The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3

We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K_i value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.     

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.     

Full-length and N-TIMP-3 display equal inhibitory activities toward TNF-alpha convertase

We previously reported that tumor necrosis factor-alpha converting enzyme (TACE) was specifically inhibited by TIMP-3 but not TIMP-1, -2, and -4. Further mutagenesis studies showed that the N-terminal domain of TIMP-3 (N-TIMP-3) retained full inhibitory activity towards TACE. Full-length TIMP-3 and N-TIMP-3 exhibited indistinguishable values for the association rate constant and inhibitory affinity constant for the active catalytic domain of TACE (k(on) approximately 10(5) M(-1) s(-1) and K(app)(i) approximately 0.20 nM). Moreover, their k(on) (approximately 10(4) M(-1) s(-1)) and K(app)(i) (approximately 1.0 nM) values with a longer form of TACE (which encompasses the complete ectodomain including disintegrin, EGF and Crambin-like domains) were also shown to be similar. Detailed kinetic analyses indicated that TIMP-3 associated more quickly and with tighter final binding with TACE devoid of these C-terminal domains. We conclude that, unlike the interaction between many MMPs and TIMPs, the C-terminal domains of TIMP-3 and TACE are not essential in the formation of a tight binary complex.     

ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.     

Dynamic characterisation of the netrin-like domain of human type 1 procollagen C-proteinase enhancer and comparison to the N-terminal domain of tissue inhibitor of metalloproteinases (TIMP)

The backbone mobility of the C-terminal domain of procollagen C-proteinase enhancer (NTR PCOLCE1), part of a connective tissue glycoprotein, was determined using 15N NMR spectroscopy. NTR PCOLCE1 has been shown to be a netrin-like domain and adopts an OB-fold such as that found in the N-terminal domain of tissue inhibitors of metalloproteinases-1 (N-TIMP-1), N-TIMP-2, the laminin-binding domain of agrin and the C-terminal domain of complement protein C5. NMR relaxation dynamics of NTR PCOLCE1 highlight conformational flexibility in the N-terminus, strand A and the proximal CD loop. This region in N-TIMP is known to be essential for inhibitory activity against the matrix metalloproteinases and suggests that this region is of equal importance for NTR PCOLCE1, although the specific functional activity of the NTR PCOLCE1 domain is still unknown. Dynamics observed within the structural core of NTR PCOLCE1 that are not observed in N-TIMP molecules suggest that although the two domains have a similar architecture, the NTR PCOLCE1 domain will show different thermodynamic properties on binding and hence the target molecule could be somewhat different from that observed for the TIMPs. ModelFree order parameters show that NTR PCOLCE1 has more flexibility than both N-TIMP-1 and N-TIMP-2.     

The specificity of TIMP-2 for matrix metalloproteinases can be modified by single amino acid mutations.

Residues 1-127 of human TIMP-2 (N-TIMP-2), comprising three of the disulfide-bonded loops of the TIMP-2 molecule, is a discrete protein domain that folds independently of the C-terminal domain. This domain has been shown to be necessary and sufficient for metalloproteinase inhibition and contains the major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs). Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and TIMP/MMP crystal structures have been altered by site-directed mutagenesis. We show, by measurement of association rates and apparent inhibition constants, that the specificity of these N-TIMP-2 mutants for a range of MMPs can be altered by single site mutations in either the TIMP "ridge" (Cys1-Cys3 and Ser68-Cys72) or the flexible AB loop (Ser31-Ile41). This work demonstrates that it is possible to engineer TIMPs with altered specificity and suggests that this form of protein engineering may be useful in the treatment of diseases such as arthritis and cancer where the selective inhibition of key MMPs is desirable.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

Total conversion of tissue inhibitor of metalloproteinase (TIMP) for specific metalloproteinase targeting: fine-tuning TIMP-4 for optimal inhibition of tumor necrosis factor-{alpha}-converting enzyme

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs (a disintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme (TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 (N-TIMP-4) with TACE and showed that N-TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N-TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N-TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K(i) values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.     

Catalytic properties of ADAM12 and its domain deletion mutants

Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.     

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3

Tumor necrosis factor-alpha converting enzyme (TACE) is an ADAM (a disintegrin and metalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal domain form of wild-type tissue inhibitor of metalloproteinase (TIMP-3; N-TIMP-3) and its mutants against full-length recombinant TACE and the truncated form of its catalytic domain. We show that the C-terminal domains of TACE substantially weaken the inhibitory action of N-TIMP-3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C-terminal domains of ADAM proteinases in influencing TIMP interactions.     

