The receptor interaction region of Tyk2 contains a motif required for its nuclear localization.

Janus kinases have so far been viewed as enzymatic intermediates that couple a variety of cell surface receptors to downstream substrates with diverse effector functions. Tyk2 is a member of this family that is involved in the interferon-alpha/beta and interleukin-12 signaling pathways via its specific interaction with the IFNAR1 and the beta1 receptor subunits. Here, we have analyzed the subcellular distribution of the wild-type Tyk2 protein and of several mutants expressed in Tyk2-deficient human cells. Direct GFP-associated fluorescence and immunostaining showed a diffuse localization of Tyk2 throughout the cell, including the nuclear compartment. The nuclear localization of Tyk2 requires a nuclear localization signal-like motif rich in arginine residues that maps within the region mediating interaction with cytokine receptors. To address the question of the role of the Tyk2 nuclear pool in interferon-alpha/beta-induced biological effects, cells expressing a membrane-targeted form of Tyk2-green fluorescent protein were analyzed for their interferon-alpha responses. Our studies demonstrate that Tyk2 can reside in the nucleus independently of receptor binding and that the nuclear pool is dispensable for the transcriptional and anti-vesicular stomatitis virus responses induced by interferon-alpha.

In vertebrates the Janus or JAK 1 proteins form a family of four tyrosine kinases (Tyk2, JAK1, JAK2, JAK3) that function in membrane-proximal signaling events initiated by a variety of extracellular factors binding to cell surface receptors. Notably, receptors that bind helical-bundled cytokines rely primarily on JAK kinases to activate and integrate signaling circuits (1,2). In such receptor complexes JAK proteins are specifically targeted to the cytoplasmic regions of transmembrane receptor subunits and are brought together through ligand-induced clustering and conformational changes of the subunits (3,4). Catalytic activation of the JAK occur via their reciprocal trans-phosphorylation of tyrosines in the activation loop of the tyrosine kinase domain. The activated enzymes then proceed to phosphorylate the receptor subunit(s) that act as recruitment sites for SH2-containing substrates, notably the STAT family of transcription factors (5,6).
The domain organization, structure, and mode of regulation of JAK family members have been extensively studied in recent years. These proteins can be subdivided into a large aminoterminal region (N), a kinase-like domain that plays a regulatory role, and a tyrosine kinase domain. Investigations from several groups including ours have identified the N region as involved in recognition and association with the receptor. Originally subdivided into 5 boxes of homology (JH7-JH3), the amino-terminal region was shown to contain a divergent band 4.1 domain, called the FERM or JEF domain, flanked by a SH2-related domain (7)(8)(9). Functional or structural information on these two domains in JAK are lacking.
The FERM domain is present in proteins of large functional diversity (10) and notably in the ERM protein family (ezrin, radixin, and moesin), where it mediates intramolecular binding as well as intermolecular association with transmembrane proteins (11). Conceivably, in JAK proteins the FERM domain carries the molecular determinant of receptor binding.
To date, all functional features of JAK proteins point to their involvement in events that take place at the level of complexes associated with the cell surface membrane. Interestingly, in a recent study JAK2 was identified as the kinase that associates with and phosphorylates the ATPase p97, a member of the AAA family (ATPases associated with different cellular activities), which is involved in membrane fusion and assembly of the transitional endoplasmic reticulum (12,13). Moreover, JAKs have been observed in the nuclear compartment. Endogenous JAK2 was found in the nucleus of rat pancreatic cells (14). Cytoplasmic as well nuclear immunoreactivity was detected for JAK1 and JAK2 ectopically expressed in Chinese hamster ovary cells. Immunogold electron microscopy confirmed the presence of endogenous JAK2 in nuclei of rat hepatocytes (15). Abundant JAK2 was detected by immunoblots on nuclear fractions prepared from rat liver cells (16). Here, we have studied the subcellular distribution of the tyrosine kinase Tyk2 in human cells, and we have addressed the question of the mechanism of nuclear import and of the potential role of the kinase in the nucleus. We demonstrate that Tyk2 is localized throughout the cell, including in the nuclear compartment, with the exclusion of nucleoli. We present evidence that nuclear localization of Tyk2 requires an Arg-rich nuclear localization signal (NLS)-like motif located within the FERM domain. A mutant form of Tyk2 anchored to the plasma membrane of Tyk2-deficient cells was shown to rescue transcriptional and anti-vesicular stomatitis virus responses to interferon (IFN)-␣, ruling out a role of the nuclear Tyk2 pool in these cytokine-induced biological activities.
