Unexpected Effects of FERM Domain Mutations on ... - Cell Press

Molecular Cell, Vol. 8, 959–969, November, 2001, Copyright 2001 by Cell Press

Unexpected Effects of FERM Domain Mutations on Catalytic Activity of Jak3: Structural Implication for Janus Kinases Yong-Jie Zhou,1 Min Chen,1 Nancy A. Cusack,3 Lida H. Kimmel,3 Kelly S. Magnuson,3 James G. Boyd,3 Wen Lin,3 Joseph L. Roberts,4 Andrea Lengi,4 Rebecca H. Buckley,4 Robert L. Geahlen,5 Fabio Candotti,2 Massimo Gadina,1 Paul S. Changelian,3 and John J. O’Shea1,6 1 Lymphocyte Cell Biology Section Arthritis and Rheumatism Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases 2 Genetics and Molecular Biology Branch National Human Genome Research Institute National Institutes of Health Bethesda, Maryland 20892 3 Department of Immunology Pfizer Global Research and Development Groton, Connecticut 06340 4 Department of Pediatrics Duke University School of Medicine Durham, North Carolina 27710 5 Department of Medicinal Chemistry and Molecular Pharmacology Purdue University West Lafayette, Indiana 47907

Summary Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity. Introduction The major kinases associated with type I and II cytokine receptors are members of the Janus family (Jak1, Jak2, Jak3, and Tyk2) (Ihle et al., 1998; Leonard and O’Shea, 1998; Gadina et al., 2001), and multiple lines of evidence point to the critical role of Jak1 and Jak3 in signaling by common ␥ chain (␥c)-containing receptors. Jak1 associates with the ligand-specific subunits of these cytokine receptors, and its deficiency results in severe combined immunodeficiency (SCID) in mice (Rodig et al., 1998). However, this kinase is ubiquitously expressed 6

Correspondence: [email protected]

and also functions in other cytokine receptors; not surprisingly, Jak1 deficiency is perinatally lethal. In contrast, Jak3 is predominantly expressed in hematopoietic cells and specifically associates with the ␥c chain of cytokine receptors (Miyazaki et al., 1994; Russell et al., 1994). Mutations of Jak3 result in autosomal recessive SCID (Jak3-SCID) in humans and mice (Macchi et al., 1995; Nosaka et al., 1995; Russell et al., 1995; Thomis et al., 1995). Clinically, infants with Jak3-SCID present with a phenotype identical to X-linked SCID, which is caused by mutations of ␥c chain, vividly illustrating the essential role of Jak3/␥c interactions in the development and function of the immune system. Despite their importance, however, detailed information on the structure of Jaks is lacking. Seven Janus homology (JH) domains have been assigned based on sequence similarities within the family. The C-terminal tyrosine kinase catalytic domain is designated as the JH1 domain. A centrally located domain (JH2) displays high sequence identity with kinase domains but lacks catalytic activity. Thus, JH2 is termed the pseudokinase domain and has essential regulatory functions (Luo et al., 1997; Chen et al., 2000; Saharinen et al., 2000; Yeh et al., 2000). The N-terminal portion of Jaks is required for receptor binding, and, for Jak3, the major receptor interaction moiety is the JH7-JH5 domain (Frank et al., 1995; Chen et al., 1997; Richter et al., 1998; Cacalano et al., 1999). We now know that this region is a band four-point-one, ezrin, radixin, moesin (FERM) homology domain (Girault et al., 1999). This domain, a 300 amino acid protein-protein interaction module, comprises three subdomains that together form a compact clovershaped structure (Hamada et al., 2000; Pearson et al., 2000). Subdomain A is an ubiquitin-like fold, and subdomain B is an acyl-coenzyme A binding protein-like fold, whereas subdomain C shares the fold of phosphotyrosine binding (PTB) or pleckstrin-homology (PH) domain. The linker between subdomains A and B, which is at the center of a hydrophobic core, plays a key role in stabilizing the overall structure of the FERM domain. This domain is a widespread module found in cytoskeletal proteins and the tumor suppressor, merlin. It is also found in phosphatases, but Jaks and Faks are the only kinases with this module. Functionally, the FERM domain is involved in localizing proteins to plasma membranes via intermolecular interactions. In addition, through intramolecular interactions between the FERM and tail domains, it can mask associations with other proteins. Because of the paucity of detailed structural information pertaining to the Jaks, we have turned to the use of naturally occurring mutations of Jak3 to provide clues to structure and function. In the present study, we analyzed SCID patient-derived and artificial mutations within the Jak3 FERM domain. As predicted, these mutations impaired the kinase-receptor interaction, but surprisingly they also abrogated catalytic activity and blocked ATP binding. Coimmunoprecipation experiments showed that, although the FERM and kinase domains from the wild-type and mutant Jak3 associate, only wild-type FERM domain increased the kinase activity when the FERM and kinase domains were expressed

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Figure 1. Patient-Derived Jak3 FERM Mutations Impair ␥c Association (A) Alignment of the FERM domains of Jak3 and ERM proteins. Sequences of the putative subdomain A and A-B linker of human Jak3 were aligned with murine radixin, human moesin, and human ezrin using the MultiAlin program (Corpet, 1988) and the Blosum62 symbol comparison table. Secondary structure elements are indicated above the sequences using the ESPript www interface with radixin as superimposed secondary structure. An asterisk above the sequence alignment indicates mutations in Jak3; purple indicates the SCID patient-derived mutations; and light blue indicates the artificial mutations. (B) Schematic structure of three FERM lobes (Hamada, et al., 2000) with predicted placement of Jak3 mutations. (C) Schematic structure of wild-type and mutant Jak3. The SCID patient-derived Jak3 mutants, Del58A, Y100C, and D169E in the FERM domain and C759R in the JH2, and the mutant K855A that has a mutation at the ATP binding-site of Jak3 kinase domain, are illustrated. (D) FERM mutants inhibit Jak3/␥c association. COS-7 cells were transfected with 5 ␮g of Tac-␥c along with the other indicated cDNAs. Cell lysates were immunoprecipitated with anti-Tac mAb and blotted with anti-Jak3 Ab (upper). The membrane was reprobed with anti-␥c Ab (middle) and lysates were immunoblotted with anti-Jak3 Ab (bottom) to ascertain equivalent expression of proteins in each condition.

