Actin-Dependent Manner Src, Paxillin, and Focal Adhesion

Ligation of HLA Class I Molecules on Endothelial Cells Induces Phosphorylation of Src, Paxillin, and Focal Adhesion Kinase in an Actin-Dependent Manner This information is current as of September 27, 2021. Yi-Ping Jin, Ram Pyare Singh, Ze-Ying Du, Ayyappan K. Rajasekaran, Enrique Rozengurt and Elaine F. Reed J Immunol 2002; 168:5415-5423; ; doi: 10.4049/jimmunol.168.11.5415

http://www.jimmunol.org/content/168/11/5415 Downloaded from

References This article cites 65 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/168/11/5415.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• 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 by guest on September 27, 2021

*average

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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ligation of HLA Class I Molecules on Endothelial Cells Induces Phosphorylation of Src, Paxillin, and Focal Adhesion Kinase in an Actin-Dependent Manner1

Yi-Ping Jin,* Ram Pyare Singh,* Ze-Ying Du,* Ayyappan K. Rajasekaran,* Enrique Rozengurt,† and Elaine F. Reed2* The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity Downloaded from blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection. The Journal of Immunology, 2002, 168: 5415–5423. http://www.jimmunol.org/ form of chronic rejection, termed accelerated transplant molecules with human anti-HLA Abs recognizing polymorphic arteriosclerosis, is the major cause of solid organ graft residues located on the class I H chain transduces activation sig- A failure after the first year posttransplantation (1, 2). nals in EC and SMC and initiates cell proliferation in a model Transplant arteriosclerosis is characterized by the proliferation of relevant to the development of transplantation-associated vascu- intimal smooth muscle cells (SMC)3 and endothelial cells (EC) in lopathies (14Ð18). Thus, engagement of class I molecules by anti- the walls of the arteries of the transplanted organ resulting in oc- HLA Abs stimulated tyrosine phosphorylation of intracellular pro- clusion of the vessels and fibrosis of the graft. Anti-HLA Abs have teins, increased fibroblast growth factor (FGF) receptor cell

long been implicated in the process of chronic rejection, as several surface expression, and enhanced proliferative responses to basic by guest on September 27, 2021 studies have shown that patients developing anti-donor HLA Abs FGF (bFGF). These results have led us to conclude that anti-HLA following transplantation are at increased risk of developing Abs can contribute to the process of chronic rejection by binding chronic rejection and graft loss (3Ð10). A consistent finding in to the surface of the endothelium and smooth muscle of the allograft arteriosclerotic lesions is Ig deposits in affected vessel walls graft and transducing signals that ultimately result in cell and within the media (2, 11, 12). In addition, allografts trans- proliferation. planted to Ig-deficient mice fail to develop fibrotic arteriopathic Numerous studies have shown that HLA class I molecules can lesions, whereas prominent fibrotic lesions occur in recipients de- transduce signals that regulate various aspects of cell metabolism, veloping humoral immunity to the allograft (13). including activation and cell growth, or cell cycle arrest and apo- Although anti-HLA Abs have been implicated in chronic rejec- ptosis (19Ð33). For example, in both resting and activated T and B tion, their precise role in the disease process is not well under- cells, Src and Syk kinases become activated following class I li- stood. In previous studies we have shown that ligation of class I gation, leading to phosphorylation of phospholipase C␥-1, generation of inositol triphosphate and diacylglycerol, and regulation of intracellular free ionized calcium (19, 32, 34). It has also been reported that phosphoinositide-3 kinase and threonine/serine ki- *Department of Pathology and Laboratory Medicine, School of Medicine, and †De- nases are involved in MHC class I-induced signal transduction in partment of Medicine, School of Medicine and Molecular Biology Institute, Univer- sity of California, Los Angeles, CA 90095 T and B cells (35). Furthermore, MHC class I ligation activates the Received for publication January 8, 2002. Accepted for publication March 28, 2002. Janus tyrosine kinase-2, leading to phosphorylation of the transcription factor STAT-3 (19, 20, 23, 36, 37). Cross-linking of class 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 I molecules has also been reported to induce apoptosis of T and B with 18 U.S.C. Section 1734 solely to indicate this fact. lymphocytes through the activation of the Src kinase p56Lck and 1 This work was supported by National Institute of Allergy and Infectious Diseases c-Jun N-terminal kinase activity (20). Although MHC class I mol- Grant RO1 AI 42819 and American Heart Association Grant 9750894A. ecules expressed by other cell types such as macrophages, mast 2 Address correspondence and reprint requests to Dr. Elaine F. Reed, Department of cells, fibroblasts, and ECs have been shown to transduce prolifer- Pathology and Laboratory Medicine, School of Medicine, University of California Immunogenetics Center, 1000 Veteran Avenue, Los Angeles, CA 90095. E-mail ad- ative signals, the intracellular signaling pathways remain to be dress: [email protected] elucidated (38). 3 Abbreviations used in this paper: SMC, smooth muscle cell; EC, endothelial cell; In the present study, we have investigated the intracellular signal FGF, fibroblast growth factor; bFGF, basic FGF; FAK, focal adhesion kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; PTK, protein transduction pathway triggered by the binding of anti-HLA class I tyrosine kinase. Abs to human ECs. This study is the first to show that binding

