Characterization of Active and Inactive Forms of the JAK2 Protein-tyrosine Kinase Produced via the Baculovirus Expression Vector System*

Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C (cid:68) 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N (cid:68) 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phospho- tyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C (cid:68) 795) was not observed; further, coproduction of rJAK2(C (cid:68) 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previ- ously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411–21414). Mutant (N (cid:68) 661)rJAK2 ex-hibited

Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C⌬795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N⌬661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C⌬795) was not observed; further, coproduction of rJAK2(C⌬795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results ( The JAK family of protein-tyrosine kinases (JAK1, JAK2, JAK3, and TYK2; EC 2.7.1.112) appears to play a crucial role in the signal transduction cascade initiated by activation of numerous cytokine receptors (2). Cytokines that signal through a common ␥ chain, such as interleukin-2, interleukin-4, and interleukin-7, trigger the tyrosine phosphorylation of JAK3 and may also induce the activation of other Janus kinases (3)(4)(5). Interferons ␣ and ␤ require both JAK1 and TYK2 in their signaling cascade (6), and interferon ␥ signaling requires both JAK1 and JAK2 (7). A number of cytokine receptors are associated with JAK2, and activation of these receptors by their cognate ligands results in the rapid tyrosine phosphorylation of JAK2. Such receptor systems include those for erythropoietin (8), interleukin-3 (9), interleukin-6 (10), leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M (11), granulocyte-macrophage colony-stimulating factor (12), growth hormone (13), and prolactin (14,15). It is noteworthy that deletion of the membrane-proximal domains of most of these cytokine receptors abrogates not only the transduction of a cytokineinitiated proliferation but also the cytokine-triggered activation of JAK2 (8,10,12,16,17). The specific mechanism by which cytokine receptor activation converts these kinases from a latent to an active state remains unclear, but in systems involving the prolactin receptor (14,15), the leukemia inhibitory factor receptor ␤ chain, and the interleukin-6 signal transducer gp130 (11), JAK2 is associated with receptor before ligand stimulation of the receptor. Thus, it does not appear that receptor association per se is sufficient to activate the kinase. The detailed enzymologic behavior of the "activated" Janus kinases has yet to be characterized, and site-directed mutagenesis studies assessing the functional importance of some of the structural domains of murine JAK2 (1) and of human TYK2 (18) have only recently appeared in the literature.
As an initial step toward understanding the mechanism of enzymatic activation of the JAK protein-tyrosine kinases, we overexpressed the rat jak2 cDNA clone in Sf21 cells via the baculovirus expression vector system. We also overexpressed two deletion mutants of JAK2 that were predicted to be active and inactive forms based on the retention and deletion, respectively, of a highly conserved tyrosine kinase motif. The characterization of the resultant protein products is described.

Construction of Recombinant Baculoviruses-
The transfer vector pBacPAK9:rJAK2 was created by first subcloning a 3.0-kilobase EcoRI fragment from the 3Ј-end of the rat jak2 clone pBK-CMV:RA3.17 (19) into the EcoRI site of pBacPAK9 (Clontech), isolating a plasmid containing the jak2 insert in the correct orientation, then substituting a 2.0-kilobase BamHI fragment from the 5Ј-end of the rat jak2 clone pBK-CMV:RA3.17 into this plasmid. The transfer vector pBacPAK9: rJAK2(C⌬795) was created by digesting pBacPAK9:rJAK2 with the restriction endonuclease StuI, religating the plasmid digest mixture, then isolating a plasmid that lacked the StuI fragment at the 3Ј-end of the jak2 coding region. The transfer vector pBacPAK9:(N⌬661)rJAK2 was created by digesting pBacPAK9:rJAK2 with the restriction endonuclease BamHI, religating the plasmid digest mixture, then isolating a plasmid that lacked the BamHI fragment at the 5Ј-end of the jak2 coding region. Recombinant baculoviruses were generated by Lipofectin-mediated cotransfection of Sf21 cells with Bsu36 I-digested BacPAK6 viral DNA (20) (Clontech no. 6144-1) and the appropriate transfer vector. Recombinant baculoviral clones were isolated from the cotransfection supernatant via limiting dilution (21) and identified by dot-blot hybridization (22) against a radiolabeled probe derived from base pairs 2050 -2640 of the rat Jak2 cDNA. The viral isolate was amplified and the titer ascertained by dot-blot hybridization of DNA from Sf21 cells infected in a limiting dilution assay.
