pp56Lck Mediates TCR ζ-Chain Binding to the Microfilament Cytoskeleton Moshe M. Rozdzial, Chris M. Pleiman, John C. Cambier and Terri H. Finkel3 This information is current as of September 23, 2021. J Immunol 1998; 161:5491-5499; ; http://www.jimmunol.org/content/161/10/5491 Downloaded from References This article cites 28 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/161/10/5491.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 23, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. pp56Lck Mediates TCR z-Chain Binding to the Microfilament Cytoskeleton1 Moshe M. Rozdzial,* Chris M. Pleiman,2* John C. Cambier,*† and Terri H. Finkel3†‡ The TCR z-chain (z) 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 z-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of z 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 z is a predominant form of the z-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents z-chain association in solution and releases z-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. z-Chain Downloaded from phosphorylation and z-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 z-chain tyrosine phosphorylation and z-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted z-cytoskeleton association, sug- gesting that z-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in z-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase. The Journal of Immunology, 1998, 161: 5491–5499. http://www.jimmunol.org/ ctivation of mature T lymphocytes is regulated by the association of specific PTKs with the ITAMs via Src homology 2 multichain TCR. The TCR, upon engagement, initiates a (SH2) domains (10, 11). A cascade of biochemical events that lead to receptor as- SH2 domains play a significant role in signal transduction in sociation with cytoskeleton (1) and culminate in the rearrangement many cell types by mediating the formation of specific heteromeric and reorientation of the cytoskeleton (2), production of lympho- protein complexes with phosphotyrosine-containing peptides (12). kines, and cellular proliferation and differentiation. The TCR con- The binding specificity of this protein-protein interaction is depen- sists of the Ag-binding subunits (ab), the CD3 complex (g-, d-, dent on both the primary sequence flanking the site of tyrosine and «-chains), and the z-chains (3). Both the CD3 components and phosphorylation and the structure of the particular SH2 domain z-chains are capable of independently mediating TCR signaling (13). Individual SH2-containing polypeptides select unique se- by guest on September 23, 2021 (4), and each contains, respectively, one and three copies of the quences, except for the Src subfamily (Src, Fyn, Lck, and Fgr), immunoreceptor tyrosine-based activation motif (ITAM),4 com- which preferentially recognizes the sequence, P-Tyr-Glu-Glu-Ile posed of the amino acid sequences YXX(L/I)X(7–8)YXX(L/I) (5). (13). Thus, SH2-containing polypeptides serve as adaptors to link As the TCR has neither intrinsic protein tyrosine kinase (PTK) nor specific effector proteins to phosphotyrosine-containing target phosphatase function, the ITAM sequences appear to be both nec- motifs. essary and sufficient for the induction of protein tyrosine phos- In T cells, biochemical and genetic studies have implicated the phorylation in T cells (6). While there is a low level of constitutive Src (pp56Lck and p59Fyn) and the Syk/ZAP-70 families of PTKs in tyrosine phosphorylation of z, even in resting T cells (7, 8), with cellular activation, demonstrating a functional interaction of these activation, tyrosine phosphorylation of z increases (9), leading to kinases with the TCR complex and/or the coreceptors CD4 and CD8. Both pp56Lck (Lck) and p59Fyn (Fyn) have been shown to z « *Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and interact with ITAMs in the - and CD3 -chains, resulting in phos- Research Center, Denver, CO 80206; †Department of Immunology, University of phorylation of the ITAM tyrosines, and leading to recruitment of Colorado Health Sciences Center, Denver, CO 80262; and ‡Departments of Pediatrics other molecules involved in T cell signal transduction, such as and Biochemistry & Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262 ZAP-70 (10, 11). Received for publication December 15, 1997. Accepted for publication July 16, 1998. Although not well studied in T cells, the cytoskeleton also plays a direct role in the regulation and compartmentalization of the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance activation process (reviewed in Ref. 1). Previously, we have shown with 18 U.S.C. Section 1734 solely to indicate this fact. that as a consequence of TCR ligation, the z-chain rapidly binds to 1 This work was supported by National Institutes of Health Grants R01 AI30575, P01 the membrane skeleton independent of receptor internalization (1). AI22295 (T.H.F.), and T32 AI00048 (M.M.R.), the University of Colorado Health Pretreatment with drugs that disrupt the actin cytoskeleton abro- 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.). gated the association of z with cytoskeleton (1, 14) and inhibited 21 g 2 Current address: Cadus Phamaceutical, 777 Old Saw Mill River Rd., Tarrytown, NY sustained Ca mobilization, IFN- (15) and IL-2 (M. M. Rozd- 12533. zial, unpublished observations) production. In addition, in T cell 3 Address correspondence and reprint requests to Dr. Terri H. Finkel, Division of hybridomas transfected with mutated or deleted z-chain chimeras, Basic Sciences, Department of Pediatrics, National Jewish Medical and Research lack of z-cytoskeleton association correlated with inhibition of late Center, 1400 Jackson St., Denver, CO 80206. activation events, such as IL-2 production (1). Recent studies have 4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; SH2, Src homology 2; GST, glutathione-S-trans- also documented the activation-dependent association of tyrosine ferase; SHIP, Src homology 2-containing inositol phosphatase. kinases with the membrane skeleton in both B (16) and T (17) Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 5492 ROLE OF pp56Lck IN z-CHAIN-MICROFILAMENT ASSOCIATION lymphocytes. These data provide direct evidence of an activation- the addition of sample buffer to the reconstituted pellets. Electrophoresis, dependent interaction between Ag receptor and the cytoskeletal immunoblotting, and densitometry were performed as previously described z z matrix that may support the interaction of signaling polypeptides (1). Amount of bound -chain was quantitated as associated per second relative to the baseline (assigned a value of 1) at the 0 time control. As the and their substrates. results for thymocytes were essentially identical to those obtained for Determination of the role of microfilaments in TCR-signaling lymph node T cells, these sets of data were pooled. cascades and molecular dissection of the z-cytoskeletal interaction 1 Effect of Mg2 is, however, hampered by the formation of a complex insoluble 1 pellet upon activation of intact cells. Therefore, we developed an T cell lysates were incubated with increasing concentrations of Mg2 with assay for analysis of z-cytoskeleton association under cell-free or without 1 mM ATP for 8 min at 37°C. Electrophoresis and immuno- blotting were performed as previously described (1). conditions, so as to be able to modify the system before pellet formation (1). Here, we demonstrate that addition of divalent cat- Challenge with alkaline phosphatase z ions and ATP to a T cell lysate leads to the phosphorylation of Cell lysates were incubated in the presence or absence of 1 U/100 mlof and association of z with microfilaments. Furthermore, we show bacterial alkaline phosphatase for 30 min and incubated with MgATP, with that, in the lysed cell system, z-cytoskeleton association is prefer- or without phosphatase inhibitors (0.2 mM VO3, 10 mM NaF), as de- entially inhibited by a recombinant Lck-SH2 peptide or by a re- scribed. The reconstituted MgATP pellets were then disrupted and incu- bated in the presence or absence of 1 U/10 ml alkaline phosphatase and duction in levels of Lck.