of anti-HLA Abs to class I molecules expressed on ECs induces quently washed with TBST and developed with an ECL obtained from tyrosine phosphorylation of Src kinases, including Src and Fyn, Amersham (Arlington Heights, IL). The phosphorylated bands were and the focal adhesion proteins focal adhesion kinase (FAK) and scanned using the GS-710 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA) and quantified using the Quantity One software program paxillin. Anti-HLA Abs mediate increased FGFR cell surface ex- (Bio-Rad). pression through a FAK-dependent pathway that requires the integrity of the actin cytoskeleton. Src kinase activity was not re- Flow cytometry analysis quired for class I-induced FGFR cell surface expression; however, it was necessary for class I-mediated redistribution of FGFRs to ECs were grown in 60-mm culture dishes and incubated with 10 ␮g/ml the nucleus. mAb W6/32 for up to 2 h. Where indicated, the cells were pretreated with inhibitors for 1 h before exposure to mAb W6/32. The cells were washed three times with HBSS and detached with 0.125% trypsin/0.05% EDTA. Materials and Methods Expression of FGFR was determined by indirect immunofluorescence on a Abs and chemicals FACScan flow cytometer as previously described (15). Briefly, ECs (0.5 ϫ 106) were incubated with a rabbit anti-FGFR polyclonal Ab (Upstate Bio- Cytochalasin D and propidium iodide were purchased from Sigma-Aldrich technology) for 30 min at 4¡C. The cells were washed three times and (St. Louis, MO). Latrunculin A was obtained from Molecular Probes (Eu- stained with a FITC-conjugated donkey anti-rabbit IgG (Jackson Immu- gene, OR). The Src kinase inhibitor PP2 and inactive analog PP3 were noResearch Laboratories). Following a 30-min incubation at 4¡C, the cells obtained from Calbiochem (La Jolla, CA). Anti-human bFGF neutralizing were washed and fluorescence was analyzed on a FACScan flow cytometer Ab and recombinant human bFGF were purchased from R&D Systems using CellQuest Software (both from BD Biosciences, Mountain View, CA). (Minneapolis, MN). The anti-phosphotyrosine mAb 4G10 and polyclonal Gates for forward and side scatter measurements were set on EC, and a rabbit anti-FGFR Ab were obtained from Upstate Biotechnology (Lake

minimum of 10,000 events was acquired. Instrument calibration was per- Downloaded from Placid, NY). The following Abs were used for immunoprecipitation: Anti- formed using CaliBRITE beads and FACScomp software (BD Biosciences). paxillin Ab (349; Transduction Laboratories, Lexington, KY); anti-FAK (2A7; Upstate Biotechnology); anti-v-Src (Oncogene Research Products, Boston, MA); and anti-Lck (3A5), rabbit polyclonal Ab against FAK (C- Immunohistochemical staining and confocal microscopy analysis 20), c-Src (N-16), Fyn (Fyn3), anti-Lyn (44), anti-extracellular signal-regulated kinase (ERK; K-23), and Flg (C-15) all from Santa Cruz Biotech- ECs were grown in four-well chamber slides (BD Biosciences) in EC nology (Santa Cruz, CA). Anti-phospho-p42/44 mitogen-activated protein growth medium until they were 80% confluent. The cell monolayers were 202 204 rinsed with PBS, fixed, and permeabilized with methanol for 20 min at (MAP) kinase (Thr /Tyr ) was purchased from Cell Signaling Tech- Ϫ http://www.jimmunol.org/ nology (Beverly, MA). Biotinylated anti-rabbit IgG, avidin-fluorescein, 20¡C. Cells were rehydrated with PBS for 10 min, incubated with 4% and fluorescent mounting medium were obtained from Vector Laboratories goat serum for1hatroom temperature to prevent nonspecific staining, and (Burlingame, CA). FITC-conjugated donkey anti-rabbit IgG was purchased incubated with the rabbit anti-FGFR-1 (anti-flg, C15) primary Ab (1/500 in from Jackson ImmunoResearch Laboratories (West Grove, PA). 4% goat serum in PBS) at 4¡C for overnight. The cells were washed three times with 0.5% Triton X-100 in PBS followed by staining with a biotin- Murine anti-HLA class I mAb ylated anti-rabbit secondary Ab (1/200 dilution in 4% goat serum in PBS; Vector Laboratories) at room temperature for 30 min. The cells were W6/32 (IgG2a), a mouse mAb reactive with a monomorphic epitope on washed three times and incubated with avidin-fluorescein (1/200 dilution in HLA class I Ags, was obtained from the American Type Culture Collection PBS; Vector Laboratories) for 30 min followed by staining with propidium (Manassas, VA). The mouse IgG used as an isotype control was supplied iodide (1 ␮g/ml; Sigma-Aldrich) for 10 min at room temperature. Slides

by Sigma-Aldrich. were mounted with fluorescent mounting medium (Vector Laboratories) by guest on September 27, 2021 and immunofluorescent staining was analyzed with OLYMPUS FLUO- Cells VIEW confocal laser scanning biological microscope (Olympus, Melville, Human aortic EC from single donors were obtained from Clonetics (San NY). Quantitation of the pixel intensities of FGFR-1 and propidium iodide Diego, CA) and maintained in EC basal medium, supplemented with 5% in the nuclei of EC were quantified by measuring the average pixel intensity FCS, 10 ng/ml human epidermal growth factor, 1 mg/ml hydrocortisone, 3 in each section using FLUOVIEW software version 2.1. Overall differences in mg/ml bovine brain extract, and 5 mg/ml gentamicin (Clonetics) at 37¡Cin fluorescent intensities between treated EC and controls was performed using ANOVA and Scheffe’s method for multiple pairwise comparisons (STATA a humidified incubator (5% CO2, 95% air). Cells from passages 3Ð8 were used. Cells were grown to a confluency of 80% and incubated for 16 h in Statistical Software, Release 7.0; Stata, College Station, TX). growth factor-free medium before use. Preparation of cell lysates and immunoprecipitation Results EC were treated with mAb W6/32 or mouse isotype control IgG for various Ligation of HLA class I molecules by anti-HLA Abs induces time points at 37¡C, washed three times with ice-cold PBS containing 1 tyrosine phosphorylation of EC proteins mM sodium orthovanadate, and lysed in buffer (containing 20 mM Tris To determine whether ligation of HLA class I molecules on EC (pH 7.9), 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, 10% glycerol, 1% ␮ with anti-HLA Abs induces tyrosine phosphorylation of intracel- Triton X-100, 10 mM NaF, 1 mM PMSF, 1 mM Na3VO4,10 g/ml apro- tinin, and 10 ␮g/ml leupeptin) for 10 min on ice. The cell lysates were lular proteins, confluent ECs were incubated with mAb W6/32 for centrifuged at 15,000 ϫ g for 10 min at 4¡C and the supernatants were various periods of time and the cell lysates were separated by ␮ collected and precleared for 2 h with 30 l of protein A/G plus agarose at SDS-PAGE, transferred to polyvinylidene difluoride membranes, 4¡C. The total protein content was measured using the Coomassie plus method (Pierce, Rockford, IL) using BSA as standard. For immunopre- and immunoblotted with the anti-phosphotyrosine mAb 4G10. Ly- cipitation, 100Ð300 ␮g of the precleared cell lysate was immunoprecipi- sates prepared from EC treated with mouse isotype IgG were used tated with 1 ␮g of specific Ab overnight at 4¡C followed by immunopre- as a negative control. As shown in Fig. 1, treatment with W6/32 cipitation with protein A/G plus agarose for one additional hour. The induced rapid tyrosine phosphorylation of a protein at an approx- immune complexes were washed two times with lysis buffer and two times imate molecular mass of 60 kDa. Tyrosine phosphorylation of the with PBS and the immune complexes were subjected to Western blotting. 60-kDa protein occurred as early as 1 min, peaked at 30 min, and Western blotting remained at high levels at 60 min. In contrast, tyrosine phosphor- Proteins in immune complexes or whole cell lysates were heated for 5 min ylation was not observed in isotype control-treated ECs (Fig. 1, A, at 95¡C in SDS sample buffer, electrophoresed on SDS polyacrylamide lane 1, and B). gels, and transferred to a polyvinylidene difluoride membrane. The mem- Previous studies from our laboratory have shown that ligation of branes were blocked using 5% nonfat dry milk in TBS (pH 7.4) containing class I molecules on the surface of ECs with anti-HLA class I Abs 0.05% Tween 20 (TBST) for2hatroom temperature, and incubated with the appropriate primary Ab overnight at 4¡Corfor2hatroom temperature. induces FGFR expression, increased bFGF ligand binding, and The blots were washed with TBST followed by incubation in HRP-con- subsequent cell proliferation (14, 16Ð18). HLA class I-mediated jugated secondary Ab for1hatroom temperature. The blots were subse- cell proliferation could be prevented by the addition of neutralizing The Journal of Immunology 5417