Solubilization and Immunoprecipitation of Recombinant Proteins-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Sf21 cells, originally derived from Spodoptera frugiperda, were grown at 27°C in IPL-41 media supplemented with 10% heat-inactivated fetal calf serum, 0.1% pluronic F-68, 50 units of penicillin/ml, and 50 g of streptomycin/ml. Sf21 cells were infected with recombinant baculovirus at a defined multiplicity of infection (m.o.i), 1 then harvested (10-min centrifugation, 3000 ϫ g) at various times post-infection (p.i.) as indicated in the figure legends. Cell pellets were washed in cold phosphatebuffered saline, pH 7.4, and then frozen and stored at Ϫ80°C. Typically, 4 ϫ 10 6 cells were solubilized by resuspension and incubation in 1 ml of lysis buffer (1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 1 g/ml pepstatin A, 2 g/ml leupeptin, 10 mM Tris-Cl, pH 7.6) for 1 h at 4°C. Nuclei and insoluble material were removed by centrifugation for 10 min at 3000 ϫ g. The lysate was incubated with 0.1 volume of 10% protein A-Sepharose CL-4B (Pharmacia Biotech Inc.) for 1 h at 4°C and centrifuged 15 min at 18000 ϫ g; the supernatant was then transferred to new vials. In some experiments, aliquots were taken at this point for SDS-PAGE and immunoblot analysis. Samples were immunoprecipitated by addition of rabbit anti-mouse JAK2 antisera (Upstate Biotechnology Inc., catalog no. 06-255) and overnight incubation at 4°C. Onetenth volume of 10% protein A-Sepharose CL-4B was added, and samples were incubated 30 min at 4°C and then centrifuged 10 min at 18,000 ϫ g. The pellet was washed three times with 1 ml of lysis buffer, then boiled in SDS-PAGE sample buffer before electrophoretic analysis.
In Vitro Kinase Assay-Infected Sf21 cells were harvested as above, but not washed with phosphate-buffered saline, then lysed and precleared as above. The lysates were incubated with rabbit anti-mouse JAK2 antisera for 2 h at 4°C; then, 0.1 volume of 10% protein A-Sepharose CL-4B was added, samples were incubated another 30 min, centrifuged 15 min at 18,000 ϫ g, and then washed twice with 1 ml of lysis buffer. The immunoprecipitated proteins were resuspended with 100 l of kinase mixture (50 mM NaCl, 5 mM MgCl 2 , 5 mM MnCl 2 , 0.1 mM Na 3 VO 4 , 250 Ci/ml carrier-free [␥-32 P]ATP, 10 mM HEPES, pH 7.4) and incubated 30 min at room temperature (ϳ21-22°C). 1 ml of lysis buffer was added to the sample, which was then centrifuged 15 min at 18,000 ϫ g, and the pellet was washed twice with 1 ml of lysis buffer. Proteins were eluted by boiling in SDS-PAGE sample buffer and then resolved by electrophoresis on a 7.5% polyacrylamide gel that was subsequently stained with Coomassie Blue, destained, dried, and autoradiographed.

RESULTS
A recombinant baculovirus was created with a transfer vector (pBacPAK9:rJAK2) that contained approximately 150 base pairs between the polyhedron promoter and the initiation codon of rat Jak2. The mutants (N⌬661)rJAK2 and rJAK2(C⌬795) were derived from restriction endonuclease digests and religation of the transfer vector pBacPAK9:rJAK2. A schematic diagram showing the JAK homology domains retained in each of the three forms, rJAK2, (N⌬661)rJAK2, and rJAK2(C⌬795), is presented in Fig. 1.
