Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8429-8433, September 1993 Medical Sciences Structure of the murine Jak2 - and its role in interleukin 3 OLLI SILVENNOINEN*t, BRUCE A. WITTHUHN*, FREDERICK W. QUELLE*, JOHN L. CLEVELAND**, TAOLIN YI*, AND JAMES N. IHLE*:t§ *Department of Biochemistry, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105; and *Department of Biochemistry, University of Tennessee, Memphis, TN 38163 Communicated by George R. Stark, May 18, 1993

ABSTRACT Interleukin 3 (IL-3) regulates the prolifera- mains that characterize many tyrosine kinases. There is tion and differentiation of hematopoietic cells. Although the extensive similarity in the N-terminal region among the JAK 1L-3 receptor chains lack kinase catalytic domins, IL-3 in- family members (10). duces tyrosine phosphorylation of cellular . To inves- A role for the tyk2 in signal transduction was estab- tigate the potential role of the JAK family of protein-tyrosine lished in studies examining interferon a (IFN-a) (12). By kinases in EL-3 signal transduction, we have obtained full- using a genetic approach, the tyk2 gene was cloned by its length cDNA clones for murine Jakl and Jak2 protein-tyrosine ability to reconstitute the cellular response to IFN-a in kcinas and prepared antiserum against the predicted proteins. mutant cells. It is hypothesized that the kinase activity of Using antisera against Jak2, we demonstrate that 1L-3 stimu- Tyk2 is activated after IFN-a binding and is responsible for lation results in the rapid and specific tyrosine phosphorylation the phosphorylation ofthe 113- and 91/84-kDa proteins ofthe of Jak2 and activates its in vitro kinase activity. IFN-stimulated gene factor 3a complex (13, 14). This com- plex associates with the IFN-stimulated gene factor 3y pro- Hematopoiesis is regulated through the interaction ofgrowth tein and activates gene expression by binding to the inter- factors with their cognate receptors (1, 2). Interleukin 3 (IL-3) feron-stimulated response element. supports the proliferation and differentiation of early pro- Jak2 is involved in the response to erythropoietin (EPO) genitors and cells committed to several myeloid lineages (3). (15). EPO stimulation induces tyrosine phosphorylation of The receptor for IL-3 is composed of an a subunit of 60-70 Jak2 and activates its in vitro autophosphorylation activity. kDa and a ,B subunit of 130-140 kDa (4). Both subunits Using a series ofEPO receptor mutants, the induction ofJak2 contain the extracellular conserved motifs found in the cy- tyrosine phosphorylation was found to correlate with the tokine receptor superfamily. The cytoplasmic domains share induction of biological responses. Jak2 was also shown to limited similarity with other cytokine receptors and lack physically associate with the membrane-proximal cytoplas- detectable catalytic domains. However, considerable evi- mic region of the EPO receptor that is required for biological dence suggests that signal transduction involves tyrosine activity. Here we report the complete structure ofthe murine phosphorylation (1, 4). Thus there has been considerable Jak2 gene. We demonstrate that Jak2 is rapidly tyrosine interest in identifying a protein-tyrosine kinase that associ- and its kinase is activated in re- ates with the receptor and is activated by IL-3 binding. phosphorylated activity Polymerase chain reactions (PCRs) have been done with sponse to IL-3. degenerative oligonucleotides to conserved tyrosine kinase domains to identify potential kinases in myeloid cells (5). By MATERIALS AND METHODS using this approach and Northern blot analysis, IL-3- dependent cells express (6) the for a limited number of Isolation of Murine Jak2 Clones. PCR, with degenerative tyrosine kinases including Lyn, Tec, c-Fes, Jaki, and Jak2. oligonucleotides corresponding to the conserved protein- Lyn kinase has been implicated in IL-3 signaling (7). How- tyrosine kinase domain, was used to amplify cDNAs from ever, we have not detected an effect of IL-3 on Lyn kinase murine bone-marrow-derived monocytes (16). The Jak2 activity or on its tyrosine phosphorylation (unpublished cDNA clone was used to screen myeloid cDNA libraries data). We have also not detected any tyrosine phosphoryla- (17-19). The longest cDNAs were subcloned and the nucle- tion or activation ofTec or c-Fes kinase activity (unpublished otide sequence was determined (20). data). Therefore, we focused on assessing the role of murine Cells and Cell Culture. The cell lines used have been Jakl and Jak2 genes in IL-3 signal transduction. described (21) and were maintained in IL-3 (25 units/ml). The JAK (; alternatively referred to as just Mouse bone-marrow-derived monocytes were grown as de- another kinase) family of kinases was detected in PCR scribed (17). amplification of tyrosine kinase domains in hematopoietic Generation of Antibodies. Synthetic peptides correspond- cells (8). The complete structure ofthe humanJAKI gene has ing to the hinge region between domains 1 and 2 (aa 758-776) been reported (9) and, recently, a partial sequence of the were coupled to keyhole limpet hemocyanin by gluteralde- murine Jak2 gene has been reported (10). Independently, a hyde and used for immunization of rabbits. A synthetic third member (tyk2) was isolated by homology screening (11). peptide to Jakl (aa 785-804) was used for competition The family is characterized by two kinase domains. The studies. C-terminal domain has all the hallmarks of protein kinases and the second domain has the hallmarks of a Abbreviations: IL-3, interleukin 3; IFN-a, interferon a; EPO, eryth- but differs significantly from both the protein-tyrosine and ropoietin. -serine/threonine kinases. There are no SH2 and SH3 do- tPresent address: Department of Pharmacology, New York Univer- sity Medical Center, School of Medicine, 550 First Avenue, New York, NY 10016. The publication costs ofthis article were defrayed in part by page charge 1To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" ¶The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L16956). 8429 Downloaded by guest on September 28, 2021 8430 Medical Sciences: Silvennoinen et al. Proc. Natl. Acad. Sci. USA 90 (1993)