as separate polypeptides. Thus, this work suggests a novel role for the Jak3 FERM domain in maintaining a functional kinase domain. Similar to ERM proteins, the Jak3 FERM domain has important functions in both inter- and intramolecular interactions. However, in contrast to ERM proteins, it appears that the Jak3 FERM domain must coordinate both types of interactions simultaneously for proper signaling capability. Reciprocally, we also demonstrated that interference with the kinase domain by an inhibitor, which alters the conformation of the lobes in the kinase domain, dramatically decreased the ability of Jak3 to bind the receptor. Taken together, our data support a model in which the FERM and kinase domains associate and reciprocally influence each other’s function and structure. Results Patient-Derived FERM Mutations Inhibit Receptor Association FERM domains are widely expressed modules important for localization of proteins to plasma membranes.

A linker between subdomains A and B (Figure 1A and 1B, indicated by green) is particularly important in stabilizing the overall structure of the domain (Hamada et al., 2000; Pearson et al., 2000). Serendipitously, we have identified a SCID patient with a mutation in a residue that would be predicted to be at the beginning of this important linker region (Y100C). This residue is conserved in murine, avian, and piscine Jak3, other Jaks, and FERM domain proteins (Girault et al., 1999). We also identified another Jak3 SCID patient with two mutations in the FERM domain: an in-frame deletion of A58 (DelA58) and a substitution of D169 (D169E). These mutations would be predicted to reside in subdomain A and subdomain B, respectively. To study each mutation in isolation and ensure that the mutated Jaks were expressed at equivalent levels, we generated the mutants by site-directed mutagenesis (Figure 1C) and expressed them in COS-7 cells, which lack endogenous Jak3. These N-terminal mutants were coexpressed with a chimeric receptor Tac-␥c construct containing the transmembrane and cytoplasmic domains of ␥c fused to the extracellular domain of the IL-2R

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␣ (IL-2R␣) chain. We employed this system because the mAb against the extracellular domain of Tac-␥c protein efficiently coprecipitates the receptor/Jak3 complex (Chen et al., 1997). As shown in Figure 1D (upper panel), wild-type Jak3 coprecipitated with ␥c (lane 1). Importantly, catalytically inactive versions of Jak3, K855A, and C759R also associated with ␥c to the same extent as wild-type Jak3 (lanes 2 and 6), indicating that catalytic function is dispensable for receptor association. This finding is consistent with our previous observations (Chen et al., 1997, 2000; Zhou et al., 1997) and will have considerable relevance in subsequent experiments described in the present study. In contrast, the Y100C mutant exhibited reduced association, as previously reported (lane 3) (Cacalano et al., 1999). In addition, the two identified mutations, Del58A and D169E, also clearly interfered with receptor association (lanes 4 and 5), although equal levels of Tac-␥c (middle panel) and Jak3 (bottom panel) were expressed in these cells. Thus, these results demonstrated that multiple residues in the Jak3 FERM domain are required for receptor binding, consistent with the known function of other FERM domains in mediating plasma membrane localization. Patient-Derived FERM Mutations Abrogate In Vitro Catalytic Activity The Y100C mutation resulted in reduced, but not a complete lack of, receptor association. Therefore, it might be anticipated that some residual signaling would be detected in this patient’s cells. However, as shown in Figure 2A, this was not the case; no detectable STAT activation was evident with cytokine stimulation (lane 4). This suggested that the Y100C mutant Jak3 might have deficits beyond its poor receptor association. We therefore then measured its catalytic activity and found that it lacked activity (Figure 2B, upper panel, lane 2), even though comparable amounts of Jak3 were present in the immunoprecipates from patient and control cells (lower panel). This explained why no residual signaling could be detected and led us to investigate whether this was a specific property of the Y100C mutant or if other FERM mutants had impaired kinase activity. Again, wildtype and mutant versions of Jak3 were expressed in COS-7 cells, and the cell lysates were immunoprecipitated with Jak3 antiserum. The in vitro kinase activity of Jak3 immune complexes was assayed by measuring autophosphorylation and phosphorylation of the exogenous substrate, GST-␥c. Wild-type Jak3 readily autophosphorylated (Figure 2C, upper panel, lanes 1 and 2) and phosphorylated GST-␥c (Figure 2D, upper panel, lanes 1 and 2). In contrast, the previously reported catalytically inactive mutants, K855A and C759R, had no kinase activity (lanes 3, 4, 11, and 12). Significantly, none of the three patient-derived mutants, Y100C, Del 58A, and D169E, had kinase activity as measured by either assay (lanes 5–10), despite the fact that equal amounts of kinase and substrate were present (Figure 2C and 2D, lower panels). Multiple FERM Mutations Inhibit Both Receptor Association and Catalytic Activity In view of the fact that the patient-derived FERM mutations inhibited in vitro kinase activity, we next made a

