pp56^Lck Mediates TCR ζ-Chain Binding to the Microfilament Cytoskeleton

Animals

All mice, except the Lck knockout mice (the kind gift of Dr. Tak Mak, Amgen, Thousand Oaks, CA) were bred in our facility or purchased from The Jackson Laboratory (Bar Harbor, ME).

In vitro reconstitution, immunoprecipitation, gel electrophoresis, and immunoblotting

Thymocytes or lymph node T cells were freshly prepared and isolated as cell suspensions from normal adult mice by pressing organs through a 200-μ nylon mesh (Bally Ribbon, Bally, PA). The cell suspensions were then washed three times in balanced salt solution and 5% FBS, solubilized with 0.5% Nonidet P-40 in a Tris-buffered saline (150 mM NaCl, 10 mM Tris, pH 7.3) solution containing protease and phosphatase inhibitors (0.2 mM VO₃, 10 mM NaF, 1 mM PMSF, and 1 mg/ml each of aprotinin, leupeptin, and α-1-antitrypsin) and centrifuged at 10,000 rpm for 10 min to pellet the preexisting detergent-insoluble material. The detergent soluble fraction was then precleared of endogenously reconstituted components following an initial warming at 37°C for 10 min, and then incubated with or without MgATP, or Mg²⁺ (0.2 mM, or as otherwise indicated) or ATP (1 mM, or as otherwise indicated) alone at 37°C for 10 min, and centrifuged at 10,000 rpm for 10 min to sediment the precipitated material. Immunoprecipitation of the detergent-soluble fraction was then performed with Sepharose-conjugated anti-ζ mAb (H146-968) (8) or, in series, with agarose-linked anti-phosphotyrosine Ab (Ab-1; Oncogene Science, Uniondale, NY) and anti-ζ mAb (H146-968). Boiled protein samples at 1–5 × 10⁷ cell equivalents/lane were separated under nonreducing conditions by one-dimensional SDS-PAGE (10%). Except where otherwise indicated, equivalent cell numbers were loaded per lane in each experiment. Electrophoretic transfer of protein onto 0.2-mm nitrocellulose filters was carried out in 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 20% methanol at room temperature and constant current (150–200 mA) for 2 h. The filters were then quenched in blotting buffer composed of 125 mM NaCl and 25 mM Tris, pH 7.6 (TS), and 5% skim milk, or with 5% crystallized BSA for phosphotyrosine detection. Following electrotransfer, the nitrocellulose filters were immunoblotted with specific Abs to ζ or phosphotyrosine (Ab-2; Oncogene Science) and washed in TS-0.05% Tween-20. Actin was detected with an anti-actin mAb (kindly provided by Dr. B. Jockusch, Braunschweig, Germany). Lck was detected with polyclonal Abs raised in rabbits against the C-terminal sequence of Lck and were the kind gift of Dr. Terry Potter (National Jewish Medical and Research Center, Denver, CO). The washed filters were incubated with [¹²⁵I]protein A (4 × 10⁵ cpm/ml) in quenching buffer for 1 h and washed as above. The blots were then dried and exposed to Kodak XAR-2 film at −70°C. Densitometry was done on a MacIntosh image scanner and analyzed with the Image 1.49 program (National Institutes of Health, Bethesda, MD) for 1-D scanning. Unless otherwise stated, all reagents were purchased from Sigma Chemical Co., St. Louis, MO.

Kinetics of ζ-cytoskeleton association

Thymocyte and lymph node T cell lysates were incubated with 0.2 mM Mg²⁺ and varying ATP concentrations for 0, 0.5, 1, 2, 4, and 8 min at 37°C, centrifuged at 10,000 rpm for 30 s, and the reaction was stopped with the addition of sample buffer to the reconstituted pellets. Electrophoresis, immunoblotting, and densitometry were performed as previously described (1). Amount of bound ζ-chain was quantitated as ζ associated per second relative to the baseline (assigned a value of 1) at the 0 time control. As the results for thymocytes were essentially identical to those obtained for lymph node T cells, these sets of data were pooled.

Effect of Mg²⁺

T cell lysates were incubated with increasing concentrations of Mg²⁺ with or without 1 mM ATP for 8 min at 37°C. Electrophoresis and immunoblotting were performed as previously described (1).

Challenge with alkaline phosphatase

Cell lysates were incubated in the presence or absence of 1 U/100 μl of bacterial alkaline phosphatase for 30 min and incubated with MgATP, with or without phosphatase inhibitors (0.2 mM VO₃, 10 mM NaF), as described. The reconstituted MgATP pellets were then disrupted and incubated in the presence or absence of 1 U/10 μl alkaline phosphatase and centrifuged at 10,000 rpm for 10 min to sediment the precipitated material. Immunoprecipitation of the detergent soluble fractions and immunoblotting was performed as previously described (1).

