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The Journal of Immunology, 1998, 161: 5491-5499.
Copyright © 1998 by The American Association of Immunologists

pp56Lck Mediates TCR {zeta}-Chain Binding to the Microfilament Cytoskeleton1

Moshe M. Rozdzial*, Chris M. Pleiman2,*, John C. Cambier*,{dagger} and Terri H. Finkel3{dagger},{ddagger}

* Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; {dagger} Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262; and {ddagger} Departments of Pediatrics and Biochemistry & Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TCR {zeta}-chain ({zeta}) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in {zeta}-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of {zeta} to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated {zeta} is a predominant form of the {zeta}-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents {zeta}-chain association in solution and releases {zeta}-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. {zeta}-Chain phosphorylation and {zeta}-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in {zeta}-chain tyrosine phosphorylation and {zeta}-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted {zeta}-cytoskeleton association, suggesting that {zeta}-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in {zeta}-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of mature T lymphocytes is regulated by the multichain TCR. The TCR, upon engagement, initiates a cascade of biochemical events that lead to receptor association with cytoskeleton (1) and culminate in the rearrangement and reorientation of the cytoskeleton (2), production of lymphokines, and cellular proliferation and differentiation. The TCR consists of the Ag-binding subunits ({alpha}ß), the CD3 complex ({gamma}-, {delta}-, and {varepsilon}-chains), and the {zeta}-chains (3). Both the CD3 components and {zeta}-chains are capable of independently mediating TCR signaling (4), and each contains, respectively, one and three copies of the immunoreceptor tyrosine-based activation motif (ITAM),4 composed of the amino acid sequences YXX(L/I)X(7, 8)YXX(L/I) (5). As the TCR has neither intrinsic protein tyrosine kinase (PTK) nor phosphatase function, the ITAM sequences appear to be both necessary and sufficient for the induction of protein tyrosine phosphorylation in T cells (6). While there is a low level of constitutive tyrosine phosphorylation of {zeta}, even in resting T cells (7, 8), with activation, tyrosine phosphorylation of {zeta} increases (9), leading to association of specific PTKs with the ITAMs via Src homology 2 (SH2) domains (10, 11).

SH2 domains play a significant role in signal transduction in many cell types by mediating the formation of specific heteromeric protein complexes with phosphotyrosine-containing peptides (12). The binding specificity of this protein-protein interaction is dependent on both the primary sequence flanking the site of tyrosine phosphorylation and the structure of the particular SH2 domain (13). Individual SH2-containing polypeptides select unique sequences, except for the Src subfamily (Src, Fyn, Lck, and Fgr), which preferentially recognizes the sequence, P-Tyr-Glu-Glu-Ile (13). Thus, SH2-containing polypeptides serve as adaptors to link specific effector proteins to phosphotyrosine-containing target motifs.

In T cells, biochemical and genetic studies have implicated the Src (pp56Lck and p59Fyn) and the Syk/ZAP-70 families of PTKs in cellular activation, demonstrating a functional interaction of these kinases with the TCR complex and/or the coreceptors CD4 and CD8. Both pp56Lck (Lck) and p59Fyn (Fyn) have been shown to interact with ITAMs in the {zeta}- and CD3{varepsilon}-chains, resulting in phosphorylation of the ITAM tyrosines, and leading to recruitment of other molecules involved in T cell signal transduction, such as ZAP-70 (10, 11).

Although not well studied in T cells, the cytoskeleton also plays a direct role in the regulation and compartmentalization of the activation process (reviewed in Ref. 1). Previously, we have shown that as a consequence of TCR ligation, the {zeta}-chain rapidly binds to the membrane skeleton independent of receptor internalization (1). Pretreatment with drugs that disrupt the actin cytoskeleton abrogated the association of {zeta} with cytoskeleton (1, 14) and inhibited sustained Ca2+ mobilization, IFN-{gamma} (15) and IL-2 (M. M. Rozdzial, unpublished observations) production. In addition, in T cell hybridomas transfected with mutated or deleted {zeta}-chain chimeras, lack of {zeta}-cytoskeleton association correlated with inhibition of late activation events, such as IL-2 production (1). Recent studies have also documented the activation-dependent association of tyrosine kinases with the membrane skeleton in both B (16) and T (17) lymphocytes. These data provide direct evidence of an activation-dependent interaction between Ag receptor and the cytoskeletal matrix that may support the interaction of signaling polypeptides and their substrates.