Plasmid Constructs-For construction of Tyk2-GFP, a BssHII-XbaI fragment that spanned Tyk2 from aa 1025 to 1187 and contained at the 3Ј end a KpnI site was amplified and swapped into pBS-Tyk2. From this construct, the 3.9-kilobase HindIII-KpnI fragment containing Tyk2 cDNA was cloned in-phase into pEGFP-N3 vector (CLONTECH). For construction of mutants R220 -221/AA and R231-233-235/AAA, Arg to Ala substitutions were introduced by polymerase chain reaction using mutagenic primers (sequences available on request) and the overlap extension method onto a 1.4-kilobase BspE1-BsiWI Tyk2 fragment, which was then swapped into pRc-Tyk2B vector (21). For construction of mutant ⌬219 -240, a deletion of 66 base pairs was introduced into a 1.4-kilobase BspEI-BsiWI Tyk2 fragment amplified by polymerase chain reaction using primers that contained 5Ј complementary extensions. The amplified BspEI-BsiWI fragment was swapped into pRc-Tyk2B. For construction of mbTyk2-GFP, a 520-base pair Tyk2 polymerase chain reaction fragment was amplified using a sense primer with a 5Ј HindIII site followed by the sequence encoding residues 1-16 of human Lck (Met-Gly-Cys-Gly-Cys-Ser-Ser-His-Pro-Glu-Asp-Asp-Trp-Met-Glu-Asn) and an antisense primer spanning the Tyk2 SalI site. The fragment was cloned into Tyk2-GFP. All fragments derived from polymerase chain reactions were fully sequenced. The BS-IL-12R␤1 plasmid and the IL-12R␤2 cDNA cloned in the pEF-BOS expression vector (22) were gifts from U. Gubler. To obtain a ␤1 expression construct, a XbaI fragment containing the IL-12R␤1 cDNA was released from pBluescript and cloned into the pSR␣-puro expression vector.
Protein Analysis-The anti-Tyk2 antibodies used for immunoprecipitation and Western blotting (polyclonal R5 and monoclonal T10 -2) have been described (17). The anti-IFNAR1 EA12 mAb (a gift from L. Runkel) was used in immunoprecipitations, and the anti-IFNAR1 64G12 mAb (a gift from P. Eid) was used in immunoblots. The antiphosphotyrosine 4G10 mAb was from Upstate Biotechnology, Inc. Preparation of cell extracts and immunoprecipitations was performed as previously described (17). Immunoblots were revealed with an ECL detection system (Amersham Pharmacia Biotech). Protein bands were quantified by scanning with the Kodak Image Station 440CF.
Fluorescence Microscopy-Cells were plated at 5 ϫ 10 4 on glass coverslips in 24-well plates 16 h before transient transfection. GFP fluorescence was monitored 24 h after transfection. Cells were fixed with 4% formaldehyde for 10 min at room temperature and evaluated. For indirect immunofluorescence, fixed cells were washed 3ϫ with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed 3ϫ, and blocked in medium with 10% serum. Anti-Tyk2 mAb T10 -2 was diluted 1:200. The fluorescein-conjugated anti-mouse secondary antibody (Sigma) was diluted 1:200. Antibodies were diluted in medium with 10% serum and incubated for 1 h. Coverslips were mounted onto glass slides with Mowiol containing 2.5% DABCO (Sigma). Visualization was with a Zeiss Axiovert 135 microscope (40ϫ oil immersion lens) equipped for epifluorescence. Images were captured with a Hamamatsu Orca II CDD camera and analyzed with the AquaCosmos software.