are distinct from the proteins phosphorylated following activation of the FGFR by bFGF. Anti-HLA class I Abs stimulate tyrosine phosphorylation of Src kinases Because we observed that anti-HLA Abs stimulate tyrosine phosphorylation of a major protein band of an approximate molecular mass of 60 kDa, we considered the possibility that the candidate protein may be a member of the Src family of tyrosine kinases. To investigate this possibility, cells were treated with mAb W6/32 for various time points and the cell lysates were immunoprecipitated with a panel of anti-Src Abs. The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with anti- phosphotyrosine mAb 4G10. As shown in Fig. 2A, anti-HLA class I Abs induced a marked increase in tyrosine phosphorylation of Src. Tyrosine phosphorylation was increased at 1 min following treatment with W6/32, peaked at 30 min, and remained at high levels thereafter. Densitometric scanning showed that anti-class I Abs (10 ␮g/ml) induced a 4-fold increase in phosphorylation of Downloaded from Src at 30 min compared with EC treated with isotype control IgG (Fig. 2A). Conﬁrmation that similar amounts of Src were recovered from lysates of anti-HLA Ab-treated and nontreated cells was obtained by blotting the immunoprecipitated proteins with the anti- Src Ab (Fig. 2A, lower panel). Ab ligation of class I molecules also

stimulated a time-dependent phosphorylation of p59 Fyn. As http://www.jimmunol.org/ shown in Fig. 2B, an increase in tyrosine phosphorylation of Fyn was detected at 1 min after the addition of 10 ␮g/ml W6/32, reaching a peak at 30 min. Immunoblotting with anti-Fyn Ab of Fyn immunoprecipitates veriﬁed that similar amounts of Fyn were recovered from lysates after anti-class I stimulation (Fig. 2B, lower panel). In contrast, no increase in tyrosine phosphorylation of p56/ p53 Lyn was observed when EC were treated with mAb W6/32 (Fig. 2C). Lck appeared to be constitutively phosphorylated in EC,

FIGURE 1. Ligation of class I molecules by anti-HLA Abs induces ty- by guest on September 27, 2021 with no observed increase in tyrosine phosphorylation above the rosine phosphorylation of EC proteins. A, Quiescent ECs were incubated with 10 ␮g/ml isotype control IgG (lane 1), 10 ␮g/ml mAb W6/32 (lanes baseline level when ECs were treated with anti-class I mAb (Fig. 2–5), or 1 ng/ml bFGF (lanes 11–14) for the times indicated. EC (lanes 2D). Immunoblotting with anti-Lyn and anti-Lck Abs confirmed 6–10) were pretreated for2hat37¡C with 1 ␮g/ml anti-human bFGF that similar amounts of protein were recovered from cell lysates neutralizing Ab followed by stimulation with mAb W6/32. B, Quiescent (Fig. 2, C and D, lower panels). ECs were incubated with 10 ␮g/ml isotype control IgG for the times in- To exclude the possibility that FGFR signaling contributes to dicated (lanes 2–5) or without isotype control (lane 1). Proteins in the cell class I-mediated tyrosine phosphorylation of Src, ECs were pre- lysates were separated by SDS-PAGE, transferred to a polyvinylidene di- treated with anti-human bFGF neutralizing Ab for 2 h before ex- fluoride membrane, and immunoblotted with anti-phosphotyrosine mAb posure to anti-HLA Abs. As presented in Fig. 2, neutralizing anti- 4G10. Arrows indicate the protein bands with increased density in W6/32- bFGF Abs had no apparent effect on class I-mediated tyrosine treated ECs compared with untreated cells. The band sizes are indicated in phosphorylation of Src or Fyn. Furthermore, treatment of ECs with kilodaltons. The results are representative of four independent experiments. recombinant human bFGF failed to stimulate tyrosine phosphorylation of Src or Fyn. During these investigations we found that anti-class I Abs stim- Abs to bFGF, suggesting that the FGFR is required for the gen- ulated increased phosphorylation of a 70-kDa protein that copre- eration of class I-mediated signals. In view of these results, ex- cipitated with anti-Src and anti-Fyn Abs (Fig. 2, A and B). To periments were performed to determine whether FGFR signaling determine whether Src kinase mediates phosphorylation of the p70 contributes to class I-mediated tyrosine phosphorylation of intra- protein, cells were preincubated in the presence of PP2, a specific cellular proteins. For this, ECs were pretreated with anti-bFGF inhibitor of Src kinase activity, before class I stimulation. Follow- neutralizing Ab for 2 h before exposure to anti-HLA Abs. The ing treatment with anti-class I Abs for various periods of time, cell addition of neutralizing anti-bFGF Abs had no effect on class I- lysates were immunoprecipitated with anti-Src Abs and the immu- mediated tyrosine phosphorylation of the 60-kDa protein (Fig. 1A, noprecipitates were analyzed by Western blotting with anti- lanes 7–10). Furthermore, treatment of ECs directly with bFGF phosphotyrosine Abs. As shown in Fig. 2E, pretreatment of EC induced tyrosine phosphorylation of proteins with an approximate with PP2 completely prevented HLA class I-mediated phosphor- molecular mass of 42Ð44 kDa (Fig. 1A, lanes 11–14). Increased ylation of the p70 protein. In contrast, increased tyrosine phos- tyrosine phosphorylation of the p42/44 proteins was detected at 10 phorylation of the p70 protein was unaffected by pretreatment of min and declined to almost baseline levels by 60 min. These results the cells with PP3, an inactive control analog of PP2. These results demonstrate that binding of anti-HLA Abs to class I Ags expressed demonstrate that binding of anti-HLA Abs to class I Ags on ECs by EC transduces signals resulting in a heavily tyrosine-phospho- transduces signals, resulting in phosphorylation of several mem- rylated 60-kDa protein. The results also indicate that the proteins bers of the Src family including Src and Fyn. Ligation of class I that become phosphorylated following exposure to anti-HLA Abs molecules also induced the phosphorylation of a p70 protein that 5418 ROLE OF Src AND FOCAL ADHESION PROTEINS IN CLASS I SIGNALING

