©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Amino-terminal Portion of the JAK2 Protein Kinase Is Necessary For Binding and Phosphorylation of the Granulocyte-Macrophage Colony-stimulating Factor Receptor Chain (*)

Yanming Zhao (1), Fred Wagner (1), Stuart J. Frank (2)(§), Andrew S. Kraft (1)(¶)

From the (1) Divisions of Hematology/Oncology and Endocrinology and the (2) Veterans Administration Medical Center, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptor stimulates JAK2 protein kinase activation, protein phosphorylation, and JAK2 association with the chain of the GM-CSF receptor. To better understand how different domains of the JAK2 function to regulate association and phosphorylation of the receptor, the minimal portion of the receptor necessary for JAK2 binding has been determined. Using glutathione S-transferase (GST) fusion proteins expressing different portions of the membrane-proximal domain of the chain, we demonstrate that JAK2 binds to amino acids 458-495, but showed little binding to fusion proteins containing amino acids 483-559, 483-530, or 458-484. The GST- 458-495 bound equally well to the wild type (WT) JAK2, a carboxyl-terminal deletion of JAK2 removing the protein kinase domain (amino acids 1000-1129), and a deletion of the kinase-like domain (amino acids 523-746). However, an amino-terminal JAK2 deletion (amino acids 2-239) markedly reduced binding to this GST-. Far Western blotting demonstrated that a GST fusion protein containing amino acids 1-294 of JAK2, but not fusion proteins containing amino acids 295-522, 523-746, or 747-1127, bound GST- 458-559. When the JAK2 WT and deletions were transiently expressed along with the and subunits of the GM-CSF receptor and the cells were treated with GM-CSF, the following results were obtained: 1) WT JAK2 phosphorylated the subunit in a GM-CSF- dependent manner, 2) the kinase-like domain deletion phosphorylated the subunit, and 3) both the kinase domain deletion and the amino-terminal deletion failed to stimulate phosphorylation of the subunit. Therefore, phosphorylation of the subunit requires the binding of JAK2 through its amino terminus.


INTRODUCTION

GM-CSF() regulates the growth and differentiation of hematopoietic cells by binding to a high affinity receptor composed of an chain and a chain. The chain (60-80 kDa) alone is sufficient for the low affinity binding of GM-CSF (1, 2), as it interacts with the chain (140 kDa) to constitute the high affinity receptor (3, 4) . The chain contains a short intracytoplasmic region of approximately 54 amino acids, which has been shown to be necessary for hormone-stimulated growth (5, 6, 7) . The intracytoplasmic portion of the chain (approximately 400 amino acids) is much larger than that of the chain. In the presence of the chain, a minimum membrane-proximal region of the chain containing 100 amino acids has been shown to be sufficient to mediate GM-CSF-stimulated cell growth (8, 9) . This near membrane-proximal region of the chain contains two stretches of amino acids, denoted box 1 and box 2 (10) , which are highly conserved in the large family of hemopoietin receptors, which includes interleukin-6, interleukin-2, erythropoietin (Epo), granulocyte colony-stimulating factor, growth hormone, and prolactin.

Although the GM-CSF receptor does not encode a protein kinase, binding of GM-CSF to its receptor stimulates the rapid tyrosine phosphorylation of specific protein substrates and the GM-CSFR chain (9, 11, 12, 13) . This GM-CSF-induced phosphorylation has been shown to be mediated by the JAK2 protein tyrosine kinase (9, 14) . The JAK family of protein kinases consists of JAK1, JAK2, JAK3, and Tyk2 (14, 15, 16, 17, 18) . These protein kinases have a molecular mass of approximately 130 kDa and contain a carboxyl-terminal kinase domain, a kinase-like (pseudokinase) domain without SH2 or SH3 domains, and a potential carboxyl-terminal phosphorylation site, VDGYFRL. JAK2 is phosphorylated, and its protein kinase activity is stimulated in response to GM-CSF (9) , Epo (19, 20) , growth hormone (21, 22) , interleukin-6 (24) , prolactin (25) , and interferon (26) . Mutagenesis experiments have demonstrated that JAK2 binds to the near membrane-proximal region of these receptors, which contains both the box 1 and box 2 sequence of amino acids. Deletion of this receptor region blocks JAK2 activation and mitogenesis by these hormones (9, 19, 22, 23, 24, 30, 31) .