Determination of the role of microfilaments in TCR-signaling cascades and molecular dissection of the {zeta}-cytoskeletal interaction is, however, hampered by the formation of a complex insoluble pellet upon activation of intact cells. Therefore, we developed an assay for analysis of {zeta}-cytoskeleton association under cell-free conditions, so as to be able to modify the system before pellet formation (1). Here, we demonstrate that addition of divalent cations and ATP to a T cell lysate leads to the phosphorylation of {zeta} and association of {zeta} with microfilaments. Furthermore, we show that, in the lysed cell system, {zeta}-cytoskeleton association is preferentially inhibited by a recombinant Lck-SH2 peptide or by a reduction in levels of Lck. Finally, as predicted by these data from the cell-free system, we show that {zeta}-cytoskeleton association is abrogated in activated T cells from Lck-deficient (knockout) mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 {alpha}-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-{zeta} mAb (H146-968) (8) or, in series, with agarose-linked anti-phosphotyrosine Ab (Ab-1; Oncogene Science, Uniondale, NY) and anti-{zeta} mAb (H146-968). Boiled protein samples at 1–5 x 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 {zeta} 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 x 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 {zeta}-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 {zeta}-chain was quantitated as {zeta} 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 pp60c-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 DH5{alpha} (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 DH5{alpha} (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 CD3{varepsilon} with anti-CD3{varepsilon} mAb (145-2C11) (21) on intact cells or by addition of MgATP to T cell lysates, as described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Divalent cations and ATP are required for reconstitution of {zeta}-cytoskeletal binding in a cell-free system

In earlier work, we found that although the coprecipitation of {zeta} and actin is enhanced under activating conditions, actin polmerization alone is insufficient to induce {zeta} cytoskeleton association (1). Reconstitution of {zeta}-cytoskeleton association required addition of MgATP to the cell lysates (1). In order to address the possibility that Mg2+ or nucleotide alone contributed to {zeta}-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. 1Go, A and B). Relative to the 5 ± 1% (SE, n = 7) of the TCR{zeta} associated with the in vitro reconstituted pellet under control conditions, upon addition of exogenous MgATP (Fig. 1GoA), an average of 30 ± 4% (SE, n = 7) of the {zeta}-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 {zeta}-cytoskeletal interaction. In the absence of Mg2+ or in the presence of chelators of divalent cations, neither {zeta} (Fig. 1GoA) nor polymerized actin (Fig. 1GoB) were found in the in vitro reconstituted pellet, further implicating microfilaments in {zeta}-cytoskeleton association. Only the lysate containing both Mg2+ and ATP (Fig. 1Go, A and B) showed any prominent association of {zeta} 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. {zeta}-Chain association with the cytoskeleton can be reconstituted in a cell-free system. A, {zeta}-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-{zeta} mAb. {zeta} 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-{zeta} and anti-actin mAbs. The disruption of {zeta} 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 {zeta}-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 {zeta}-cytoskeleton association, increasing concentrations of Mg2+ were added to cell lysates in the presence (Fig. 1GoC, lanes 8–12) or absence (Fig. 1GoC, lanes 3–7) of 1 mM ATP. Mg2+ concentrations of 1–2 mM (Fig. 1GoC, lanes 5 and 6) were optimal in inducing some {zeta}-cytoskeleton association, but only about 6% of that stimulated in the presence of ATP (Fig. 1GoC, lanes 10 and 11), and were comparable to amounts induced by 1 mM ATP alone (Fig. 1GoC, lane 1). Concentrations above 2 mM Mg2+ were inhibitory to {zeta}-cytoskeleton association (Fig. 1GoC, lane 7). The level of Mg2+ used in most of the studies here, 0.2–0.25 mM, is not sufficient to induce {zeta} to translocate to the pellet (Fig. 1GoC, lane 3). {zeta}-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 {zeta}-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 {zeta}-cytoskeleton association is dependent on regulation of both the Mg2+ and ATP levels, as shown below.