The Amino-terminal Region Directs Tyk2 into the Nucleus-
The subcellular distribution of the Tyk2 was investigated in human HT-1080 fibrosarcoma cells by indirect immunofluorescence using a high affinity mAb directed to an epitope within the amino-terminal portion of the protein. A weak specific staining appeared distributed in both the cytoplasmic and the nuclear compartments in wild-type cells as compared with Tyk2-deficient 11,1 cells (data not shown). To improve the rather weak signal, immunostaining experiments were performed on an 11,1-derived clone stably reconstituted with wildtype Tyk2 expressed at a 3-fold higher level with respect to the endogenous protein (21). Staining in these cells appeared in denser regions of the cell surface, diffuse in the cytoplasm, and it was clearly detected in the nucleus (Fig. 1A, top left panel). The same distribution was obtained upon using the mAb p5D4 directed to the vesicular stomatitis virus epitope fused at the carboxyl terminus of the protein (data not shown). These results suggested that Tyk2 could reside within the nuclear compartment. No detectable change in the localization profile was observed upon stimulation of the cells with IFN-␣. Given the mass of the protein (134 kDa), its nuclear import may represent an active process relying on a discrete region(s) of the protein.
To narrow down the region contributing to nuclear import, we analyzed the localization of truncated Tyk2 mutants stably Tyk2 Nuclear Localization expressed in 11,1 cells. The functional characterization of these mutant proteins has been previously reported (17,23). The 70-kDa N protein lacks the two kinase domains and encompasses the amino-terminal region of Tyk2. Its overall distribution resembled that of the full-length protein, with nuclear staining slightly more intense and clear nucleolar exclusion (Fig. 1A, left panel). A 42-kDa mutant form encompassing residues 1-384 was exclusively nuclear (Fig. 1A, left panel), suggesting that an active process of nuclear import overcomes the passive diffusion of this small protein across the nuclear pore. The ⌬1-287 mutant is an amino-terminal truncated version of Tyk2 spanning residues 288 -1187. Its distribution profile was strikingly different, being cytoplasmic and perinuclear with a clear exclusion from the nucleus (Fig. 1A, left panel). Similar staining profiles were obtained from the analysis of 11,1 cells transiently transfected with the three constructs (Fig. 1A, middle panels).
To substantiate these findings and rule out possible localization artifacts due to the immunostaining procedure, we fused GFP at the carboxyl terminus of Tyk2. To assess the functionality of Tyk2-GFP, the construct was stably expressed in 11,1 cells, which are unresponsive to IFN-␣ due to Tyk2 deficiency. Since these cells contain an integrated IFN-inducible gpt construct, rescuing of the IFN-␣ response can be assessed by their ability to survive in HAT or 6-thioguanine when supplemented with the cytokine (24). Clones expressing Tyk2-GFP were as responsive to IFN-␣ as cells expressing wild-type Tyk2 (data not shown). Thus, the addition of the GFP moiety did not alter the function of Tyk2. As shown in Fig. 1A (right panel), Tyk2-GFP was found in the nucleus with the exclusion from nucleoli. On the other hand, the truncated ⌬1-287 mutant fused to GFP was clearly excluded from the nucleus (Fig. 1A, right panel). These results demonstrated that Tyk2 is distributed in all cellular compartments, including the nucleus, and the presence of the 287 amino-terminal residues is required for nuclear import.
Nuclear Localization of Tyk2 Requires an Arginine-rich Motif in the N Region-Classical nuclear NLS motifs are short se-quences with positively charged residues grouped in a single or a bipartite cluster (25,26). To identify a potential NLS in Tyk2, we scanned the first 287 residues and found an arginine-rich stretch within the JH6 box. Alignment of the four JAK proteins showed a conservation of basic residues in this region that was less evident in JAK1 ( Fig. 2A). To prove that this motif was required for the nuclear translocation of Tyk2, it was disrupted by point mutations or by a small deletion (Fig. 2B). In mutant designated R220 -221/AA, adjacent Arg-220 and -221 were changed to Ala, in mutant R231-233-235/AAA, Arg-231, -233, and -235 were changed to Ala, and in the ⌬219 -240 mutant 22 aa spanning the potential NLS were deleted. After transient expression in 11,1 cells, these mutants showed distinct subcellular distributions (Fig. 2C). Although the R220 -221/AA mutant was distributed as the wild-type protein, the R231-R233-R235/AAA mutant was significantly less nuclear. The ⌬219 -240 mutant was totally excluded from the nucleus. These data demonstrate that this motif plays a critical role in the nuclear localization of Tyk2.
To determine whether the NLS-like motif can function as a genuine nuclear-targeting element, the 22-amino acid motif (aa 219 -240) was moved from its natural location to the carboxyl terminus of Tyk2, and localization was assessed. Nuclear staining was not restored. Likewise, when the same motif was fused to the amino terminus of the GFP protein (wt22-GFP), no appreciable change in distribution was observed as compared with the diffusing GFP (data not shown). Therefore, the 22amino acid motif per se did not act as a NLS when removed from its natural context.
