Temperature-sensitive ZAP70 mutants degrading through a proteasome-independent pathway. Restoration of a kinase domain mutant by Cdc37.

CD8 deficiency is an autosomal recessive form of severe combined immunodeficiency diseases characterized by the absence of CD8(+) T lymphocytes and impaired T cell functions. We identified two novel mis-sense mutations in the zap70 genes of a CD8-deficiency patient. One mutation (P80Q) affects a residue in an SH2 domain and another (M572L) in the kinase subdomain XI. Both mutations cause a degradation of ZAP70 protein in a temperature-sensitive manner through an ATP-dependent and proteasome-independent pathway. We further demonstrated that Cdc37, a protein kinase-specific chaperone, bound to M572L but not P80Q mutant and restored the expression of the M572L mutant when overexpressed. The restoration of M572L mutant by Cdc37 required the function of HSP90. These results indicate that Cdc37 in conjunction with HSP90 functions as a molecular chaperone for a temperature-sensitive kinase domain mutant of ZAP70.

CD8 deficiency is an autosomal recessive form of severe combined immunodeficiency diseases (SCIDs) 1 and is associated with defects in the ZAP70 protein tyrosine kinase (PTK), which plays a pivotal role in signal transduction through the T cell receptor (TCR) (1)(2)(3)(4)(5). Upon TCR stimulation, ZAP70 is recruited to tyrosine-phosphorylated immunoreceptor tyrosinebased activation motifs (ITAMs) within the cytoplasmic domains of TCR subunits; this is an essential step for ZAP70 activation and subsequent cellular signaling pathways (6 -8). Association of ZAP70 with the TCR is mediated by an interaction between the two SH2 domains of the ZAP70 molecule arranged in tandem and the two phosphorylated tyrosine residues in the ITAM (9 -13).
All of the mutations in the ZAP70 molecule that have been reported to cause SCID in humans cluster around the kinase subdomain VIII, resulting in instability of the ZAP70 protein (3)(4)(5). In general, misfolded proteins are recognized by cellular proteins, chaperones and proteases. Molecular chaperones assist in the proper folding of misfolded polypeptide and render it functional, whereas proteases eliminate such a polypeptide (14). It is thus likely that the counterbalance between chaperones and proteases determines the stability of mutant ZAP70 molecules. With respect to proteases, it is widely accepted that the ubiquitin-proteasome system is involved in degradation of abnormal proteins (15,16). However, the contribution of proteasomes to the instability of mutant ZAP70 proteins is yet to be determined.
We show here that two novel mis-sense mutations in the zap70 genes of a CD8-deficiency patient cause degradation of ZAP70 protein in vivo in a temperature-sensitive (ts) manner through an ATP-dependent and proteasome-independent pathway. We also show that overexpression of Cdc37 (17,18), a protein kinase-specific molecular chaperone, preferentially restores the expression of a kinase-domain mutant of the ZAP70 molecule even at the nonpermissive temperature.

EXPERIMENTAL PROCEDURES
Cell Culture and Immunoblot Analysis-The HTLV-1-transformed T cell lines SN, established from a CD8 deficiency patient, and KO, derived from a healthy donor, were maintained in RPMI 1640 medium supplemented with antibiotics, 5 ϫ 10 Ϫ5 M 2-mercaptoethanol, 10 mM Hepes, 10% fetal calf serum, and IL-2 (10 units/ml). A cDNA library was constructed from mRNA of SN cells using -ZAP II (Stratagene) and screened with a probe comprising the KpnI/HpaI fragment of the human zap70 cDNA. DNA sequencing was performed with dRhodamine Terminator Cycle Sequencing Ready Reaction on a PRISM 310 automated sequencer (Applied Biosystems). Informed consent was obtained from the patient's parents prior to the experiments. Jurkat cells, a human T cell line, were maintained in RPMI 1640 medium with the supplements described above except for IL-2. Jurkat cells (5 ϫ 10 7 cells/ml) were homogenized with a Dounce homogenizer in P-buffer solution (20 mM imidazole-HCl, pH 6.8, 100 mM KCl, 20 mM EGTA, 2 mM MgCl 2 , and 2 mM dithiothreitol). The lysate was centrifuged at 15,000 ϫ g for 15 min, and the supernatant was mixed with equal volume of P-buffer solution containing 2 mM ATP and 40% glycerol to obtain total lysate. Total lysate was further centrifuged at 500,000 ϫ g for 5 h to obtain S500 (supernatant) and P500 (pellet) fractions. Immunoblotting was performed as described (19).