A time course of protein production in Sf21 cells infected with the baculovirus-expressing rJAK2 is shown in Fig. 2. The cells were harvested at various times p.i., solubilized in a buffer containing 1% Triton X-100, pre-cleared with protein A-Sepharose CL-4B, then immunoprecipitated with antiserum directed against a synthetic peptide DSQRKLQFYEDKHQLPAPK(C) corresponding to amino acid residues 758 -776 of murine and rat JAK2. At the saturating levels of virus used for this experiment, production of rJAK2 was observed as early as 27 h p.i. (Fig. 2, panel A), while tyrosine phosphorylation of rJAK2 was not detected at this time point (Fig. 2, panel B). Tyrosine phosphorylation of rJAK2 was first observed in this experiment at 51 h p.i. (Fig. 2, panel B), at a stage when rJAK2 production had increased (Fig. 2, panel A). The level of rJAK2 production and tyrosine phosphorylation of rJAK2 appeared to plateau at 71 h p.i. and was stable through 123 h p.i. The molecular mass deduced from the rat jak2 cDNA sequence is 130 kDa, but the apparent molecular mass of the native rat JAK2 is 120 kDa (23,24), which corresponds to the apparent molecular mass of the recombinant rat JAK2 produced via baculovirus expression vector system. The JH1 domain contains a motif that is recognized as a protein-tyrosine kinase consensus domain (25). Recombinant baculovirus producing rJAK2(C⌬795), a mutant of rat JAK2 containing amino acid residues 1 through 795 and thus lacking the JH1 domain and most of the JH2 domain, was created with the initial intention of testing whether active rJAK2 could transphosphorylate an inactive rJAK2(C⌬795). A time course of protein production in Sf21 cells infected with baculovirus producing rJAK2(C⌬795) is presented in Fig. 3. The molecular mass of rJAK2(C⌬795) deduced from its cDNA is 91 kDa. As seen in panel A, a 90-kDa protein was produced and recognized by anti-JAK2 antisera (Fig. 3, panel A) but not by the antiphosphotyrosine monoclonal antibody (Fig. 3, panel B). As in Fig. 2, Sf21 cells infected with recombinant baculovirus producing rJAK2 contained a 120-kDa protein that was recognized by both polyclonal anti-JAK2 antisera and by the monoclonal antiphosphotyrosine antibody. When Sf21 cells were co-infected with equal M.O.I. levels of both baculoviruses, the production of both rJAK2 and rJAK2(C⌬795) was detected by anti-JAK2 immunodecoration (Fig. 3, panel A), but neither form was recognized by antiphosphotyrosine antibody (Fig. 3, panel B). One interpretation of these data is that the inactive rJAK2(C⌬795) and rJAK2 form an inactive oligomer, an interpretation originally proposed by Zhuang et al. (1), to account for similar observations obtained in a COS cell expression system. Another concept that derives from such an interpretation is that the amino-terminal portion of the enzyme may contain an inhibitory or regulatory domain.
We sought to test this concept, as well as to determine if sufficient information was contained in the JH1/JH2 domains to confer catalytic activity, by constructing the recombinant baculovirus that produced (N⌬661)rJAK2. (N⌬661)rJAK2, which contained amino acid residues 661 through 1132 and thus retained all of JH1 but only 60% of JH2, was predicted to be a 472-amino acid protein with a molecular mass of 55 kDa. This protein comigrated with rabbit immunoglobulin G heavy chain, and therefore immunodecoration of (N⌬661)rJAK2 with anti-JAK2 anti-sera following anti-JAK2 immunoprecipitation was obscured. A demonstration of the time course of (N⌬661)rJAK2 production was achieved by anti-JAK2 immunodecoration of detergent-solubilized proteins that had not been immunoprecipitated before electrophoresis (Fig. 4, panel  A). Under these conditions, an endogenous cross-reactive 97-kDa protein was also observed. More importantly, the (N⌬661)rJAK2 protein was detected as a 56-kDa protein (or as a doublet of protein bands) between 27 and 49 h post-infection (Fig. 4, panel A) and became tyrosine phosphorylated by 49 h p.i. (Fig. 4, panel B). As the infection progressed, a distinct 62-kDa protein in the anti-JAK2 immunoprecipitation complex was detected by anti-phosphotyrosine immunodecoration at 72 and 93 h p.i. (Fig. 4, panel B). A Coomassie-stained band was observed at this mobility in immunoblots obtained from cells infected with baculoviruses producing rJAK2, rJAK2(C⌬795), or (N⌬661)rJAK2 (data not shown), but tyrosine phosphorylation of the 62-kDa protein was only observed in immunoblots from cells infected with baculovirus producing (N⌬661)rJAK2. It is not yet clear whether the common Coomassie-detectable band, which was not detectable by anti-JAK2 (data not shown), was a non-phosphorylated version of the tyrosine-phosphorylated 62-kDa protein or whether it coincidentally comigrated with it.