CCCCGCAACAAGATCGCAACTOT T?CCCTCCC 33 9 9 C AAAOOOTAAGAAGACAAGTTOGACGATTATGGCAACTVCACAAAACGGAAGTTCTTVVV 1 L833 X G v R R X V G D Y G Q L H X t t V L L So CAGAAGAACAGCCCTTTTTTTCCCTCCCGCGAAGCCCAATGTTCTCAAAAAAGCTCTAG 93 a t AAAGTCCTA0ATAAAOCACATAGGAACTATTCAGAG CVVCVVGAAGCAGA TCoV 11l893 X V L D X a H R P Y S I S f T 9 a a S n 600 ATGAATCGCCTGCCCTTACAATCACAGAAATOOAGGCAACCTCCACATCTCCTGTACAT 1S3 a a AT0MACTCA.TTCTCACAGCA&VTTTTTTVVCAATTATGGT0VCV0V0TCVT0GA0A0 14 0 N A C L T N T t A T S T S P V H 20 L9S3 N S Q L S H X H L V L P Y c v c v C e a 620

213 t 9 CAGAATGCTGATATTCCTGGAAGTGCTAATTCTGTGAACCAGATAGAGCCAGTCCTTCAA 2 Q P G D I P G S A N S V X Q I t P V L Q 40 GAOAACATTCTOOTTCAGAAATVTTOAAAAmTTTATCACTOGATACATACCTGAAAAG 1013 9 H I L V Q a r v X f G S L D T T L X X 640

t 9 CTGTATCT0TACCATTCTCTTGGGCCCAA GAAGGCAGAGTATCTGAAGTTTCCAAGT0GA 273 ssecasaaasatatsalw^auaX C V Y L r H S L G Q A G Y L K * P S G 60 ~~~~~~~~~~~~~211073 10 X 9 * I 0 S L V R L 0 V a R Q L a V a 660

333 c a c CGCTATGCTTCAGAAOAAATTTGTGT0GCTGCTTCTAAAGCTTGTGGTATTACGCCTCTG 2 t Y V A I C V A A S K A C C I T P V 80 AVCATTTTCTA0AA0AAAAATCCCTTATTCATVC0AATV0VOeVCVAAAAATATccT 1133 n V. r L X t K S L I H C P V C a K H t L 6$0

393 TATCATASATATGTTTCCGTTAATOAGTGAAACCGAAAGCATCTGGTACCCACCCAATCAT 2 Y N N A L N S t T R I W Y P N H 100 CfTATCA0AGAAGAAGOACA00AGAOOc0AACCCACCTTTCATCAAACTTAGTGATcT 1193 L I R L * D a R T ¢ 11 PP * I X L S D P 700

?TCTTCCACATAGACGAGTCAACCAGOCATCACATACTCTACAGCATAAGGCTCTAC?TC 453 a c ATTAcATTACAGTACTACC0AACAVCATTCTTCAGAAVACAATACC 2 V F H I 0 S T R H 0 L Y R I R F Y 120 t2S3 I S I t V L P D S IL 9 a I P w V p 720

CCTCATTOGTACTOTAGTO?CAGCACCAGAACCTACAGATACGCAGTGTCCCGGOGGGC S13 9 aca PN W Y C S 0 S S R T Y R Y C V S a G A 140 CCT0AAVOCATTOAOAATCCTAAAAATCVCAATCTOOCAACAOACAAGTO0ACVOOS 1313 p a C I a H P X P L 0 L a T D K W S 7 a 740 > csc 573 CAAGCTCCTCTGCTTGAT0ACTTTTCATGTCTTACCTTVVV0CVCAGTGGC00CAT0AT 2 A P L L D D V N S Y L F A Q w R N 0 160 ACCACTCT0V00AOATCTATGGA0GAO.ATAAOCCCCCOAGTCCCVGATTCTCAA 1373 L W * I C S C e D K P L 3 A L D S S P T T Q 760 TTTGTTCAcOGATGOATAAAAGTACCTG?GACTCATCAAACTCAGGAAGAOVOVCTTC00 633 9 AGAAAGCTGCAGTTCTATCAAGATAAGCATCACTTCCTCCACCCAAGTGCACAGACTTA ;2433 f V H C W I K V P V T H t Q Y C L 0 180 R K L Q r Y D K H Q L P A P K W T t L 780