To determine the regions of the JAK2 protein kinase that regulate interaction with and phosphorylation of the GM-CSFR chain, we have mapped the domain of the membrane-proximal region of the chain, which binds the JAK2 protein. Using this short sequence, expressed as a GST fusion protein and amino, carboxyl, and pseudokinase deletion mutants of the JAK2 protein kinase, we have determined the region of JAK2 necessary for interaction with the chain. By expressing these mutants in vivo, we demonstrate that specific portions of JAK2 protein kinase regulate hormone-inducible chain phosphorylation.


MATERIALS AND METHODS

Plasmid Construction

cDNAs encoding GST fusion proteins with the intracytoplasmic portion of the GM-CSFR subunit were generated by ligating the PCR fragments of GM-CSFR subunit (a gift of Dr. A. Miyajima, DNAX Research Institute, Palo Alto, CA) into the BamHI/EcoRI site of the pGEX2T vector (Pharmacia Biotech Inc., Alameda, CA). The PCR products correspond to amino acid numbers of the GM-CSFR :458-495 (Box 1 plus), 483- 559 (Box 2), 458-484 (Box 1), 458-559 (Box 1 and 2), and 483-530 (Box bb). The GST fusion proteins were expressed in Escherichia coli, and the proteins were purified as described (6) .

The murine JAK2 cDNA was a gift from Dr. J. Ihle (St. Jude Children's Hospital Memphis, TN). The JAK2 amino-terminal deletion (ATD) was generated in Bluescript SK vector (Stratagene, La Jolla, CA) by replacing the DNA fragment of JAK2 from the ATG start to the first EcoRI site (amino acid 294) with a PCR fragment with a NotI site and a ATG start codon inserted at the 5` end, and spanning amino acids 240-294. The pseudokinase domain (PSKD) deletion was generated by cutting the JAK2 cDNA at the two internal BglII sites, removing the fragment that encodes amino acids (aa) 523-746, and religating the cDNA. The carboxyl-terminal deletion (CTD) was generated by cleaving the cDNA with NdeI and ApaI and removing aa 932-1129 of the carboxyl terminus. This fragment was replaced with a PCR fragment spanning aa 932-999, to which NdeI and ApaI site, as well as a stop codon, had been added. Each cDNA was removed from Bluescript SK and inserted into the NotI/ApaI site of the PRC/CMV vector (Invitrogen, San Diego, CA). cDNA sequencing confirmed the presence of the mutations and the fidelity of the remaining regions subjected to PCR.

GST fusion proteins expressing four fragments of JAK2 (GST-J1, aa 1-294; GST-J2, aa 295-522; GST-J3, aa 523-746; GST-J4, aa 747-1127) were generated by PCR and inserted into the BamHI/EcoRI site of the pGEX2T vector. The sequence of the PCR products was confirmed by sequencing.

Overexpression of JAK2 Deletions in CV-1 Cells

CV-1 cells were maintained in DMEM containing 10% newborn calf serum. 8 10 CV-1 cells in 10-cm dishes were infected with vvT7 pol vaccinia virus (multiplicity of infection = 5) (a gift of Dr. Mark Mulligan, UAB, Birmingham, AL) in 2 ml of DMEM without serum at 37 °C for 1 h. After infection, the cells were incubated in serum-containing media for 2 h and transfected with the various JAK2 cDNAs using Lipofectin (Life Technologies, Inc.; 3-7 µg of plasmid plus 20-40 µl of Lipofectin reagent/10-mm dish) for 4 h. Twenty-four hours after transfection the cells were lysed, and the lysate was used as a source of expressed JAK2 proteins.