To demonstrate the relative intensities of the anti-phosphotyrosine signal of rJAK2 and of (N⌬661)rJAK2, as well as to test whether co-expression of these two forms would influence each other's phosphotyrosine state, co-infection studies were carried out (Fig. 5). Sf21 cells were infected with varying pro- portions of baculoviruses producing rJAK2 and (N⌬661)rJAK2, then harvested and detergent-solubilized 88 h post-infection. The presence of rJAK2 was determined via Western blot analysis of proteins that had not been immunoprecipitated before electrophoresis (Fig. 5, panel A). As the proportion of baculovirus producing rJAK2 increased, so did the intensity of the 120-kDa rJAK2 protein band. The decrease in intensity of the 56-kDa (N⌬661)rJAK2 protein band did not correlate as well with the decrease in the proportion of (N⌬661)rJAK2-producing baculovirus. 2 It was still apparent that the amount of rJAK2 produced at an M.O.I. of 18 or 20 equaled or exceeded the amount of (N⌬661)rJAK2 produced at an M.O.I. of 2, and overall, the amounts of anti-JAK2 cross-reactive material were comparable between the two proteins. This was not the case, however, when the anti-phosphotyrosine signals were compared (Fig. 5, panel B). A 120-kDa phosphotyrosine signal was first observed at an rJAK2 M.O.I. of 15, slightly intensified at an rJAK2 M.O.I. of 18, and was clearly visible at an rJAK2 M.O.I. of 20. In contrast, a very intense phosphotyrosine signal was apparent in all samples in which (N⌬661)rJAK2 was produced, and this intensity only slightly decreased when the (N⌬661)rJAK2 M.O.I. was reduced to 2. The very intense signal was a combination of two phosphotyrosine bands, as demonstrated by the data in Fig. 4. It should be noted that other minor bands (migrating at approximately 135, 105, and 45 kDa) were detectable as phosphotyrosine-containing proteins in the anti-JAK2 immunoprecipitate from (N⌬661)rJAK2-producing cells.
The variable intensity of a phosphotyrosine signal that cor-related to the retention or loss of a putative catalytic domain suggested that the rJAK2 and (N⌬661)rJAK2 proteins were active enzymes but that the rJAK2(C⌬795) was not. In an attempt to verify that these proteins were indeed active enzymes and not merely substrates for phosphorylation, in vitro kinase assays were conducted with proteins purified by immunoprecipitation (Fig. 6). This assay demonstrated incorporation of radioactive phosphorus into the 120-kDa protein when rJAK2 was incubated with [␥-32 P]ATP and a conspicuously greater incorporation of radioactive phosphorus into the 56-kDa protein when (N⌬661)rJAK2 was incubated with [␥-32 P]ATP. However, no autophosphorylation of the 90-kDa protein was observed upon incubation of the rJAK2(C⌬795) with [␥-32 P]ATP, as was anticipated. This demonstration of autophosphorylation verified that the rJAK2 and (N⌬661)rJAK2 were active enzyme forms and that the rJAK2(C⌬795) was an inactive form.