ATVGCGGT0TTAOACATCATGAGAATACGCAAGOACAAACACCAGACTCCACTOOC=TC 693 CCAAACCTTATAAATAATTGCATCGACTATCACCAGAT?TCAGCCCCCCTTTCAGAGCT 2493 N A V L D N R I A K t KD Q T P L A V 200 A N L I N N C H DV Y P D r R P A r A 800

GtCATCCGTGATCTTAACAGCCTGtTTACTCCAGATTATGAACCACTAACACAAAATGAC 255 3 TATAACTCTGTCAOCTACAAGACATTCTTACCAAAGTCcCTTCGAGCOAAcATCCAAGAC 753 V I R D L N S L r T PD VY L L T I N D 820 Y N S V S Y K T f L P K C V R A K I Q D 220

ATVCTACCAAACATCAGAATACGGTCCCTAGGGTTTTCTGGTGCTTTTGAA0ACAGGCAC 2613 TATCACATTTTAACCCGGAAGCGAATCAGGcACAGATTTCGCAGATTCATTCACCAATTC 613 N L P N N R I G A L C r S C A r C D R D 840 N I L T R K R I R Aa R R I Q Q 240 CCTACACACGTTGAAGACAGACACTTGAAGTTTCTACAGCAGCTTGGCAAAGVTAACTTC 2673 P T Q r t R H L K r L Q Q LC K C N 860 AGTCAAT0VAAAGCCACTCCCAGGAACCTAAAACTTAAGTATCTTATAAACCTOGAAACC 673 S Q C A t A R L K L K Y L I L T 260 G0GAGTGTGGAGATGTGCCGCTATGACCCCCTCCAGGACMCACTGGCCACGVGCTCGCT 2733 G S V N C R Y D P L Q D N T G t V V A 680 CVCCAGVCTGCCTTCrACACAGAACAGTTTVAATAAAGAATCT0CAAGA0VCTVCA 933 L S A Y t Q V K S A C P S 250 Q CTOAAGAAACTCCACCACAGCACTGAAGAGCACCCCGAGACTTGAGAGCCAGAGCGACG 2793 V K K L Q H SVt t tH L RD r t R I I t 900 GGTGAG0AOATTV0ACAVVAAC0AAACOGTOGAATTCAOV00VCAAGA 993 033e VAT I I I T GS G G I Q W S a 300 ATCCTGA.AATCCTTGCACCATGACAACATCGTCAAGTACAAGGCAGTGTVCTACACSCCC 26S3 I L K S L Q H D N I V K Y K C V C Y S A 920 GCGAAACATAAGGAAAOOA0ACACT0ACAOAACAG0ACOTACA0TTATATTGT0ATTTC 1053 C H S 3 T L T Q 0 V Q L Y C D 320 CGGCGGCCCAACCTVAAGATTAATTATGAATAVTTACCATATGCAACGTTACGAGACTAT 2913 e RR N L R LINS Y L P Y C S L D Y 940 t CCTGATATTAVTTCATTCAGTAVAATT CAACAaccAT COAA AAAAGTAGA 1113 P D I I D V S I Q A N Q a C S a S a 340 CTCCAAAAACATAAAGAACCCATAGATCACAAAAAACTTCTTCAATACACATCTCAGATA 2973 L Q K HKK R I D H K K L L Q Y S Q I 960 9 9Ca C99a9 9 C ATTGTAACT0TCCATAAACAACATG0TAAAGrTTCGACATAGAACrTAGCTCATTAAAA 1173 S V t V H K Q D 0 x V L I L S S L K 360 TGCAACGGCATGGAATATCTTGCTACAAAAAGGTATATCCACAGCGACCTGGCAACAAGC 3033 D A R V C K 0 N t Y L C T K R Y IH R L T 980

t AAGCC'tTCTCATTCGTCTCATTAATTGACGGGCATTACACACTAACTGCGGAVCGCCAC 1233 3093 A L S V S L I 0 G Y Y R L T A 0 A N 380 AACATATTGGTCOAAAATCAGAACAGGOTTAAAATAGGCAGACTTCGCATAACCAAAGTC N I L V t N t N R V K I C D C L T K V 1000