The NLS-like Motif Is Part of the IFNAR1 Interaction Domain of Tyk2-Since the triple alanine substitution or the deletion of aa 219 -240 partially or totally impaired the ability of Tyk2 to accumulate in the nucleus, we asked the question of whether these changes affected the functionality of Tyk2 in the IFN-␣-signaling pathway. To this aim, using the HAT/6-thioguanine survival test (24)  clones (clones 10 and 20) displayed a 10-fold reduced IFN-␣2 sensitivity as compared with control cells, whereas a third clone (clone 2) was fully sensitive. Interestingly, quantification of the exogenous protein expressed in each clone revealed a 2-3-fold higher level in clone 2 with respect to the others. Thus, higher protein levels appeared to compensate for the partial impairment of this mutated protein. Conversely, cells expressing the ⌬219 -240 protein were IFN-unresponsive and indistinguishable from Tyk2-deficient 11,1 cells. These data, summarized in Table I, demonstrated a partial loss of function of the weakly nuclear R231-233-235/AAA mutant and a complete loss of function of the ⌬219 -240 protein.
We had previously shown that Tyk2 sustains the level of expression of the IFN-␣ receptor chain IFNAR1 in human fibrosarcoma cells (17,18). Hence, we tested whether the ⌬219 -240 mutant protein was still able to perform this chaperone-like function. For this, we monitored by immunoblot the expression level of IFNAR1 in these stable transfectants. As shown in Fig. 3A, the IFNAR1 level in a representative clone expressing ⌬219 -240 was as low as in Tyk2-deficient 11,1 cells. This finding demonstrates that Tyk2 deleted of the NLS-like motif had lost its ability to interact with IFNAR1.
Deletion of the NLS Motif Impairs Tyk2 Interaction with the IL-12R ␤1 Chain-Tyk2 has been shown to be activated by IL-12 and to interact with the ␤1 component of the IL-12 receptor (27,28). We therefore asked whether the deletion of the NLS-like motif impairs Tyk2 activation by IL-12. Since the IL-12 receptor is restricted to specific hematopoietic cell types (29), we engineered its expression in our cellular system. We derived from 11,1 cells a Tyk2-positive clone (S␤␤T23) that expresses at the cell surface the components of the IL-12R, the ␤1 and the ␤2 chains (Fig. 4A). To assess correct function of the ectopic receptor complex in these cells, we measured Tyk2 phosphorylation after treatment with IL-12. As predicted, in S␤␤T23 cells, Tyk2 was activated not only by IFN-␣ but also by IL-12 (Fig. 4B). The more robust phosphorylation seen upon IL-12 treatment is likely to reflect the overexpression of IL-12 receptors with respect to the level of endogenous IFN-␣ receptors. Interestingly, in the absence of added cytokine, Tyk2 was basally phosphorylated in S␤␤T23 cells but not in the control WT cells (Fig. 4B, compare lanes 1 and 4), most likely as a consequence of its constitutive association with the IL-12R ␤1 chain. Having shown that reconstituted IL-12 receptors are functional, we asked whether the ⌬219 -240 mutant could be activated in response to IL-12. For this, we used S␤␤4 cells, also expressing the IL-12R subunits (Fig. 4A) but lacking Tyk2. S␤␤4 and 11,1 cells were transiently transfected with either the wild-type Tyk2 or the ⌬219 -240 mutant, and phosphorylation was monitored. Wild-type Tyk2 was more basally phosphorylated in S␤␤4 cells as compared with 11,1 cells (Fig. 4C,  lanes 1 and 5) as a result of its association with the IL-12 receptors. This was not the case with the ⌬219 -240 mutant (Fig. 4C, lanes 3 and 7). Furthermore, IL-12 treatment induced hyperphosphorylation of wild-type Tyk2, whereas the phosphorylation level of ⌬219 -240 remained unchanged, indicating that the ⌬219 -240 mutant failed to be activated in response to IL-12.
