The Journal of Immunology

Focal Adhesion Negatively Regulates Lck Function Downstream of the T Receptor

Nicole M. Chapman,* Sean F. Connolly,* Erin L. Reinl,†,1 and Jon C. D. Houtman*,†

Focal adhesion kinase (FAK) is a critical regulator of signal transduction in multiple cell types. Although this is activated upon TCR engagement, the cellular function that FAK plays in mature human T cells is unknown. By suppressing the function of FAK, we revealed that FAK inhibits TCR-mediated signaling by recruiting C-terminal Src kinase to the membrane and/or receptor complex following TCR activation. Thus, in the absence of FAK, the inhibitory of Lck and/or Fyn is impaired. Together, these data highlight a novel role for FAK as a negative regulator TCR function in human T cells. These results also suggest that changes in FAK expression could modulate sensitivity to TCR stimulation and contribute to the progression of malig- nancies and autoimmune diseases. The Journal of Immunology, 2013, 191: 6208–6221.

uman T cells control the extent of the adaptive immune trans autophosphorylation of Y394, a residue found in the activation response following infection and in many pathological loop of the kinase domain (8, 11). Importantly, increases in Lck H conditions (1–6). T cells are activated upon stimulation Y394 and decreases in Y505 phosphorylation are correlated with of the TCR by peptide-bound MHC complexes in combination enhanced Lck activity (11). Thus, Lck activity is dictated by the with one or more costimulatory receptors (2). Constitutively active balance of Lck Y394 and Y505 phosphorylation, and perturbations Lck is recruited to the TCR complex after Ag stimulation, where it in the phosphorylation ratio of these two residues can increase or phosphorylates ITAMs found in multiple TCR subunits (2, 7, 8). decrease TCR-induced signaling and T cell activation. This event is critical for ZAP70 activation (2). The adaptor pro- To phosphorylate Lck Y505, cytoplasmic Csk is recruited to the teins, linker for activation of T cells (LAT) and SLP-76, are then T cell membrane, a process that is vital for its function (12, 16–18). phosphorylated by ZAP70. Together, LAT and SLP-76 recruit and The current model is that in unstimulated T cells Csk binds to control the activation of multiple effector such as phos- phospho-Y317 on phosphoprotein associated with glycosphingolipid- pholipase C (PLC)-g1 and PI3K, thereby triggering downstream enriched microdomains (PAG), also known as Csk-binding protein signaling events like influx and Akt activation (2, 9, 10). (18, 19–23). This interaction localizes Csk to the plasma membrane TCR activation culminates in transcriptional and morphological and enhances its catalytic function, which allows Csk to phosphor- changes that regulate production, receptor expression, ylate Lck Y505 (24). Upon TCR activation, PAG/Csk-binding protein

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. and the migratory properties of T cells (2). is dephosphorylated, after which Csk is transiently displaced from The phosphorylation of Lck Y394 and Y505 controls Lck en- detergent-insoluble membrane lipid rafts (18, 22). This transient dis- zymatic activity to prevent inappropriate T cell responses. Lck Y505 placement allows CD45 to dephosphorylate Lck Y505, resulting in phosphorylation stabilizes the protein in a closed, inactive confor- the enhanced enzymatic function of Lck (11, 25). Within 5 min after mation, which limits TCR function (11–15). This residue is TCR activation, Csk reassociates with lipid rafts, presumably because phosphorylated by C-terminal Src kinase (Csk) and dephosphory- PAG Y317 is rephosphorylated (18, 19). However, contrary to this lated by CD45 (11). The activity of Lck is also enhanced by the model, Lck Y505 phosphorylation remains unchanged or increases after TCR stimulation (7, 8, 12, 26). Furthermore, the observation that PAG-deficient T cells do not have enhanced T cell activation suggests http://classic.jimmunol.org *Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, that alternative mechanisms exist to regulate Csk’s recruitment to the IA 52242; and †Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242 membrane after TCR activation (20, 21, 27). Therefore, the mecha- 1Current address: Department of Obstetrics and Gynecology, Washington University nisms that regulate Csk’s recruitment to the membrane after TCR School of Medicine, St. Louis, MO. stimulation are not clear. Received for publication June 17, 2013. Accepted for publication October 11, 2013. Actin cytoskeletal responses are essential for cytokine release This work was supported by American Heart Association Predoctoral Fellowship and cellular spreading downstream of the TCR (28, 29). Focal

Downloaded from 11PRE7390070 (to N.M.C.), National Institutes of Health Predoctoral Training Grant adhesion kinase (FAK) is phosphorylated by Lck and/or Fyn upon in Immunology T32AI007485 (to N.M.C. and S.F.C.), American Heart Association TCR induction (30, 31). Previously, FAK was found to control Scientist Development Grant 0830244N, and National Institutes of Health Grant R01 CA136729 (to J.C.D.H.). cellular processes linked to actin polymerization. In line with this Address correspondence and reprint requests to Prof. Jon C.D. Houtman, University role, inhibiting FAK’s expression or function in T cells, B cells, of Iowa, 375 Newton Road, Building 2234 MERF, Iowa City, IA 52242. E-mail macrophages, and neutrophils impaired actin-dependent processes address: [email protected] such as adhesion or spreading (31–35). Thus, the observation that The online version of this article contains supplemental material. FAK regulates actin-dependent responses is likely to have im- Abbreviations used in this article: Csk, C-terminal Src kinase; FAK, focal adhesion portant implications in TCR function. However, because FAK is kinase; hAPBT, human activated peripheral blood T cell; LAT, linker for activation of T cells; MFI, mean fluorescence intensity; miRNA, microRNA; PAG, phosphoprotein expressed at low levels in human T cells compared with B cells associated with glycosphingolipid-associated microdomains; PLC, ; (36), it may serve an alternative function downstream of the TCR. Pyk2, proline-rich 2; SH2, Src homology 2; TIRF, total internal In this study, we show that the transient knockdown of FAK reflection fluorescence. results in enhanced or extended TCR-induced signal transduction, Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 cytokine production, and CD69 expression in Jurkat E6.1 cells and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301587 The Journal of Immunology 6209