t t C t a t CATTACCTCTrCCAAAGkGGTCGCTCCCCCAGCTGTGCTCGAGAACATACACACCAACTCC 1293 3153 H Y L C X V A P P A V L N I N S N C 400 VV0CCGCAGGACAAAGAATACTACAAAGTAAAGGAGCCAGGCAAAGCCCCATATTCTCG L P Q DKK Y Y t V K t P G t S P I W 1020 t c a a CACGCCCAATATCAATGGATTTTGCCATTAGCAAACTAAACAAGGCGGGTAACCACACT 13S3 H P t N D A I S K L K K A c N Q T 420 TACGCACCT0AATCCTGCAGAGACCCAAGTTTCTGTCGCCTCCACATGTTCGAGCTTT 32 13 Y A P t 5 L T t 5 K P S V A S D V W 5 F 1040 9 a t t c c GGACTATATOTGCTACGATGCAGCCCTAAGCACTTCAACAAATACTTTCTGACCTTTOCT 1413 L Y V L R C S P K o r N K Y r L T A 440 COACTOGTTCTATACCAACVTTCACATACATCGAGAAGACTAAAAGTCCACCCGVCCAA 3273 C V V L 2 L Y I K S K S P P V 1060 t a CTTGAGCGAGAAAATGTCATTGAATATAAACACTGTTTGATTACGAAGAATGAGAATOGA 1473 V R N V I Y K H C L t T N N G 460 TTTATGOAVAJTVGCAATGATAAACAACGCCAAATGATTOTGTTCCATTTCATAGAG 3333 R I 0 H D K Q C Q I V N L 2 1080 9 t gt GAATACAACCTCAGCOGGACTAAGAGGAACTTCAGTAACCTTAAGGACCTTTTGAATTGC 1533 Y N L S C T K A N S N L K 0 L L N C 480 CTACTCAAGCACAACGOAAGATTOCCAAGOCCAGAAGGATVGCCCAGATGAOATTTATOTO 3393 1100 C t t TACCACATCGAAACTGTCCGCCACACAGTATCATCTTCCAGTTTACCAAATGCTGCCCC 1S93 Y Q t V R S 0 5 I I V Q T C C P S00 34S3 1120 9 t 9 CCAAAGCCAAAAGATAAATCAAACCTVTCCGTCTTCACAACAAATGGTATTTCTGATGTT 1653 P K P K D K S N L L V r R T N G I S 0 V S20 TTCCACACACATCCCATCCOCAACATATAGGCCACCGAA CCCACVCATTCACTCAGAC 3S13 v C W I K S 0 T v 1129

C CACATCTCACCAACATTACAGAGGCATAATAATCTGAATCAAATAGCTGTTCACAAAATC 1713 Q I S P T L N N V N Q N V I 540 ACCAAGCAGACTTCCACAACCACAACAAACTCTCTACCCVVCVCTACACATCCTTAT 3573 L 9 a AGAATCAA-ATTTAlATATTTAATGAAAGTCTTGGCCAAGGTACTTTTACAAAAAT1TTTT 1773 C (a) (a) (a) R U Z D L I F N * S L C Q C T F T X I 560 CATGATVCCAGCTACCCAGAAGAAACTGTGACGCCCTCTGCTCAAAGCTTTVCTTC

FIG. 1. (Legend appears at the bottom of the opposite page.) Downloaded by guest on September 28, 2021 Medical Sciences: Silvennoinen et al. Proc. Natl. Acad. Sci. USA 90 (1993) 8431

In Vitro Translation and Transcription. Full-length Jakl or A B C D Jak2 cDNAs were inserted into pBSK (Stratagene) to make transcripts with T3 RNA polymerase. Approximately 3 ,g of KDa : RNA was used in translation reaction mixtures (Stratagene) in the presence of 35S translabel (NEN). The products were 215 - divided equally and either subjected to SDS/PAGE directly or immunoprecipitated with Jakl or Jak2 antisera. Compe- FIG. 2. In vitro translation and titions were preformed by incubating peptides (100 pug/ml) immunoprecipitation of Jak2. Capped Jak2 mRNA was used in with antisera for 1 h at 4°( prior to immunoprecipitation. 105 - in vitro translation reaction mix- In Vito Kinase Assays. Immunoprecipitated proteins on tures and products labeled with protein A-Sepharose (Pharmacia) were washed with kinase [35S]methionine. After the reac- buffer (50 mM NaCI/5 mM MgCl2/5 mM MnCl2/0.1 mM 69 - tions, Jak2 was immunoprecipi- Na3VO4/10 mM Hepes, pH 7.4) and incubated for 30 min at tated from reaction mixtures in the room temperature with an equal volume of kinase buffer absence of competing peptides containing ([32P]ATP at 0.25 mCi/ml. After extensive wash- (lane A) or in the presence of the to ing, proteins were eluted with sample buffer for SDS/PAGE peptide which the antiserum 43 - was raised (lane B), to an irrele- were and separated on 7% gels. 32P-containing proteins vant peptide (lane C), or to a pep- visualized by autoradiography. In vitro-phosphorylated Jak2 tide corresponding to the same was isolated from gel slices and the phosphoamino acid region of Jakl. The immunopre- content was determined (22). cipitates were resolved by SDS/ PAGE.