DISCUSSION
The tyrosine phosphorylation of JAK2 overexpressed via baculovirus expression vector system has been previously reported (12), and it appeared that the tyrosine phosphorylation occurs constitutively in such an overexpression system. By observing the time course of rJAK2 protein production (Fig. 2) and by observing JAK2 production at a fixed post-infection harvest time under varied multiplicities of infection (Fig. 5), 2 A plausible explanation for this is that the (N⌬661)rJAK2-producing baculovirus produces (N⌬661)rJAK2 more efficiently than does the rJAK2-producing baculovirus due to differences in the 5Ј-untranslated region. Since the rJAK2-producing baculovirus and the rJAK2(C⌬795)producing baculovirus do not differ in the 5Ј-untranslated region, one would not anticipate such a discrepancy in protein production levels when cells are infected at equal M.O.I. levels. Indeed, the data in Fig. 3, panel A, support this contention. Cells were harvested at 27, 49, 72, and 93 h p.i. Cellular contents were solubilized as described under "Materials and Methods." Panel A, following sample pre-clearance with protein A-Sepharose CL-4B but prior to immunoprecipitation, aliquots equivalent to 1.5 ϫ 10 5 cells were subjected to SDS-PAGE. The resolved proteins were transferred to Immobilon-P membrane and then immunodecorated with anti-JAK2. The black arrow points to the (N⌬661)rJAK2. Panel B, cellular contents were immunoprecipitated with anti-JAK2, aliquots equivalent to 2 ϫ 10 6 cells subjected to SDS-PAGE, transferred to Immobilon-P membrane, and then immunodecorated with anti-phosphotyrosine. The black arrow points to (N⌬661)rJAK2, and the white arrow points to an unidentified 62-kDa protein. i. Cellular contents were solubilized as described under "Materials and Methods." Panel A, following sample pre-clearance with protein A-Sepharose CL-4B but prior to immunoprecipitation, aliquots equivalent to 1 ϫ 10 5 cells were subjected to SDS-PAGE. The resolved proteins were transferred to Immobilon-P membrane and then immunodecorated with anti-JAK2. Panel B, cellular contents were immunoprecipitated with anti-JAK2, aliquots equivalent to 1 ϫ 10 6 cells subjected to SDS-PAGE, transferred to Immobilon-P membrane, and then immunodecorated with anti-phosphotyrosine. In both panels, the black arrow points to (N⌬661)rJAK2 and the white arrow points to the rJAK2. one can amend this interpretation. It appears that once the JAK2 production exceeds a critical level, tyrosine autophosphorylation occurs spontaneously.
A simple, plausible explanation of this observation is that monomers of JAK2 begin to form catalytically competent oligomers (possibly simple dimers) once the JAK2 concentration approaches a value that shifts the JAK2(oligomer)/ JAK2(monomer) equilibrium to a detectable amount of oligomer. This explanation is currently plausible because it is consistent with several pieces of information. First, the observation that inactive JAK2 mutants are capable of inhibiting autophosphorylation of wild-type JAK2 (initially observed by Zhuang,et al. (1) and demonstrated here in Fig. 3) could be explained by the formation of an inactive hetero-oligomer. Second, it has also been shown that JAK2-related receptors such as growth hormone receptor (26,27) dimerize subsequent to ligand binding. Activation of tyrosine kinase activity is mediated by dimerization of JAK2-related receptors such as prolactin receptor (28) and gp130 (29), which are associated with JAK2 before ligand binding (11,14,15), leading to the presumption that bringing the JAK2 proteins into close physical proximity is crucial for the "activation" of the JAK2. Finally, there is evidence that correlates a significant increase in jak2 mRNA (19) in the Nb2-SP pre-T lymphoma cell line (as compared to the parental Nb2-11C cell line) to an increase of tyrosine-phosphorylated JAK2 protein, 3 a phenomenon that may be relevant to the loss of absolute prolactin dependence in the Nb2-SP cell line (30).
The concept that the oligomeric form of JAK2 is the catalytically active form must be verified by additional independent experiments before it is acceptable as a part of the mechanism of enzymatic activation. It should be noted that there is no evidence yet to support transphosphorylation or inter-JAK2 phosphorylation; the full-length rJAK2 does not phosphorylate the inactive rJAK2(C⌬795), nor does the "hyperactive" (N⌬661)rJAK2 appear to phosphorylate rJAK2. These data are admittedly inconclusive since we have not shown that rJAK2(C⌬795) contains suitable phosphorylation sites nor have we demonstrated that (N⌬661)rJAK2 and rJAK2 can form hetero-oligomers. Without a "transphosphorylation mechanism," many scenarios remain that would link oligomerization to activation. These include cooperativity between multiple catalytic sites (e.g. the proton-translocating ATP synthase (31)), catalytic sites formed at subunit interfaces (e.g. lipoamide dehydrogenase (32)), or relief of interactions between inhibitory domains and catalytic domains (e.g. CaM kinase II (33)). The last scenario may also help to explain the apparent hyperactivity of the (N⌬661)rJAK2 mutant.