Figure 2. Mutations in Jak3 FERM Domain Disrupt Kinase Activity (A) Jak3 FERM mutant fails to mediate IL-2-dependent STAT activation. EBV-transformed human B cell lines from a healthy individual (lanes 1 and 2) or a patient homozygous for the Y100C mutation (lanes 3 and 4) were incubated with IL-2 as indicated. Nuclear extracts were then prepared and EMSA was performed by incubating 10 ␮g of protein with a labeled oligonucleotide derived from the GAS-like element in the CD23 promoter. (B) The patient-derived FERM mutation abrogates Jak3 kinase activity. In vitro kinase assays of Jak3 were performed on immune complexes from a normal individual (lane 1) and the patient with the Y100C mutation (lane 2) at room temperature for 5 min. Samples were then separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography (upper) and blotting with antiJak3 Ab (bottom). (C and D) Patient-derived FERM domain mutations abrogate kinase activity. COS-7 cells were transfected with 5 ␮g of the indicated cDNAs. The cells were lysed and immunoprecipitated with anti-Jak3 Ab. In vitro kinase activity of Jak3 was measured in the presence of an exogenous substrate, GST-␥c at room temperature for 5 and 10 min, respectively. Samples were then separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography. Autophosphorylation of Jak3 (C) and phosphorylation of GST-␥c (D) are shown in the upper panels. Membranes were reprobed with anti-Jak3 Ab (C) and anti-GST Ab (D) to verify equal loading (bottom).

series of artificial mutations involving conserved residues in this domain. Because of the structural importance of the A-B linker and the identification of a patient mutation in this region (Figure 1), we focused most of our attention here. This linker has a highly conserved tyrosine residue (Y105 in Jak3) within this hydrophobic core, and, consequently, we made two point mutations of this residue (Y105F and Y105A). We also generated alanine-scanning mutants that affected multiple residues in this linker region (RFYF, amino acids 103-106, designated RFYF103AAAA) and a conservative substitution of tyrosine 100 (Y100F). Following these conserved residues is a less well-conserved area and a mutation was also created here (NW108AA). Finally, several other residues outside of the A-B linker are nearly invariant in FERM domain proteins, including the tryptophan residue corresponding to Jak3 W81 in subdomain A. Adjacent to W81 is a less well-conserved region that we also mutated (designated PPSH83AAAA). In light of the structural features of the FERM domain, we investigated

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Figure 3. Multiple FERM Mutations Inhibit Jak3 Catalytic Activity and ␥c Association Mutations of conserved residues in the Jak3 FERM domain disrupt catalytic activity (A and C) and receptor binding (B). (D) The Jak3 N terminus is required for catalytic activity. COS-7 cells were transfected with 5 ␮g of the indicated cDNAs. Cell lysates were immunoprecipitated with anti-Jak3 Ab, and in vitro kinase activity was assessed at room temperature for 10 min ([A], [C], [D], and top). Membranes were probed with anti-Jak3 antibody for equal loading (bottom). (B) Cell lysates were immunoprecipitated with anti-Tac mAb and blotted with anti-Jak3 Ab (upper), followed by reblotting with anti-␥c Ab (middle). Expression levels of various Jak3 proteins were detected by immunoblotting with antiJak3 Ab (bottom).

whether these mutations selectively affected catalytic activity or receptor association or affected both functions equivalently. As shown in Figure 3A (upper panel), multiple mutations within the FERM domain nearly abrogated in vitro kinase activity. These include the W81A and the Y100A mutants (lanes 3 and 4). The single mutations, Y105A (lane 5) or Y105F (Figure 3C, upper panel and lane 4), retained normal kinase activity, but the alanine scanning mutant encompassing this conserved motif, RFYF103AAAA, lost kinase activity (lane 8). This was also true for the adjacent PPSH83AAAA mutation (lane 7). In contrast, mutation of the less well-conserved residues in the A-B linker, NW108AA (amino acids 108-109), had no effect on kinase activity (lane 6). All mutants were efficiently expressed (lower panel). We next examined the ability of each of the artificial Jak3 FERM mutants to associate with ␥c (Figure 3B). Strikingly, there was nearly complete concordance between alteration of kinase activity and receptor association. That is, mutations that affected one property also affected the other; with one exception, none of these mutants had selective activity. As was illustrated in Figure 3B, the kinase inactive W81A, Y100A (upper panel, lanes 4 and 5) and the alanine scanning mutants failed to bind ␥c (upper panel, lanes 8 and 9), whereas the kinase active Y105A and NW108AA mutants bound normally (upper panel, lanes 6 and 7). These data suggest that proper structure of the Jak FERM domain is required for receptor association and for preservation of normal catalytic activity. Our previous study demonstrated that the patientderived mutation of Y100C interfered with Jak3/␥c association but a conservative substitution Y100F did not (Cacalano et al., 1999). Therefore, we were interested in determining whether this Y100F mutant had normal catalytic function. As shown in Figure 3C, the Y100F mutant had no in vitro kinase activity (upper panel, lane 3); this is the only mutant identified with selective properties. However, phosphorylation of Y100 is not likely to