RESULTS ever, clear differences in the amino acid composition and The tyrosine kinases expressed in IL-3-dependent cells were spacing in critical kinase subdomains I, II, VI, and VIII (24) identified by PCR using degenerative oligonucleotides cor- raise the possibility that this domain may have a regulatory responding to the conserved regions of the tyrosine kinase function or an unknown substrate specificity. domain (5). One ofthe most frequently isolated cDNA clones Although the N terminus ofthe JAK family proteins is less was identical to Jak2 (16). Expression analysis indicated that homologous than the kinase domains (36-39%o vs. 49-56%), Jak2 was widely expressed and prompted us to obtain there are several stretches of homology. Database searches full-length cDNA clones. Screening ofmyeloid cDNA librar- with the N-terminal sequence did not show homology with ies allowed the isolation of several overlapping clones, the other proteins. Close comparisons of the region immediately longest (4 kb) of which contained the entire coding region of N-terminal to the kinase-like domain reveal some similarity Jak2. The nucleotide sequence of Jak2 contains an open to SH2 domains. reading frame of3387 bp and the 5' end has three stop codons To biochemically characterize the Jak2 protein, anti- before the first ATG (Fig. 1). Although the first ATG does not peptide antisera were prepared against a region that was fulfill the Kozak consensus flanking sequences, it is imme- unique for Jak2 (aa 758-776). To assess the reactivity, diately followed by an ATG codon in the typical translation immunoprecipitations were done with in vitro-synthesized initiation environment (23). The 5' end does not contain an Jak2 (Fig. 2). In vitro translation of Jak2 RNA yielded a obvious signal peptide. The compiled size of the 3' untrans- 130-kDa protein (data not shown). This protein was immu- lated region of the Jak2 clones is 0.9 kb, which would noprecipitated by the Jak2 anti-peptide antiserum (Fig. 2, correspond to a 4.4-kb transcript. The Jak2 open reading lane A) but not by an irrelevant antiserum (data not shown). frame encodes 1129 aa with a calculated molecular mass of Immunoprecipitation was competed by the homologous pep- 130 kDa. Hydrophilicity analysis failed to identify transmem- tide (lane B) but not by an irrelevant peptide (lane C) or by brane regions. During our studies, a partial sequence ofJak2 a peptide that is the homologous region ofJakl (lane D). The was published (10), which lacked the first 143 aa. A compar- Jak2 antiserum did not immunoprecipitate in vitro- ison ofthe sequences indicates 71 nt differences in the coding synthesized Jakl (data not shown). Lastly, the Jak2 antise- region, resulting in 9 aa changes (Fig. 1). Our cDNA clones rum immunoprecipitated a 130-kDa protein from in vivo- did not contain a 7-aa insert at position 711 found in a cDNA methionine-labeled cells, which was competed by the homol- clone described by Harpur et al. (10). ogous peptide (data not shown). The murine Jak2 gene is closely related to the human tyk2 IL-3 stimulation induces tyrosine phosphorylation of sev- and JAKI genes (42% and 43% identities, respectively). We eral cellular substrates including the IL-3 receptor subunit have also obtained full-length cDNA clones for the murine (3, 25). We therefore determined whether Jak2 was a sub- Jaki gene (data not shown), which has 45.5% identity to Jak2 strate (Fig. 3). Western blot analysis of cell lysates with a at the nucleotide level in the coding region. The Jak2 protein monoclonal antibody against phosphotyrosine (4G10) de- has a 600-aa N terminus that lacks obvious SH2 or SH3 tected the appearance of several proteins after IL-3 stimu- domains. After this is a kinase-related domain (domain 2) and lation including a broad band at 130-140 kDa, a minor band a C-terminal kinase domain (domain 1). The C-terminal at 70 kDa, and major bands at 55 kDa, 50 kDa, and 38 kDa. kinase domain contains the conserved residues in subdo- When extracts were immunoprecipitated with Jak2 antise- mains VI-VIII that are characteristically found in tyrosine rum, a 130-kDa protein was readily detected in stimulated kinases (24). Subdomain VIII, which may contribute to cells. Also of note are proteins of 110 kDa, 70 kDa, and 60 substrate recognition, contains a unique FWY amino acid kDa that coimmunoprecipitated with Jak2. These have been motiffound in all Jak family members. Domain 2 begins at aa consistently seen in Jak2 immunoprecipitations. Immunopre- 543 and the structural subdomains can be identified. How- cipitation with Jakl antiserum consistently detected a weak