Phosphorylation of the N-terminal and C-terminal CD3-epsilon-ITAM tyrosines is differentially regulated in T cells

Tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3 chains is crucial for the recruitment of protein tyrosine kinases and effector molecules into the T cell receptor. Thus, phenylalanine substitution at the N-terminal tyrosine residue of the CD3-epsilon-ITAM abolished signal transduction functions of this ITAM, including phosphorylation at the C-terminal ITAM tyrosine, and its association with ZAP-70. In contrast, mutation at the C-terminal tyrosine of CD3-epsilon-ITAM did not prevent phosphorylation at the N-terminal tyrosine, nor its association with Lck, or p85 PI 3-K regulatory subunit. In contrast to the ZAP-70/diphosphorylated CD8-epsilon-ITAM interaction, the Lck/monophosphorylated CD8-epsilon-ITAM interaction was sensitive to octylglucoside, an agent that disrupts Lck interaction with membrane rafts. Therefore, association of Lck with membrane rafts seems to be essential for stabilization of Lck/CD3-epsilon protein-protein interactions. Overall, the data suggest that the sequential and coordinated phosphorylation of CD3-epsilon-ITAM tyrosines provides to CD3-epsilon the potential to interact with multiple downstream effectors and signaling pathways.     

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.     

CD4 ligation excludes the Carma1-Bcl10-MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells

The antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, induces CD4/ZAP-70 reorganization and ceramide release in membrane rafts. Here, we investigated whether CD4/ZAP-70 compartmentalization could be mediated by an effect of 13B8.2 on the Carma1–Bcl10–MALT1 complex in membrane rafts. We report that treatment of CD3/CD28-activated Jurkat T cells with 13B8.2, but not rituximab, excluded Carma1–Bcl10–MALT1 proteins from GM1⁺ membrane rafts and concomitantly decreased NF-κB activation. Fluorescence confocal imaging confirmed that Carma1–Bcl10 and Carma1-MALT1 co-patching, observed in GM1⁺ membrane rafts following CD3/CD28 activation, were abrogated after a 24 h-treatment with 13B8.2. The CD4/ZAP-70 compartmentalization in membrane rafts induced by 13B8.2 is thus related to Carma1–Bcl10–MALT1 raft exclusion.     

ZAP-70 Tyrosine Kinase Is Required for LFA-1–dependent T Cell Migration

The ZAP-70 tyrosine kinase is essential for T cell activation by the T cell receptor. We show that ZAP-70 is also required for migration of T cells that is dependent on the integrin LFA-1. Invasion of TAM2D2 T cell hybridoma cells into fibroblast monolayers, which is LFA-1–dependent, was blocked by overexpression of dominant-negative ZAP-70 and by piceatannol but not by herbimycin A. The Syk inhibitor piceatannol blocks the Syk homologue ZAP-70, which is expressed by TAM2D2 cells, with the same dose dependence as the inhibition of invasion. Dominant-negative ZAP-70 completely inhibited the extensive metastasis formation of TAM2D2 cells to multiple organs upon i.v. injection into mice. Migration of TAM2D2 cells through filters coated with the LFA-1 ligand ICAM-1, induced by 1 ng/ml of the chemokine SDF-1, was blocked by anti–LFA-1 mAb and also abrogated by dominant-negative ZAP-70 and piceatannol. In contrast, migration induced by 100 ng/ml SDF-1 was independent of both LFA-1 and ZAP-70. LFA-1 cross-linking induced tyrosine phosphorylation, which was blocked by dominant-negative ZAP-70 and piceatannol. We conclude that LFA-1 engagement triggers ZAP-70 activity that is essential for LFA-1–dependent migration.     

Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1

The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 A resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between beta-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.     

Evidence for Restricted Reactivity of ADAMDEC1 with Protein Substrates and Endogenous Inhibitors

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1′ position. Cys³⁹², present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys³⁹² for a Ser increased the reactivity with α₂-macroglobulin but not with casein or Cm-Tf. Substitution of Asp³⁶² for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.     

TIMP-3 inhibits aggrecanase-mediated glycosaminoglycan release from cartilage explants stimulated by catabolic factors

Aggrecanases are considered to play a key role in the destruction of articular cartilage during the progression of arthritis. Here we report that the N-terminal inhibitory domain of tissue inhibitor of metalloproteinases 3 (N-TIMP-3), but not TIMP-1 or TIMP-2, inhibits glycosaminoglycan release from bovine nasal and porcine articular cartilage explants stimulated with interleukin-1alpha or retinoic acid in a dose-dependent manner. This inhibition is due to the blocking of aggrecanase activity induced by the catabolic factors. Little apoptosis of primary porcine chondrocytes is observed at an effective concentration of N-TIMP-3. These results suggest that TIMP-3 may be a candidate agent for use against cartilage degradation.     

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.     

ZAP-70 Protein Tyrosine Kinase Is Constitutively Targeted to the T Cell Cortex Independently of its SH2 Domains

ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH₂-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639–649), ZAP-70 may be localized close to its downstream targets.