Immunoprecipitation of Src-family kinases

T cell lysates were incubated in the presence or absence of polyclonal Abs to Fyn (courtesy of Dr. Terry Potter), Lck (courtesy of Dr. Terry Potter), or pp60^c-Src (Upstate Biotechnology, Lake Placid, NY) for 30 min at 4°C. The tyrosine kinases were depleted from solution by immunoprecipitation with protein A-Sepharose (Pharmacia, Uppsala, Sweden) for 1 h at 4°C. Depleted lysates were incubated with or without MgATP, and electrophoresis and immunoblotting were performed as described.

Competition with exogenous SH2 peptide

Cell lysates were incubated for 2 h in the absence or presence of glutathione-S-transferase (GST) or GST fusion proteins containing the SH2 domain of the Src family kinases, Fyn (residues 144–255) (18), Lck (residues 117–239), or the SHIP phosphatase (residues 0–114, kindly provided by Kazuhiro Nakamura (National Jewish Medical and Research Center, Denver, CO). GST fusion proteins were prepared as described (18) and isolated using glutathione-Sepharose beads (Pharmacia). Cell lysates were also incubated for 2 h in the presence or absence of increasing concentrations of exogenous Fyn-SH2 peptide containing the Fyn residues 144–255 (18) (our unpublished data), which span the 300 base pairs encoding the SH2 domain of the kinase. Preparation of other regions of the Fyn PTK has been described previously (18). Following incubation in the absence or presence of the Fyn-SH2 peptide, lysates were incubated with MgATP, as described above. Control lysates were incubated with a concentration of BSA equivalent to the highest concentration of SH2 peptide used.

Constructs

DNA fragments containing the portion encoding the SH2 domain of Fyn (residues 144–255) were amplified with the PCR using the following primers: Fyn 144–255, 5′-CAGTCAGAATTCGATGGAGTCAACTGGAGCCA-3′ and 5′-CAGTCAGAATTCTCCAGGTTTGTGGGGTAC-3′. The PCR products were then ligated into pGEX-3X (Pharmacia) and transfected into Escherichia coli DH5α (Life Technologies, Gaithersburg, MD). The Fyn-SH2 peptide was subsequently eluted by cleavage from the beads with 30 μg of factor Xa (Boehringer Mannheim, Indianapolis, IN) and dialyzed into PBS. For the generation of GST fusion proteins containing the SH2 domains of Lck (residues 117–239), Fyn (residues 144–255), or SHIP (residues 0–114), PCR was used to amplify cDNAs encoding the SH2 domains. The oligo pair used for amplifying the mouse Lck SH2 domain was: 5′ oligo, 5′-GATATCGCGAAAGCAAACAGCCTG-3′, and 3′ oligo, 5′-GAATTCCCATTCGTCCTCCCACCATGG-3′. The amino acids encoded in this fragment span 117–239. The protein fragment contains 10 amino acids on either side of the SH2 domain to stabilize folding of the domain. The PCR product was obtained by amplifying from the mLck cDNA (kindly provided by Roger M. Perlmutter, University of Washington, Seattle, WA), which was then subcloned into pCR2.1 (Invitrogen, Carlsbad, CA). The 5′ and 3′ amplifying oligos contain EcoRV and EcoRI restriction sites. The sequence was confirmed by dideoxynucleotide-sequence analysis using Sequenase (United States Biochemical, Cleveland, OH). The fragment containing the Lck SH2 domain was cleaved with EcoRV (blunt) and EcoRI and cloned into pGEX-3X (Pharmacia) cut with SmaI (blunt) and EcoRI. The following primers were used for the SHIP SH2 0–114: 5′ primer, 5′-GGAATTCATGCCTGCCATGGTCCCT-3′; 3′ primer, 5′-TTTTCCTTTTGCGGCCGCTCATCAATAGCATCCTC-3′. After digesting with the restriction enzymes, BamHI and EcoRI (for GST-Lck SH2) and EcoRI and NotI (for GST-SHIP SH2), the resulting fragments were ligated into pGEX-3X (Pharmacia) and pGEX-5X (Pharmacia), respectively, and transfected into E. coli DH5α (Life Technologies) and purified with glutathione-Sepharose beads (Pharmacia).

Lck and Fyn-deficient mice

Thymocytes and lymph node T cells from wild-type and Lck-knockout (19) or Fyn-knockout (20) mice were isolated and activated by ligation of CD3ε with anti-CD3ε mAb (145-2C11) (21) on intact cells or by addition of MgATP to T cell lysates, as described.