FIGURE 2. Anti-HLA class I Abs induce tyrosine phosphorylation of Src family protein kinases. A and B, Quiescent EC were treated with 10 ␮g/ml isotype control IgG (lane 1), 10 ␮g/ml mAb W6/32 (lanes 7–11), or 1 ng/ml bFGF (lanes 12–13) for the times indicated. EC (lanes 2–6) were pretreated for2hat37¡C with 1 ␮g/ml anti-human bFGF neutralizing Ab followed by incubation with 10 ␮g/ml anti-HLA class I mAb W6/32 for the times indicated. C and D, EC were treated with 10 ␮g/ml Downloaded from mAb W6/32 (lanes 2–5) or 1 ng/ml bFGF (lanes 11–14) for the times indicated. EC (lanes 6–10) were pretreated for2hat37¡C with 1 ␮g/ml anti- human bFGF neutralizing Ab followed by incubation with 10 ␮g/ml anti-HLA class I mAb W6/32 for the times indicated. The precleared cell lysates were immunoprecipitated with anti-Src (N-16, A), anti- http://www.jimmunol.org/ Fyn (Fyn3, B), anti-Lyn (44, C), and anti-Lck (3A5, D) Abs. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine Ab 4G10. Quantiﬁcation of phosphorylation was performed by scanning densitometry. Val- ues shown are expressed as the percentage of the maximal increase in tyrosine phosphorylation value above control (nonstimulated) values. The results of

one of three experiments are shown. E, Quiescent by guest on September 27, 2021 ECs were pretreated for1hat37¡C in the presence (ϩ) or absence (Ϫ) of PP2 or PP3, followed by stimulation with 10 ␮g/ml anti-HLA class I mAb W6/32 for 10 min. The results of one of three independent experiments are presented.

coprecipitates with Src and is of unknown identity. Phosphoryla- escent cells were stimulated with different concentrations of tion of the p70 protein was completely blocked by the speciﬁc Src W6/32 for 10 min and lysed, and the extracts were immunopre- kinase inhibitor PP2, suggesting that phosphorylation of this pro- cipitated with anti-paxillin or anti-FAK Abs. The immunoprecipi- tein results from the action of Src kinases. tates were subsequently analyzed by immunoblotting with the anti- phosphotyrosine Ab 4G10. As shown in Fig. 3A, treatment with Anti-HLA class I Abs stimulate tyrosine phosphorylation of FAK mAb W6/32 stimulated an increase in tyrosine phosphorylation of and paxillin in EC paxillin in a dose-dependent manner. Stimulation of phosphoryla- Growth factor receptor and integrin stimulation of ECs and SMCs tion was observed at concentrations of anti-HLA Ab ranging from is known to promote the recruitment of Src family protein tyrosine 0.625 to 10 ␮g/ml with maximal effects (86% increase) at 10 ␮g/ kinases (PTKs) into a signaling complex with focal adhesion pro- ml. To determine the kinetics of class I-induced phosphorylation of teins (39). Therefore, we investigated the possibility that the major paxillin, cells were treated for different periods of time with anti- substrates for Src tyrosine phosphorylation reside within focal ad- HLA Abs. As shown in Fig. 3B, increased paxillin phosphorylation hesion proteins FAK and paxillin. To determine whether anti-HLA was detected as early as 1 min after the addition of anti-class I Abs, Abs stimulate the phosphorylation of focal adhesion proteins, qui- reaching a maximum after 10 min. Densitometric scanning showed The Journal of Immunology 5419

FIGURE 3. Ligation of class I molecules on EC stimulates tyrosine phosphorylation of paxillin. A, ECs treated with different doses of mAb W6/32 for 10 min. B, ECs treated with 10 ␮g/ml W6/32 for the indicated times. C, ECs pretreated for 2 h with 1 ␮g/ml anti-human bFGF neutralizing Ab and stimulated with 10 ␮g/ml W6/32 or 1 ng/ml bFGF. The precleared cell lysates were immunoprecipitated with anti-paxillin Ab (349), sep-

arated by SDS-PAGE, and immunoblotted with Downloaded from the anti-phosphotyrosine Ab 4G10. Quantiﬁcation of paxillin phosphorylation was performed by scanning densitometry. Values shown are expressed as the percentage of the maximal increase in paxillin tyrosine phosphorylation value above control (nonstimulated) values. Results represent one of ﬁve independent experiments. http://www.jimmunol.org/ by guest on September 27, 2021