be the explanation for the alternation of Jak3 catalytic activity, as Y100 does not appear to be a major autophosphorylation site in Jak3 (Y.-J.Z. et al., unpublished data). Altogether, these results suggested the possibility that as with ERM proteins, the FERM domain in Jaks has two functions: intermolecular interactions (receptor association) and intramolecular interactions (maintenance of the proper catalytic activity). The alternative was that the Jak3 FERM mutations distort the conformation of this region of the molecule and cause it to aberrantly impinge upon the kinase domain, although in normal circumstances these regions do not interact. If the latter model was correct, we reasoned that the N-terminal regions of the molecule would be dispensable for the kinase activity. To distinguish these possibilities, we tested a series of N-terminal deletion mutants (Figure 3D). Significantly, none of the FERM deletion mutants had in vitro kinase activity (upper panels and lanes 3–5). For comparison, wild-type Jak3 and the kinase inactive mutant K855A were included (lanes 1 and 2). These data suggested that, in fact, the N terminus is required for preservation of normal catalytic activity. Patient-Derived FERM Mutants Bind ATP Poorly Our data indicated that natural and artificial FERM domain mutations abolished in vitro catalytic activity, strongly suggesting that FERM domain provides structural support for a functional kinase domain. To confirm and extend the results of the in vitro kinase assays, we next determined if wild-type Jak3 could be labeled with a reactive ATP analog, 14C-5⬘-p-fluorosulfonylbenzoyl adenosine (14C-FSBA), which covalently alkylates a conserved lysine residue within the ATP binding cleft of many kinases (Colburn et al., 1987). We first ascertained that wild-type Jak3 specifically bound 14C-FSBA in vitro. As shown in Figure 4, this was the case (Figure 4A, upper panel and lane 1), and the binding was competed by excess unlabeled ATP (lanes 2–5 and Figure 4B). We therefore next investigated whether the patient-derived


Figure 4. FERM Mutants Do Not Bind an ATP Analog, FSBA Transfected COS-7 cells were lysed and immunoprecipitated with anti-Jak3 Ab. (A and B) 14C-FSBA binds specifically to Jak3. (C and D) Catalytically inactive Jak3 mutants are unable to bind 14C-FSBA. The immune complexes from cells transfected with wild-type and mutant Jak3 were incubated with 14CFSBA at 30⬚C for 60 min using the conditions for a standard in vitro kinase assay with or without excess unlabeled ATP. Samples were then washed and subjected to SDS-PAGE and autoradiography ([A], [C], and upper). The radioactivity of each labeled protein was quantitated by Phosphoimager analysis and was depicted as counts per minute of 14CFSBA labeled protein (B and D). The level of expression in each sample was determined by blotting with anti-Jak3 Ab ([A] and [C] bottom panel).

FERM mutants bound FSBA, with the binding of 14CFSBA to wild-type Jak3 shown in lane 1 (Figure 4C, upper panel, and Figure 4D). Mutation of the Jak3 ATP binding site, K855A, which significantly impaired binding of 14C-FSBA, is shown as a control (Figure 4C, upper panel, lane 2, and Figure 4D). Interestingly, all patientderived FERM mutants (Y100C, Del58A, and D169E; Figure 4C, upper panel, lanes 3–5, and Figure 4D) and a patient-derived JH2 mutant, C759R, bound less 14C-FSBA (Figure 4C, upper panel, lane 6, and Figure 4D) than the K855A mutant, although all were expressed at equivalent levels (Figure 4C, lower panel). Given that the FERM domain mutants were unable to bind ATP, it was not surprising that none had efficient in vitro catalytic activity. The Jak3 Amino Terminus Binds the Catalytic Domain and Can Regulate Its Function The preceding data pointed to the possibility of interactions between the kinase domain and the amino terminus; therefore, it was important to directly test this possibility. To this end, plasmids encoding varying N-terminal portions of Jak3 were coexpressed with a separate plasmid encoding the isolated Flag-tagged catalytic domain (Flag-JH1). The Jak3 mutants were immunoprecipitated with an anti-Jak3 N-terminal peptide antibody. Their ability to associate with JH1 kinase domain was assessed by immunoblotting with anti-Flag antibody (Chen et al., 2000). As shown in Figure 5A (upper panel), expression of constructs encoding the Jak3 FERM domain (StuI, lane 3) and the FERM and SH2 domains (NcoI, lane 2) showed that these polypeptides coimmunoprecipitated Flag-JH1; note that the FERM domain was not expressed as abundantly as the FERM-SH2 construct (middle panel). Interestingly, however, a longer construct comprising the pseudokinase domain, SH2, and FERM domains did not associate with the kinase domain (lane 1). The specificity of the interaction was also supported by the lack of association of ␥c with JH1 (lane 4). Next, we asked whether the binding of the amino terminus could influence the activity of the isolated kinase domain. Varying amounts of the FERM domain construct were coexpressed with a plasmid encoding

the isolated kinase domain, Flag-JH1. The kinase domain was immunoprecipitated and subjected to in vitro kinase assays. As shown in Figure 5B and 5C, the coexpression of the FERM domain clearly enhanced kinase activity. When 0.125 ␮g of the FERM domain plasmid was coexpressed, mean catalytic activity increased about 2.3-fold from 28,170 cpm to 63,610 cpm (n ⫽ 2, Figure 5B, lanes 3–6), even though the amount of kinase domain was constant (middle panel). Interestingly though, this effect was diminished when the FERM domain was expressed at very high levels (Figure 5C, lane 4). The levels of expression of the FERM domain are shown in the lower panel. We next asked what the consequence would be of having a patient-derived mutation in the isolated FERM domain on the ability of this segment to interact with the kinase domain. When a FERM domain containing the Y100C mutation was coexpressed with the kinase domain, the domains still associated (Figure 5D, lane 2), but the mutant FERM domain failed to upregulate kinase activity (Figure 5E). These data provide further support for the contention that functionally relevant interactions exist between the Jak3 amino terminus and kinase domain, and this can be disrupted with patient mutations. Jak3/␥c Association Is Blocked by a Kinase Inhibitor To further explore the idea that intramolecular interactions exist between the FERM and kinase domains of Jak3, we next considered the possibility that alteration of the conformation of the kinase domain would affect receptor binding. Although FERM domains in other proteins mediate intramolecular interactions, the idea that kinase-FERM domain interaction is important for Jak function seemed unlikely for several reasons. As shown in Figure 1C, the catalytic activity of Jak3 is not required for receptor association; the Jak3 N terminus is necessary and sufficient for binding ␥c, and this association is not phosphorylation dependent (Chen et al., 1997). Indeed, it is a consistent finding for Jaks that removal of the kinase domain does not block receptor association (Frank et al., 1995; Richter et al., 1998; Cacalano et al., 1999). Nonetheless, if the N-terminal and C-terminal domains of Jak3 interact, perturbation of the kinase