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



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  		FIGURE 2. Reconstituted {zeta}-cytoskeletal binding requires ATP and is associated with tyrosine phosphorylation of the {zeta}-chain. A, ATP is a specific substrate for reconstitution of {zeta}-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-{zeta} mAb. {zeta} 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 {zeta}-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-{zeta} or anti-phosphotyrosine mAb on duplicate blots. Data are representative of at least three experiments.

 
To examine the amount of ATP required for {zeta}-cytoskeleton association and the kinetics of this association, cell lysates were incubated with increasing concentrations of MgATP. Fig. 3GoA 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 {zeta} binding above background was observed below 0.1 mM and appeared optimal at 1 mM ATP and above (Fig. 3Go, B and D). At concentrations of 1 mM ATP and above, optimal association of {zeta} and the actin cytoskeleton in this lysed cell system was very rapid (30 s at 37°C; Fig. 3Go, A and B) and was dependent upon temperature and lysate concentration (data not shown). The kinetics of association of tyrosine phosphorylated {zeta} with the pellet were similar to that observed for {zeta}-cytoskeleton association (data not shown). The kinetics of actin polymerization were also similar, although with a lower threshold of MgATP concentration (Fig. 3Go, A and C).



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  		FIGURE 3. Reaction kinetics and time course of {zeta}-actin association in a lysed cell system. A, Representative ATP concentration dependence of relative {zeta} 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-{zeta} 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 {zeta}-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 ({square}), 0.5 ({lozenge}), 1.0 ({circ}), or 2.5 ({triangleup}) 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-{zeta} mAb (B) or with an anti-actin mAb (C). The amount of {zeta}-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 {zeta}-cytoskeleton association and actin polymerization as a function of ATP concentration in a cell-free system. Rate measurements, calculated as the relative amount of {zeta} bound per second ({zeta} 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 Km for ATP (0.5 mM) of {zeta} association (F) and the apparent Km for ATP (0.9 mM) of actin polymerization (G), in this reconstituted system.

 
The differential dependence on MgATP concentration for {zeta}-cytoskeleton association and actin polymerization was borne out by rate measurements of relative amount of {zeta} bound to the cytoskeletal pellet and relative actin polymerized with increasing ATP concentration. These rate measurements showed that both {zeta} association and actin polymerization were saturable and followed Michaelis-Menton kinetics (Fig. 3Go, D and E, respectively). Double reciprocal plots of these data (Fig. 3Go, F and G) permitted the calculation of the apparent Michaelis-Menton constant for ATP of the {zeta}-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 Km was calculated from the x-intercept. Thus, {zeta}-cytoskeleton association had an apparent Km for ATP almost twice that measured for actin polymerization, suggesting that actin polymerization occurs at a lower ATP threshold and perhaps prior to {zeta}-chain association. Interestingly, the reaction kinetics at ATP concentrations greater than or equal to 0.5 mM (Fig. 3Go, B and C) showed a biphasic peak, for reasons not yet understood. In addition, the degree of {zeta} association at an ATP concentration greater than 1.0 mM decreased after an initial peak (Fig. 3GoB; 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. 3GoC). Likewise, at ATP concentrations below 1.0 mM (Fig. 3GoA), 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, {zeta}-chain associates with the cytoskeleton in a cell-free system with rate and extent similar to that seen following TCR ligation on intact cells.

{zeta}-Cytoskeleton association is dependent upon tyrosine phosphorylation of the {zeta}-chain

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



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  		FIGURE 4. Dephosphorylation of the {zeta}-chain inhibits {zeta}-cytoskeleton association in solution and releases {zeta}-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). {zeta}-Association with the cytoskeleton decreases in the presence of increasing concentrations of AP (MgATP pellet), while {zeta}-chain coordinately increases in the supernatant (released {zeta}). The addition of phosphatase inhibitors (0.2 mM VO3, 10 mM NaF), block the release of {zeta}-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 {zeta}-chain phosphorylation that leads to {zeta}-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 {zeta}-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. 5Go) and Fyn (data not shown) associate with the insoluble pellet from intact cells activated by TCR ligation (Fig. 5GoA, lane 2) and with the reconstituted pellet in the presence of MgATP (Fig. 5GoB, 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. 5Go, lower panels), suggesting that activated Lck and phosphorylated {zeta}-chain colocalize to the actin cytoskeleton in response to TCR ligation.