that anti-class I Abs (10 ␮g/ml) induced a 3-fold increase in phos- or FAK was not affected by pretreatment with the inactive analog phorylation of paxillin at 10 min compared with EC treated with PP3 (data not shown). isotype control IgG (Fig. 4A). Class I-mediated induction of pax- Because tyrosine-phosphorylated FAK and paxillin are found in illin phosphorylation was not altered by pretreatment of the cells focal contacts associated with cell membrane ruffles and stress with anti-bFGF neutralizing Ab. As shown in Fig. 3C, lane 3, fibers (40), experiments were designed to evaluate whether dis- pretreatment of EC with anti-bFGF neutralizing Ab followed by ruption of the actin cytoskeleton could prevent tyrosine phosphor- class I ligation resulted in a 2.6-fold increase in paxillin phosphorylation of these focal adhesion proteins in response to anti-class I ylation at 10 min compared with EC treated with isotype control. Abs. For this, cells were pretreated with 2.5 ␮M cytochalasin D or Moreover, treatment of ECs directly with bFGF did not induce 1 ␮M latrunculin A for 1 h and then stimulated with mAb W6/32 significant differences in tyrosine phosphorylation of paxillin in for 10 min. As shown in Fig. 5, both latrunculin A and cytocha- five independent experiments (Fig. 3C). lasin D completely blocked W6/32-induced tyrosine phosphoryla- Anti-HLA class I Abs also stimulated tyrosine phosphorylation tion of FAK and paxillin. These results indicate that the integrity of FAK (Fig. 4B). Densitometric scanning showed a 40% increase of the actin cytoskeleton is required for class I-induced tyrosine in FAK phosphorylation in cells treated with mAb W6/32 com- phosphorylation of FAK and paxillin. pared with control cells (Fig. 4B). Immunoblotting with anti-FAK Abs of FAK immunoprecipitates verified that similar amounts of BFGF induces phosphorylation of ERK in ECs FAK were recovered from cell lysates after HLA class I Ab treat- As indicated in Fig. 1, exposure of EC to bFGF resulted in the ment. Thus, class I ligation induces a rapid and parallel increase in phosphorylation of heavily tyrosine-phosphorylated proteins at the the tyrosine phosphorylation of the focal adhesion proteins FAK approximate molecular mass of 42Ð44 kDa. To determine whether and paxillin. the candidate proteins are the p44/p42 ERK, whole cell lysates To determine whether Src tyrosine kinase activity is required for were immunoblotted with an anti-phospho-ERK Ab. Treatment class I-induced phosphorylation of paxillin and FAK, ECs were with recombinant human bFGF induced rapid and transient acti- treated in the presence or absence of PP2 followed by stimulation vation of p44ERK1/p42ERK2 in ECs (Fig. 6, upper panel). BFGF- with mAb W6/32. As shown in Fig. 5B, pretreatment with PP2 induced ERK phosphorylation reached maximum levels at 10 min blocked class I-induced phosphorylation of paxillin. Exposure of and declined by 30 min. W6/32 failed to induce phosphorylation of EC to PP2 also abrogated class I-induced phosphorylation of FAK ERK alone or augment phosphorylation of ERK when added to the (Fig. 5A). In contrast, class I-mediated phosphorylation of paxillin cells in combination with bFGF. Confirmation that similar 5420 ROLE OF Src AND FOCAL ADHESION PROTEINS IN CLASS I SIGNALING

FIGURE 6. FGFR ligation stimulates phosphorylation of ERK. ECs were treated with isotype control IgG (C), FGF (1 ng), 10 ␮g/ml mAb W6/32 (W), FGF (10 ng) plus W (10 ␮g/ml mAb W6/32), or FGF (10 ng) for the time points indicated. Cell lysates were fractionated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated with anti-phospho-ERK polyclonal Ab. Upper panel, The blots were incubated in HRP-conjugated secondary Ab, developed with an ECL, and exposed to x-ray film to visualize the proteins. Lower panel, Immunoblotting with FIGURE 4. HLA class I induced tyrosine phosphorylation of FAK and anti-ERK was performed to confirm equal loading of proteins. The results paxillin in EC. ECs were incubated in the presence (ϩ) or absence (Ϫ)of are representative of three independent experiments. 10 ␮g/ml W6/32 for 10 min and the cell lysates were prepared and precleared for 2 h with protein A/G plus agarose at 4¡C. The precleared cell lysates were immunoprecipitated with anti-paxillin Ab (349, A) or anti- FAK (2A7, B) overnight at 4¡C. The immunoprecipitates were analyzed by

SDS-PAGE and immunoblotted with the anti-phosphotyrosine Ab 4G10. the addition of PP2 inhibited class I-mediated induction of Downloaded from Quantification of paxillin and FAK phosphorylation was performed by FGFR-1 nuclear labeling (Fig. 7D). Quantitative analysis of the scanning densitometry. Values shown are expressed as the percentage of confocal nuclear immunofluorescence data was performed by mea- the maximal increase in paxillin and FAK tyrosine phosphorylation value suring the levels of FGFR fluorescence (Fig. 7, green) and pro- above control (nonstimulated) values. The results of one of five indepen- pidium iodide nuclear fluorescence (Fig. 7, red) in each optical dent experiments are presented. section. The average nuclear fluorescence intensity of these two