GST Fusion Protein Binding Assay

Equal amounts of GST- fusion proteins immobilized on glutathione-Sepharose (Sigma) were washed once in TNE buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 2 mM EDTA) containing 1 mM sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 mM sodium fluoride and once in TNE buffer without inhibitors. Bis(sulfosuccinimidyl) suberate cross-linker (Pierce) was added to the final concentration of 2.5 mM. The cross-linking reaction was allowed to proceed on ice for 15 min and quenched with 10 mM ammonium acetate for an additional 10 min on ice. CV-1 cell lysates (see above) were used as a source of mutated or WT JAK2 proteins. Approximately 10 µg of the Jak2 proteins (as judged by Western blots) were incubated at 4 °C for 30 min with the cross-linked GST- fusion proteins immobilized on glutathione-Sepharose. Resins were extensively washed in lysis buffer, and associated proteins were then eluted in Laemmli sample buffer. Eluted proteins were separated on SDS-polyacrylamide (10%) gels and immunoblotted with various antisera.

Immunoprecipitation and Western Blotting

An antibody to the JAK2 protein was raised by injecting an SDS gel-purified fusion protein of glutathione S-transferase and aa 747-1129 of JAK2 every 3 weeks into a New Zealand White rabbit. The methods for immunogen injection have been described (6) . This antibody was used for all immunoprecipitations. The GM-CSFR antibody was raised in a similar manner to an external domain of the receptor, as described previously (6) . The PY20 mouse monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). An additional JAK2 antibody directed at residues(758-776) was purchased from Upstate Biotechnology, Inc. (UBI, Lake Placid, NY) and was used for Western blotting.

Immunoprecipitations were carried out by lysing the cells in TNE buffer containing inhibitors (see above). The cell lysate was then microcentrifuged for 15 min to remove debris. Antibodies were added into the supernatant and incubated at 4 °C for 1 h. The JAK2 and GM-CSF antibodies produced in our laboratories were added at 1:40 dilution. 30 µl of protein A-Sepharose (Pharmacia Biotech Inc.) slurry was used to adsorb immune complexes, and the beads were washed five times in lysis buffer prior to elution in Laemmli sample buffer. The eluted proteins were electrophoresed in 8% SDS-PAGE and, for Western blots, electrophoretically transferred to a polyvinylidene difluoride membrane filter (Millipore, Bedford, MA). The blotted filter was incubated in TBS (20 mM Tris, pH 7.6, 137 mM NaCl) containing 3% bovine serum albumin (Fraction V, Sigma) for 1 h. The filter was incubated with either an anti-phosphotyrosine monoclonal antibody (PY20), polyclonal anti-GM-CSFR antibody, or UBI anti-JAK2 antibody at concentrations of 1 µg/ml (1:100 dilution) and 1 µl/ml, respectively. Bound antibodies were visualized using either peroxidase-conjugated rabbit anti-mouse immunoglobulin and the enhanced chemiluminescence (ECL) system (Amersham Corp.) or by biotinylated protein A (Amersham) and a streptavidin alkaline phosphatase conjugate (BRL) along with the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate detection system (Promega, Madison, WI).

Far Western Blotting

The GST GM-CSFR 458-559 (Boxes 1 and 2) was biotinylated (32) at a protein concentration of 1-3 mg/ml at room temperature for 2 h with biotinamidocaproate N-hydroxysuccinimide ester (Sigma) in 100 mM sodium borate, pH 8.8. The biotinylated protein was purified by gel filtration.