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Figure 5. The Jak3 FERM Domain Binds the Kinase Domain and Can Regulate Its Activity (A and D) Jak3 FERM domain associates with the JH1 domain. COS cells were transfected with Flag-JH1 (1 ␮g, [A]), together with 1 ␮g of each Jak3 C-terminal deletion mutant [lanes 1–3: J3(⌬JH1): FERM-SH2-JH2-aa 1–813; J3(NcoI): FERM-SH2-aa 1–530; and J3(StuI): FERM-aa 1–192] and Tac-␥c (lane 4) or transfected with Flag-JH1 (1 ␮g, [D]), together with 1 ␮g of J3(StuI) (lane 1), the J3(StuI) cDNA containing Y100C mutation and Tac-␥c (lane 3), respectively. Lysates were immunoprecipitated with a Jak3 N-terminal anti-peptide Ab (lanes 1–3 in [A] and lanes 1 and 2 in [D]) or anti-Tac-mAb (lane 4 in [A] and lane 3 in [D]), and blotted with anti-Flag mAb (upper panel). Expression of the Jak3 deletion mutants (second panel), Tac-␥c (third panel), and Flag-JH1 (bottom panel) was assessed by blotting with indicated antibodies. (B, C, and E) Overexpression of the FERM domain modulates kinase activity. COS cells were transfected with the kinase domain construct, Flag-JH1 (0.5 ␮g) in the absence (lanes 1 and 2 in [B] and lane 1 in [C] and [E]) or presence of the indicated amounts of the FERM domain constructs, J3(StuI) (lanes 3–8 in [B], lanes 2–4 in [C] and lanes 2–3 in [E]) or J3(StuI/Y100C) (lanes 4 and 5 in [E]). Lysates were immunoprecipitated with anti-Flag mAb and then subjected to an in vitro kinase assay ([B], upper; [C and E], inserts). The level of expression was determined by blotting with indicated Ab ([B], middle and bottom). Incorporated radioactive phosphate was quantitated by Phosphoimager analysis and is expressed as counts per minute of 32P-labeled protein (C and E).

domain in the context of the intact molecule might alter the ability of the N terminus to bind ␥c. Specifically, it has been well established that kinase inhibitors that intercalate between the lobes of the kinase domain alter their conformation (Al-Obeidi and Lam, 2000). Analysis of Lck-staurosporine complexes, for instance, showed that this inhibitor interacts with the ATP binding cleft and induces a dramatic conformational change in the N- and C-terminal portions of the kinase domain (Zhu et al., 1999). We, therefore, investigated whether staurosporine could influence Jak3/receptor interactions, assuming that the catalytic domain and N terminus communicate with each other. To address this point, we first developed a system to evaluate the binding ability of purified recombinant proteins in vitro. As shown in Figure 6A, when GST-␥c or GST was coated onto ELISA plates, purified recombi-

nant Jak3 specifically bound to GST-␥c (closed diamonds), but not to GST (open squares). Further evidence for the specificity of this assay was the finding that Jak3 binding to ␥c was found to be competitively inhibited by a peptide corresponding to the Jak3 binding region of a wild-type ␥c subunit (Figure 6B, open diamonds). In contrast, a ␥c peptide containing the X-SCID point mutation L271Q (Figure 6B, open squares), previously shown to disrupt Jak3/␥c association (Russell et al., 1994), did not inhibit binding. To determine whether the behavior of recombinant Jak3 and ␥c in this assay were the same as in the immunoprecipitation assay as we previously reported (Chen et al., 1997), we tested the binding ability of ␥c to Jak3 and to a C-terminal deletion mutant of Jak3. As expected, the JH7-JH3 mutant bound ␥c nearly as well as the full-length Jak3, indicating that the kinase and


Figure 6. Staurosporine Binding Disrupts Jak3/␥c Association (A) Recombinant Jak3 and ␥c associate in vitro. ELISA plates were coated with GST (open squares) or GST-␥c (closed diamonds) proteins and then incubated with purified Jak3 protein for various times. After washing, anti-Jak3 Ab binding was used to measure Jak3/␥c association. (B) Inhibition of Jak3/␥c binding by a ␥c peptide. The recombinant Jak3/␥c proteins were incubated as described in (A); but prior to the addition of anti-Jak3 Ab, a wild-type ␥c-peptide (open diamonds) or a SCID patient-derived ␥c-peptide (open squares) was added to disrupt the association. As a control, GST and Jak3 incubated with a wild-type ␥c peptide is shown (closed squares). After washing, we used antiJak3 Ab to detect the Jak3/␥c association. (C) A Jak3 mutant lacking the kinase and pseudokinase domains binds to ␥c. (D) Staurosporine inhibits binding of the full-length Jak3 to ␥c, but has no effect on a Jak3 mutant lacking the kinase and pseudokinase domains. The recombinant Jak3 and GST-␥c proteins were incubated as described in (A); but prior to the addition of anti-Jak3 Ab, varying concentrations of staurosporine were added. (E) Staurosporine inhibition of Jak3/␥c association is competed by ATP. Jak3 and GST-␥c were incubated without (open diamonds) or with 0.075 ␮M (closed diamonds), 0.15 ␮M (open squares), or 0.3 ␮M staurosporine (SP, closed squares) in the presence of indicated concentrations of ATP.