CD4 human activated peripheral blood T cells (hAPBTs). Using gies. The HRP-conjugated secondary Abs and streptavidin-HRP were from total internal reflection fluorescence (TIRF) microscopy and immu- Jackson ImmunoResearch Laboratories. The IRdye 800CW- or IRdye 680- noprecipitations, we found that Csk recruitment to the membrane labeled secondary Abs were obtained from Licor Biosciences. Protein A/G agarose beads were bought from Santa Cruz Technologies. Anti-human and TCR complex following TCR induction requires FAK ex- rIL-2, anti-human IFN-g, biotin-conjugated anti-human IL-2, and biotin- pression. After TCR activation, FAK-deficient cells also displayed conjugated anti-human IFN-g Abs were purchased from eBioscience. PE decreased Lck Y505 phosphorylation and increased Lck Y394 anti-human ab TCR, PE/Cy5 anti-CD69, Dylight 488 goat anti-mouse and TCRz-chain phosphorylation. Together, these findings demon- IgG1, DyLight 488 donkey anti-rabbit IgG, anti-CD3 (OKT3), and anti- CD4 (RPA-T4) Abs were obtained from BioLegend. The anti-Akt pS473, strate that FAK negatively regulates TCR signaling by controlling Alexafluor 568 anti-rabbit, and Alexafluor 568 anti-mouse IgG1 Abs and Lck activation. the magnetic Dynabeads were from Invitrogen. The goat anti-mouse IgG Ab was acquired from Rockland International. The FAK inhibitor, PF- Materials and Methods 573228, was purchased from Tocris Bioscience. Criterion Precast 4–15% polyacrylamide gels were purchased from Bio-Rad. Oligonucleotides were Human samples generated at Integrated DNA Technologies. All chemicals used were re- All human subject studies were in compliance with the Declaration of search grade and purchased from a variety of sources. Helsinki. Healthy donors between the ages of 18 and 55 y old were recruited by the DeGowin Blood Center at the University of Iowa’s Hospitals and Plasmids Clinics. These donors provided written consent for their blood products to The Luc microRNA (miRNA) cassette was a gift of A. McCaffrey (TriLink be used in research projects conducted at the University of Iowa in com- BioTechnologies). Sequences for Fak-specific miRNAs were selected (Sup- pliance with the University of Iowa’s Institutional Review Board. Whole plemental Table I) and used to replace the Luc targeting sequence, as pre- blood was passed through leukocyte reduction system cones (37), and viously described (39). Two copies of the Luc and Fak 3183 miRNAs were these completely deidentified cones were then provided to investigators at PCR amplified and ligated into the pVetLeGFP vector developed by the the University of Iowa. Because the blood products from LRS cones were University of Iowa’s Gene Vector Transfer Core. The Luc miR- and Fak normally discarded, we did not require further Institutional Review Board miR-GFP reporter lentiviruses were generated by the Gene Vector Transfer approval to use these blood samples. Core at the University of Iowa. Reagents and Abs Transfection and stimulation of Jurkat E6.1 cells RPMI 1640, L-glutamine, penicillin-streptomycin, and PBS were acquired Jurkat E6.1 T cells were grown to a density of 3–4 3 105 cells/ml in from Life Technologies. The FBS was obtained from Atlanta Biologicals complete RPMI (RPMI 1640 supplemented with 10% FBS, 2 mM L-glu- and Denville. Human rIL-2 used for ELISA was purchased from R&D tamine, 50 U/ml penicillin, and 50 mg/ml streptomycin). Cells were trans- Systems, whereas the human rIL-2 used in growth media was obtained fected with Luc or Fak miRNA or the various FAK knockdown/re- from AIDS Research and Reference Reagent Program, Division of AIDS, expression vectors (5 mg per 5 3 106 cells) using the Amaxa Cell Line National Institute of Allergy and Infectious Diseases, National Institutes of Nucleofector Kit V (Lonza) and program X-001 and then incubated for Health: human rIL-2 from M. Gately (Hoffman-La Roche) (38). The anti- 72 h. Prior to stimulation, cells were grown to a density of 2–5 3 105 phospho–LAT tyrosine 191, anti-actin and anti-FAK Abs, polyvinylidene cells/ml. Cells were stimulated with 10 mg/ml soluble anti-CD3 for various difluoride membrane, and polybrene were obtained from Millipore. The times, as previously described (9, 40–43). anti–SLP-76 Ab was purchased from Antibody Solutions. The anti-Lck pY505, anti–SLP-76 pY128, anti-CD247 pY142, anti–proline-rich tyrosine Isolation and growth of CD4 hAPBT kinase 2 (Pyk2), anti-Csk, anti-Grb2, and anti-LAT Abs were acquired from BD Biosciences. The anti–PLC-g1 pY783, anti–PLC-g1, anti-Lck, PBMCs were obtained from LRS cones (37), as previously described (44).

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. and anti-Src pY416 Abs were purchased from Cell Signaling Technolo- CD4 T cells were purified from nonadherent PBMCs by negative selection

FIGURE 1. miRNAs repress FAK expression in human T cells. (A) were transfected with either a control (Luc) miRNA or various miRNAs specific for FAK. Whole-

http://classic.jimmunol.org cell lysates were taken 72 h later, and the expression of FAK and actin was analyzed by immunoblotting. (B) Jurkat T cells were transfected with luciferase- or FAK-specific miRNAs. Whole-cell lysates were prepared at the indicated times, and FAK and

Downloaded from actin expression were analyzed by immunoblotting. The densitometric ratio of FAK expression compared with actin expression is shown below the graph. (C) Whole-cell lysates from Jurkat E6.1 T cells transfected with miRNAs against luciferase or FAK were analyzed by immunoblot for FAK, Pyk2, LAT, SLP-76, PLC-g1, Lck, Csk, and Grb2 and actin ex- pression 72 h after transfection. (D) The normalized expression of FAK, Pyk2, LAT, SLP-76, PLC, PLC-g1, Lck, Csk, or Grb2 is shown as the mean of two to three experiments 6 SD. 6210 FAK INHIBITS TCR FUNCTION

using the human CD4 T cell isolation (Stem Cell Technologies). Cells Analysis of immunoblotting were routinely .95% pure. Human CD4 T cells then were activated with anti-CD3 and anti-CD28 beads in the presence of 100 U/ml IL-2. The relative intensity of bands was determined using the gel-plotting macro of ImageJ or the analysis software in the Odyssey v3.0 program. Nor- FAK knockdown and stimulation of CD4 hAPBT malization of the phospho-protein intensity to the actin intensity was conducted, as we have described previously (40–43). Upon normalization, Purified CD4 T cells were activated for 3 d with the Ab-coated beads. The the percentage of maximal Luc phosphorylation was calculated as follows: 6 cells were then resuspended at 2 3 10 cells/ml in complete RPMI plus 100 % maximum Luc phosphorylation = (normalized intensity of time point 4 U/ml rIL-2 and 8 mg/ml polybrene. Luc or Fak miRNA-containing lenti- normalized intensity of maximum Luc time point) 3 100%. The average viruses were then incubated with the cells (multiplicity of infection 2) for percentage from each least three independent experiments 6 SEM for 24 h. The virus was removed, and the cells were incubated for 48–72 h. every time point was then plotted using Microsoft Excel. These cells were then stimulated by cross-linking with 2 mg/ml anti-CD3 and 2 mg/ml anti-CD4, as previously described (40, 41, 43). For imaging Correlation analyses experiments, CD4 T cells were activated for 5–7 d with the Dynabeads as + above, and transfections were carried out as described for the Jurkat cells. Human CD4 T cells were isolated by negative selection and activated using anti-CD3– and anti-CD28–coated beads in the presence of rIL-2. On Immunoblotting the indicated days, equal numbers of these cells were lysed using hot 23 sample buffer and were boiled and sonicated, as described above. The Proteins in cellular lysates were separated by PAGE using 4–15% Criterion expression of FAK, Csk, phospho-Lck Y505, and/or Pyk2 was detected by gels and transferred to polyvinylidene difluoride membranes. The mem- immunoblotting and normalized to actin expression in each lane. These branes were blocked at room temperature in TBST (10 mM Tris [pH 8.0], normalized values obtained from each independent experiment were used 150 mM NaCl, and 0.05% Tween 20) with 5% nonfat dry milk, 1% BSA, to produce the scatter plots shown in Fig. 10E and 10G. To generate the or in SEA BLOCK Blocking Buffer (Thermo Scientific) diluted 1:1 in graphs shown in Fig. 10F and 10H, equal numbers of freshly isolated CD4+ PBS. Immunoblotting was performed, as described (9, 41, 43). Ab binding T cells (unstimulated CD4 T cell), unstimulated Jurkat E6.1 cells, unsti- was detected using ECL and a Fiji imager or using the Licor Odyssey mulated HuT78 T cells, and CD4+ T cells that were activated for 6 d using Infrared Imager. Pan Abs do not always recognize the phosphorylated the anti-CD3– and anti-CD28–coated beads plus rIL-2 (activated CD4 forms of TCR signaling proteins, including those for LAT and SLP-76 T cell) were lysed with hot 23 sample buffer. Each of these samples was (40). Therefore, stripped membranes were reprobed with a pan-actin Ab to loaded in duplicate onto a polyacrylamide gel, and immunoblotting was ensure equivalent protein loading. used to detect differences in FAK, Csk, and phospho-Lck Y505 expression by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. FIGURE 2. FAK controls the magnitude of early TCR-mediated signaling in Jurkat T cells. Control and FAK-deficient Jurkat cells were stimulated with soluble anti-TCR for various times. (A) Total changes in TCR- induced phosphorylation were analyzed by immunoblotting with antiphosphotyrosine (Y). (B) The expression of FAK, PLC-g1 SLP-76, Lck, Csk, LAT, or actin was ana- http://classic.jimmunol.org lyzed by immunoblotting. (C) The site-specific phosphorylation of LAT Y191, SLP-76 Y128, and PLC-g1 Y783 in TCR-stimulated control or FAK-deficient Jurkat T cells was analyzed by immunoblotting. (D) The per- centage of phosphorylation for LAT Y191, PLC-g1 Y783, and SLP-76 Y128 compared