values is represented in Fig. 7G. The average nuclear fluorescence http://www.jimmunol.org/ amounts of ERK were recovered from lysates of anti-HLA Ab- intensity of propidium iodide was similar in both treated and nontreated and nontreated cells was obtained by blotting the immu- treated EC (Fig. 7, AÐF). The average nuclear fluorescence inten- noprecipitated proteins with anti-ERK Ab (Fig. 6, lower panel). sity of FGFR was 18 in cells treated with mAb W6/32 alone or PP3 and mAb W6/32. In contrast, the average fluorescence intensity of the Role of Src and focal adhesion proteins in class I-mediated nuclei of cells treated with isotype control, PP2, or PP3 was 8. These FGFR translocation in ECs differences were highly significant ( p Ͻ 0.0001), indicating that class In previous studies we have shown that ligation of class I mole- I signaling stimulates the translocation of FGFR-1 to the nucleus. cules on the surface of EC by anti-HLA Abs stimulates FGFR-1 Moreover, these results suggest that HLA class I-mediated redistri- by guest on September 27, 2021 redistribution from cytoplasmic stores to the nucleus and cell sur- bution of FGFR-1 to the nucleus is mediated by Src kinases. face (41). To determine whether Src tyrosine kinase is involved in Because these studies clearly support a role of Src tyrosine ki- HLA class I-mediated redistribution of FGFR-1 to the nucleus, EC nases in the redistribution of FGFRs within the cell, it was of were treated for 2 h with PP2 and the cells were labeled with interest to determine whether Src mediates class I-induced accu- anti-FGFR-1 Abs and optically sectioned and analyzed by confo- mulation of FGFRs on the cell surface. For these experiments, ECs cal immunofluorescence microscopy. Quiescent EC show diffuse were pretreated with PP2 for 2 h before the addition of anti-class cytoplasmic FGFR-1 staining and low FGFR-1 nuclear staining I Abs, and cell surface expression of FGFRs was determined by (Fig. 7A). However, after treatment with W6/32 for 30 min, the FACS analysis (Fig. 8A). Treatment with mAb W6/32 for 10 min majority of cells show intense nuclear FGFR-1 fluorescence (Fig. stimulated a 4-fold increase in FGFR surface expression (Fig. 8A) 7B). Similarly, class I-stimulated EC pretreated with PP3 show a compared with cells treated with isotype control IgG. Pretreatment marked increase in nuclear FGFR-1 labeling (Fig. 7F). In contrast, of cells with PP2 followed by stimulation with anti-class I Abs had no appreciable effect on class I-induced FGFR surface expression. These results indicate that Src family PTKs mediate HLA class I-induced FGFR-1 translocation to the nucleus. However, Src tyrosine kinase activity does not appear to be required for the redistribution of FGFR to the cell surface. The results presented above suggest that different signaling pathways are responsible for the redistribution of FGFR to distinct subcellular compartments. We next examined whether the focal adhesion proteins FAK and paxillin participate in class I-induced FGFR translocation to the cell surface. For this, ECs were pretreated for 1 h with 1 ␮M latrunculin A or 2.5 ␮M cytochalasin D, concentrations that disrupt the actin cytoskeleton and the assembly FIGURE 5. Class I-mediated phosphorylation of FAK and paxillin is of focal adhesions in EC, and then stimulated with 10 ␮g/ml mAb Src dependent and requires an intact cytoskeleton. ECs were pretreated for W6/32 for another 2 h. The cells were analyzed by flow cytometry for ϩ Ϫ ␮ ␮ 1hat37¡C in presence ( ) or absence ( )of25 M PP2, 2.5 M FGFR surface expression using a polyclonal anti-FGFR Ab. Treat- cytochalasin D, or 1 ␮M latrunculin A, and stimulated with 2.5 ␮g/ml mAb W6/32 for 10 min. Precleared cell lysates were immunoprecipitated with ment of ECs with either latrunculin A (Fig. 8B) or cytochalasin D anti-FAK (2A7, A), or anti-paxillin Ab (349, B). The immunoprecipitates were (Fig. 8C) inhibited FGFR expression in response to class I Abs. Thus, analyzed by SDS-PAGE and immunoblotted with anti-phosphotyrosine Ab our results indicate that the actin cytoskeleton is involved in class 4G10. One of three representative experiments is presented. I-mediated translocation of FGFRs to the cell surface. The Journal of Immunology 5421 Downloaded from http://www.jimmunol.org/

FIGURE 8. The actin cytoskeleton is involved in class I-induced FGFR surface expression in EC. Quiescent ECs were pretreated for1hat37¡C in the presence or absence of 25 ␮M PP2 (A), 1 ␮M latrunculin A (B), or 2.5 ␮M cytochalasin D (C), followed by incubation with 10 ␮g/ml anti- HLA class I mAb W6/32 for 2 h. The cells were incubated with anti-FGFR polyclonal Ab (Upstate Biotechnology) and stained with a FITC-conju-

