1992

7. Vigon I, Florindo C, Fichelson S, Guenet J-L, Mattei M, Souyri M-G, Cosman D, Gisselbrecht S: Characterization of the murine Mpl protooncogene, a member of the hematopoietic cytokine receptor family: Molecular cloning, chromosomal location and evidence for a function in cell growth. Oncogene 8:2607, 1993

8. Skoda RC, Seldin DC, Chiang M-K, Peichel CL, Vogt TF, Leder P: Murine c-mpl: A member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal. EMBO J 12:2645, 1993

9. Benit L, Courtois G, Charon M, Varlet P, Dusanter-Fourt I, Gisselbrecht S : Characterization of mpl cytoplasmic domain se- quences required for myeloproliferative leukemia virus pathogenicity. Virology 68:5270, 1994

10. Russell SM, Johnston JA, Noguchi M, Kawamura M, Bacon CM, Friedman M, Berg M, McVicar DW, Witthuhn BA, Siven- noinen 0, Goldman AS, Schmalstieg FC, Ihle JN, O’Shea JJ, Leo-

nard WJ: Interaction of IL-2Rp and yc Chains with Jakl and Jak3: Implications for XSCID and XCID. Science 266: 1042, 1994

1 1 . Miyazaki T, Kawahara A, Fujii H, Nakagawa Y, Minami Y, Liu 2-J, Oishi I, Silvennoinen 0, Witthuhn BA, Ihle JN, Taniguchi T: Functional activation of Jakl and Jak3 by selective association with IL-2 receptor subunits. Science 266:1045, 1994

12. Noguchi M, Nakamura Y, Russell SM, Ziegler SF, Tsang M, Cao X, Leonard WJ: Interleukin-2 receptor y chain: A functional component of the interleukin-7 receptor. Science 262: 1877, I993

13. Methia N, Louache F, Vainchenker W, Wendling F: Oligo- deoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 82:1395, 1993

14. Gurney AL. Carver-Moore K, de Sauvage FJ, Moore MW: Thrombocytopenia in c-mpl-deficient mice. Science 265: 1445, 1994

15. Miyajima A, IOtamura T, Harada N, Yokota T, Arai K: Cy- tokine receptors and signal transduction. Annu Rev Immunol 10:295. 1992

16. lhle JN , Witthuhn BA, Quelle FW, Yamamoto K, Their- felder WE, Kreider B, Silvennoinen 0: Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci 19:222, 1994

17. Ziemecki A, Harpur AG, Wilks AF: JAK protein tyrosine kinases: Their role in cytokine signaling. Trends Cell Biol 4:207, 1994

18. Muller M, Briscoe J, Laxton C, Guschin D, Ziemecki A, Silvennoinen 0, Harpur AG, Barbieri G, Witthuhn BA, Schindler C, Pellegrini S, Wilks AF, Ihle JN, Stark GR, Kerr IM: The protein tyrosine kinase JAKl complements defects in interferon-alp and -y signal transduction. Nature 366:129, 1993

19. Velasquez L, Fellous M, Stark GR, Pellegrini S: A protein kinase in the interferon (YIP signaling pathway. Cell 70:313, 1992

20. Watling D, Guschin D, Muller M, Silvennoinen 0, Witthuhn BA, Quelle FW, Rogers NC, Schindler C, Stark GR, Ihle JN, Kerr IM: Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-? signal transduction pathway. Nature 366:166, 1993

21. Johnston JA, Kawamura M, Kirken RA, Chen Y-Q, Blake TB, Shibuya K, Ortaldo JR, McVicar DW, O’Shea JJ: Phosphoryla- tion and activation of the Jak-3 Janus kinase in response to interleukin-2. Nature 370:151, 1994

22. Witthuhn FA, Quelle FW, Silvennoinen 0, Taolin Y, Tang B, Miura 0, Ihle JN: JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74:227, 1993

23. Quelle FW, Sat0 N, Witthuhn BA, Inhorn RC, Eder M, At- sushi M, Griffin JD, Ihle JN: JAK2 associates with the ,Bc chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 14:4335, 1994

24. Stahl N. Boulton TG, Farmggella T, Ip NY, Davis S, Wit- thuhn BA, Quelle F W , Silvennoinen 0, Barbieri G, Pellegrini S, Ihle JN, Yancopoulos GD: Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-11-6 ,B receptor components. Science 263:92, 1994

25. Bacon CM, McVicar DW, Ortaldo JR, Rees RC, O’Shea JJ, Johnston JA: Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: Differential use of Janus family tyrosine kinases by IL-2 and L-12. J Exp Med 181:399, 1995

26. Avanzi GC, Brizzi MF, Gianotti J, Ciarletta A, Yang Y, Peg- oraro L, Clark SC: M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J Cell Physiol 145:458, 1990

27. Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miya- gawa K, Pia0 Y-F, Miyazona K, Urabe A, Takaku F: Establishment




TPO AND JAKZ ACTIVATION 3451

and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, and erythropoeitin. J Cell Physiol 140:323, 1989

28. Kawamura M, McVicar DW, Johnston JA, Blake T, Chen Y- Q, La1 BK, Lloyd AR, Kelvin DJ, Staples EJ, Ortaldo JR, O’Shea JJ: Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc Natl Acad Sci USA 91:6374, 1994

29. Zeigler FC, de Sauvage F, Widmer HM, Keller CA, Donahue C, Schreiber RD, Malloy B, Hass Philip, Eaton D, Matthews W In vitro megakaryocytopoietic activity of c-mpl ligand (TPO) on puri- fied murine hematopoietic stem cells. Blood 84:4045, 1994

30. Avanzi GC, Lista P, Giovinazzo B, Miniero R, Saglio G, Benetton G, Coda R, Cattoretti G, Pegoraro L: Selective growth

response to L-3 of human leukaemic cell line with megakaryoblastic features. Br J Haematol 69:359, 1987

31. Miura 0, Nakamura N. Quelle W , Witthuhn BA, Ihle JN, Aoki N: Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo. Blood 84:1501, 1994

32. Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, Russell SM, Drago J, Noguchi M, Grinberg A, Bloom ET, Paul WE, Katz, Love PE, Leonard WJ: Defective lymphoid development in mice lacking expression of the common cytokine receptor y chain. Immu- nity 2:238, 1995

33. DiSanto J P , Muller W, Guy-Grand D, Fischer A, Rajewsky K Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor y chain. Proc Natl Acad Sci USA 92:377, 1995




1995 85: 3444-3451

O'SheaPJ Tortolani, JA Johnston, CM Bacon, DW McVicar, A Shimosaka, D Linnekin, DL Longo and JJ the Janus kinase, JAK2Thrombopoietin induces tyrosine phosphorylation and activation of

http://www.bloodjournal.org/content/85/12/3444.full.htmlUpdated information and services can be found at:

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American


http://www.bloodjournal.org/content/85/12/3444.full.html

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





thrombopoietin induces tyrosine phosphorylation and activation

Documents