Downloaded from with the luciferase miRNA-transfected cells was calculated, as described in Materials and Methods. The mean of three to four experiments 6 SEM is shown. *p # 0.05, **p # 0.01. The Journal of Immunology 6211

in each sample. The densitometric intensity for FAK, Csk, and phospho- 0.05% sodium azide), and samples were collected on an Accuri flow cytom- Lck Y505 in each lane was then normalized to actin expression. To gen- eter. The mean fluorescence intensity was determined using Cflow Plus anal- erate the plots shown in Fig. 10F and 10H, the average expression of the ysis program. Percentage of TCR surface expression was determined using the duplicate samples was first calculated. Next, the average of these duplicate following equation: percentage of surface expression = (mean fluorescence values from each of the four independent samples was determined and intensity [MFI] at time point) 4 (MFI at 0 min) 3 100%. graphed. The Pearson correlation coefficient and linear regression analysis were used to evaluate the relationship between FAK or Csk expression and CD69 expression Lck Y505 phosphorylation. Control and FAK-deficient Jurkat cells were suspended at 1 3 107 cells/ml Immunoprecipitations in RPMI 1640 and stimulated with various doses of anti-CD3 for 4 h at 37˚C. Cells were washed with FACS buffer and stained on ice with Control or FAK-deficient cells were stimulated with 2 mg/ml soluble anti- PE/Cy5 anti-human CD69 for 30 min. Samples were collected using an CD3 for various times. These cells were lysed with immunoprecipitation Accuri flow cytometer. The MFI of each sample was determined using the lysis buffer, and Csk was then immunoprecipitated using 2 mg anti-Csk Ab Accuri Cflow Plus software. The data were normalized such that CD69 and protein A/G beads, as described (9, 40). To precipitate the TCR expression at 0 min was equal to 1, and the average normalized value from complex from CD4 hAPBTs, the cells were treated with soluble anti-CD3 three experiments 6 SEM was plotted using Microsoft Excel. and anti-CD4 with or without anti-mouse IgG, and the samples were lysed as above. The protein A/G beads were then added to the supernatants and Cellular imaging m 3 rotated overnight. Bound proteins were eluted using 25 l hot 2 buffer, Control or Fak-deficient T cells were stimulated on glass chamber slides and samples were boiled at 95˚C for 4 min. coated with anti-CD3 (45). Cells were fixed with 3.2% paraformaldehyde Cytokine production and permeabilized with 0.25% Triton X-100. After blocking, primary Abs against ZAP70 pY319 or total Csk were then diluted in SEA BLOCK Control and FAK-deficient cells were washed and resuspended at 6 3 105 buffer prepared in PBS and incubated overnight at 4˚C. Secondary Abs cells/ml in complete RPMI and stimulated with various doses of plate-bound were also incubated overnight at 4˚C. The images were obtained using the OKT3 for 24 h. The production of IL-2 or IFN-g was determined by stan- Leica AM TIRF MC imaging system found in the University of Iowa’s dard tetramethylbenzidine sandwich ELISA. The absorbance at 490 nm was Central Microscopy Core. Images from 6–12 randomly selected fields per measured using the Epoch plate reader (Biotek). Data were then normalized treatment were obtained using the 1003 oil objective and a TIRF align- to account for the variations between human donors and/or independent ment of 150 nm at room temperature. The GFP-TIRF or mCherry-TIRF experiments. The relative amount of cytokine that was produced in each laser intensity and exposure times were set using the control cells stimu- sample was determined using this formula: percentage of maximum cytokine lated for 3 min for the Jurkat cells or 7.5 min for CD4 hAPBTs, and these value in Fak miRNA-treated sample = (concentration of sample 4 maximum settings were kept constant for the remaining samples. Alternatively, the concentration in Fak miRNA-treated sample) 3 100%. The mean 6 SEM cells were stained using FITC-conjugated phalloidin overnight at 4˚C and from three to four independent experiments was then calculated. imaged using the 1003 oil objective and the GFP-epifluorescence filter. All images were acquired using the Leica AF software and processed using TCR downregulation the Fiji software package. 7 Cells were resuspended at 1 3 10 cells/ml in RPMI 1640 and incubated for 10 Imaging analysis min at 37˚C. The cells were then stimulated with 1 mg/ml soluble OKT3 for various times, transferred to ice, and fixed using 1% ice-cold methanol-free All image analyses were performed using the Coloc 2 plug-in for the Fiji formaldehyde for 5 min. Cells were then stained with PE anti-human ab on software package. To determine percentage of colocalization, 25 randomly ice for 30 min. Cells were washed with FACS buffer (PBS, 5% FBS, and selected cells from each of the four independent Jurkat or three independent by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd.

FIGURE 3. The kinetics or extent of TCR signaling is altered in CD4 hAPBTs that lack FAK. (A) CD4 http://classic.jimmunol.org hAPBTs were transduced with a len- tivirus containing miRNAs against luciferase (Luc) or FAK. The cells were then stimulated by soluble anti- CD3 and anti-CD4 cross-linking for various times. The site-specific phos- phorylation of LAT Y191, SLP-76 Downloaded from Y128, and AKT S473 was analyzed by immunoblotting. The expression of FAK, Pyk2, and actin was also examined. (B) Percentage of phos- phorylation for LAT Y191, SLP-76 Y128, and Akt S473 relative to the luciferase miRNA-transduced cells is depicted. Data are shown as mean of three experiments 6 SEM. *p # 0.05, **p # 0.01. 6212 FAK INHIBITS TCR FUNCTION