Divalent cations and ATP are required for reconstitution of ζ-cytoskeletal binding in a cell-free system

In earlier work, we found that although the coprecipitation of ζ and actin is enhanced under activating conditions, actin polmerization alone is insufficient to induce ζ cytoskeleton association (1). Reconstitution of ζ-cytoskeleton association required addition of MgATP to the cell lysates (1). In order to address the possibility that Mg²⁺ or nucleotide alone contributed to ζ-cytoskeleton association in vitro, thymic cell lysates were first cleared of preexisting detergent insoluble material and then incubated with Mg²⁺ or EDTA in the presence or absence of ATP (Fig. 1⇓, A and B). Relative to the 5 ± 1% (SE, n = 7) of the TCRζ associated with the in vitro reconstituted pellet under control conditions, upon addition of exogenous MgATP (Fig. 1⇓A), an average of 30 ± 4% (SE, n = 7) of the ζ-chain was decreased from solution and cosedimented with a secondary pellet, representing a sixfold increase over background and similar to that observed in activated lymph node T cells, in vivo (1). Ca²⁺ and Mn²⁺ (data not shown) substituted for Mg²⁺ in inducing the ζ-cytoskeletal interaction. In the absence of Mg²⁺ or in the presence of chelators of divalent cations, neither ζ (Fig. 1⇓A) nor polymerized actin (Fig. 1⇓B) were found in the in vitro reconstituted pellet, further implicating microfilaments in ζ-cytoskeleton association. Only the lysate containing both Mg²⁺ and ATP (Fig. 1⇓, A and B) showed any prominent association of ζ with the pellet, suggesting that the association observed following TCR ligation on intact cells is an ATP-dependent interaction that requires divalent cations.

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FIGURE 1.

ζ-Chain association with the cytoskeleton can be reconstituted in a cell-free system. A, ζ-Cytoskeleton association in a cell-free system in the presence of Mg²⁺, ATP, and EDTA. Thymocytes were lysed and cleared of the preexisting detergent-insoluble pellet. The detergent-soluble supernatants were incubated in the absence or presence of 0.2 mM Mg²⁺ (Mg²⁺; lanes 2 and 5 or lanes 1, 3, 4, and 6, respectively), 5 mM EDTA (EDTA; lanes 1 and 4 or lanes 2, 3, 5, and 6, respectively), or 5 mM ATP (ATP; lanes 1, 2, and 3 or lanes 4, 5, and 6, respectively). The cell lysates were then incubated at 37°C and centrifuged to separate the polymerized pellet from the cleared supernatant. The pellets were separated by PAGE and Western blotted with anti-ζ mAb. ζ associated with the MgATP pellet (lane 4) only under conditions that included both ATP and Mg²⁺ without EDTA. Optimal association occurred with pH in the acidic range (pH 6) (data not shown). Data are representative of at least three experiments. B, EDTA disrupts actin polymerization in a lysed cell system. The detergent-soluble supernatants were incubated in the absence or presence of 2.0 mM Mg²⁺ (Mg²⁺; lanes 1, 2 and 3), 5 mM EDTA (EDTA; lanes 1 and 2 or 3, respectively), or 2.5 mM ATP (ATP; lane 1, or lanes 2 and 3, respectively). Following incubation at 37°C and centrifugation, the pellets were separated by PAGE and Western blotted with anti-ζ and anti-actin mAbs. The disruption of ζ association with the MgATP pellet (lane 3) was correlated with the disruption of actin polymerization under conditions that included EDTA. Data are representative of at least three experiments. C, Reconstituted ζ-cytoskeletal binding is dependent on the regulation of both Mg²⁺ and ATP levels. T cell lysates were incubated with increasing concentrations of Mg²⁺ (0.25, 0.5, 1.2, and 4 mM) in the absence (lanes 3–7) or presence (lanes 8–12) of 1 mM ATP. Lane 1 represents lysate incubated in 1 mM ATP alone, and lane 2 is devoid of exogenous reagents. Data are representative of at least three experiments.

To determine the individual contribution of Mg²⁺ to in vitro induced ζ-cytoskeleton association, increasing concentrations of Mg²⁺ were added to cell lysates in the presence (Fig. 1⇑C, lanes 8–12) or absence (Fig. 1⇑C, lanes 3–7) of 1 mM ATP. Mg²⁺ concentrations of 1–2 mM (Fig. 1⇑C, lanes 5 and 6) were optimal in inducing some ζ-cytoskeleton association, but only about 6% of that stimulated in the presence of ATP (Fig. 1⇑C, lanes 10 and 11), and were comparable to amounts induced by 1 mM ATP alone (Fig. 1⇑C, lane 1). Concentrations above 2 mM Mg²⁺ were inhibitory to ζ-cytoskeleton association (Fig. 1⇑C, lane 7). The level of Mg²⁺ used in most of the studies here, 0.2–0.25 mM, is not sufficient to induce ζ to translocate to the pellet (Fig. 1⇑C, lane 3). ζ-Cytoskeleton association was over an order of magnitude greater with the inclusion of both Mg²⁺ and 1 mM ATP, at all Mg²⁺ concentrations tested. Thus, although Mg²⁺ or ATP alone are capable of insolubilizing some ζ-chain (presumably as a result of an interaction with endogenous stores of ATP or Mg²⁺, respectively), together, ATP and Mg²⁺ have a synergistic effect. The observed ζ-cytoskeleton association is dependent on regulation of both the Mg²⁺ and ATP levels, as shown below.