FIG. 1 (on oppositepage). Sequence ofthe murine Jak2 cDNA. The sequence oftheJak2 open readingframe and noncodingregions is shown. Nucleotide and amino acid differences from the published partial Jak2 cDNA sequences (10) are shown. The ATG codons are indicated (*). The symbol > above nt 522 designates the 5' end of the reported Jak2 sequence. The symbol A at nt 2226 indicates the location of the 7-aa insert as detected (10). The nucleotides shown in parentheses in the 3' noncoding region were present in the previous studies (10) but not detected in our studies. Downloaded by guest on September 28, 2021 8432 Medical Sciences: Silvennoinen et al. Proc. Natl. Acad Sci. USA 90 (1993) Antiserum: TCL aJAK2 aIL3Rf3 aJAK1 r- F l r - 1 N~o *No IL3: - + - + - + +

_~+4 , _q + _ + KDa r- +- + kDa 215 - 215 -

_~ c - ~- Jak2 105 - 105 -

70 69 -

43- 43 - Autoradiography

Probed with anti-PTyr ** , 4- Jak2 FIG. 3. IL-3 stimulation ofthe tyrosine phosphorylation ofJak2. Probed with Anti-Jak2 DA-3 cells were deprived of growth factors for 14 hr, either unstimulated (-) or stimulated with IL-3 (10 units/ml) (+), and FIG. 5. IL-3 stimulation activates Jak2 in vitro kinase activity in lysed, and extracts were incubated with the indicated antisera. The immunoprecipitates. DA3 cells were removed from growth factors immunoprecipitates or total cell lysates (lanes TCL) were resolved and were either unstimulated (-) or stimulated with IL-3 for 10 min by SDS/PAGE. The proteins were transferred to nitrocellulose and (+). Extracts were immunoprecipitated with normal rabbit serum the blots were probed with the 4G10 monoclonal antibody against (NRS) orJak2 antiserum in the absence ofcompeting peptide (aJak2) phosphotyrosine. The positions of the migration of standards are or in the presence of the peptide (30 ug/ml) to which the antiserum indicated on the left. Antisera: aJAK2, anti-JAK2; aIL3R,B, anti- was raised (aJak2 + Jak2 peptide) or in the presence ofan equivalent IL-3 receptor , chain; aJAK1, anti-JAK1. amount of the peptide that corresponds to the comparable region of Jakl (aJak2 + Jakl peptide). The immunoprecipitates were used for band at 130 kDa, indicating that Jakl may also be a substrate. in vitro kinase assays and the products were resolved by SDS/ The inducible tyrosine phosphorylation of the chain, PAGE, transferred to nitrocellulose, and detected by autoradiogra- IL-3p phy (Upper). The blots were subsequently probed with the Jak2 which migrates slightly above Jak2, is shown in Fig. 3. Thus antiserum (Lower). the broad band seen in total cell lysates consists ofboth Jak2 and the IL-313 chain. stimulated but not unstimulated cells. The kinetics are illus- To further establish Jak2 phosphorylation, cells were stim- trated in Fig. 4B. Jak2 tyrosine phosphorylation was maximal ulated with IL-3 and the phosphotyrosine-containing fraction at 5 min after IL-3 stimulation and, subsequently, decreased. was isolated on Sepharose beads containing anti-phosphoty- These kinetics are comparable to the general pattern of rosine monoclonal antibody 1G2. Jak2 was readily detected tyrosine phosphorylation (26). During this period, there was on Westem blots of the IG2-isolated proteins (Fig. 4A) from no change in the levels of Jak2 as assessed by Western blot analysis with Jak2 antiserum (Fig. 4B). B To assess the effect on Jak2 kinase activity, cells were 0 5 10 30 60 120 stimulated with IL-3 for 10 min, Jak2 was immunoprecipi- A TCL IG2 tated and in vitro kinase assays were performed. No in vitro ..,- kinase activity was detected in immunoprecipitates with 1L3: - + -+ normal serum from unstimulated or stimulated cells (Fig. 5). However, immunoprecipitates with Jak2 antiserum con- C tained kinase activity and resulted in the phosphorylation of ::. :II : a protein of 130 kDa that comigrated with Jak2. In contrast, the 130-kDa band was not detected with unstimulated cells. Phosphoamino acid analysis of the 130-kDa band demon- FIG. 4. Specificity and kinetics ofJak2 tyrosine phosphorylation strated the presence ofpredominantly phosphotyrosine (data after IL-3 stimulation. Cell extracts were prepared from unstimulated not shown). Interestingly, there were no other major protein cells (lanes - and 0) or from cells stimulated with IL-3 at 10 units/ml bands phosphorylated in these in vitro reactions. The Jak2 for 10 min (+) or