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  		FIGURE 5. pp56Lck 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 CD3{varepsilon} with anti-CD3{varepsilon} (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 {zeta}-chain upon TCR ligation (22) and is, along with Fyn, a candidate kinase involved in the tyrosine phosphorylation of {zeta}-chain leading to {zeta}-cytoskeleton association (23). To differentiate the role of various protein tyrosine kinases in {zeta}-cytoskeleton association, we examined T cell lysates in which levels of specific Src family kinases were decreased by immunoprecipitation (Fig. 6Go). {zeta}-Cytoskeleton association was abrogated in the reconstituted pellets in which levels of Lck (Fig. 6GoA, lane 4, and 6B) were reduced. In contrast, in T cell lysates in which levels of either Fyn (Fig. 6GoA, lane 6) or pp60c-Src (Fig. 6GoA, lane 8) were reduced, {zeta}-chain still associated with the reconstituted pellet upon MgATP addition. This inhibition of {zeta}-cytoskeleton association is not the result of coimmunoprecipitation of {zeta}-chain with Lck, nor are the levels of soluble {zeta}-chain in the cell lysate affected (data not shown). Titration of the anti-Lck Ab (Fig. 6GoB) showed that inhibition of {zeta}-cytoskeleton association (Fig. 6GoB, bottom panel) was directly correlated with both the loss of Lck from the cytoskeletal pellet (Fig. 6GoB, middle panel) and with the amount of Lck immunoprecipitated from solution (Fig. 6GoB, top panel), with a corresponding decrease in tyrosine phosphorylation of the {zeta}-chain in both pellet and supernatant fractions (data not shown). Thus, in this cell-free system, Lck plays a crucial role in inducing both {zeta}-chain tyrosine phosphorylation and {zeta}-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, {zeta}-cytoskeleton association, respectively (data not shown), suggesting that Fyn is not involved in {zeta}-cytoskeleton association and that c-Src may act on upstream regulators to inhibit the association.



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  		FIGURE 6. Immunoprecipitation of pp56Lck from the cell lysate inhibits {zeta} 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 {zeta} association with the cytoskeleton is inversely correlated with the amount of pp56Lck 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-{zeta} (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 {zeta}-cytoskeleton association in intact thymocytes and lymph node T cells from mice lacking Lck (Lck knockouts) (19) (Fig. 7Go) or Fyn (Fyn knockouts) (20) (Fig. 8Go). 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 CD3{varepsilon} on T cells from Lck- mice did not induce {zeta}-chain association with the insoluble pellet (Fig. 7GoA). In contrast, crosslinking of CD3{varepsilon} on T cells from both Fyn-negative (Fyn-) and wild-type (Fyn+) mice induced association of {zeta}-chain with the insoluble pellet (Fig. 8GoA). Analysis of immunoprecipitated {zeta}-chain by antiphosphotyrosine immunoblotting confirmed that the {zeta}-chain from Lck+, but not Lck-, T cells was tyrosine phosphorylated following TCR ligation (Fig. 7GoA). These data suggest that tyrosine phosphorylation is required for binding of {zeta} to the cytoskeleton, and implicate Lck, rather than Fyn, as the kinase involved in this {zeta}-chain tyrosine phosphorylation. Interestingly, the addition of MgATP to T cell lysates from either Lck- (Fig. 7GoB) or Fyn- (Fig. 8GoB) mice induced {zeta}-chain phosphorylation and {zeta}-cytoskeleton association, suggesting that other Src family kinases may substitute for Lck in this cell-free assay and that all other requisite components for {zeta}-cytoskeleton association are present in the Lck- mice. These data suggest that tyrosine phosphorylation is required for {zeta}-cytoskeletal binding, and implicate Lck as the kinase involved in {zeta}-cytoskeleton association in vivo.