To determine the nucleotide specificity of ζ-cytoskeleton association, T cell lysates were incubated in the presence or absence of Mg²⁺ with ATP, GTP, ADP, or the nonhydrolyzable ATP analog, AMPPNP (Fig. 2⇓A). In the absence of EDTA, ATP alone induced ζ-cytoskeleton association, presumably in concert with endogenous divalent cations. ζ-Chain association occurred predominantly with the addition of MgATP, suggesting that ATP is specifically used as a substrate for the ζ-cytoskeletal interaction, possibly as a phosphoryl donor in a kinase reaction. Indeed, the level of tyrosine phosphorylated ζ increased in the cell lysate and was a prominent component of the pellet after incubation with MgATP (Fig. 2⇓B). Under nonphosphorylating conditions there was minimal ζ binding to the pellet, although actin polymerization was induced (Fig. 2⇓B). Thus, we postulate that in this cell-free system, MgATP supports in vitro phosphorylation of tyrosine residues on the ζ-chain, which then binds (directly or indirectly) to polymerized actin.

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FIGURE 2.

Reconstituted ζ-cytoskeletal binding requires ATP and is associated with tyrosine phosphorylation of the ζ-chain. A, ATP is a specific substrate for reconstitution of ζ-cytoskeleton association in vitro. Cell lysates were incubated in the absence (lane 1) or presence of 0.5 mM Mg²⁺ (Mg, lane 2) or in 5 mM ATP (lanes 3 and 4), AMP-PNP (lanes 5 and 6), ADP (lanes 7 and 8) or GTP (lanes 9 and 10), with or without Mg²⁺, respectively. The cell lysates were then incubated at 37°C and centrifuged to isolate the pellets, which were separated by PAGE and Western blotted with anti-ζ mAb. ζ associated predominantly with the MgATP pellet (lane 4), relative to untreated (lane 1), Mg²⁺-treated (lane 2), and other nucleotide-treated lysates (lanes 5–10). Data are representative of at least two experiments. B, Tyrosine-phosphorylated ζ-chain is a component of the insoluble pellet reconstituted with MgATP. Thymocytes were lysed, incubated with 2 mM Mg²⁺ (lanes 1–4) and 2.5 mM ATP (lanes 2 and 4), and centrifuged to sediment the reconstituted pellet (lanes 1–4). The pellets were separated by PAGE and Western blotted with anti-ζ or anti-phosphotyrosine mAb on duplicate blots. Data are representative of at least three experiments.

To examine the amount of ATP required for ζ-cytoskeleton association and the kinetics of this association, cell lysates were incubated with increasing concentrations of MgATP. Fig. 3⇓A shows the data of a time course experiment using two ATP concentrations, differing by an order of magnitude, as representative of the aggregate data. Little or no ζ binding above background was observed below 0.1 mM and appeared optimal at 1 mM ATP and above (Fig. 3⇓, B and D). At concentrations of 1 mM ATP and above, optimal association of ζ and the actin cytoskeleton in this lysed cell system was very rapid (30 s at 37°C; Fig. 3⇓, A and B) and was dependent upon temperature and lysate concentration (data not shown). The kinetics of association of tyrosine phosphorylated ζ with the pellet were similar to that observed for ζ-cytoskeleton association (data not shown). The kinetics of actin polymerization were also similar, although with a lower threshold of MgATP concentration (Fig. 3⇓, A and C).

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FIGURE 3.

Reaction kinetics and time course of ζ-actin association in a lysed cell system. A, Representative ATP concentration dependence of relative ζ association and actin polymerization in the insoluble pellet at 37°C. Thymocytes were lysed and incubated with 0.2 mM Mg²⁺ and 0.25 or 2.5 mM ATP for 0 (control), 0.5, 1, 2, 4, and 8 min at 37°C. The pellets were separated by PAGE and Western blotted with anti-ζ or actin mAb. The rate of association with the reconstituted pellet was dependent on the ATP concentration. Data are representative of at least four experiments. B and C, ATP concentration dependence of relative ζ-cytoskeleton association and actin polymerization over time. Thymocytes or lymph node T cells were lysed and incubated with 0.2 mM Mg²⁺ and 0.25 (□), 0.5 (◊), 1.0 (○), or 2.5 (▵) mM ATP for 0, 0.5, 1, 2, 4, or 8 min at 37°C. The reaction was stopped by boiling the precipitate in SDS sample buffer after rapid centrifugation. The pellets were separated by PAGE and Western blotted with anti-ζ mAb (B) or with an anti-actin mAb (C). The amount of ζ-cytoskeleton association (B) or actin polymerization (C) relative to baseline at the 0 time control (arbitrarily assigned a value of 1) was determined by densitometry for values below saturation. Since the data for thymocytes and lymph node T cells were similar for each ATP concentration, the data are representative of at least four pooled experiments. D and E, Rate of ζ-cytoskeleton association and actin polymerization as a function of ATP concentration in a cell-free system. Rate measurements, calculated as the relative amount of ζ bound per second (ζ bound/sec, D) and actin polymerized per second (actin polym/sec, E), were determined from the linear portion of the time course plots (presented in B and C, respectively), for each ATP concentration. Each data point was generated from at least four experiments. F and G. Double reciprocal plots of data presented in D and E. The lines were obtained by least squares method (r = 0.98). The x-intercepts represent the apparent K_m for ATP (0.5 mM) of ζ association (F) and the apparent K_m for ATP (0.9 mM) of actin polymerization (G), in this reconstituted system.