pseudokinase domains are not required for ␥c binding (Figure 6C). We next added staurosporine to this assay to assess whether it influenced Jak3/␥c binding. Surprisingly, despite the results in Figure 6C and previous data, this appeared to be the case. As shown in Figure 6D, staurosporine blocked wild-type Jak3 binding to ␥c in a dosedependent manner (IC50 of 5 nM, open diamonds). Given this unanticipated result, it was important to document that this inhibitory effect involved the kinase domain and was not simply due to a nonspecific effect. We addressed this concern in two ways. First, staurosporine failed to block the JH7-JH3 mutant binding to ␥c (open squares, dotted line), indicating that the inhibitory effect of drug required the presence of the kinase domain. Second, if the effect of staurosporine were via its binding in the ATP binding cleft of Jak3, we expect that ATP would compete with staurosporine and restore Jak3

binding to ␥c. This too was the case (Figure 6E); ATP reversed the staurosporine inhibition of Jak3/␥c binding in a dose-dependent manner (closed diamonds, closed squares, and open squares), whereas the ATP by itself had no effect (open diamonds). Together, these data argued that staurosporine mediated its effect by interacting with the ATP binding cleft at the C terminus of Jak3, resulting in alteration of the N terminus/␥c interaction indirectly by an effect on the kinase domain. It should be noted that we do not know at present if the pseudokinase domain might also bind ATP or ATP analogs, so staurosporine could also bind this domain. To further confirm the effect of staurosporine on Jak3/ ␥c association, we used a complementary approach. The association of Jak3 and ␥c had originally been identified by coprecipitation of native proteins from cell lysates. We therefore sought to ascertain whether the effect of staurosporine on recombinant proteins would

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Figure 7. Staurosporine Inhibits Association of Prebound Jak3/␥c Proteins from Cells COS-7 cells were cotransfected with the indicated cDNAs and were lysed 36 hr after transfection. (A) Cell lysates were immunoprecipitated with anti-Tac mAb, and Jak3/␥c complexes were incubated without (lanes 1 and 6) or with staurosporine (lanes 2–5) or the Src kinase inhibitor PP2 (lanes 7–10) at indicated concentrations at room temperature for 1 hr. The immune complexes were then washed, subjected to SDS-PAGE, transferred to nitrocellulose, and detected by blotting with antiJak3 Ab (upper). The same membrane was reprobed with anti-␥c Ab (middle). Cell lysates containing Jak3 and each mutant were also detected by blotting with anti-Jak3 Ab (bottom). (B) The cell lysates were immunoprecipitated with anti-Jak3 Ab, and in vitro kinase activity was performed in the absence (lanes 1 and 6) or in the presence of various concentrations of staurosporine (lanes 2–5) or PP2 (lanes 7–10). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography (upper). Membranes were probed with anti-Jak3 Ab to ascertain equal loading (bottom).

also be evident on the association of the native proteins and could interrupt the prebound complex of Jak3/␥c isolated from cells. As shown in Figure 7A, Jak3/␥c association was observed in the absence of staurosporine (upper panel, lane 1) but was disrupted by staurosporine in a dose-dependent manner (upper panel, lanes 2–5). As a positive control, the effect of staurosporine on the catalytic activity of Jak3 was assessed by an in vitro kinase assay (Figure 7B). As expected, Jak3 kinase activity was evident in the absence of staurosporine (upper panel, lane 1), but was inhibited in its presence (upper panel, lanes 2–5). It should be noted that kinase activity is more sensitive to staurosporine than receptor association. As a further control, a selective Src kinase inhibitor, PP2, which has no effect on the catalytic activity of Jak3 (Figure 7B, lanes 6–10) (Zhou et al., 2000), also had no effect on Jak3/␥c association (Figure 7A, lanes 6–10). Taken together, these data clearly suggest that intramolecular interactions exist between the catalytic and FERM domains of Jak3. The alteration in the catalytic domain structure of Jak3 by staurosporine may very well induce a conformational change of the FERM domain and block its ability to bind the receptor.

In the present study, we identified an additional function of the Jak FERM domain, namely preservation of proper catalytic function. Both natural and artificial FERM domain mutations unexpectedly inhibited catalytic activity and ATP binding. Thus, two mechanisms contribute to the disease pathogenesis in SCID patients with FERM mutations: impaired Jak3/␥c association and inactivation of catalytic activity. Even more unexpected though was the finding that a competitive inhibitor of Mg-ATP, staurosporine, disrupted Jak3 binding to ␥c, despite clear evidence that the kinase domain is dispensable for this association. This raises the question of why these domains, which reside at the opposite ends of the molecule, would reciprocally affect each other’s functions. For some kinases, the noncatalytic domains mediate subcellular distribution and protein-protein interactions. For example, the N termini of Tec family protein kinases have PH domains, which mediate membrane targeting. Mutation of this portion of the protein can interfere with catalytic function by disrupting its binding to phospho-

Discussion In the present study, we explored the functional consequences of SCID patient-derived mutations involving conserved residues in the FERM domain of Jak3. Even prior to the recognition that Jak N terminus comprises a FERM domain, it was documented that this region mediates receptor association (Chen et al., 1997; Cacalano et al., 1999). Now it is clear that like other FERM domains, a major function of the Jak3 FERM domain is to mediate localization to plasma membranes—in this case by binding to the cytokine receptor, ␥c. Only one other family of tyrosine kinase, the FAK family, has a FERM domain, and Fak has been reported to associate in vitro with the epidermal growth factor receptors via this domain (Sieg et al., 2000). So, it appears that plasma membrane localization and binding to transmembrane proteins is a general property of FERM domains.