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  		FIGURE 7. Induction of {zeta}-cytoskeleton association is abrogated in intact T cells from pp56Lck 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-CD3{varepsilon} ({alpha}-CD3{varepsilon}) 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-{zeta} 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 {zeta}-cytoskeleton association nor {zeta} 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-{zeta} and anti-actin mAbs. {zeta}-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. {zeta}-Cytoskeleton association is unaffected in intact T cells or T cell lysates from pp59Fyn 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-CD3{varepsilon} ({alpha}-CD3{varepsilon}) 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-{zeta} mAb (1 ). Both Fyn+- and Fyn--activated lymph node T cells and thymocytes (lanes 4 and 8, respectively) showed increases in {zeta}-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-{zeta} mAb. {zeta} 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 {zeta}-cytoskeleton association

Since SH2 domains are the specific downstream targets of tyrosine-phosphorylated proteins, including the {zeta}-chain, we analyzed {zeta}-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. 9GoA) 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 {zeta}-cytoskeleton association. In comparison with the other GST-fusion proteins, GST-Fyn SH2 or GST-SHIP SH2 (Fig. 9GoA, lanes 3 and 6), or with MgATP (Fig. 9GoA, lane 2) or GST alone (Fig. 9GoA, lane 4), the GST-Lck SH2 fusion protein preferentially inhibited {zeta}-cytoskeleton association (Fig. 9GoA, lane 5). Both the GST-Fyn SH2 and the GST-Lck SH2 fusion proteins also inhibited Lck association with the reconstituted pellet (Fig. 9GoA, lanes 3 and 5), yet only GST-Lck SH2 domain resulted in the concomitant inhibition of {zeta}-cytoskeleton association, suggesting that {zeta}-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in {zeta}-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 {zeta}-cytoskeleton association. A, GST fusion protein containing the Lck SH2 domain preferentially inhibits {zeta}-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 {zeta} 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 {zeta}-cytoskeleton association by 50 to 70% relative to the other fusion proteins. B, Recombinant Fyn-SH2 peptide inhibits {zeta}-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). {zeta}-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 {zeta}-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. 9GoB, {zeta}-cytoskeletal binding diminished with increasing concentrations of the SH2 peptide, relative to MgATP alone (Fig. 9GoB, lane 3) or to BSA (Fig. 9GoB, 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 {zeta}-cytoskeleton association by Fyn-SH2 domains suggests that Src SH2-containing polypeptides are involved, either directly or indirectly, in the interaction of {zeta} with the microfilament cytoskeleton.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that TCR{zeta}-chain can be induced to associate with the actin cytoskeleton in a cell-free system (1). Here, we expanded our characterization of this system, and, using this cell-free system, examined the role of Lck and tyrosine phosphorylation in {zeta}-cytoskeleton binding. This reconstituted system is specific for ATP and divalent cations since other nucleotides and ATP analogs did not substitute for ATP in supporting cytoskeletal binding. The results of kinetic studies showed that {zeta}-cytoskeleton association has an apparent Km for ATP of 0.9 mM, providing evidence that this is a saturable enzymatic reaction that is affected by concentration and/or temperature and that ATP is utilized as a substrate as a possible phosphoryl donor in kinase reactions. Indeed, tyrosine-phosphorylated {zeta}-chain was a predominant form of {zeta} that associated with the cytoskeleton. Dephosphorylation of {zeta}-chain by alkaline phosphatase inhibited or reversed the {zeta}-cytoskeletal association, suggesting that early phosphorylation events regulate this interaction.

In the lysed cell system presented here, as in intact cells, {zeta} cosedimented with actin ( Figs. 1–3GoGoGo) and the {zeta}-cytoskeleton association was disrupted by cytochalasin D (1) and EDTA. These data suggest that an intact microfilament array is necessary for compartmentalization of {zeta}-chain with the cytoskeletal pellet and that microfilaments associate, either directly or indirectly, with the {zeta}-chain following T cell activation. This association is, however, not driven by actin polymerization, since microfilaments can be isolated with minimal bound {zeta}-chain, under conditions that polymerize filaments in the absence of ATP (Figs. 1Go and 2Go). In addition, the reconstituted {zeta}-cytoskeleton association is dynamic over time (Fig. 3Go) and {zeta} constructs with deletions in the terminal tyrosine do not cosediment with cytoskeleton (1), arguing that this in vitro interaction is specific and not due to protein trapping in the insoluble pellet. Interestingly, the interaction of {zeta}-chain with actin is associated with increased actin polymerization both in the cell-free system and in intact cells, suggesting that induced actin polymerization may require the involvement of receptor association (24), analogous to that seen following interaction of integrins on platelets and fibroblasts with their substrates. These data suggest that the properties of the reconstituted {zeta}-cytoskeleton association mimic those observed following TCR ligation on intact cells. This lysed cell system may therefore be useful as a model system for the biochemical study of the mechanisms and molecules involved in the interaction between TCR and the cytoskeleton.