The differential dependence on MgATP concentration for ζ-cytoskeleton association and actin polymerization was borne out by rate measurements of relative amount of ζ bound to the cytoskeletal pellet and relative actin polymerized with increasing ATP concentration. These rate measurements showed that both ζ association and actin polymerization were saturable and followed Michaelis-Menton kinetics (Fig. 3⇑, D and E, respectively). Double reciprocal plots of these data (Fig. 3⇑, F and G) permitted the calculation of the apparent Michaelis-Menton constant for ATP of the ζ-cytoskeletal binding (0.9 mM) and actin polymerization (0.5 mM) in this lysed cell system. The line of best fit was determined by linear regression (r = 0.98, for both sets of data) and the K_m was calculated from the x-intercept. Thus, ζ-cytoskeleton association had an apparent K_m for ATP almost twice that measured for actin polymerization, suggesting that actin polymerization occurs at a lower ATP threshold and perhaps prior to ζ-chain association. Interestingly, the reaction kinetics at ATP concentrations greater than or equal to 0.5 mM (Fig. 3⇑, B and C) showed a biphasic peak, for reasons not yet understood. In addition, the degree of ζ association at an ATP concentration greater than 1.0 mM decreased after an initial peak (Fig. 3⇑B; 2.5 mM ATP), as was seen previously in intact cells (1), and is correlated with a decrease in actin from the pelleted material over time (Fig. 3⇑C). Likewise, at ATP concentrations below 1.0 mM (Fig. 3⇑A), or at temperatures equal to or below 25°C (data not shown), the kinetics of association were two to five times that observed in intact cells, peaking between 2 and 4 min. Thus, in the presence of divalent cations and physiologic concentrations of ATP, ζ-chain associates with the cytoskeleton in a cell-free system with rate and extent similar to that seen following TCR ligation on intact cells.

ζ-Cytoskeleton association is dependent upon tyrosine phosphorylation of the ζ-chain

Because tyrosine phosphorylation of the ζ-chain appeared to correlate with microfilament association, we asked whether agents that dephosphorylate the ζ-chain, such as alkaline phosphatase, would disrupt ζ-cytoskeleton association. Fig. 4⇓A shows that alkaline phosphatase added directly to the in vitro reconstituted MgATP pellet (Fig. 4⇓A, lane 2) or to the cell lysate prior to activation (Fig. 4⇓A, lane 3) disrupted ζ-cytoskeleton association relative to the control MgATP pellet (Fig. 4⇓A, lane 1). This disruption by the phosphatase was dose dependent, was correlated with the increasing release of dephosphorylated ζ-chain from the pellet, and was inhibited by the inclusion of phosphatase inhibitors in the buffer (Fig. 4⇓B). These data suggest that ζ-chain must be phosphorylated in order to bind, directly or indirectly, to the cytoskeleton.

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FIGURE 4.

Dephosphorylation of the ζ-chain inhibits ζ-cytoskeleton association in solution and releases ζ-chain from the bound state. A, Thymocytes were lysed and incubated with 0.2 mM Mg²⁺ and 5 mM ATP (MgATP, lanes 1–3), in the absence (lane 1) or presence (lanes 2 and 3) of bacterial alkaline phosphatase (AP) added at 1 U/100 μl to the resuspended reconstituted pellet (lane 2) or at 10 U/100 μl to the cell lysate prior to incubation with MgATP (lane 3). Data are representative of at least two experiments. B, Thymocytes were lysed and incubated in the presence of 0.2 mM Mg²⁺ and 2.5 mM ATP (MgATP) with increasing concentrations of bacterial alkaline phosphatase (AP) at 8 U (lane 1), 16 U (lane 2), 32 U (lane 3). ζ-Association with the cytoskeleton decreases in the presence of increasing concentrations of AP (MgATP pellet), while ζ-chain coordinately increases in the supernatant (released ζ). The addition of phosphatase inhibitors (0.2 mM VO₃, 10 mM NaF), block the release of ζ-chain from the reconstituted pellet (MgATP pellet + P-ase inhibitors). Data are representative of at least two experiments.