gated donkey anti-rabbit IgG. Cell fluorescence was analyzed on a FAC- by guest on September 27, 2021 Scan flow cytometer using CellQuest Software (BD Biosciences). Gates for forward and side scatter measurements were set on EC, and a minimum of 10,000 events was acquired. Solid thin line, FGFR expression in EC treated with mAb W6/32; solid thick line, FGFR expression in EC pretreated with inhibitor followed by stimulation with mAb W6/32; dashed line, FGFR expression in EC treated with the isotype control. The results are representative of three independent experiments. FIGURE 7. Ligation of class I molecules by anti-HLA Abs increases FGFR-1 association with the nucleus. Quiescent ECs were treated with 10 report were aimed at elucidating the signal transduction machinery ␮ ␮ g/ml mouse IgG for 30 min (A); 10 g/ml anti-HLA class I mAb W6/32 initiated following ligation of class I molecules by anti-HLA Abs for 30 min (B); 12.5 ␮M PP2 for 30 min (C), 12.5 ␮M PP2 for 30 min on human ECs. As described in our earlier investigations, Ab li- followed by stimulation with 10 ␮g/ml W6/32 for 30 min (D); 12.5 ␮M PP3 for 30 min (E); and 12.5 ␮M PP3 for 30 min followed by stimulation gation of class I molecules expressed on the surface of ECs and with 10 ␮g/ml W6/32 for 30 min (F). The monolayers were fixed, perme- SMCs induces cell proliferation by up-regulating FGFR expres- abilized, blocked, and labeled with the anti-FGFR-l Ab. The cells were sion and, as a result, enhances the binding of bFGF to the cell incubated with biotinylated anti-rabbit secondary Ab followed by staining (14Ð18). Therefore, our attention was focused on determining the with avidin-fluorescein and propidium iodide. FGFR-1 fluorescence was signaling proteins mediating class I-induced FGFR expression in analyzed using an OLYMPUS FLUOVIEW confocal laser scanning bio- human ECs. Our studies show for the first time that anti-HLA Ab logical microscope. The fluorescence intensities of FGFR (green) and pro- binding to class I molecules on ECs results in the phosphorylation pidium iodide (red) in EC were quantified by measuring the average pixel of Src, paxillin, and FAK. intensity in each section using FLUOVIEW software and the data was plotted The Src family of PTKs consists of at least nine members, in- (G). A minimum of 40 cells was analyzed in each section. In F, the bar ϭ 5 cluding Src p60, Yes p62, Lck p56, Lyn p56/p53, Fyn p59, Fgr ␮m. The results are representative of three independent experiments. p55, Hck p56/59, Blk p55, and Rak (42, 43). Src family members have been shown to play important roles in regulating complex signal transduction pathways and are differentially expressed in a Discussion wide variety of tissues. Consequently, we examined whether class HLA class I signaling pathways have been implicated in the pro- I stimulation induces tyrosine phosphorylation of distinct members liferation of vascular ECs, SMCs, T cells, and B cells, as well as of the Src PTK family. Our studies revealed that Fyn and Src apoptosis of activated T and B cells (19Ð33). Although the mech- become immediately phosphorylated on tyrosine residues in re- anisms and molecules involved in the HLA-mediated apoptosis sponse to class I ligation. These results are consistent with previ- have been recently suggested (20, 23), information on HLA class ous studies documenting the ability of class I molecules to phos- I-mediated cell proliferation is sparse. The studies described in this phorylate members of the Src family in T and B cells (36, 44). In 5422 ROLE OF Src AND FOCAL ADHESION PROTEINS IN CLASS I SIGNALING human B cells, class I ligation results in the phosphorylation of volved in the induction of cell proliferation (62Ð64). In previous p53/56lyn, whereas p56Lck tyrosine kinase is activated in human T studies, we reported that anti-HLA Abs transduce signals that re- cells following class I ligation (36). Our studies showed that treat- sult in the redistribution of FGFRs to the cell surface and nucleus ment of EC with anti-class I Abs induced the phosphorylation of a (15Ð18, 41). These studies have clearly demonstrated that HLA 70-kDa protein that coimmunoprecipitated with anti-Src and anti- class I-induced redistribution to the cell surface results in increased Fyn Abs. Pretreatment of cells with the Src tyrosine kinase inhib- ligand binding and initiation of cell proliferation through the MAP itor PP2 specifically blocked class I-induced tyrosine phosphory- kinase pathway. However, the function of the class I-targeted nu- lation of the p70 protein. Thus, our results suggest a Src PTK- clear FGFR-1 is still unknown. In the current study, we observed dependent pathway in the tyrosine phosphorylation of this that class I initiates different signaling cascades that target the unidentified 70-kDa protein in response to class I stimulation. FGFR to distinct intracellular locations. We found that Src kinase The results presented in this report also demonstrate that Ab inhibitor PP2 had no effect on class I-mediated induction of FGFR ligation of HLA class I molecules rapidly induces phosphorylation expression on the surface of EC, whereas this inhibitor completely of paxillin and FAK. Paxillin serves as an adapter protein that blocked class I-induced FGFR translocation to the nucleus. These provides multiple docking sites at the plasma membrane for an results indicate a Src-dependent pathway for class I-induced nu- array of signaling and structural proteins, including the tyrosine clear translocation of FGFR-1 and a Src-independent pathway for kinases Src and FAK (45). FAK is a nonreceptor tyrosine kinase class I-induced FGFR plasma membrane expression. FACS studies that plays a key role mediating integrin signaling and cell adhesion suggest that the integrity of the actin cytoskeleton is required for (46). FAK, in association with Src, have been shown to stimulate productive class I signaling and FGFR-1 translocation. Thus, treat- the phosphorylation of paxillin at two main sites, tyrosine 31 and ment of EC with either latrunculin A or cytochalasin D blocked FAK Downloaded from tyrosine 18 (47Ð52). Together, paxillin, Src, and FAK form a sig- and paxillin phosphorylation, as well as FGFR-1 translocation to the naling complex that transduces signals to downstream molecules, cell surface. Our data show that inhibition of Src kinase, which is thereby stimulating gene expression (reviewed in Refs. 45, 53, and important for the phosphorylation of paxillin, inhibits the nuclear 54). This suggests that class I ligation stimulates Src phosphory- translocation but not the plasma membrane translocation of FGFR, lation and binding to FAK, resulting in the formation of a FAK-Src whereas the disruption of the actin cytoskeleton prevented class complex, enhanced FAK PTK activity, and subsequent phosphor- I-mediated plasma membrane FGFR translocation. The actin cy- http://www.jimmunol.org/ ylation of paxillin. Consistent with this interpretation, we found toskeleton is regulated by a variety of pathways, including Rho, Rac, that treatment of EC with PP2 blocked class I-mediated phosphor- Cdc42, Rho-associated kinase, and profilin, as well as the tyrosine ylation of paxillin and FAK, indicating a Src PTK-dependent path- phosphorylation of FAK, CAS, and paxillin. We hypothesize that the way in the tyrosine phosphorylation of these focal adhesion pro- role of paxillin in actin cytoskeleton organization is likely to be subtle teins in response to class I stimulation. Our data also show that (it is one of multiple pathways), whereas cytochalasin D disrupts the treatment of EC with either cytochalasin D or latruncalin A, at whole actin cytoskeleton network. Thus, cytochalasin D leads to more concentrations known to disrupt the actin cytoskeleton, abrogated severe consequences than inhibition of paxillin phosphorylation. class I-mediated increases in tyrosine phosphorylation of paxillin Together, our previous results (14Ð18, 41, 65) and current find- and FAK. These results indicate that an intact cytoskeleton is re- ings are consistent with a model in which anti-HLA Ab-mediated by guest on September 27, 2021 quired for class I-induced tyrosine phosphorylation of FAK and clustering of class I molecules stimulates the organization of pax- paxillin and suggest that actin-dependent clustering of molecules is illin, Src, and FAK into cell matrix adhesions, which act in concert required to trigger class I signals. as signaling units. These protein phosphorylation events activated The mechanism whereby class I molecules transduce signals is by class I signaling stimulate the translocation of FGFR from cy- unknown. Although the H chain of class I contains serine, threo- toplasmic stores to the nucleus and cell surface. Class I-induced nine, and tyrosine residues that can be phosphorylated, studies up-regulation of FGFR augments FGF binding and triggers a series characterizing class I signal transduction using constructs lacking of downstream events including activation of the ERK/MAP ki- most of the cytoplasmic tail have shown that signal transduction nase pathway, cyclin E-dependent kinase activity, and Rb inacti- does not require this portion of the molecule (55). This suggests vation, causing the EC to proliferate (65). Class I-dependent trans- that there is no direct interaction between class I molecules and location of FGFR-1 to the nucleus of EC may also play a direct Src, paxillin, and FAK. Thus, class I molecules probably associate role in regulating gene transcription and cell proliferation. with other molecules that have the capacity to transduce signals or Further studies are required to determine the effect of class I generate intracellular messengers. In this respect, class I molecules signaling in an in vivo allograft model. However, our in vitro stud- have been shown to interact with various peptide hormone recep- ies support the hypothesis that anti-HLA Abs may contribute to the tors that function as tyrosine kinases, such as the insulin receptor process of chronic rejection by binding to the endothelium and and the epidermal growth factor receptor (56Ð58). The data pre- smooth muscle of the graft and initiating protein tyrosine phosphor- sented in this paper support a model in which class I molecules ylation events that stimulate FGFR translocation and subsequent cell associate with a coreceptor to form a signaling complex initiated proliferation. Because signaling through class I molecules can induce by Src-dependent binding of Src family PTKs to FAK and paxillin. cell proliferation in vitro, agents that block this process may be useful Elucidation of the coreceptor requires further studies; however, it in the prevention and treatment of chronic allograft rejection. is tempting to speculate that this molecule may be a member of the integrin family, because it is well established that Src, paxillin, and References FAK are activated following engagement of integrins with the ex- 1. Kreis, H. A., and C. Ponticelli. 2001. Causes of late renal allograft loss: chronic tracellular matrix (39, 46, 59). allograft dysfunction, death, and other factors. Transplantation 71:SS5. FGFRs are generally known as plasma membrane proteins that 2. Paul, L. C. 2001. Immunologic risk factors for chronic renal allograft dysfunction. Transplantation 71:SS17. send signals to the nucleus principally via the MAP kinase and the 3. Davenport, A., M. E. Younie, J. E. Parsons, and P. T. Klouda. 1994. Develop- Janus kinase-STAT pathways (60, 61). However, over the past few ment of cytotoxic antibodies following renal allograft transplantation is associ- years, data has accumulated to suggest that nuclear targeting and ated with reduced graft survival due to chronic vascular rejection. Nephrol. Dial. Transplant. 9:1315. action of FGFs and FGFRs could occur as well. This alternative or 4. Costa, A. N., M. P. Scolari, S. Iannelli, A. Buscaroli, G. L. D’Arcangelo, complementary nuclear signaling pathway also appears to be in- B. Brando, F. Indiveri, M. Savi, L. C. Borgnino, L. B. DeSanctis, et al. 1997. The The Journal of Immunology 5423