We observed that Lck and Fyn, but not Src, are components of the detergent insoluble pellet from intact cells and of the reconstituted cytoskeletal pellet from lysed cells. This compartmentalization of Lck and Fyn, like {zeta}-chain, is enhanced under conditions of TCR ligation or reconstitution following incubation with MgATP. Compartmentalization of these specific Src family kinases with {zeta}-chain and with the cytoskeletal fraction suggested an involvement of Lck or Fyn in the tyrosine phosphorylation-induced protein-protein interaction. We showed that reduction of Lck, but not Fyn or Src, inhibited the induction of {zeta}-cytoskeleton association. We also showed that TCR ligation of T cells from Lck-, but not from Fyn-, mice failed to induce {zeta}-chain phosphorylation or {zeta}-cytoskeleton association. These results suggest that Lck is required for the tyrosine phosphorylation of {zeta}-chain leading to microfilament binding. That {zeta}-cytoskeleton association can be reconstituted in cell lysates from Lck- mice suggests there is a redundancy of kinase activity (22, 25, 26) in the reconstituted system, and that the machinery, components, and pathways leading to {zeta}-cytoskeletal interaction are intact in these mice, except for the lack of Lck. Why then did depletion of Lck from wild-type (Lck+) cell lysates inhibit {zeta}-cytoskeleton association (Fig. 6Go)? We hypothesize that an important adaptor/effector in the pathway leading to {zeta}-cytoskeleton association is depleted by coimmunoprecipitation with Lck. As discussed below, a polypeptide containing an SH3 domain may function as just such a molecule. In summary, our data suggest that Lck is required both in vitro and in vivo as a kinase and/or effector in the pathways leading to {zeta}-cytoskeletal interaction.

The data presented here also show that the Lck SH2 peptide preferentially inhibited the {zeta}-cytoskeletal interaction and that the SH2 peptide from Fyn was also inhibitory, in a dose-dependent manner. The Fyn SH2 peptide was inhibitory at higher concentrations than Lck, suggesting that SH2-containing proteins are critical to the formation of this heteromeric protein complex, but retain their binding specificities. Since actin does not have SH2 domains, intermediary signaling molecules and/or cytoskeletal-binding proteins containing these motifs must bridge the {zeta}-actin interaction. These intermediary proteins might, in turn, bind to actin via SH3 domains (12). That an SH2 domain of Lck, and to a lesser extent, of Fyn, inhibits the interaction, suggests that these kinases are involved as 1) adaptor/bridging proteins for the {zeta}-actin association, 2) activated kinases that regulate other molecules in the binding pathway, or 3) regulators of the availability of phosphorylated tyrosines on the signaling ITAMs for other molecules, such as ZAP-70. Recent studies have shown that the SH2 domain of Lck is essential for signal transduction events following TCR ligation (23) including the tyrosine phosphorylation of the {zeta}-chain and IL-2 production (27).

These data are consistent with a model in which {zeta} binds to actin, via an SH2-containing intermediary protein, subsequent to its tyrosine phosphorylation by Lck. This interaction may stabilize actin polymerization and the formation of a signaling complex. We postulate that these nascent {zeta}-cytoskeletal complexes, which we call "cytoskeletal organizing centers," serve to compartmentalize and anchor activated enzymes critical for T cell signal transduction. Another intriguing possibility comes from recent data demonstrating that forces applied to cell surface receptors anchored to the cytoskeleton quickly propagate to the cell interior (28). Thus, Ag-receptor ligation could translate into mechanical control of DNA and gene expression and regulation.