Lck is the tyrosine kinase responsible for the ζ-chain phosphorylation that leads to ζ-cytoskeleton association

Dephosphorylation by alkaline phosphatase is not specific to phosphorylated tyrosines. We therefore sought to determine the involvement of the Src family members in ζ-cytoskeleton association. Lck, Fyn, and c-Src were probed for association with the cytoskeleton in intact activated thymocytes, and with the detergent insoluble pellet following reconstitution in cell lysates. Lck (Fig. 5⇓) and Fyn (data not shown) associate with the insoluble pellet from intact cells activated by TCR ligation (Fig. 5⇓A, lane 2) and with the reconstituted pellet in the presence of MgATP (Fig. 5⇓B, lane 2). In contrast, c-Src does not appear to cosediment with either the insoluble or reconstituted pellets (data not shown). Under both conditions, Lck is tyrosine phosphorylated (Fig. 5⇓, lower panels), suggesting that activated Lck and phosphorylated ζ-chain colocalize to the actin cytoskeleton in response to TCR ligation.

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FIGURE 5.

pp56^Lck compartmentalizes to the cytoskeleton upon TCR ligation of intact T cells and in a cell-free system. A, Thymocytes were lysed and the detergent-insoluble pellet isolated, following cross-linking of CD3ε with anti-CD3ε (145-2C11) mAb and goat anti-mouse (GAM) polyclonal Ab (lane 2), or treated with GAM alone (lane 1). B, Alternatively, cell lysates were incubated in the presence of 0.5 mM Mg²⁺ (lane 1) or 0.5 mM Mg²⁺ and 5 mM ATP (lane 2). The cell lysates were then fractionated into reconstituted pellet fractions (lanes 1 and 2). Subsequently, all pellets were separated by PAGE and Western blotted with Abs specific for Lck. Inductive association of Lck with the cytoskeleton occurs in the presence of MgATP (B, lane 2) or cell activation (A, lane 2), with a concomitant depletion from the lysate supernatant (data not shown). Data are representative of at least five experiments.

Lck phosphorylates ζ-chain upon TCR ligation (22) and is, along with Fyn, a candidate kinase involved in the tyrosine phosphorylation of ζ-chain leading to ζ-cytoskeleton association (23). To differentiate the role of various protein tyrosine kinases in ζ-cytoskeleton association, we examined T cell lysates in which levels of specific Src family kinases were decreased by immunoprecipitation (Fig. 6⇓). ζ-Cytoskeleton association was abrogated in the reconstituted pellets in which levels of Lck (Fig. 6⇓A, lane 4, and 6B) were reduced. In contrast, in T cell lysates in which levels of either Fyn (Fig. 6⇓A, lane 6) or pp60^c-Src (Fig. 6⇓A, lane 8) were reduced, ζ-chain still associated with the reconstituted pellet upon MgATP addition. This inhibition of ζ-cytoskeleton association is not the result of coimmunoprecipitation of ζ-chain with Lck, nor are the levels of soluble ζ-chain in the cell lysate affected (data not shown). Titration of the anti-Lck Ab (Fig. 6⇓B) showed that inhibition of ζ-cytoskeleton association (Fig. 6⇓B, bottom panel) was directly correlated with both the loss of Lck from the cytoskeletal pellet (Fig. 6⇓B, middle panel) and with the amount of Lck immunoprecipitated from solution (Fig. 6⇓B, top panel), with a corresponding decrease in tyrosine phosphorylation of the ζ-chain in both pellet and supernatant fractions (data not shown). Thus, in this cell-free system, Lck plays a crucial role in inducing both ζ-chain tyrosine phosphorylation and ζ-cytoskeleton association in response to addition of MgATP. In contrast, titration of Fyn and Src in the cell lysates, by immunoprecipitation with increasing Ab concentrations, resulted in no effect on, or potentiated, ζ-cytoskeleton association, respectively (data not shown), suggesting that Fyn is not involved in ζ-cytoskeleton association and that c-Src may act on upstream regulators to inhibit the association.

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FIGURE 6.

Immunoprecipitation of pp56^Lck from the cell lysate inhibits ζ association with the cytoskeleton. A, T cell lysates from resting cells were incubated in the presence of 50 μg normal rabbit serum (lanes 1 and 2) or Abs to Lck (lanes 3 and 4), Fyn (lanes 5 and 6), or Src (lanes 7 and 8). The tyrosine kinases were immunoprecipitated from solution with protein A-Sepharose, and incubated in MgATP (even lanes) or Mg²⁺ alone (odd lanes), as described. Data are representative of at least three experiments. B, Inhibition of ζ association with the cytoskeleton is inversely correlated with the amount of pp56^Lck in the cell lysate. T cell lysates were incubated in the absence (lane 1) or presence of 80 μg normal rabbit serum (lane 2) or polyclonal Abs to Lck (lanes 3–6) at increasing Ab concentrations of 10 μg (lane 3), 20 μg (lane 4), 40 μg (lane 5), or 80 μg (lane 6). Lck was subsequently immunoprecipitated from solution with protein A-Sepharose. Lysates were then incubated with MgATP (lanes 2–6) or Mg²⁺ alone (lane 1) and processed as described. The protein A immunoprecipitates (top panel) and reconstituted cytoskeletal pellets (middle and lower panels) were resolved on Western blots with anti-Lck (top and middle panels) or anti-ζ (lower panel) Abs. Complete depletion of the Src family kinases by immunoprecipitation was not feasible, presumably due to protection of a subset of protein in micelles (data not shown). Data are representative of at least three experiments.