Bacteria expressing the four GST-JAK2 fusion proteins were lysed and the extract run on a 10% SDS-PAGE gel and transferred to nitrocellulose. The filter was blocked in TBST (TBS plus 1% Triton X-100) containing 2% nonfat dry milk. The filter was then incubated for 2 h in the same buffer containing 1 µg/ml biotintylated GST fusion protein, followed by extensive washing in TBST followed by streptavidin-conjugated alkaline phosphatase (Boehringer Mannheim) in TBST at a dilution of 1:5000 for 1 h. The blot was further developed with the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate detection system (Promega) (32) .


RESULTS AND DISCUSSION

The JAK2 Binding Region in GM-CSF Receptor

The membrane-proximal intracytoplasmic region of hematopoietin receptors, including GM-CSFR , has been shown to be necessary for JAK2 protein binding (9) . To map in detail the minimal amino acid sequence necessary for physical association with JAK2, we generated GST fusion proteins containing short stretches of the membrane-proximal region of GM-CSF receptor (Fig. 1A). The fusion proteins were expressed in E. coli and then purified on glutathione-Sepharose beads. These beads were then cross-linked with a non-cleavable protein cross-linker to increase protein binding efficiency (data not shown). Because simple transfection of CV-1 cells with JAK2 cDNA did not yield a large amount of protein, these cells were first infected with a vaccinia virus that produces the T7 polymerase (27, 28) and then transfected with the JAK2 cDNA containing the T7 promoter. This system allows for massive overexpression of the JAK2 protein. CV-1 extracts were then incubated with the GST fusion protein on beads.


Figure 1: The binding of WT JAK2 to GM-CSFR fusion constructs. A, structure of the GST fusion constructs. The amino acids that are conserved among hematopoietin, receptors denoted as box 1 and box 2, are shown as a closed area, and the hatched area contains the stretches of amino acids between these two boxes. The stippled area indicates the glutathione S-transferase protein. B, extracts of CV-1 cells expressing WT JAK2 were incubated with equivalent amounts of GST fusion proteins bound to glutathione-Sepharose beads. The binding reaction was carried out as described under ``Materials and Methods.'' The beads were then boiled in Laemmli's buffer and transferred, and the blot was probed with an anti-JAK2 Ab. The JAK2 is shown by an arrow. The molecular sizes are shown on the y axis in kilodaltons.



Because hematopoietin receptors contain two conserved stretches of amino acids in the membrane-proximal region, identified as box 1 and box 2, it was of particular interest to examine the role of each of these sequences in JAK2 binding. The various fusions tested are diagrammed in Fig. 1A. The results (Fig. 1B) demonstrate that JAK2 protein binding requires aa 458-495 (construct box 1 plus), which includes box1 and 14 amino acids between box 1 and 2 of the GM-CSFR . Increasing the length of the fusion protein including aa 458 to 559 (construct box 1 and 2) also allowed this binding, but deletion of 11 amino acids (458-484, construct box 1) including box 1 plus 3 amino acids markedly reduced the binding of JAK2, suggesting that amino acids 484-495 play a crucial role in this interaction. However, neither the region between box 1 and 2 (aa 483-530, construct bb) nor the region between the boxes combined with box 2 (aa 483-559, construct box 2) mediate significant JAK2 binding. Therefore, JAK2 binding requires the membrane-proximal region including box 1 and the next 14 amino acids.

Previous studies have demonstrated the importance of the membrane-proximal domain of many growth factor receptors for JAK2 binding. In the Epo receptor the region between box 1 and box 2 was necessary for Epo-dependent growth, activation of JAK2 kinase activity, and JAK2 binding (19) . The results presented on the GM-CSF receptor demonstrate that while a portion of the region between box 1 and 2 is necessary for JAK2 binding the sequence of amino acids between box 1 and 2 does not alone bind this protein kinase. It has been demonstrated that tryptophan (amino acid 494 in the GM-CSFR ), which is conserved in all hematopoietin receptors, is important for both interleukin-6 and Epo-dependent growth and signal transduction. This conserved tryptophan is found in the box 1 plus fusion protein but not in the box 1 protein, suggesting that it might also play an important role in the binding of JAK2 to the GM-CSF receptor. Although it is also found in box bb, this tryptophan is not sufficient alone to mediate binding.