CD4 hAPBT experiments were analyzed. The Manders correlation coef- down of FAK in Jurkat cells did not affect Pyk2 expression, nor were ficients were then determined and plotted using GraphPad Prism 5. Any cell Lck, Csk, SLP-76, LAT, Grb2, and PLC-g1 protein levels altered , that had a Costes p value 0.9 was excluded from further analysis to (Fig. 1C, 1D). Thus, the Fak 3183 miRNA selectively and potently reduce the likelihood of false-positive colocalization. The mean channel intensity from these cells was also recorded to determine the amount of inhibits FAK expression in Jurkat cells. Csk and ZAP70 phosphotyrosine 319 staining. Stimulated cells that were FAK negatively regulates TCR-induced signaling in Jurkat stained with the secondary Abs alone served as the negative control for these experiments. cells and CD4 hAPBTs Statistical analysis Because FAK appears to integrate receptor-mediated signals that control actin reorganization in immune cells (32–35), we examined Statistical analysis was performed in Microsoft Excel using a two-tailed t actin polymerization upon TCR induction. Jurkat cells expressing test assuming equal variance. the Luc or FAK-specific miRNAs were stimulated on glass chamber slides treated with anti-TCR and stained to detect actin filaments Results that form upon T cell spreading (45). As shown in Supplemental miRNAs repress FAK expression in human T cells Fig. 1A and 1B, the intensity of phalloidin staining and the degree To elucidate whether FAK is required for TCR function in mature of cellular spreading as measured by changes in cell area were human T cells, we transiently suppressed FAK expression using comparable in control and FAK-deficient Jurkat cells. Therefore, it miRNAs. Seven RNA interference sequences were selected and used does not appear that TCR-induced actin remodeling that drives to replace the endogenous targeting sequences of human miR30 (39). cellular spreading requires FAK. We then tested the silencing efficacy of these sequences by tran- The actin cytoskeleton also regulates TCR expression on resting siently transfecting the Jurkat E6.1 human T cell line. By 72 h after and activated T cells (29). As such, we next examined TCR levels transfection, only the sequence corresponding to nt 3183–3202 by flow cytometry. As shown in Supplemental Fig. 1C, the steady- inhibited FAK protein expression by .90% compared with the state expression of the TCR was comparable in both cell lines. control sequence against firefly luciferase (Luc miRNA) (Fig. 1A). Additionally, no differences in the loss of TCR surface expression As shown in Fig. 1B, FAK knockdown was detectable at 48 h and were observed following receptor activation in the control or FAK- maximal suppression occurred at 72–96 h after transfection. deficient cells (Supplemental Fig. 1D). Thus, basal TCR expression Pyk2 is also expressed in human T cells and is activated by TCR and ligand-induced TCR downregulation are normal in the absence stimulation (43, 46). Pyk2 shares 45% amino acid identity to FAK of FAK. andisoverexpressedinFAK2/2 cells, where it compensates for The actin cytoskeleton network also regulates signaling down- multiple cellular functions (47–49). We found that transient knock- stream of the TCR (28, 29). Therefore, we next investigated whether by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd.

FIGURE 4. TCR-induced effector func- tions are enhanced in FAK-deficient T cells. (A) CD69 expression in control or FAK- depleted Jurkat cells stimulated for 4 h with the indicated doses of soluble OKT3. The graph shows the mean fold change over the http://classic.jimmunol.org unstimulated samples 6 SEM from three in- dependent experiments. (B) Control or FAK- deficient Jurkat cells were stimulated for 24 h with various doses of plate-bound anti- TCR, and IL-2 production was measured by ELISA. The mean normalized value 6 SEM from three independent experiments Downloaded from is shown. (C) CD4 hAPBTs were transduced with luciferase (Luc) or Fak-specific miRNAs. Cells were then stimulated with various con- centrations of plate-bound anti-TCR for 24 h. The production of IL-2 and IFN-g was measured by ELISA. The mean normalized value 6 SEM of at least three independent replicates is shown. The Journal of Immunology 6213

TCR-induced signaling was altered in the absence of FAK. To that proteins was strikingly different, however. When FAK expression end, control and FAK-deficient Jurkat cells were stimulated for was silenced in Jurkat cells, the site-specific phosphorylation of LAT, various times with a soluble, agonistic anti-TCR Ab. We then ex- PLC-g1, and SLP-76 was enhanced by 2- to 3-fold at 1–5 min after amined broad changes in early TCR-induced signaling using an TCR stimulation (Fig. 2C, 2D). These data demonstrate that FAK antiphosphotyrosine immunoblot. Surprisingly, TCR-inducible sig- suppresses the level of early TCR-mediated signaling in Jurkat cells. naling appeared to be enhanced in the FAK-deficient Jurkat cells as In Jurkat cells, many proximal signaling molecules including measured by an antiphosphotyrosine immunoblot (Fig. 2A). This PLC-g1 and Pyk2 have exaggerated TCR-induced activation com- global increase in tyrosine phosphorylation was not due to altered pared with hAPBTs (40). Because of these differences, we also protein expression of Lck, LAT, SLP-76, PLC-g1, or Csk after TCR examined whether FAK deficiency altered TCR-induced signaling induction (Fig. 2B). To confirm these findings, we employed a in primary human T cells. Transducing CD4 hAPBTs with GFP- quantitative method to examine changes in TCR-induced signaling expressing lentiviruses containing the Luc- or FAK-specific miR- using phospho-specific Abs and immunoblotting (42). Following NAs led to .75% silencing of FAK expression while having no activation with the stimulatory anti-TCR Ab, the site-specific effect on Pyk2 protein levels (Fig. 3A), demonstrating that this phosphorylation of LAT Y191, PLC-g1Y783,andSLP-76Y128 suppression vector is also highly specific in primary human CD4 was examined. As shown in Fig. 2C and 2D, the phosphorylation T cells. FAK-deficient CD4 hAPBTs initially displayed lower LAT kinetics of LAT, SLP-76, and PLC-g1 were comparable between Y191 phosphorylation after TCR activation. However, the phos- Luc and Fak miRNA-treated Jurkat cells, with maximum phos- phorylation of LAT appeared to last longer in FAK-deficient cells, phorylation occurring at 1–2 min after stimulation and returning to although this change was not significant (Fig. 3B). The TCR-inducible baseline by 15 min. The magnitude of the phosphorylation of these phosphorylation of SLP-76 was also significantly extended in the

FIGURE 5. Recruitment of Csk to membrane and TCR complex is im- paired in FAK-deficient Jurkat T cells. (A) Cells were stimulated on anti- TCR–coated glass chamber slides for various times and stained with Abs to detect ZAP70 phosphotyrosine 319

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. and total Csk. Cells were also stained with secondary Abs alone (US) as a negative control. The images are representative of four independent experiments. The white scale bar is equal to 5 mm. (B) For every ex- periment, 25 cells from each time point were analyzed using the Fiji analysis software. The intensity of Csk and ZAP70 phosphotyrosine 319 http://classic.jimmunol.org staining from 100 cells analyzed from four independent experiments was plotted. The horizontal line depicts the mean value (top graphs). The Manders correlation coefficients were used to calculate Csk and phospho- ZAP70 Y319 colocalization. The

Downloaded from Manders coefficients from 100 total Jurkat cells analyzed from four in- dependent experiments were plotted. The horizontal line represents the mean value (bottom graphs). The p values reflect the statistical differ- ences between the control and FAK- deficient cells at the indicated times. n.s. means p . 0.05. 6214 FAK INHIBITS TCR FUNCTION