To confirm these results in vivo, we analyzed ζ-cytoskeleton association in intact thymocytes and lymph node T cells from mice lacking Lck (Lck knockouts) (19) (Fig. 7⇓) or Fyn (Fyn knockouts) (20) (Fig. 8⇓). Thymocytes and lymph node T cells from Lck-negative (Lck⁻) mice are reduced about 10-fold in number compared to wild-type (Lck⁺) mice, express CD3 (although at reduced levels), and have measurable proliferative responses (19). Crosslinking of CD3ε on T cells from Lck⁻ mice did not induce ζ-chain association with the insoluble pellet (Fig. 7⇓A). In contrast, crosslinking of CD3ε on T cells from both Fyn-negative (Fyn⁻) and wild-type (Fyn⁺) mice induced association of ζ-chain with the insoluble pellet (Fig. 8⇓A). Analysis of immunoprecipitated ζ-chain by antiphosphotyrosine immunoblotting confirmed that the ζ-chain from Lck⁺, but not Lck⁻, T cells was tyrosine phosphorylated following TCR ligation (Fig. 7⇓A). These data suggest that tyrosine phosphorylation is required for binding of ζ to the cytoskeleton, and implicate Lck, rather than Fyn, as the kinase involved in this ζ-chain tyrosine phosphorylation. Interestingly, the addition of MgATP to T cell lysates from either Lck⁻ (Fig. 7⇓B) or Fyn⁻ (Fig. 8⇓B) mice induced ζ-chain phosphorylation and ζ-cytoskeleton association, suggesting that other Src family kinases may substitute for Lck in this cell-free assay and that all other requisite components for ζ-cytoskeleton association are present in the Lck⁻ mice. These data suggest that tyrosine phosphorylation is required for ζ-cytoskeletal binding, and implicate Lck as the kinase involved in ζ-cytoskeleton association in vivo.

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FIGURE 7.

Induction of ζ-cytoskeleton association is abrogated in intact T cells from pp56^Lck knockout mice, but is rescued in the reconstituted cell-free system. A, Isolated lymph node T cells from wild-type (Lck⁺) or Lck-negative (Lck⁻) mice were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of an anti-CD3ε (α-CD3ε) mAb (145-2C11) and cross-linked with a secondary goat anti-mouse (GAM) Ab or treated with GAM (lanes 1 and 3) alone. The cells were then lysed in nonionic detergent and centrifuged to separate the detergent-insoluble pellet fractions. The pellets were separated by PAGE and Western blotted with anti-ζ or anti-phosphotyrosine mAbs (1 ). In comparison with Lck⁺ T cells (compare lane 2 with lane 1), activated T cells from Lck⁻ mice (lane 4) showed neither increase in ζ-cytoskeleton association nor ζ tyrosine phosphorylation relative to nonactivated controls (lane 3). Identical results were obtained in analyses of thymocytes and splenic T cells from Lck⁻ mice (data not shown). B, Isolated thymocytes from Lck⁺ or Lck⁻ mice were lysed and cleared of the preexisting detergent insoluble pellet. The detergent soluble supernatants were incubated in the presence of 0.2 mM Mg²⁺ (Mg²; lanes 1 and 2) or 0.2 mM Mg²⁺ and 2.5 mM ATP (ATP; lanes 3 and 4, respectively). The cell lysates were then incubated at 37°C and centrifuged to separate pellet from the supernatant. The pellets were separated by PAGE and Western blotted with anti-ζ and anti-actin mAbs. ζ-Associated with the MgATP pellets from both Lck⁺ and Lck⁻ mice. Identical results were obtained in analyses of lymph node and splenic T cells from Lck⁻ mice (data not shown).

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FIGURE 8.

ζ-Cytoskeleton association is unaffected in intact T cells or T cell lysates from pp59^Fyn knockout mice. A, Isolated lymph node T cells (LN; upper panel) and thymocytes (Thy; lower panel) from wild-type (Fyn⁺; lanes 1–4) or Fyn-negative (Fyn⁻; lanes 5–8) mice were incubated in the absence (lanes 1 and 5) or presence (lanes 2 and 6) of an anti-CD3ε (α-CD3ε) mAb (145-2C11) and cross-linked with a secondary goat anti-mouse (GAM) Ab (lanes 4 and 8) or treated with GAM (lanes 3 and 7) alone. The cells were then lysed in nonionic detergent and centrifuged to separate the detergent-insoluble pellet fractions. The pellets were separated by PAGE and Western blotted with anti-ζ mAb (1 ). Both Fyn⁺- and Fyn⁻-activated lymph node T cells and thymocytes (lanes 4 and 8, respectively) showed increases in ζ-cytoskeleton association relative to nonactivated controls. B, Isolated lymph node T cells (LN; upper panel) and thymocytes (Thy; lower panel) from Fyn⁺ or Fyn⁻ mice were lysed and cleared of the preexisting detergent-insoluble pellet. The detergent-soluble supernatants were incubated in the absence (lanes 1 and 4) or presence of 0.2 mM Mg²⁺ (Mg²; lanes 2 and 5) or 0.2 mM Mg²⁺ and 2.5 mM ATP (ATP; lanes 3 and 6). The cell lysates were then incubated at 37°C and centrifuged to separate the pellet from the supernatant. The pellets were separated by PAGE and Western blotted with anti-ζ mAb. ζ associated with the MgATP pellets from both Fyn⁺ and Fyn⁻ lymph node T cells (LN; upper panel) and thymocytes (Thy; lower panel).