Plasmid Constructs-A cDNA clone for wild-type ZAP70 tagged with the Myc-epitope (EQKLISEEDL) at its C terminus was obtained from Jurkat cells using reverse transcriptase-PCR and cloned into the HindIII and XbaI sites of pcDNA3.1ϩ (Invitrogen). P80Q and M572L mutants were created by PCR-based site-directed mutagenesis (Quick Change TM , Stratagene). Dominant-negative ZAP70 (ZAP70⌬C) molecules were constructed by subcloning the SH2 domains (HindIII and HincII fragment) into the HindIII and EcoRV sites of * This work was supported by Grants-in-aid for Scientific Research on Priority Areas 09276233 and 10172229 from the Ministry of Education, Science, Sports and Culture of Japan, the National Grant-in-aid for the Establishment of High-tech Research Center in a Private University, a grant-in-aid from the Yamanouchi Foundation for Research on Metabolic Disorders, a grant from Daiichi Pharmaceutical Co. Ltd., and a grant from the Japan Society for the Promotion of Science (JSPS-RFTF 97L00701). 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 U.S.C. Section 1734 solely to indicate this fact.
In Vitro Pull-down Assay-Biotinylated ZAP70 was generated in vitro using the TNT T7 rabbit reticulocyte lysate system (Promega) in the presence of Transcend tRNA (Promega). GST-Cdc37 and biotinylated ZAP70 were incubated in 100 l of P-buffer solution containing 5 mM EDTA at 30°C for 30 min, followed by incubation with 15 l of glutathione-Sepharose (Amersham Pharmacia Biotech) at 4°C for 2 h. After washing with 2ϫ diluted P-buffer solution containing 0.1% Tween 20, bound ZAP70 proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with streptavidin-HRP (Amersham Pharmacia Biotech). Either vehicle or 1 g of a synthetic phosphopeptide corresponding to CD3-ITAM that was N-terminally biotinylated (13) was preadsorbed to 20 l of streptavidin beads (Sigma), followed by incubation with 35 S-labeled ZAP70 molecules for 1 h in 100 l of binding buffer (20 mM Hepes, pH 7.5, 137 mM NaCl, 2 mM dithiothreitol, 1 mM vanadate, 1% aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin A). After washing with Tris-buffered saline containing 0.05% Tween 20, bound ZAP70 proteins were resolved by SDS-PAGE and analyzed on an BAS2000 (Fujix) image analyzer.

Novel Mis-sense Mutations in the zap70 Genes of a CD8
Deficiency Patient-An HTLV-1-transformed T cell line, SN, established from a CD8 deficiency patient (22) expressed no ZAP70 protein (Fig. 1A) even though the cell line expressed a normal size zap70 mRNA at the usual levels (data not shown). Molecular cloning and sequencing of zap70 cDNAs from SN cells uncovered two mutations (Fig. 1B): a C to A transition at position 448, resulting in a substitution of glutamine for proline at residue 80 (P80Q) in the N-terminal SH2 domain (numbering according to Ref. 6), and an A to T transition at position 1923, causing the substitution of leucine for methionine at residue 572 (M572L) in the kinase subdomain XI (23). This methionine residue is well conserved among PTKs. Genomic DNA analysis showed that the patient was heterozygous for these mutations; the C to A mutation was paternal, and the A to T mutation was maternal (Fig. 1C). A healthy sibling of the patient carries the paternal mutation.