absence of FAK (Fig. 3B). We next determined whether Akt that end, we measured differences in CD69 expression and cytokine phosphorylation was altered, because this kinase regulates many production. As shown in Fig. 4A, Jurkat cells treated with the T cell functions, including cytokine production (50). Interestingly, Luc miRNA showed a modest upregulation in the expression of the phosphorylation of Akt S473 was significantly enhanced in CD69 when stimulated with 100 ng/ml soluble anti-TCR. CD69 FAK-deficient cells compared with the control cells (Fig. 3B). expression in these cells had increased CD69 by ∼2-fold after Together, these data reveal that TCR-induced signaling is en- stimulation with 250 ng/ml anti-TCR. By comparison, CD69 up- hanced and/or prolonged in the absence of FAK, suggesting that regulation in FAK-deficient cells was similar to the control cells FAK feedback inhibits TCR-mediated signaling in human T cells. following stimulation with 2- to 4-fold lower doses (25–50 ng/ml) of anti-TCR. The control and Fak miRNA-treated cells showed FAK controls the threshold of TCR activation in human T cells similar CD69 responses at 250 ng/ml anti-TCR (Fig. 4A). We also We next addressed whether the changes in TCR-induced signaling found FAK-deficient Jurkat cells produced 2- to 3-fold more IL-2 described above altered downstream functions in human T cells. To production after stimulation with 0.5–1 mg/ml anti-TCR (Fig. 4B). by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 6. Csk membrane and TCR localization are altered in FAK-deficient CD4 hAPBTs. (A) CD4 hAPBTs were transfected with the Luc or FAK- specific miRNAs and incubated for 72 h. The cells were stimulated for 7.5 min with anti-TCR coated onto glass chamber slides. These cells were then stained with anti–phospho-ZAP70 Y319 and anti-Csk, followed by the appropriate secondary Abs. Representative cells from three separate human donors are shown. The white scale bar is equal to 5 mm. (B) The intensity of Csk and ZAP70 pY319 staining and colocalization between these proteins was analyzed as in Fig. 9. The values from 75 cells taken from three independent experiments are shown, and the mean values are represented by the horizontal lines. The p values reflect the statistical differences between the control and FAK-deficient cells at the indicated times. n.s. means p . 0.05. The Journal of Immunology 6215

FAK-deficient CD4 hAPBTs secreted ∼5-fold more IL-2 and ∼2- CD4 hAPBTs. At the 0-min time point, the addition of the cross- fold more IFN-g following stimulation with 0.25–0.5 mg/ml anti- linking Ab caused Csk to associate with the TCR-CD4 complex. TCR. No significant differences were observed at the 1 mg/ml This increase in Csk’s association with the TCR-CD4 complex dose (Fig. 4C). Thus, TCR induction occurs at lower doses of could be the result of postlysis stimulation. These data may also stimulation in the absence of FAK, suggesting that this protein indicate that, upon cross-linking CD3 with CD4, the association fine-tunes the responsiveness of T cells to TCR activation. between Csk and CD3 is stabilized (57), allowing us to detect the interaction by immunoprecipitation. We also attempted to verify The localization of Csk is altered in FAK-deficient cells these interactions using TIRF microscopy; however, FAK-specific In other cell types, FAK associates with Csk (51–53). TCR-induced Abs detected many nonspecific bands and could therefore not be signaling is enhanced in Csk-deficient human T cells, and these used for imaging studies (Supplemental Fig. 2 and N. Chapman cells are also more responsive to TCR induction (12, 13). Because and J. Houtman, unpublished observations). These data show that this phenotype is strikingly similar to what we observed in FAK- FAK is recruited to the TCR-CD4 complex, where it associates deficient human T cells, we investigated whether FAK controlled with Lck and its inhibitor Csk. Thus, FAK could regulate the Csk function in human T cells. Csk is active when it is recruited to function of these . the plasma membrane (16–18). To assess whether Csk was differ- FAK, Csk, and Lck formed a stable complex in human T cells, entially recruited to the T cell membrane after TCR stimulation, we and FAK-deficient T cells displayed hyperactive TCR function used TIRF microscopy. This type of microscopy will only excite similar to Csk-deficient T cells. Therefore, FAK could serve as proteins labeled with fluorochromes when they are within 100–200 a scaffolding protein to recruit Csk to its substrate, Lck. To test nm from the cell-chamber slide interface, and is therefore a useful this possibility, we stimulated control and FAK-deficient Jurkat way to track the recruitment of cytoplasmic proteins to the mem- cells with soluble anti-TCR for various times and performed Csk brane (54). As seen in Fig. 5, Csk was detectable in the membranes immunoprecipitations. We found that a cellular fraction of Lck was of TCR-stimulated control Jurkat cells at 3 min after receptor ac- bound with Csk in TCR-stimulated cells. Strikingly, the TCR- tivation, and its membrane expression increased over time. These inducible association between Lck and Csk was greatly reduced results are consistent with previous membrane fractionation studies in the absence of FAK (Fig. 7B). Thus, FAK facilitates complex (17, 18). Csk was also found in the membrane of the control CD4 formation between Csk and Lck after TCR induction. hAPBTs after TCR stimulation (Fig. 6). Strikingly, there was a The TCR-inducible phosphorylation and function of Lck are significant decrease in the intensity of Csk staining after TCR altered in FAK-deficient T cells stimulation in FAK-deficient Jurkat cells and CD4 hAPBTs (Figs. 5, 6). We also analyzed phosphor-ZAP70 Y319 staining as an internal The recruitment of Csk to the membrane, the TCR, and/or Lck was staining control, and we observed no detectable differences in the defective in FAK-deficient T cells (Figs. 5, 6). In Csk-deficient amount of phospho-ZAP70 Y319 that was present in the membrane T cells, the phosphorylation of Lck Y394 and Y505 is enhanced fractions (Figs. 5, 6). The latter observation is most likely due to the and reduced, respectively, and these alterations result in enhanced fact that ZAP70 turnover at the TCR is very rapid at these times Lck activity (12, 13). To examine whether the defects in Csk lo- (55). We did not select for FAK-deficient cells in these assays; calization were correlated with differences in Lck phosphoryla-

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. therefore, those cells that had high Csk intensity were most likely tion, we stimulated control or FAK-deficient Jurkat cells and CD4 still FAK sufficient. These results show that Csk membrane re- hAPBTs and examined the phosphorylation of Lck Y394 and cruitment is defective in FAK-deficient T cells. Y505. We used an anti-Src pY416 Ab to detect changes in Lck Csk interacts with the TCR complex (56, 57). Therefore, we next Y394 phosphorylation, which recognizes all Src kinases when determined whether the Csk and TCR association was mediated by FAK. Using phospho-ZAP70 Y319 as a surrogate marker for the TCR (55), we found that a small fraction (15–20%) of membrane- associated Csk colocalized with phosphorylated ZAP70 at 3–7.5 min after TCR stimulation. Similarly, 25–30% of phosphorylated http://classic.jimmunol.org ZAP70 colocalized with Csk in Jurkat cells (Fig. 5B, bottom graphs). When FAK expression was suppressed, the amount of phosphorylated ZAP70 that colocalized with Csk was significantly decreased at 5–7.5 min after stimulation (Fig. 5). Similar results were also obtained using CD4 hAPBTs, although there was sub- stantially more colocalization between Csk and phosphorylated