stimulated for the indicated times. (A) The peptide blocked precipitation of kinase activity, whereas a phosphotyrosine-containing protein fraction was isolated by binding peptide to the corresponding region of Jakl had no effect. to and elution from lG2-coupled Sepharose beads; the 1G2 mono- clonal antibody against phosphotyrosine is as described (26, 33). The proteins were resolved by SDS/PAGE and transferred to nitrocel- DISCUSSION lulose, and the blots were probed with the Jak2 antiserum. Alterna- Our studies provide the complete sequence of the murine tively, the total cell lysate (TCL) was electrophoresed and trans- ferred, and the blots were probed with the Jak2 antiserum. (B and C) Jak2 as indicated by three criteria. (i) The coding sequence The cell extracts were immunoprecipitated with the Jak2 antiserum, initiates at the same site as the published sequences ofhuman the precipitates were resolved by SDS/PAGE and transferred to tyk2 and JAKI. (ii) The first ATG is preceded by stop codons nitrocellulose, and the blots were probed with the 4G10 monoclonal in all reading frames. (iii) The size of the compiled cDNAs is antibody (B) or the Jak2 antiserum (C). consistent with the observed transcript. Our sequence varies Downloaded by guest on September 28, 2021 Medical Sciences: Sflvennoinen et aL Proc. Natl. Acad. Sci. USA 90 (1993) 8433 from the published partial sequence (10). Since a portion of 3y-related DNA binding proteins including ICSBP, IRE1, the previous sequence was obtained from PCR-amplified IRF2, and possibly Myb (32). DNAs, some differences may be PCR artifacts. IL-3 stimulation induces tyrosine phosphorylation (3, 27). We thank Linda Snyder, Brenda Simmons, and Cynthia Miller for One ofthe substrates is Jak2 (3). Among the tyrosine kinases excellent technical assistance in these studies. This work was in IL-3-dependent cells, there was a remarkable specificity supported in part by the National Cancer Institute Cancer Center for Jak2. In particular, no changes in phosphorylation ofLyn, Support (CORE) Grant P30 CA21765, by Grants RO1 DK42932 Tec, or c-Fes have been seen (data not shown). However, we (J.N.I.) and DK44158 (J.L.C.) from the National Institute of Diabe- consistently see a low level of Jakl phosphorylation after tes and Digestive and Kidney Diseases and by the American Leba- IL-3 stimulation. Therefore, Jakl may share sufficient sim- nese Syrian Associated Charities (ALSAC). to Jak2 to weakly associate with the IL-3 receptor ilarity 1. Metcalf, D. (1989) Nature (London) 339, 27-30. complex. Alternatively, since there is considerable sequence 2. Clark, S. C. & Kamen, R. (1987) Science 236, 1229-1237. homology between Jakl and Jak2 at the potential autophos- 3. Ihle, J. N. (1992) in Interleukins: Molecular Biology and Im- phorylation site, Jakl may be a substrate for Jak2. However, munology, ed. Kishimoto, T. (Karger, Basel), pp. 65-106. we have not detected activation of Jakl in vitro kinase 4. Miyajima, A., Kitamura, T., Harada, N., Yokota, T. & Arai, K. activity (data not shown). (1992) Annu. Rev. Immunol. 10, 295-331. IL-3 stimulation results in the activation of Jak2 in vitro 5. Wilks, A. F. (1991) Methods Enzymol. 200, 533-546. kinase activity. The C-terminal protein-tyrosine kinase do- 6. Mano, H., Mano, K., Tang, B., Koehler, M., Yi, T., Gilbert, main ofJak2 contains the characteristic autophosphorylation D. J., Jenkins, N. A., Copeland, N. G. & Ihle, J. N. (1993) site of tyrosine kinases (24). Oncogene 8, 417-424. The requirement for IL-3 binding for kinase activity indi- 7. Torigoe, T., O'Connor, R., Santoli, D. & Reed, J. C. (1992) cates that Jak2 is highly regulated in cells, consistent with a Blood 80, 617-624. major role in growth regulation. The primary substrate ofthe 8. Wilks, A. F. (1989) Proc. Natl. Acad. Sci. USA 86, 1603-1607. 9. Wilks, A. F., Harpur, A. G., Kurban, R. R., Ralph, S. J., in vitro kinase reactions was Jak2. In particular, there was no Zurcher, G. & Ziemiecki, A. (1991) Mol. Cell. Biol. 11, 2057- detectable phosphorylation of immunoglobulins nor is eno- 2065. lase a substrate for Jak2 (data not shown), indicating thatJak2 10. Harpur, A. G., Andres, A. C., Ziemiecki, A., Aston, R. R. & may have a strict substrate specificity. The requirement for Wilks, A. F. (1992) Oncogene 7, 1347-1353. receptor activation and the substrate specificity may account 11. Firmbach-Kraft, I., Byers, M., Shows, T., Dalla-Favera, R. & for the inability to demonstrate Jakl protein-tyrosine kinase Krolewski, J. J. (1990) Oncogene 5, 1329-1336. activity in previous studies (9). 