The results described above showed that deletion of the NLS-like motif in Tyk2 not only impaired its nuclear localization but also prevented its interaction with cytokine receptor  chains. To determine whether these two apparently unrelated properties, nuclear localization and receptor interaction, could be functionally dissociated, we tested whether other mutations within the receptor binding region of Tyk2 similarly affected subcellular localization. For this, the localization of three Tyk2 mutants that retain an intact NLS-like motif but lack the structural determinant for IFNAR1 binding was monitored. Mutant ⌬1-51 lacks residues 1-51, mutant ⌬JH4 lacks the JH4 homology box, and mutant T (1-518)J contains the aminoterminal 518 residues of Tyk2 (JH7 to JH4) fused to the carboxyl-terminal portion of JAK1 (17,18). Immunofluorescence staining revealed the presence of all three mutant proteins in both the cytoplasmic and nuclear compartments (Fig. 1B), demonstrating that the integrity of the receptor binding domain is not required for nuclear import. These results, summarized in Table II, suggest that the subcellular localization of Tyk2 is receptor-independent.
A Membrane-localized Form of Tyk2 Is Functional-A key question is whether the nuclear Tyk2 pool plays a role toward IFN-␣/␤-induced biological effects. To circumvent the problem of the positional and functional overlapping of the NLS-like motif and the receptor binding domain, a membrane-anchored form of Tyk2-GFP was generated by fusing to its amino terminus residues 1-16 of human Lck, a member of the Src family of tyrosine kinases. This sequence promotes membrane binding by allowing myristoylation and palmitoylation (30). The subcellular localization of this protein (mbTyk2-GFP) was monitored both by direct GFP visualization and immunostaining. In transiently or stably transfected cells expressing mbTyk2-GFP, the fluorescence heavily decorated the plasma membrane (Fig.  5). Confocal microscopy confirmed the absence of nuclear fluorescence (data not shown). The ability of the modified protein to sustain the IFNAR1 receptor in stable transfectants was analyzed by immunoblot and by fluorescence-activated cell sorter. Immunoblot analysis of one representative clone is shown in Fig. 3B. The level of IFNAR1 in mbTyk2-GFP-expressing cells was ϳ50% of the level present in Tyk2-GFP-expressing cells. These transfectants were monitored for their transcriptional response to IFN-␣ by testing their dose-dependent survival in HAT-containing medium. No appreciable difference in the IFN sensitivity (1 pM-1 nM range) was detected. A more complex biological effect of IFN-␣, i.e. its ability to induce in sensitive cells a state of resistance to viral infection, was measured in the two clones. The mbTyk2-GFP-expressing cells and the WT-GFP control cells were comparably sensitive to IFN when challenged with the vesicular stomatitis virus; 50% antiviral protection was obtained after treatment with 5-15 pM IFN-␣2. DISCUSSION In this study, we provide evidence that the tyrosine kinase Tyk2 is localized in both the nuclear and cytoplasmic compartments. Tyk2 resides in the nucleus provided that an Arg-rich motif, mapping around aa 219 -240 within the FERM domain, is present. Although the motif resembles a bipartite NLS, point mutations in the first basic cluster (R220 -221/AA) were with-out effect. Alanine substitutions of R231-233-235 in the second cluster partially impaired nuclear localization. Deletion of the 22-aa motif totally abolished nuclear localization. When moved out of its natural context or when fused to GFP, this motif did not act as a NLS, suggesting that it is required but not sufficient and that other regions in Tyk2 possibly contribute to its strength (26,31).
The capacity of exogenous Tyk2 to sustain the IFNAR1 protein level and to rescue IFN-␣ signaling in Tyk2-deficient cells correlated with its ability to reside in the nucleus. This apparent paradox can be reconciled, given that the NLS-like motif maps within the JH6 box, which was previously shown to be part of the IFNAR1 interaction region (18). Thus, disruption of the NLS-like sequence is expected to affect binding to IFNAR1 as well as nuclear localization. The IL-12R ␤1 binding domain in Tyk2 has not been mapped. Yet, the finding that the mutant deleted of the NLS-like motif failed to be activated in response to IL-12 strongly suggests that the same region in Tyk2 is involved in interaction with both IFNAR1 and the ␤1 chain. Furthermore, our data showed that the receptor-engaged wildtype Tyk2 pool is more prone to phosphorylation (Fig. 4) with respect to the non-engaged one, probably due to a different conformation and/or localization.