Downloaded from ZAP70 in these cells (Fig. 6). Jurkat cells are of thymocyte origin and do not express CD4 (40), which may explain these differences. These data indicate that Csk recruitment to the TCR complex is also impaired in the absence of FAK. The results described above suggest that FAK associates with Csk at the TCR. To test this possibility, we stimulated CD4 hAPBTs for various times and pulled down the TCR/CD4 complex using the stimulatory anti-CD3 and anti-CD4 Abs in the absence or presence of an anti-mouse cross-linking Ab. We found that FAK and Csk FIGURE 7. FAK forms a complex with Csk and Lck in human T cells. (A) Activated CD4 T cells were stimulated by anti-CD3 and anti-CD4 cross- coimmunoprecipitated with the TCR-CD4 complex (Fig. 7A). linking for various times. The TCR/CD4 complex was then immunoprecip- Consistent with published data (11, 51, 58, 59), Lck was also itated, and the expression of FAK, Csk, and Lck was analyzed by immu- associated with the TCR-CD4 complex (Fig. 7A). Interestingly, noblotting. Data are representative of three independent experiments. (B) FAK and Lck were found to coimmunoprecipitate with the TCR- Control or Fak-deficient Jurkat T cells were stimulated for various times with CD4 complex in the absence of cross-linking Ab, suggesting that anti-CD3. Csk was immunoprecipitated, and its association with Lck was these proteins constitutively associate with CD3 and/or CD4 in assessed by immunoblotting. Data are representative of three experiments. 6216 FAK INHIBITS TCR FUNCTION

they are phosphorylated on their activating sites. Consistent with comparable in the control and FAK-deficient T cells .5 min after previous reports (7, 8, 12), both Lck Y394 and Y505 were phos- receptor activation. Because ITAM phosphorylation appears to be phorylated in unstimulated Jurkat cells and CD4 hAPBTs, and the limiting factor that mediates ZAP70 recruitment to the TCR TCR stimulation enhanced the phosphorylation of these sites in the (60), these data may also explain why no significant differences in control cells. Strikingly, the levels of Lck Y505 induced by TCR phospho-ZAP70’s membrane recruitment were observed by TIRF activation were significantly reduced in the FAK-deficient Jurkat microscopy (Figs. 5, 6). Collectively, these results suggest that cells and CD4 hAPBTs. The phosphorylation of Lck Y394, the Lck’s function is enhanced in FAK-deficient T cells and that FAK- autophosphorylation site, was also increased by ∼2-fold in the deficient T cells are more poised for TCR activation, resulting in FAK-deficient cells (Fig. 8). This result suggests that Lck’s enzy- increased TCR-dependent signaling and function. matic activity is enhanced in FAK-deficient cells. Thus, the defects in Csk recruitment observed in FAK-deficient cells are correlated FAK expression correlates with Lck Y505 phosphorylation in with decreased Lck Y505 and increased Y394 phosphorylation. human T cells In Csk-deficient Jurkat cells, TCRz-chain phosphorylation occurs FAK expression has been reported to increase upon T cell activa- with faster kinetics and is modestly elevated at early times after tion (61). We found that FAK protein levels were upregulated TCR stimulation (13). Therefore, we also examined whether TCRz by 3- to 10-fold in primary CD4 T cells that were stimulated for Y142 phosphorylation was altered in FAK-deficient Jurkat cells. 1–4 d with anti-CD3 and anti-CD28, whereas Pyk2 levels remained In the Jurkat cells expressing the Luc-specific miRNA, the site- unchanged (Fig. 10A, 10B). The human T cell lines Jurkat E6.1 specific phosphorylation of TCRz Y142 was detected in unstimu- and HuT78 also expressed slightly more FAK protein relative to lated cells and increased by ∼40% after 15 min of stimulation (Fig. primary CD4 T cells (Fig. 10C, 10D), indicating that FAK-dependent 9A, 9B). By comparison, the phosphorylation of TCRz Y142 was functions may be differentially regulated in these cell lines com- enhanced by ∼1.5-fold in the FAK-deficient T cells at 0–5 min pared with primary human CD4 T cells. after TCR stimulation (Fig. 9A, 9B). Thus, FAK-deficient T cells Like FAK expression, Lck Y505 phosphorylation was also in- have higher levels of TCRz phosphorylation in resting conditions creased in activated CD4 hAPBTs (Fig. 10A, 10B). Basal Lck and early after TCR stimulation. TCRz Y142 phosphorylation was Y505 phosphorylation was also augmented in HuT78 T cells and by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. FIGURE 8. The phosphorylation of Lck is altered in FAK-deficient human T cells. (A) Luciferase (Luc) or Fak miRNA-containing Jurkat T cells were stimulated as in Fig. 2. The site-specific phosphorylation of Src Y416 (equivalent to Lck Y394) and Lck Y505 was then analyzed by immunoblotting. (B) Quantification http://classic.jimmunol.org of three to four experiments exam- ining the phosphorylation of Lck in control and FAK-deficient Jurkat cells. Data are shown as mean 6 SEM. (C) Control or Fak-deficient primary human CD4 T cells were stimulated as in Fig. 3. (D) Percentage of phos- Downloaded from phorylation of Src Y416 and Lck Y505 in stimulated control or FAK- depleted primary human CD4 T cells was determined. Data are depicted as mean of three experiments 6 SEM. *p # 0.05, **p # 0.01. The Journal of Immunology 6217

mediated signaling is enhanced or extended and that TCR-induced effector functions are augmented in Jurkat cells and CD4 hAPBTs that lack FAK. It appears that FAK controls the function of the Lck by recruiting Csk to the membrane and/or receptor complex in TCR- stimulated cells (Supplemental Fig. 3). These studies reveal that FAK regulates Csk activity and Lck/Fyn function downstream of the TCR and, to our knowledge, demonstrate for the first time that FAK is a negative regulator of TCR function. While this manuscript was under review, Wiemer et al. (31) pub- lished a report examining how FAK controlled CD4 T cell activation. To do so, FAK’s function was impaired using PF562271, an in- hibitor that suppresses the catalytic function of both FAK and its related kinase, Pyk2. In contrast to the results we obtained using FAK-deficient human CD4 T cells, treatment with PF562271 ap- peared to suppress proximal TCR-mediated signaling and prolif- eration. Wiemer et al. (31) used doses of PF562271 that were substantially higher than those required to suppress FAK’s enzy- matic activity in vitro and in cell-based assays (62). This is a major limitation of their study, as higher doses of PF562271 have been reported to suppress the in vitro catalytic functions of Pyk2, cyclin-dependent kinases, Fyn, and Lck (62). To confirm the results of their inhibitor experiments, this group also conditionally deleted FAK in murine CD4 T cells, which resulted in a ∼65% knockdown of FAK protein expression. In contrast to the results obtained with PF562271, anti-CD3– and anti-CD28–induced proliferation was FIGURE 9. TCRz-chain phosphorylation is altered in FAK-deficient normal to elevated in these FAK-deficient murine CD4 T cells (31). T cells. (A) Control or FAK-deficient Jurkat T cells were stimulated as in Therefore, it is likely that the differences seen in this published Fig. 2. The phosphorylation of TCRz Y142 was then assessed by immu- noblotting. (B) Quantification of four independent experiments examining study and ours are due to off-target effects of PF562271. TCRz Y142 phosphorylation in the control and FAK-deficient Jurkat cells. The activation of Lck and Fyn is tightly regulated to prevent The graph shows the mean 6 SEM. inappropriate T cell activation. This regulation is achieved by altering Lck’s localization or phosphorylation (11, 63). Lck Y505 Jurkat cells compared with primary human T cells (Fig. 10C, plays a seminal role in inhibiting Lck activity: the in vitro kinase 10D). Thus, it appeared that Lck Y505 phosphorylation correlated activity of Lck is reduced when Lck Y505 is phosphorylated, and with FAK expression. To investigate this possibility, linear re- T cells that express a Lck Y505F mutant are hyperactivated (64–