The NH-terminal Domain of Jak2 Contains the GM-CSF Receptor Binding Region

A similar approach was used to examine which region(s) of the JAK2 protein are important for interaction with the GST GM-CSF receptor fusion (aa 458-495). Either the wild type or the three JAK2 mutants (Fig. 2A) lacking either the amino-terminal 239 residues (ATD), the carboxyl-terminal 130 residues of the protein kinase domain (CTD) removing the phosphotransferase motif, or a significant portion of the kinase-like domain (223 amino acids out of 283 predicted) (PSKD) were expressed in CV-1 cells (Fig. 2B). These four proteins were then individually incubated with the box1 plus fusion protein. The PSKD, CTD, and WT JAK2 each bound to the box 1 plus fusion as well as the WT JAK2 (Fig. 2C), whereas no binding was detected when these constructs were incubated with GST protein (data not shown). Notably, deletion of the amino terminus of JAK2 (ATD) markedly reduced the binding with the GST GM-CSF receptor fusion, suggesting that the JAK2 GM-CSF receptor interaction domain is in the amino terminus of the molecule (Fig. 2C).


Figure 2: The binding of wild type and JAK2 mutants to the box 1 plus GST fusion protein. A, structure of the wild type and JAK2 mutants. The kinase-like domain of the WT JAK2 is closed, while the protein kinase domain is denoted with stripes. The ATD is from amino acid 2 to 239. The CTD is from 1000 to 1129, and the PSKD is from 523 to 746. B, the WT and deletion constructs were transfected into vaccinia-infected CV-1 cells. Extracts of the cells were run on an SDS-PAGE gel, transferred, and probed with anti-JAK2. The molecular sizes in kilodaltons (kDa) are shown on the y axis. Arrows denote the four proteins. C, extracts shown in B were mixed with box 1 plus fusion protein beads, and binding reactions were conducted as described under ``Materials and Methods.'' The beads were then boiled in Laemmli's buffer, run on an SDS-PAGE, transferred, and Western blotted with an anti-JAK2 antibody.



To demonstrate the physical association between the amino terminus of JAK2 and the GM-CSF box 1 plus box 2 amino acids, we generated GST fusion proteins, each containing approximately one-fourth of the JAK2 protein (GST-J1, aa 1-294; GST-J2, aa 295-522; GST-J3, aa 523-746; and GST-J4, aa 747-1129). Western blotting with an anti-GST antibody (Fig. 3A) demonstrated that these GST fusion proteins have molecular masses ranging from approximately 50 to 68 kDa. To map the region of JAK2 that associates with the box 1 plus box 2 region of receptor, a far Western blot was carried out. The GST-JAK2 fusion proteins in bacterial lysate were run on an SDS-PAGE gel and transferred to nitrocellulose. This blot was then probed with a biotin-labeled GST GM-CSF (aa 458-559; construct box 1 and 2) and the protein interaction identified by developing the filter with streptavidin-conjugated alkaline phosphatase. Only the GST-J1 fusion protein bound the probe, demonstrating that amino acids 1-294 of JAK2 contain GM-CSF receptor protein binding domain.


Figure 3: Interaction of the amino terminus of JAK2 with GM-CSFR 458 to 559 (construct box 1 and 2). A, bacterial extracts expressing GST-JAK2 fusion proteins (lane1, GST-J1 aa 1-294; lane2, GST-J2 aa 295-522; lane3, GST-J3, aa 523-746; and lane4, GST-J4 aa 747-1129) were run on an SDS-PAGE gel. This gel was then transferred and probed with an anti-GST antibody 1:1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). B, a Western blot identical to that described in panelA was probed with biotin labeled GM-CSFR 458 to 559 (construct box 1 and 2) and the far Western blot developed with streptavidin-conjugated alkaline phosphatase. The arrow indicates the binding of the labeled probe to GST-J1. Some nonspecific color reaction is seen between lanes3 and 4.