Both Mutations Render ZAP70 Protein Unstable in Vivo-Protein expression was not detectable in a human T cell line Jurkat transfected with Myc-tagged mutant cDNAs, whereas transfection of the wild-type (WT) cDNA resulted in expression of a Myc-tagged ZAP70 protein of the expected size ( Fig. 2A). However, these mutant ZAP70 proteins were produced at a level comparable to the WT protein in an in vitro reticulocyte translation system (see Figs. 2C and 3C), suggesting that the mutant proteins are unstable and degraded in vivo. In fact, pulse-chase experiments showed that whereas WT ZAP70 protein was stable with a half-life of Ͼ4 h, P80Q as well as M572L mutant proteins were rapidly degraded with a half-life of approximately 1 h (Fig. 2B).
Mutant ZAP70 Proteins Are Degraded in an ATP-dependent and Proteasome-independent Manner-Specific inhibitors for proteasomes such as lactacystin and the peptide aldehyde MG132 failed to block the proteolysis of ZAP70 mutants, whereas these inhibitors blocked degradation of ␤-catenin, a known target of the ubiquitin-proteasome pathway (Fig. 2C). When incubated with total cell lysates in an in vitro degradation assay system, both M572L and P80Q mutant proteins prepared by an in vitro translation system were degraded in an ATP-dependent manner, whereas WT ZAP70 protein remained stable (Fig. 2D). The proteolytic activity was recovered from the S500 fraction (Fig. 2E, upper panel), which was devoid of proteasomes (Fig. 2E, lower panel). Other protease inhibitors, including E64 (10 M), TPCK (100 M), TLCK (100 M), leupeptin (20 g/ml), and pepstatin A (20 g/ml), had no effect on the degradation of ZAP70 mutants (data not shown). Although tripeptidyl peptidase II (TPPII) activity is ATP-dependent and Temperature-sensitive zap70 Mutations 34516 is enhanced by lactacystin (24), AAF-chloromethylketone, a specific inhibitor for TPPII, failed to block degradation (data not shown). These results, collectively, indicate that the ZAP70 mutants are degraded by a novel ATP-dependent and proteasome-independent pathway that remains to be identified.
P80Q and M572L Mutants Are Temperature-sensitive-Because the in vitro translation reaction was performed at 30°C, it was possible that the mutant proteins are stable at low temperatures. Indeed, both mutants turned out to be ts. Both P80Q and M572L mutant proteins were readily detected in transfected cells at a temperature lower than 33°C (Fig. 3A). Jurkat cells transfected with WT or mutant zap70 cDNAs were cultured at 30°C, stimulated with pervanadate, a potent activator of intracellular PTKs, and assayed for ZAP70 kinase activities in vitro using tubulin as a substrate (25). The WT and P80Q mutant ZAP70 were similarly activated by pervanadate as indicated by autophosphorylation of ZAP70 and phosphorylation of tubulin (Fig. 3B). On the other hand, the activity of the M572L mutant was partially impaired. Although pervanadate induced the same level of tyrosine phosphorylation of the WT and M572L mutant proteins in vivo (Fig. 3B, lower panel), the level of kinase activity determined by autophosphorylation and phosphorylation of tubulin was approximately 50% that of WT ZAP70 (Fig. 3B, upper panel). These results demonstrate the importance of the conserved methionine residue at the kinase subdomain XI for PTK activity as well as the stability of the ZAP70 protein.
We next examined the SH2 function of the mutant ZAP70 proteins. The 35 S-labeled P80Q mutant ZAP70 protein produced in vitro at 30°C bound to the phosphorylated ITAM peptide with an affinity comparable to the WT and M572L mutant in an in vitro pull-down assay (Fig. 3C). Note that the P80Q mutant synthesized in vitro bound to the phosphorylated ITAM peptide even at 37°C, the nonpermissive temperature, indicating that the protein is stable at 37°C once synthesized and properly folded. To examine whether the SH2 domains of the P80Q mutant bind to ITAMs in vivo, we constructed a dominant-negative form of ZAP70 (ZAP70⌬C), consisting of two SH2 domains, that sequesters endogenous ZAP70 from phosphorylated ITAMs (26). When expressed at 30°C, the ZAP70⌬C containing the P80Q mutation blocked the TCRinduced signaling pathway as well as the ZAP70⌬C consisting of the WT SH2 domains (Fig. 3D). However, such a blocking effect by the P80Q mutant was not observed at 37°C. Thus, the defect resulting from the P80Q mutation is likely due to the rapid degradation of mutant polypeptide and not to a lack of SH2 function.