We have addressed the question of the role of nuclear Tyk2 by studying the function of a membrane-bound form of the protein modified by amino-terminal myristoylation and palmitoylation. Given the properties of the Lck-derived motif, the mbTyk2 protein should be directed to discrete microdomains of the plasma membrane, the lipid rafts (37). Accordingly, this modification did not appear to greatly affect the ability of the kinase to associate with the IFN-␣/␤ receptor, which at least in part is localized in caveolin-rich membrane domains (38). The full rescuing capacity of the mbTyk2 protein excludes a role of this kinase in the nuclear import mechanism of the activated STAT proteins (32)(33)(34)(35)(36). The possibility that nuclear Tyk2 intervenes in the dephosphorylation of nuclear STAT, for example by activating a nuclear tyrosine phosphatase (39), was tested by comparing the duration of STAT1 and STAT2 phosphorylation in wild-type and mbTyk2-expressing cells upon long term IFN-␣ treatment. STAT dephosphorylation occurred

Tyk2 Nuclear Localization
with similar kinetics in both cell types (data not shown), ruling out a role of nuclear Tyk2 in STAT inactivation.
A number of cytokines and cytokine receptors, including the IFN families, have been found in the nuclear compartment of a variety of cell types (14, 15, 40 -44). These findings have no clear biological significance yet, but they raise the possibility that JAK proteins could reach the nucleus when complexed to receptors. This is unlikely, since it was found through the study of mutant forms that nuclear localization of Tyk2 did not require the integrity of the receptor binding domain (Table II).
Our data support a model where the enzyme can reside in the nucleus independently of its association to cytokine receptors. Little is known of the post-activation fate of JAK proteins. Dephosphorylation by tyrosine phosphatases and catalytic inactivation through the binding of cytokine-induced suppressor of cytokine signaling proteins can down-regulate JAK activity (21,45). Ligand-induced activation of the receptor-kinase complex may be followed by receptor endocytosis and dissociation of the kinase from the receptor, an event that may cause unmasking of the NLS and consequent transport to the nucleus. We do not favor this model since we did not detect accumulation of Tyk2 in the nucleus in response to IFN-␣ or IL-12, even in cells overexpressing IL-12 receptors.
Given that the known function of Tyk2 is exerted at the membrane, one possibility is that nuclear sequestration of Tyk2 is a mechanism that controls and/or limits its availability. This mechanism has been described for a number of proteins (26,46,47). In such a scenario, the nucleus might serve as a reserve compartment for inactive protein. One interesting example of a regulated sorting mechanism through the nucleus has been recently described for the yeast Ste5 scaffold protein, involved in the mitogen-activated protein kinase cascade (48). A Ste5 mutant that cannot transit to the nucleus was found to be unable to localize at the periphery, where it performs its function. The possibility that the fraction of Tyk2 that is not engaged with receptors is directed into the nucleus via the exposed NLS is not supported by our data, since Tyk2 mutants that are unable to interact with IFNAR1, such as ⌬1-51 and ⌬JH4, are not exclusively nuclear. This suggests that the nuclear-cytoplasmic partitioning of the protein is completely, or in large part, independent of the receptor and may rely upon intramolecular interactions or interactions with anchoring molecules that in turn could modulate the exposure of the region containing the NLS-like motif. Consistent with this model, the smaller truncation mutant (aa 1-384), spanning the JH7 to JH5 regions, is wholly nuclear. Import of Tyk2 in the nucleus may be counterbalanced by an active export mechanism. So far, we have been unable to demonstrate leptomycin D-sensitive nuclear export or to locate an obvious leucine-rich export sequence.
Through the functional study of the membrane-targeted Tyk2 form, we have found that nuclear Tyk2 is dispensable for IFN-␣-induced transcriptional and anti-vesicular stomatitis virus responses. We obviously cannot rule out a potential role of Tyk2 in nuclear events induced by other IFN subtypes or upon infection with other viruses in activities that are cell typerestricted or redundantly performed by other kinases. JAK proteins are now widely recognized as participants of a variety of signaling cascades, and the role of Tyk2 is not limited to IFN-␣/␤and IL-12-induced pathways (49 -51). The nuclear Tyk2 pool may be mobilized or locally activated in response to specific stimuli in a cell cycle-dependent manner or according to the physiological state of the cell. Many proteins phosphorylated on tyrosine have been found in the nucleus (52). Some of these represent culmination of cytoplasmic events, as is the case for the translocated STATs, but it will be important to establish whether tyrosine phosphorylation cascades involving JAK kinases can occur within the nucleus.