Binding of JAK2 to the GM-CSFR Is Required for GM-CSFR Tyrosine Phosphorylation

We sought to determine whether the Jak2 mutants had functional consequences on GM-CSFR phosphorylation in vivo. After infection with the vaccinia virus vvT7 pol (27, 28) , we transiently expressed the WT JAK2 and the JAK2 mutants along with GM-CSF and subunits in CV-1 cells (Fig. 4A). Because the receptor cDNA expression plasmid contains the T7 polymerase initiation site, the receptor is also overexpressed (Fig. 4C). In the absence of JAK2, no tyrosine phosphorylation of the GM-CSFR is seen (Fig. 4D). Overexpression of the WT JAK2 or PSKD JAK2 mutant stimulated tyrosine phosphorylation of the GM-CSFR in the absence of added GM-CSF, suggesting that overexpression of JAK2 is sufficient to activate its protein kinase activity (Fig. 4D). The absence of a significant portion of the PSKD did not inhibit phosphorylation.


Figure 4: Regulation of GM-CSFR phosphorylation. A, CV-1 cells were infected with vaccinia virus and then transfected with the WT JAK2 or the kinase-like domain (PK, PSKD), carboxyl-terminal (CD), or amino-terminal (AD) deletion JAK2 mutants as well as the and GM-CSF receptors. After 24 h, the JAK2 was immunoprecipitated. The immunoprecipitate was run on an SDS-PAGE gel and Western blotted with JAK2 antisera. The amino-terminal deletion mutant is shown by an arrow. Molecular sizes are shown in kilodaltons on the y axis. B, the Western blot in A was stripped and reprobed with anti-Tyr(P) antibody. The amino-terminal deletion is shown by an arrow. C, one-third of the extracts described in A were immunoprecipitated with an antibody to the GM-CSF receptor. The immunoprecipitate was run on an SDS-PAGE gel and Western blotted with the same antibody. D, the Western blot in C was stripped and probed with an anti-Tyr(P) antibody. The receptor is shown with an arrow.



CTD, lacking the COOH-terminal 130 amino acids that include the phosphotransferase motif (PXXWYXPE), was not detectably phosphorylated in these cells (Fig. 4B). This kinase-deficient Jak2 was not able to promote tyrosine phophorylation of the GM-CSFR (Fig. 4D) despite its ability to interact with the box 1 plus fusion protein (Fig. 2C) in vitro. In contrast, when overexpressed the ATD can be phosphorylated on tyrosine to a similar extent as the WT JAK2 (Fig. 4B, arrow), but it failed to phophorylate GM-CSFR (Fig. 4D), indicating that the phosphorylation of the GM-CSFR requires the amino terminus of JAK2.

To examine the potential regulatory role of GM-CSF in mediating GM-CSFR phosphorylation, an identical experiment was performed without vaccinia virus vvT7 pol infection. Twenty-four hours after transfection, cells were serum-starved for 8 h and stimulated with human GM-CSF (10,000 units/ml) for 10 min. A Western blot of the immunoprecipitated GM-CSFR with anti-phosphotyrosine Ab demonstrates that transfection of either the ATD or the CTD of JAK2 did not mediate phosphorylation of the receptor, even in the presence of GM-CSF (Fig. 5A). GM-CSF induced tyrosine phosphorylation of the GM-CSFR when WT JAK2 was coexpressed. Coexpression of the GM-CSFR with the PKSD JAK2, in contrast, resulted in tyrosine phosphorylation of the GM-CSFR independent of GM-CSF treatment. Reprobing of these Western blots with the GM-CSFR antisera demonstrates equivalent expression of the receptor (Fig. 5B). Since the expression level of JAK2 is a critical regulator of its phosphorylation, it is not possible from these experiments to determine whether the pseudokinase domain plays a specific role in regulating the phosphorylation of JAK2. Recently it has been shown that a protein kinase-like domain of atrial natriuretic peptide is capable of binding a protein phosphatase (29) . It is possible that a phosphatase binds to the JAK2 kinase-like domain and is inhibited by GM-CSF addition.