In conclusion, we have developed methods that reconstitute the {zeta}-cytoskeleton interaction in a cell-free system. Our findings indicate that 1) intact cell membranes are not required for this interaction, 2) the intracellular machinery required for this interaction shows redundancy but remains intact under cell-free conditions, and 3) the interaction is regulated by tyrosine phosphorylation, through the involvement of the Src-family kinase, Lck, and its SH2 domain. The in vitro reconstituted system predicted a critical role for a particular Src-family kinase, a role that was confirmed in vivo in our studies of mutant mice. In intact cells, Lck was required for TCR-induced tyrosine phosphorylation of {zeta} and for {zeta}-cytoskeleton association. This cell-free system thus provides a powerful tool with which to further define and map the {zeta}-actin interaction. In particular, this system will facilitate analysis of postulated inhibitors of the interaction, without the associated difficulties of introducing peptides, Abs, or vectors into cells. In turn, in vivo use of such inhibitors identified in our in vitro system will facilitate analysis of the role of {zeta}-cytoskeleton association in downstream events of T cell signal transduction.


   Acknowledgments
 
We thank Dr. B. Jockusch for the gift of anti-actin mAb, Dr. T. Potter for the gift of anti-Lck and anti-Fyn polyclonal antibodies, Kazuhiro Nakamura for the gift of GST-SHIP SH2, Sarah Johnson for the SH2 polypeptides, Dr. Tak Mak for Lck knockout mice, and V. Angkachatchai for assistance with time course experiments.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R01 AI30575, P01 AI22295 (T.H.F.), and T32 AI00048 (M.M.R.), the University of Colorado Health Sciences Center Cancer Center (M.M.R. and T.H.F.), the Arthritis Foundation, the Bender Foundation, and the Eleanore and Michael Stobin Trust (T.H.F.). Back

2 Current address: Cadus Phamaceutical, 777 Old Saw Mill River Rd., Tarrytown, NY 12533. Back

3 Address correspondence and reprint requests to Dr. Terri H. Finkel, Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Back

4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; SH2, Src homology 2; GST, glutathione-S-transferase; SHIP, Src homology 2-containing inositol phosphatase. Back