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. gression and Pearson correlation analyses were performed. As 66). Defective TCR-inducible Lck Y505 phosphorylation has been shown in Fig. 10E, there was a strong linear relationship between observed in hyperresponsive CD4 T cells isolated from patients Lck Y505 phosphorylation and FAK expression (p = 0.093), and with acute coronary syndromes (67). Thus, deregulated Lck Y505 the Pearson correlation between these proteins trended toward phosphorylation can promote T cell hyperactivation. In this study, significance (p = 0.062). Similarly, a significant linear relationship we showed that the TCR-inducible phosphorylation of Lck Y505 (*p=0.025) and Pearson correlation (*p = 0.025) were observed was significantly decreased in the absence of FAK. This defect when we compared Lck Y505 phosphorylation and FAK expres- was also accompanied by an increase in Lck Y394 autophosphor- sion in human nonstimulated and activated CD4 T cells, unsti- ylation (8) and enhanced TCR-induced function. Our data support mulated Jurkat cells, and unstimulated HuT78 T cells (Fig. 10F). the idea that Lck Y505 phosphorylation is vitally important to http://classic.jimmunol.org Thus, changes in FAK expression appear to serve as a rheostat to inhibit TCR-induced responses and demonstrate that FAK is a tune both basal and TCR-inducible Lck Y505 phosphorylation in critical regulator of Lck Y505 phosphorylation downstream of the primary human CD4 T cells and transformed human T cell lines. TCR. This inhibitory function may be achieved by suppressing Lck’s We also examined whether Csk expression and Lck Y505 catalytic function and/or limiting its accessibility to its protein sub- phosphorylation were correlated. Unlike FAK and Lck pY505 strates, including the TCRz-chain (14, 15). expression, Csk expression did not change after TCR stimulation Several reports have demonstrated that Csk regulates TCR func-

Downloaded from (Fig. 10A, 10B), and the expression of the Lck pY505 and Csk tion in both the basal and activated states. Indeed, knocking down was not correlated as determined by linear regression analysis Csk or inhibiting its function increases TCR-dependent signaling (p=0.93) or Pearson correlation (p = 0.79; Fig. 10G). Jurkat cells and effector responses (12, 13, 18), demonstrating that this kinase and HuT78 T cells expressed more Csk protein at basal levels than is indispensable for proper mature T cell function. Csk been re- primary CD4 T cells (Fig. 10C). Therefore, the linear relationship ported to bind to the plasma membrane, where it exerts its inhibi- between Csk and Lck Y505 phosphorylation was stronger in these tory effects on TCR function (12, 16–18). We have confirmed by cells (p = 0.076). However, the Pearson correlation was not sig- high-resolution TIRF imaging that Csk is brought to the membrane nificant (Fig. 10H; p = 0.076). Therefore, unlike FAK, Csk ex- following TCR activation. Our data suggest that FAK regulates the pression is not increased after human CD4 T cells are activated, membrane localization and function of Csk in activated T cells. We and differences in Csk expression do not positively correlate with found that the translocation of Csk to the membrane following TCR enhanced Lck Y505 phosphorylation in human T cells. activation was dependent upon FAK. This reduction in Csk protein found in the membrane was correlated with increased Lck Y394 Discussion and TCRz-chain phosphorylation, decreased Lck Y505 phosphor- In this study, we evaluated the role that FAK plays in TCR-induced ylation, and increased sensitivity to TCR induction. However, we did effector function in human T cells. Surprisingly, we found that TCR- not see an increase in the magnitude of ZAP70 Y319 or Erk1/Erk2 6218 FAK INHIBITS TCR FUNCTION

FIGURE 10. FAK expression cor- relates with the phosphorylation of Lck tyrosine 505. (A) Primary hu- man CD4 T cells were activated in culture with magnetic beads coated with anti-CD3 and anti-CD28 in the presence of 100 U/ml rIL-2 for the indicated times. The expression of FAK, Pyk2, Csk, Lck Y505, or actin was assessed by immunoblotting. (B) The expression of the proteins was normalized to actin expression. The fold change in protein expression relative to day 0 stimulation was then calculated. The data are shown as mean of three experiments 6 SEM. (C) Equal numbers of freshly isolated CD4 T cells (unstimulated [US] CD4 T cell), CD4 T cells activated for 6 d with anti-CD3– and anti-CD28– coated beads (CD4 hAPBT), unsti- mulated HuT78 T cells, or unsti- mulated Jurkat E6.1 T cells were lysed. Immunoblotting was then used to examine FAK, phospho-Lck Y505, Lck, Csk, or actin expression. (D)Rel- ative expression of the various proteins was calculated as in (B). Fold change in protein expression relative to US CD4 T cell sample was calculated, and the mean of four experiments 6 SEM is shown. (E–H) Linear regres- sionandPearsoncorrelationanalyses were used to examine the relationship between Lck Y505 phosphorylation and average FAK expression or aver- age Csk expression in CD4 T cells (E,

by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. G) or in CD4 T cells and human T cell lines (F, H).

phosphorylation (data not shown). These observations are strikingly phospho-Y317 on PAG (22, 23). This interaction allows Csk to similar to those made using Csk-deficient Jurkat cells or human phosphorylate Lck Y505 to suppress tonic TCR signals to prevent CD4 T cells (13). Thus, FAK and Csk appear to cooperatively in- aberrant T cell development or activation (12, 13, 24, 71). After hibit TCR function following its activation. TCR stimulation, the current paradigm suggests that PAG is de- We also demonstrated that FAK associated with Csk at the ac- phosphorylated, which subsequently releases Csk from the plasma http://classic.jimmunol.org tivated TCR. Using TIRF microscopy, we also found that a small membrane (18, 19, 22). After Csk moves into the , this fraction of the membrane-associated Csk colocalized with phos- model then states that CD45 must dephosphorylate Lck Y505 to phorylated ZAP70. Thus, Csk may be actively sequestered from induce T cell activation (11, 25, 72). Csk is then recruited back the activated TCR complex, as was recently reported in murine into the plasma membrane once activated Fyn phosphorylates CD8 T cells (56). The Csk and phosphorylated ZAP70 interaction PAG Y317. Indeed, PAG overexpression inhibits TCR-dependent was significantly reduced in FAK-deficient Jurkat cells and CD4 signaling and function (19, 22), supporting the idea that PAG is

Downloaded from hAPBTs. Therefore, it appears that FAK regulates Csk recruitment an important regulator of Csk function in T cells. to or retention at the TCR, where Csk could inhibit the pool of Although this model persists as the mode by which Csk feedback active Lck that is found at the stimulated receptor (14, 58, 59). inhibits TCR signaling, several more recent publications have Further work is needed to address how FAK regulates Csk’s func- challenged this paradigm. First, we and others have demonstrated tion. We hypothesize that FAK Y397 serves to localize Csk to the that Lck Y505 phosphorylation does not decrease after TCR ac- plasma membrane after TCR induction. Interestingly, this site has tivation. Instead, Lck Y505 phosphorylation remains unchanged or the consensus-binding motif for the Src homology 2 (SH2) domain modestly increases upon TCR stimulation (7, 8, 12, 26). Second, of Csk (68). The SH2 domain of Csk has been reported to bind to despite the fact that Lck Y505 is hyperphosphorylated in CD45- FAK (51, 53); therefore, Csk may directly interact with FAK Y397. deficient T cells, Lck’s catalytic function is actually increased in Furthermore, FAK may be recruited to the plasma membrane and/or the absence of CD45, most likely because CD45 also dephos- TCR by directly binding Lck/Fyn or the TCR coreceptor CD4 (69, phorylates Lck Y394 (64, 72–74). Finally, Lck is catalytically 70). These hypotheses will be addressed in future experiments. active when it is phosphorylated on both Y394 and Y505 (8). The current model states that Csk’s function is regulated by the Thus, Lck Y505 dephosphorylation does not appear to be required plasma-membrane anchored protein, PAG. According to this model, to induce Lck’s catalytic function; however, it may serve to lo- Csk is associated with PAG via its SH2 domain, which binds to calize Lck to the TCR, where it can induce T cell activation (14). The Journal of Immunology 6219