Figure 5: GM-CSF-dependent phosphorylation of the GM-CSF receptor. A, CV-1 cells were transfected with the and GM-CSF receptors, and JAK2 WT and the mutants. After 24 h, the medium was removed and the cells were starved for 8 h in DMEM plus 0.5% bovine serum albumin. The cells were treated with 10,000 units/ml GM-CSF for 10 min and then lysed and immunoprecipitated with the GM-CSF receptor antibody. The immunoprecipitate was run on an SDS-PAGE gel, transferred, and probed with anti-Tyr(P) antibody. B, the blots shown in A were stripped and reprobed with the GM-CSF receptor antibody.



In this communication, we demonstrate that the amino-terminal 239 amino acids of JAK2 are necessary for binding to a 36-amino acid domain in the membrane-proximal region of the GM-CSFR . This JAK2 binding is necessary for the phosphorylation of the GM-CSFR receptor in vivo.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK44741 (to A. S. K.) and R29-DK-46395 (to S. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an Early Career Development Award from the American Federation for Clinical Research.

To whom correspondence should be addressed. Tel.: 205-934-4436; Fax: 205-975-6911.

The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; Epo, erythropoietin; PCR, polymerase chain reaction; ATD, amino-terminal domain; CTD, carboxyl-terminal domain; PSKD, pseudokinase domain; aa, amino acid(s); DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; WT, wild type.


ACKNOWLEDGEMENTS

We thank Dr. J. Ihle and Dr. A. Miyajima for providing the cDNA clones that were necessary for this work. We appreciate the help of Dr. B. Weaver and Patsy Spitzer in editing and preparing this manuscript.