Figure 8. A Model for Interactions of the Jak3 FERM Domain with the Kinase Domain and ␥c


lipids in the plasma membrane (Qiu and Kung, 2000). Subdomain C of FERM domains is similar to PH or PTB domains and in the case of radixin can bind phosphatidylinositol 4,5-bisphosphate (Hamada et al., 2000). While the Jak FERM domain targets Jaks to the plasma membrane, it is also clear experimentally that neither receptor binding nor plasma membrane localization is required for Jaks to phosphorylate their substrates. Additionally, the Jak3 kinase domain has activity in isolation (Zhou et al., 1997; Chen et al., 2000; Figure 5). For these reasons, we believe that interference with membrane localization is not the major mechanism underlying defective catalytic activity resulting from Jak3 FERM mutations. The N terminus of mitogen activated protein kinases serves a different function (Eblen et al., 2000); it is essential for proper substrate recognition and interaction with upstream kinases. Mutation of the N terminus of ERK2 interferes with its kinase activity, but this has been explained by the inability of MEK to bind and activate the mutant ERKs. However, this does not appear to be the case for Jaks, as expression of the isolated JH1 can mediate phosphorylation of relevant substrates (Chen et al., 2000); interaction with at least some substrates is not dependent upon noncatalytic domains. While the structure of Jaks has still not been elucidated, the crystal structure of Src family members indicates that intramolecular interactions between the catalytic domain and the SH2 and SH3 domains are of major importance in regulating their catalytic activity (Xu et al., 1997; Bjorge et al., 2000). By analogy with Src kinases, it was reasonable to speculate that intramolecular interactions between catalytic and noncatalytic domains of the Jaks regulate their catalytic function. We considered the possibility that the mutations at the Jak3 FERM domain interfered with kinase activity by aberrantly forcing the N terminus to impinge upon the kinase domain, because removal of the N terminus inhibited catalytic activity (Figure 3D). Interestingly, physical association of the domains was detected by coimmunoprecipitation of the FERM domain even if it included a patient mutation (Figure 5). However, the wild-type FERM domain also had the capacity to enhance in vitro activity of the isolated kinase domain. Thus, it would seem more reasonable to propose that the FERM domain has a permissive role in stabilizing the conformation of the activated wildtype kinase. There are other examples of positive regulation of kinases through the binding peptides outside of the kinase domain. One such example is the A helix of the Dictyostelium protein kinase A (PKA). In this case, the A helix is linearly distant from the kinase domain but is nonetheless modeled to interact with the hydrophobic core between the lobes of the kinase domain (Veron, et al., 1993). The A helix has conserved tryptophan and hydrophobic residues, and it is tempting to speculate that the region of W81 in the Jak3 FERM domain might bind the Jak kinase domain. Additionally, the noncatalytic C terminus of PKA also interacts with the kinase domain. Furthermore, the C terminus of PKA can bind the kinase domain of another kinase, PDK1. PDK1 can also bind another PKA-homologous peptide termed the PDK1-interacting fragment of protein kinase C-related kinase 2 (PRK2). These peptides bind a hydrophobic pocket in the PDK1 kinase domain, positively regulating

its catalytic activity and promoting substrate interaction (Biondi, et al., 2000). In the case of Fak, for example, deletion of the FERM resulted in a kinase that is hyperphosphorylated (Schlaepfer and Hunter, 1996). This suggests that the FERM domain of Fak can also regulate activity of the kinase domain, but may do so somewhat differently from the Jaks. Another important finding of our study is that there appears to be two-way communication between the FERM and catalytic domains of the Jaks (Figure 8). Not only do perturbations in the FERM domain regulate kinase activity, but alteration of the conformation of the kinase domain can also alter receptor interactions (Figure 6D). Intramolecular interactions have been documented to occur in ERM proteins such that the FERM and tail domains bind and interfere with intermolecular associations. Phosphorylation of the tail disrupts this intramolecular interaction, allowing the tail to bind actin filaments and the FERM domain to bind integral membrane proteins. Thus there is clear precedent for FERM domains to function in intra- and intermolecular interactions, and our present results suggest that the Jak FERM domain also has both functions. For the Jaks, though, this antagonist model of ERM proteins does not appear to be entirely apt. Irrespective of activation, the Jaks appear to be constitutively associated with cytokine receptors. However, it is also true that activation can promote Jak/receptor interactions (Russell et al., 1994). The present data also suggest that altering the structure of the kinase domain can cause Jaks to dissociate from the receptor. It is conceivable that endogenous inhibitors (perhaps SOCS family members) might do the same. Finally, the data have another very pragmatic implication. Because the absence of Jak3 has such clear but delimited consequences, it has become a popular pharmaceutical target (O’Shea et al., 2000). Enthusiasm for the development of specific tyrosine kinase inhibitors has been further spurred by the development of an effective and safe Bcr-Abl inhibitor used to treat chronic myelogenous leukemia (Druker and Lydon, 2000). Our present data argue that a potent Jak3 inhibitor could have two salutary properties: inhibition of enzymatic activity and dissociation from ␥c. Experimental Procedures Cells and Antibodies COS-7 cells and EBV-transformed human B cells were maintained as previously described (Zhou et al., 2000). Samples from Jak3 mutation patients were obtained under an IRB approved NIH protocol (99-AR-0004). Rabbit polyclonal antisera against Jak3, ␥c, and glutathione S-transferase (GST) or monoclonal antibodies (mAbs) against Tac (7G7) and Flag were described previously (Chen et al., 1997, 2000). Plasmids, Mutagenesis, and Transfection The cDNA expression constructs, pME18sJak3, pME18sK855A, Nand C-terminal deletion mutants, and Flag-JH1 were generated previously (Chen et al., 1997, 2000). Mutagenesis of amino acid residues in Jak3: Del58A, W81A, Y100C, Y100F, Y100A, D169E, Y105A, Y105F, C759R, J3(StuI/Y100C), NW108AA, PPSH83AAAA, and RFYF103AAAA was performed using the Transformer Site-Directed Mutagenesis Kit according to the manufacturer’s instructions (Clontech, San Francisco, CA). The cytoplasmic region of human IL-2R␥c subunit was subcloned into the pGEX-2T vector (Amersham, Piscataway, NJ) to express the GST-␥c fusion protein, while the full-length