Received for publication December 15, 1997. Accepted for publication July 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rozdzial, M. M., B. Malissen, T. H. Finkel. 1995. Tyrosine phosphorylated T cell receptor-{zeta} chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity 3:623.[Medline]
  2. Omann, G. M., R. A. Allen, G. M. Bokoch, R. G. Painter, A. E. Traynor, L. A. Sklar. 1987. Signal transduction and cytoskeletal activation in the neutrophil. Physiol. Rev. 67:285.[Free Full Text]
  3. Ashwell, J. D., R. D. Klausner. 1990. Genetic and mutational analysis of the T cell receptor. Annu. Rev. Immunol. 8:139.[Medline]
  4. Wegener, A-M. K., F. Letourneur, A. Hoeveler, T. Brocker, F. Luton, B. Malissen. 1992. The T cell receptor/CD3 complex is composed of at least two autonomous transduction molecules. Cell 68:83.[Medline]
  5. Cambier, J. C.. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ITAM/YXXL). Immunol. Today 16:110.[Medline]
  6. Beaufils, P., D. Choquet, R. Z. Mamoun, B. Malissen. 1993. The (YXXL/I)2 signalling motif found in the cytoplasmic segments of the bovine leukemia virus envelope protein and Epstein-Barr virus latent membrane protein 2A can elicit early and late lymphocyte activation events. EMBO J. 12:5105.[Medline]
  7. Nakayama, T., A. Singer, E. D. Hsi, L. E. Samelson. 1989. Intrathymic signalling in immature CD4+CD8+ thymocytes results in tyrosine phosphorylation of the T-cell receptor {zeta}-chain. Nature 341:651.[Medline]
  8. Rozdzial, M. M., R. T. Kubo, S. L. Turner, T. H. Finkel. 1994. Developmental regulation of the TCR {zeta}-chain. J. Immunol. 153:1563.[Abstract]
  9. Hsi, E. D., J. N. Siegel, Y. Minami, E. T. Luong, R. D. Klausner, L. E. Samelson. 1989. T cell activation induces rapid tyrosine phosphorylation of a limited number of cellular substrates. J. Biol. Chem. 264:10836.[Abstract/Free Full Text]
  10. Wange, R. L., N. Isakov, Jr T. R. Burke, A. Otake, P. P. Roller, J. D. Watts, R. Aebersold, L. E. Samelson. 1995. F2(Pmp)2-Tam {zeta} 3, a novel competitive inhibitor of the binding of ZAP-70 to the T cell antigen receptor, blocks early T cell signaling. J. Biol. Chem. 270:944.[Abstract/Free Full Text]
  11. Isakov, N., R. L. Wange, W. H. Burgess, J. D. Watts, R. Aebersold, L. E. Samelson. 1995. ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine based activation motifs with varying affinity. J. Exp. Med. 181:375.[Abstract/Free Full Text]
  12. Koch, C. A., D. Anderson, M. F. Moran, C. Ellis, T. Pawson. 1991. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252:668.[Abstract/Free Full Text]
  13. Sonyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, et al 1993. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol. 14:2777.[Abstract/Free Full Text]
  14. Caplan, S., S. Zeliger, L. Wang, M. Baniyash. 1995. Cell-surface-expressed T-cell antigen-receptor {zeta}-chain is associated with the cytoskeleton. Proc. Natl. Acad. Sci. USA 92:4768.[Abstract/Free Full Text]
  15. Valitutti, S., M. Dessing, K. Aktories, H. Gallati, A. Lanzavecchia. 1994. Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy: role of T cell actin cytoskeleton. J. Exp. Med. 181:577.[Abstract/Free Full Text]
  16. Jugloff, L. S., J. Jongstra-Bilen. 1997. Cross-linking of the IgM receptor induces rapid translocation of IgM-associated Ig {alpha}, Lyn, and Syk tyrosine kinases to the membrane skeleton. J. Immunol. 159:1096.[Abstract]
  17. Caplan, S., M. Baniyash. 1996. Normal T cells express two T cell antigen receptor populations, one of which is linked to the cytoskeleton via {zeta} chain and displays a unique activation-dependent phosphorylation pattern. J. Biol. Chem. 271:20705.[Abstract/Free Full Text]
  18. Pleiman, C. M., M. R. Clark, L. K. Timson Gauen, S. Winitz, K. M. Coggeshall, G. L. Johnson, A. S. Shaw, J. C. Cambier. 1993. Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-{gamma}2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase. Mol. Cell. Biol. 13:5877.[Abstract/Free Full Text]
  19. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K.-U. Hartmann, A. Veillette, et al 1992. Profound block in thymocyte development in mice lacking p56Lck. Nature 357:161.[Medline]
  20. Stein, P. L., H. M. Lee, S. Rich, P. Soriano. 1992. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70:741.[Medline]
  21. Leo, O., M. Foo, D. H. Sachs, L. E. Samelson, J. A. Bluestone. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA 84:1374.[Abstract/Free Full Text]
  22. Van Oers, N. S., N. Killeen, A. Weiss. 1996. LCK regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP 70 in murine thymocytes. J. Exp. Med. 183:1053.[Abstract/Free Full Text]
  23. Straus, D. B., A. C. Chan, B. Patai, A. Weiss. 1996. SH2 domain function is essential for the role of the Lck tyrosine kinase in T cell receptor signal transduction. J. Biol. Chem. 271:9976.[Abstract/Free Full Text]
  24. Melamed, I., G. P. Downey, K. Aktories, C. M. Roifman. 1991. Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. J. Immunol. 147:1139.[Abstract]
  25. Goves, T., P. Smiley, M. P. Cooke, K. Forbush, R. M. Perlmutter, C. J. Guidos. 1996. Fyn can partially substitute for Lck in T lymphocyte development. Immunity 5:417.[Medline]
  26. van Oers, N. S. C., B. Lowin-Kropf, D. Finlay, K. Connolly, A. Weiss. 1996. {alpha}ßT cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429.[Medline]
  27. Lewis, L. A., C. D. Chung, J. Chen, J. R. Parnes, M. Moran, V. P. Patel, M. C. Miceli. 1997. The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the {zeta}-chain and IL-2 production. J. Immunol. 159:2292.[Abstract/Free Full Text]
  28. Maniotis, A., C. S. Chen, D. E. Ingber. 1997. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc. Natl. Acad. Sci. USA 94:849.[Abstract/Free Full Text]



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