REFERENCES
  1. Gearing, D. P., King, J. A., Gough, N. M., and Nicola, N. A. (1989) EMBO J. 8, 3667-3676 [Abstract]
  2. Park, L. S., Martin, U., Sorensen, R., Luhr, S., Morrissey, P. J., Cosman, D., and Larsen, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4295-4299 [Abstract]
  3. Kitamura, T., Sato, N., Arai, K.-I., and Miyajima, A.(1991) Cell 66, 1165-1174 [Medline] [Order article via Infotrieve]
  4. Tavernier, J., Devos, R., Cornelis, S., Tuypens, T., Heyden, J. V., Fiers, W., and Plaetinck, G.(1991) Cell 66, 1175-1184 [Medline] [Order article via Infotrieve]
  5. Polatskaya, A., Zhao, Y., Lilly, M. B., and Kraft, A. S.(1993) Cell Growth Diff. 4, 523-531 [Abstract]
  6. Polatskaya, A., Zhao, Y., Lilly, M. B., and Kraft, A. S.(1994) J. Biol. Chem. 269, 14607-14613 [Abstract/Free Full Text]
  7. Weiss, M., Yokoyama, C., Naugle, C., Druker, B., and Sieff, C. A. (1993) Blood 82, 3298-3306 [Abstract]
  8. Sakamaki, K., Miyajima, I., Kitamura, T., and Miyajima, A.(1992) EMBO J. 11, 3541-3549 [Abstract]
  9. Quelle, F. W., Sato, N., Witthuhn, B. A., Inhorn, R. C., Eder, M., Miyajima, A., Griffin, J. D., and Ihle, J. N.(1994) Mol. Cell. Biol 14, 4335-4341 [Abstract]
  10. Murakami, M., Narazaki, M., Hibi, M., Yawata, H., Yasukawa, K., Hamaguchi, M., Taga, T., and Kishimoto, T.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11349-11353 [Abstract]
  11. Eder, M., Griffen, J. D., and Ernst, T. J.(1993) EMBO J. 12, 1647-1656 [Abstract]
  12. Watanabe, S., Mui, A. L., Muto, A., Chen, J. X., Hayashida, K., Yokota, T., Miyajima, A., and Arai, K.-I.(1993) Mol. Cell Biol. 13, 1440-1448 [Abstract]
  13. Duronio, V., Clark-Lewis, I., Federsppiel, B., Wieler, J. S., and Shrader, J. W.(1992) J. Biol. Chem. 267, 21856-21863 [Abstract/Free Full Text]
  14. Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Cleveland, J. L., Yi, T., and Ihle, J. N.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8429-8433 [Abstract/Free Full Text]
  15. Wilks, A. R., Harpur, A. G., Kurban, R. R., Ralph, S. J., Zurcher, G., Ziemiecki, A.(1991) Mol. Cell. Biol. 11, 2057-2065 [Medline] [Order article via Infotrieve]
  16. Harpur, A. G., Andres, A. C., Ziemiecki, A., Aston, R. R., and Wilks, A. S.(1992) Oncogene 7, 1347-1353 [Medline] [Order article via Infotrieve]
  17. Firmbach-Kraft, I., Byers, M., Shows, T., Dalla-Favera, R., and Krolewski, J. J.(1990) Oncogene 5, 1329-1336 [Medline] [Order article via Infotrieve]
  18. Kawamura, M., McVicar, D. W., Johnston, J. A., Blake, T. B. Chen, Y.-Q., Lal, B. K., Lloyd, A. R., Kelvin, D. J., Staples, J. E., Ortaldo, J. R., and O'Shea, J. J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6374-6378 [Abstract]
  19. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N.(1993) Cell 74, 227-236 [Medline] [Order article via Infotrieve]
  20. Zhuang, H., Patel, S. V., He, T.-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M.(1994) J. Biol. Chem. 269, 21411-21414 [Abstract/Free Full Text]
  21. Argetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., and Carter-Su, C.(1993) Cell 74, 237-244 [Medline] [Order article via Infotrieve]
  22. VanderKuur, J. A., Wang, X., Zhang, L., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C.(1994) J. Biol. Chem. 269, 21709-21717 [Abstract/Free Full Text]
  23. Frank, S. J., Gilliland, G., Kraft, A. S., and Arnold, C. S.(1994) Endocrinology 135, 2228-2239 [Abstract]
  24. Narazaki, M., Witthuhn, B. A., Yoshida, K., Silvennoinen, O., Yasukawa, K., Ihle, J. N., Kishimoto, T., and Taga, T.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2285-2289 [Abstract]
  25. Lebrun, J.-J., Ali, S., Sofer, L., Ullrich, A., and Kelly, P. A.(1994) J. Biol. Chem. 269, 14021-14026 [Abstract/Free Full Text]
  26. David, M., Petricoin, E. F., Igarashi, K.-I., Feldman, G. M., Finbloom, D. S., and Larner, A. C.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7174-7178 [Abstract]
  27. Elroy-Stein, O., Fuerst, T., and Moss, B.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6126-6130 [Abstract]
  28. Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and Fuerst, T. R.(1990) Nature 348, 91-92 [CrossRef][Medline] [Order article via Infotrieve]
  29. Chinkers, M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079 [Abstract/Free Full Text]
  30. Soiropoulos, A., Perrot-Applanat, M., Dinerstein, H., Pallier, A., Postel-Vinay, M.-C., Finidori, J., and Kelly, P. A.(1994) Endocrinology 135, 1292-1298 [Abstract]
  31. Colosi, P., Wong, K., Leong, S. R., and Wood, W. I.(1993) J. Biol. Chem. 268, 12617-12623 [Abstract/Free Full Text]
  32. Mayer, B., Jackson, P. K., and Baltimore, D.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 627-631 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.