12. Velazquez, L., Fellous, M., Stark, G. R. & Pellegrini, S. (1992) We have shown (15) that Jak2 is tyrosine-phosphorylated Cell 70, 313-322. and activated after EPO stimulation. Moreover, these studies 13. Fu, X. Y. (1992) Cell 70, 323-335. demonstrated that Jak2 physically associates with a mem- 14. Schindler, C., Shuai, K., Prezioso, V. R. & Darnell, J. E., Jr. brane-proximal region ofthe cytoplasmic (1992) Science 257, 809-813. domain ofthe EPO 15. Witthuhn, B., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, receptor that is essential for function. However, like the EPO B., Miura, 0. & Ihle, J. N. (1993) Cell 74, 227-236. receptor, the (3 subunit of the IL-3 receptor is rapidly 16. Wilks, A. F. (1989) Proc. Natl. Acad. Sci. USA 86, 1603-1607. tyrosine-phosphorylated and it can be hypothesized that this 17. Yi, T. L. & Willman, C. L. (1989) Oncogene 4, 1081-1087. phosphorylation is mediated by Jak2. For the EPO receptor, 18. Morishita, K., Parker, D. S., Mucenski, M. L., Jenkins, N. A., tyrosine phosphorylation occurs at sites in the cytoplasmic Copeland, N. G. & Ihle, J. N. (1988) Cell 54, 831-840. C-terminal end and this region is not required for mitogene- 19. Bartholomew, C. & Ihle, J. N. (1991) Mol. Cell. Biol. 11, sis. 1820-1828. The ability of IL-3 and EPO to activate Jak2 suggests the 20. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. be Acad. Sci. USA 74, 5463-5467. possibility that Jak2 may a component in the signal 21. Ihle, J. N. & Askew, D. (1989) Int. J. Cell Cloning 1, 1-30. transducing pathways of several cytokine receptors. In pre- 22. Cooper, J. A., Sefton, B. M. & Hunter, T. (1983) Methods liminary experiments, granulocyte-macrophage and granu- Enzymol. 9, 387-402. locyte colony-stimulating factors were found to induce the 23. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. tyrosine phosphorylation of Jak2. This is consistent with 24. Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, studies showing that these factors induce comparable pat- 42-52. terns of tyrosine phosphorylation (3). We have also observed 25. Sorensen, P., Mui, A. L. F. & Krystal, G. (1989)J. Biol. Chem. tyrosine phosphorylation of Jak2 in response to IFN-y in a 264, 19253-19258. macrophage cell line (unpublished data). 26. Isfort, R., Abraham, R., Huhn, R. D., Frackelton, A. R., Jr., The hematopoietic & Ihle, J. N. (1988) J. Biol. Chem. 263, 19203-19209. growth factor receptors are members of 27. Ihle, J. N. (1990) in Peptide Growth Factors and Their Recep- a receptor superfamily that includes growth hormone recep- tors, eds. Sporn, M. & Roberts, A. (Springer, New York), pp. tor, the prolactin receptor, ciliary neurotropic factor recep- 541-575. tor, and others (28). Moreover, the receptors for IFN have 28. Bazan, J. F. (1992) Science 257, 410-413. been speculated to have evolved from the same progenitor. 29. Artgetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, The recent studies implicating Tyk2 in IFN-a signaling (12) B. A., Silvennoinen, O., Ihle, J. N. & Carter-Su, C. (1993) Cell and Jak2 in signaling by IL-3, EPO (15), granulocyte and 74, 237-244. granulocyte-macrophage colony-stimulating factor, IFN-y, 30. Schindler, C., Fu, X.-Y., Improta, T., Aebersold, R. & Damell, and growth hormone (29) suggest that the JAK family kinases J. E., Jr. (1992) Proc. Natl. Acad. Sci. USA 89, 7836-7839. 31. Fu, X.-Y., Schindler, C., Improta, T., Aebersold, R. &Darnell, are involved in the signal-transducing pathways utilized by J. E., Jr. (1992) Proc. Natl. Acad. Sci. USA 89, 7840-7843. the cytokine/IFN superfamily of receptors. If correct, it is 32. Veals, S. A., Schindler, C., Leonard, D., Fu, X.-Y., Aeber- further tempting to speculate that the JAK family of kinases sold, R., Darnell, J. E., Jr., & Levy, D. E. (1992) Mol. Cell. may regulate gene expression through comparable pathways Biol. 12, 3315-3324. involving family members related to theIFN-stimulated gene 33. Frackelton, A. R., Jr., Ross, A. H. & Eisen, H. N. (1983) Mol. factor 3a proteins (30, 31) and theIFN-stimulated gene factor Cell. Biol. 3, 1343-1352. Downloaded by guest on September 28, 2021