The SH2 domain of Lck PTK preferentially inhibits ζ-cytoskeleton association

Since SH2 domains are the specific downstream targets of tyrosine-phosphorylated proteins, including the ζ-chain, we analyzed ζ-cytoskeleton association using competitive inhibition by synthetic SH2 domains. T cell lysates were incubated in the absence or presence of 10 μM of GST or GST fusion proteins (Fig. 9⇓A) containing the SH2 domain of the Src-family kinases Fyn or Lck, or the SHIP phosphatase, as a non-Src-family SH2 control domain. These lysates were subsequently incubated with MgATP and analyzed for ζ-cytoskeleton association. In comparison with the other GST-fusion proteins, GST-Fyn SH2 or GST-SHIP SH2 (Fig. 9⇓A, lanes 3 and 6), or with MgATP (Fig. 9⇓A, lane 2) or GST alone (Fig. 9⇓A, lane 4), the GST-Lck SH2 fusion protein preferentially inhibited ζ-cytoskeleton association (Fig. 9⇓A, lane 5). Both the GST-Fyn SH2 and the GST-Lck SH2 fusion proteins also inhibited Lck association with the reconstituted pellet (Fig. 9⇓A, lanes 3 and 5), yet only GST-Lck SH2 domain resulted in the concomitant inhibition of ζ-cytoskeleton association, suggesting that ζ-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in ζ-chain activation motifs and the SH2 domain of the Lck protein tyrosine kinase.

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FIGURE 9.

The SH2 domain of Src family tyrosine kinases inhibits ζ-cytoskeleton association. A, GST fusion protein containing the Lck SH2 domain preferentially inhibits ζ-cytoskeleton association. Thymocyte cell lysates were incubated for 2 h in the absence (lanes 1 and 2) or presence of 10 μM GST (lane 4) or GST fusion proteins containing the SH2 domain of Fyn (lane 3), Lck (lane 5), or the SHIP phosphatase (lane 6). Subsequently, these lysates were incubated in the presence of 0.2 mM Mg²⁺ (lane 1), or 0.2 mM Mg²⁺ and 1 mM ATP (lanes 2–6), and processed as described. These data are representative of two experiments. The mean amount of ζ cosedimenting with the pellet relative to baseline (assigned a relative density value of 1) was 8.9 for the MgATP pellet, 6.7 for GST alone, 7.6 for GST-Fyn SH2, 3.2 for GST-Lck SH2, and 8.9 for GST-SHIP SH2. The Lck SH2 fusion protein, therefore, inhibited ζ-cytoskeleton association by 50 to 70% relative to the other fusion proteins. B, Recombinant Fyn-SH2 peptide inhibits ζ-cytoskeleton association. Thymocytes were lysed and incubated in the absence (lane 1) or presence of 0.5 mM Mg²⁺ (Mg, lane 2), 0.5 mM Mg²⁺ and 5 mM ATP (MgATP, lane 3), MgATP and 80 μM BSA (MgATP + BSA, lane 4), or MgATP with increasing concentrations of recombinant Fyn-SH2 peptide ([SH2]) at 10 μM (lane 5), 20 μM (lane 6), 40 μM (lane 7), and 80 μM (lane 8). ζ-Association with the cytoskeleton decreases in the presence of increasing concentrations of Fyn-SH2 peptide. Data are representative of at least three experiments.

To determine if ζ-cytoskeleton association may be inhibited by the Fyn SH2 domain at higher inhibitory thresholds, due to lower binding specificity, T cell lysates were incubated in the absence or presence of increasing concentrations of the SH2 domain of Fyn (residues 144–255). As shown in Fig. 9⇑B, ζ-cytoskeletal binding diminished with increasing concentrations of the SH2 peptide, relative to MgATP alone (Fig. 9⇑B, lane 3) or to BSA (Fig. 9⇑B, lane 4), but at a 4- to 8-fold higher concentration requirement than with the Lck SH2 peptide. Since the phosphotyrosine-binding sites of the Src family members are conserved (13), with the Lck and Fyn SH2 domains sharing 60% sequence homology, the dose-dependent inhibition of ζ-cytoskeleton association by Fyn-SH2 domains suggests that Src SH2-containing polypeptides are involved, either directly or indirectly, in the interaction of ζ with the microfilament cytoskeleton.