There are three pieces of evidence that suggest that the (N⌬661)rJAK2 mutant is both hyperphosphorylated and hyperactive. These data are as follows: 1) the exaggerated antiphosphotyrosine signal intensity of this form relative to that of the full-length protein (Fig. 5), 2) the significant increase in radiolabel incorporation observed in in vitro kinase assay autoradiography (Fig. 6), and 3) the appearance of a novel 62-kDa protein that is recognized by anti-phosphotyrosine, but not by anti-JAK2, on Western blots (Figs. 4 and 5). Although the variance in the 5Ј-untranslated region (which does not vary between rJAK2 and rJAK2(C⌬795)) may have slightly increased expression of (N⌬661)rJAK2, the apparent equal intensity of the anti-JAK2 signal in samples that have obvious differences in anti-phosphotyrosine signal intensities (Fig. 5) leads to the conclusion that (N⌬661)rJAK2 is hyperphosphorylated relative to rJAK2. One plausible explanation for hyperactivity is that an inhibitory or regulatory domain is contained within the amino-terminal portion of the enzyme. This model is consistent with data from Zhuang, et al. (1) that show that kinase-deficient JAK2 mutants inhibit autophosphorylation of the wild-type JAK2 in the COS cell expression system, as we also show in the baculoviral expression vector system (Fig. 3). The concept of such an inhibitory or regulatory domain requires additional experimental support, since alternative interpretations for the "trans-inhibitory" properties of an inactive JAK2 still exist.
If such inhibitory or regulatory domains exist in JAK2, one might expect other Janus kinases to possess them as a consequence of the high degree of sequence conservation in these proteins. Expression of TYK2 mutants ⌬TK (lacking JH1) and ⌬KL (lacking JH2) in cells derived from the 2fTGH cell line resulted in the generation of inactive kinases (18), which is consistent with the presence of an inhibitory domain in the amino-terminal domains JH3 through JH7. A useful test of the inhibitory/regulatory hypothesis will be to determine if expression of only the TK (JH1), KL (JH2), or combined KL/TK (JH2/JH1) domains is sufficient to produce an active kinase.
The nature and relevance of the 62-kDa band observed in Fig. 4 pose intriguing questions. Although identification of this protein is in progress, 4 the identification process is not yet complete. Thus, there remains some ambiguity as to whether this protein is virally encoded, is produced in response to viral infection, or whether it is a constitutive cellular product. It is also unknown whether the appearance of an intense phosphotyrosine signal associated with the 62-kDa protein in (N⌬661)rJAK2 (but not rJAK2) immunoprecipitation complexes is due to an alteration in substrate specificity or whether the apparent increase in catalytic turnover of the (N⌬661)rJAK2 mutant increased the phosphotyrosyl forms of the 62-kDa protein beyond the capacity of endogenous phosphatases.
The dual hypotheses that the oligomeric form of JAK2 is the catalytically active form and that there is an inhibitory or regulatory domain in the amino terminus of the enzyme are currently under investigation in our laboratory. These hypoth- 3  with recombinant baculoviruses expressing one of the following: human prolactin receptor (hPRLR), (N⌬661)rJAK2, rJAK2(C⌬795), or rJAK2. Infected cells were harvested 112 h p.i. and then assayed for in vitro kinase activity as described under "Materials and Methods." Immunoprecipitated equivalents of 2 ϫ 10 6 Sf21 cells were loaded in each gel lane; the dried gel was exposed to film for 9 h. The black arrow points to the full-length (120 kDa) rJAK2 in the gel autoradiograph, and the white arrow points to the (56 kDa) (N⌬661)rJAK2 mutant. eses do, however, allow for the speculative prediction that dysfunctional regulation of JAK2 expression may be responsible for factor-independent and receptor-independent proliferation and hence may predict circumstances for JAK2-mediated oncogenesis.