Cdc37 in Conjunction with HSP90 Restores the Expression of M572L Mutant-It has been suggested that mutant proteins, which are incompletely or improperly folded when synthesized, are degraded unless folding intermediates are compromised by their cofactor(s), namely chaperone(s) (27,28). One such chaperone is Cdc37, which stabilizes various PTKs by interacting with their kinase domains (17,18). Indeed, co-transfection with Cdc37 increased the expression level of the WT ZAP70 protein in a dose-dependent manner (Fig. 4A). In contrast, only a slight effect was observed when the ZAP70⌬C lacking the kinase domain was co-expressed with Cdc37, indicating that Cdc37 functions as a kinase domain-specific chaperone for ZAP70. Expression levels of the M572L mutant at both permissive and nonpermissive temperatures were also remarkably augmented by co-transfection with Cdc37 (Fig. 4B). Although not shown, co-expression of Cdc37 did not affect the half-life of the M572L Temperature-sensitive zap70 Mutations 34517 mutant at the nonpermissive temperature, indicating that Cdc37 restored the expression of M572L mutant protein by assisting its folding process and not by blocking degradation pathway(s). In contrast, Cdc37 had a marginal effect on the P80Q mutant. It is possible that the P80Q mutation leads to conformational changes, preventing interaction between the kinase domain of the ZAP70 and Cdc37. In fact, whereas both WT ZAP70 and the M572L proteins bound GST-Cdc37, interaction of the P80Q mutant with GST-Cdc37 was marginal and observed only with an excess amount of GST-Cdc37 (Fig. 4C). Such differences in ability to bind Cdc37 would account for the preferential effect of Cdc37 on the M572L mutant. Several studies have revealed that Cdc37 acts in concert with another chaperone, HSP90 (17,18). Inhibition of HSP90 function with geldanamycin reduced the expression of both the WT and the M572L mutant even in the presence of Cdc37 (Fig.  4D). These results clearly indicate that HSP90 is required for the expression of ZAP70 protein. HSP90 is a highly abundant protein (28), but yet the expression of the M572L mutant depends on Cdc37. Therefore Cdc37 is likely the rate-limiting component and contributes to the proper folding of the M572L mutant with the aid of the HSP90 function.
With the exception of the ts-type I oculocutaneous albinism (29), spontaneous ts mutations have rarely been reported in the human. We provide evidence here that ts mutations of the zap70 gene are involved in human SCIDs. The fact that these mutants have kinase activities at 30°C, the permissive temperature, raises the possibility that the T cell defects could be rescued at a lower temperature. In fact, although the patient's peripheral blood mononuclear cells failed to respond to stimulation via the TCR at 37°C, CD4 ϩ T cells with activated and/or memory phenotypes were found to accumulate in his dermis (22). It is known that Syk PTK complements the function of ZAP70 in the thymus, where the expression level of Syk is much higher than in the periphery. It is thus possible that in this patient autoreactive T cells that would have been negatively selected under normal conditions were actually positively selected. Because Syk expression is greatly reduced upon migration to the periphery, peripheral CD4 ϩ T cells in the patient would have become unresponsive to antigenic stimulation. Because local body temperature within the skin is lower, functional ZAP70 proteins may have been expressed with the help of endogenous Cdc37 and HSP90 chaperones, subsequently inducing T cell activation. It will be of interest to examine the possibility that Cdc37 in concert with HSP90 also rescues other mis-sense mutants reported in CD8 deficiencies (3)(4)(5) and other SCIDs involving Btk and JAK3 PTKs (30,31).