Several reports have also called into question the relevance of Disclosures the PAG-Csk axis in regulating TCR signaling events. Csk’s SH2 The authors have no financial conflicts of interest. domain associates with PAG, and Csk’s enzymatic function is enhanced when bound to PAG (19, 22); however, the loss of PAG does not appear to limit Csk function. Consistent with this idea, References 1. Lloyd, C. M., and E. M. Hessel. 2010. Functions of T cells in asthma: more than genetic deletion of PAG in murine T cells did not dramatically just T(H)2 cells. Nat. Rev. Immunol. 10: 838–848. alter Csk’s localization to lipid rafts or inhibit T cell development 2. Smith-Garvin, J. E., G. A. Koretzky, and M. S. Jordan. 2009. T cell activation. or activation (20, 21). Similarly, knocking down PAG in human Annu. Rev. Immunol. 27: 591–619. 3. Jutel, M., and C. A. Akdis. 2011. T-cell subset regulation in atopy. Curr. Allergy T cells modestly increased early TCR-mediated signaling events, Asthma Rep. 11: 139–145. but actually suppressed downstream effector functions (27, 75). 4. Ja¨ger, A., and V. K. Kuchroo. 2010. Effector and regulatory T-cell subsets in These results are in sharp contrast to Csk-deficient T cells that autoimmunity and tissue inflammation. Scand. J. Immunol. 72: 173–184. 5. Atalar, K., B. Afzali, G. Lord, and G. Lombardi. 2009. Relative roles of Th1 and have developmental defects and enhanced TCR-induced activation Th17 effector cells in allograft rejection. Curr. Opin. Organ Transplant. 14: 23–29. (12, 13, 71). Additionally, after it is transiently displaced, Csk 6. Lahoute, C., O. Herbin, Z. Mallat, and A. Tedgui. 2011. Adaptive immunity in atherosclerosis: mechanisms and future therapeutic targets. Nat. Rev. Cardiol. 8: appears to be recruited back into the plasma membrane before 348–358. PAG is rephosphorylated (18, 19). These data strongly suggest that 7. Dong, S., B. Corre, K. Nika, S. Pellegrini, and F. Michel. 2010. T cell receptor PAG is not the primary regulator of Csk function in activated signal initiation induced by low-grade stimulation requires the cooperation of LAT in human T cells. PLoS One 5: e15114. T cells. It is possible that other membrane adaptors like LIME 8. Nika, K., C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, or Dok1 may recruit Csk to the membrane in the absence of PAG P. Polzella, V. Cerundolo, O. Dushek, et al. 2010. Constitutively active Lck kinase (12, 76). Alternatively, PAG may regulate Csk’s function in unsti- in T cells drives antigen receptor signal transduction. Immunity 32: 766–777. 9. Cruz-Orcutt, N., and J. C. Houtman. 2009. PI3 kinase function is vital for the mulated T cells to suppress tonic TCR signaling, whereas other function but not formation of LAT-mediated signaling complexes. Mol. Immunol. mechanisms may control Csk’s function following TCR activa- 46: 2274–2283. 10. Shim, E. K., S. H. Jung, and J. R. Lee. 2011. Role of two adaptor molecules SLP- tion. Moreover, PAG may only regulate Csk’s function at specific 76 and LAT in the PI3K signaling pathway in activated T cells. J. Immunol. 186: cellular locations distinct from sites of TCR activation. Our data 2926–2935. strongly suggest that FAK is a protein that recruits Csk to the TCR 11. Salmond, R. J., A. Filby, I. Qureshi, S. Caserta, and R. Zamoyska. 2009. T-cell receptor proximal signaling via the Src-family kinases, Lck and Fyn, influences complex after Ag stimulation in mature CD4 T cells and that this T-cell activation, differentiation, and tolerance. Immunol. Rev. 228: 9–22. process suppresses TCR-induced function. 12. Schoenborn, J. R., Y. X. Tan, C. Zhang, K. M. Shokat, and A. Weiss. 2011. Immune cell activation via Ag receptors is intricately linked to Feedback circuits monitor and adjust basal Lck-dependent events in T cell re- ceptor signaling. Sci. Signal. 4: ra59. protective immunity against pathogens and in the development of 13. Vang, T., H. Abrahamsen, S. Myklebust, J. Enserink, H. Prydz, T. Mustelin, allergic disease and autoimmunity (77–80). Like the TCR, signal M. Amarzguioui, and K. Tasken. 2004. Knockdown of C-terminal Src kinase by ε siRNA-mediated RNA interference augments T cell receptor signaling in mature transduction downstream of the BCR and Fc R is initiated by T cells. Eur. J. Immunol. 34: 2191–2199. members of the Src family kinases and suppressed by Csk (11, 81, 14. Rossy, J., D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus. 2013. Con- 82). Our data support a model wherein changes in FAK expression formational states of the kinase Lck regulate clustering in early T cell signaling. Nat. Immunol. 14: 82–89. regulate the activity of Lck by recruiting its inhibitor Csk to the 15. Stirnweiss, A., R. Hartig, S. Gieseler, J. A. Lindquist, P. Reichardt, L. Philipsen, TCR complex. Interestingly, we found that FAK associated with L. Simeoni, M. Poltorak, C. Merten, W. Zuschratter, et al. 2013. T cell activation results in conformational changes in the Lck to induce its by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. both Lck and the TCR/CD4 complex, an interaction that was activation. Sci. Signal. 6: ra13. previously reported for the related proteins Lyn and IgM-BCR 16. Cloutier, J. F., L. M. Chow, and A. Veillette. 1995. Requirement of the SH3 and (83). Thus, FAK could similarly inhibit BCR function by re- SH2 domains for the inhibitory function of tyrosine p50csk in T . Mol. Cell. Biol. 15: 5937–5944. cruiting Csk to the receptor complex to block Lyn activation. We 17. Ota´hal, P., S. Pata, P. Angelisova´,V.Horejsı´, and T. Brdicka. 2011. The effects of also showed that changes in FAK expression regulated the Ag membrane compartmentalization of csk on TCR signaling. Biochim. Biophys. sensitivity of the TCR. Similar to our observations, the magnitude Acta 1813: 367–376. ε 18. Torgersen, K. M., T. Vang, H. Abrahamsen, S. Yaqub, V. Horejsı´, B. Schraven, of Fc R-mediated signaling was higher in cells that expressed low B. Rolstad, T. Mustelin, and K. Taske´n. 2001. Release from tonic inhibition of levels of FAK (84). Thus, the expression of FAK may also fine- T cell activation through transient displacement of C-terminal Src kinase (Csk) tune FcεR-mediated signaling. Together, these data suggest that from lipid rafts. J. Biol. Chem. 276: 29313–29318. 19. Davidson, D., M. Bakinowski, M. L. Thomas, V. Horejsi, and A. Veillette. 2003. http://classic.jimmunol.org FAK may be a universal negative regulator of Ag receptor sig- Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipid naling, a finding that would have broad implications in the treat- raft-associated transmembrane adaptor. Mol. Cell. Biol. 23: 2017–2028. 20. Dobenecker, M. W., C. Schmedt, M. Okada, and A. Tarakhovsky. 2005. The ment of a variety of human disorders. ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogen- Collectively, our data demonstrate that FAK is a negative reg- esis and T-cell development and function. Mol. Cell. Biol. 25: 10533–10542. ulator of TCR function in human T cells. The fact that FAK ex- 21. Xu, S., J. Huo, J. E. Tan, and K. P. Lam. 2005. Cbp deficiency alters Csk lo- calization in lipid rafts but does not affect T-cell development. Mol. Cell. Biol. pression appears to fine-tune TCR activation suggests that FAK 25: 8486–8495.

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