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human Jak3 and JH7-JH3 mutant were subcloned into pFASTBAC vector to express GST-Jak3 and GST-JH7/JH3 fusion proteins, respectively. A chimeric receptor, Tac-␥c (␣/␥/␥), consists of the IL2R␣ (extracellular domain) and IL-2R␥c (transmembrane and cytoplasmic domains) and was cloned into the pME18s vector. For transient transfection, COS-7 cells were transfected with 5 ␮g of each cDNA by a FuGENE6 Transfection Reagent according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN). Electrophoretic Mobility-Shift Assays, Immunoprecipitation, Immunoblotting, and Immune Complex Kinase Assays EMSA was performed by using an oligonucleotide corresponding to a GAS (IFN-␥ activation site)-like element found in the CD23 promoter (5⬘-GATCAAGACCATTTCTAAGAAATCTATC-3⬘) as previously reported (Zhou et al., 1997). For immunoprecipitation, human B cell lines or transfected COS-7 cells were washed once with icecold phosphate-buffered saline (PBS) containing 2 mM Na3VO4 and lysed in detergent-containing buffers as previously described (Zhou et al., 2000). Lysates were clarified by centrifugation, and the supernatants were immunoprecipitated using each indicated Abs. The immune-complexes were washed and used for immunoblotting Jak3/␥c binding, in vitro kinase assay, or 14C-5⬘-FSBA (NEN, Boston, MA) affinity labeling. In Vitro Kinase Assays Jak3 immune complexes were washed one additional time with 100 mM NaCl and 10 mM HEPES (pH 7.5), and then resuspended in 50 ␮l of kinase reaction buffer containing 1 ␮Ci [␥-32P]ATP (Amersham, Arlington Heights, IL) with or without an exogenous substrate GST␥c (2 ␮g) or staurosporine (Sigma, St. Louis, MO) or PP2 (Calbiochem, La Jolla, CA), as previously described (Zhou et al., 2000). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography or immunoblotted with the indicated Abs. The radioactivity incorporated by Jak3 was quantified using a STORM Phosphoimager (Molecular Dynamics, Sunnyvale, CA). Affinity Labeling of ATP Binding Site of Jak3 with an Adenosine Analog 14 C-FSBA was dissolved in DMSO and incubated with immunoprecipitated Jak3 at a final concentration of 3.5 nM in 50 ␮l of reaction mixture (20 mM Tris/HCl [pH 7.5], 5 mM MgCl2, 5 mM MnCl2, and 1 ␮M ATP). After incubation at 30⬚C for 60 min, the samples were washed to remove unbound FSBA, boiled in sample buffer, and electrophoresed on SDS-polyacrylamide gels. FSBA binding was measured by autoradiography and Phosphoimager analysis. For specificity controls, the same reaction mixtures were incubated with 50-fold excess concentrations of cold ATP for 30 min at room temperature, prior to addition of 14C-FSBA (Colburn et al., 1987). The Jak3/␥c Binding ELISA The GST-Jak3 fusion proteins were produced in Sf9 cells, purified on a glutathione column, and the GST moiety removed by thrombin cleavage. The GST-␥c fusion protein was expressed in E. coli, purified using glutathione sepharose, and eluted with free glutathione. These purified recombinant proteins were used for Jak3/␥c binding assays (P.S.C. et al., unpublished data). In brief, Nunc Maxisorp 96well plates were coated with 50 ␮l of glutathione-bovine serum albumin (GSH-BSA, 2 ␮g/ml in PBS) and incubated overnight at 4⬚C. Plates were then coated with 50 ␮l of GST or GST-␥c constructs (10 ␮g/ml in PBS) and decanted plates were blocked with 50 ␮l/ well of milk (5% in PBS). Prior to the binding assay, plates were washed three times with PBST (PBS plus 0.5% Tween-20) and Jak3 (50 ␮l/well, 1:20 dilution in the assay buffer: 50 mM HEPES [pH 7.3], 125 mM NaCl, 24 mM MgCl2, and 1 mM Na3VO4) was added. After 30 min binding at room temperature, plates were washed three times and incubated with the primary Ab (anti-Jak3 Ab). The HRPconjugated secondary antibody was then added and detected by adding 50 ␮l TMB (Kirkegard and Perry Laboratories, Gaithersburg, MD). Adding 0.09 M H2SO4 stopped color development and the absorbance was read at OD450 on a SpectroMax 340 96-well plate reader (Molecular Devices, Sunnyvale, CA). To study the in vitro inhibition of Jak3/␥c binding, potential blocking reagents (staurosporine or ␥c peptides) were added and

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