presence of posttransplant HLA-specific IgG antibodies detected by enzyme- 33. Bregenholt, S., M. Ropke, S. Skov, and M. H. Claesson. 1996. Ligation of MHC linked immunosorbent assay correlates with specific rejection pathologies. Trans- class I molecules on peripheral blood T lymphocytes induces new phenotypes and plantation 63:167. functions. J. Immunol. 157:993. 5. Cherry, R., H. Nielsen, E. Reed, K. Reemtsma, N. Suciu-Foca, and C. C. Marboe. 34. Geppert, T. D., M. C. Wacholtz, S. S. Patel, E. Lightfoot, and P. E. Lipsky. 1989. 1992. Vascular (humoral) rejection in human cardiac allograft biopsies: relation Activation of human T cell clones and Jurkat cells by cross-linking class I MHC to circulating anti-HLA antibodies. J. Heart Lung Transplant. 11:24. molecules. J. Immunol. 142:3763. 6. Reed, E. F., B. Hong, E. Ho, P. E. Harris, J. Weinberger, and N. Suciu-Foca. 35. Pedersen, A. E., S. Skov, S. Bregenholt, M. Ruhwald, and M. H. Claesson. 1999. 1996. Monitoring of soluble HLA alloantigens and anti-HLA antibodies identifies Signal transduction by the major histocompatibility complex class I molecule. heart allograft recipients at risk of transplant-associated coronary artery disease. APMIS 107:887. Transplantation 61:566. 36. Pedersen, A. E., S. Bregenholt, S. Skov, M. L. Vrang, and M. H. Claesson. 1998. 7. Fenoglio, J., E. Ho, E. Reed, E. Rose, C. Smith, K. Reemstma, C. Marboe, and Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal N. Suciu-Foca. 1989. Anti-HLA antibodies and heart allograft survival. Trans- transduction in B lymphoma cells. Exp. Cell Res. 240:144. plant. Proc. 21:807. 37. Pedersen, A. E., S. Bregenholt, B. Johansen, S. Skov, and M. H. Claesson. 1999. 8. Suciu-Foca, N., E. Reed, C. Marboe, P. Harris, P. X. Yu, Y. K. Sun, E. Ho, MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein E. Rose, K. Reemtsma, and D. W. King. 1991. The role of anti-HLA antibodies tyrosine and serine/threonine kinases. Exp. Cell Res. 251:128. in heart transplantation. Transplantation 51:716. 38. Tscherning, T., and M. H. Claesson. 1994. Signal transduction via MHC class-I 9. Suciu-Foca, N., E. Reed, V. D. D’Agati, E. Ho, D. J. Cohen, A. I. Benvenisty, molecules in T cells. Scand. J. Immunol. 39:117. R. McCabe, J. M. Brensilver, D. W. King, and M. A. Hardy. 1991. Soluble HLA 39. Sieg, D. J., C. R. Hauck, and D. D. Schlaepfer. 1999. Required role of focal adhesion antigens, anti-HLA antibodies, and antiidiotypic antibodies in the circulation of kinase (FAK) for integrin-stimulated cell migration. J. Cell Sci. 112:2677. renal transplant recipients. Transplantation 51:593. 40. Casamassima, A., and E. Rozengurt. 1998. Insulin-like growth factor I stimulates 10. McKenna, R. M., S. K. Takemoto, and P. I. Terasaki. 2000. Anti-HLA antibodies tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin: role of after solid organ transplantation. Transplantation 69:319. phosphatidylinositol 3Ј-kinase and formation of a p130Cas⅐Crk complex. J. Biol. 11. Azuma, H., and N. L. Tilney. 1994. Chronic graft rejection. Curr. Opin. Immunol. Chem. 273:26149. 6:770. 41. Bian, H., and E. F. Reed. 2001. Anti-HLA class I antibodies transduce signals in 12. Paul, L. C., and B. Fellstrom. 1992. Chronic vascular rejection of the heart and

endothelial cells resulting in FGF receptor translocation, down-regulation of Downloaded from the kidney: have rational treatment options emerged? Transplantation 53:1169. ICAM-1 and cell proliferation. Transplant. Proc. 33:311. 13. Russell, P. S., C. M. Chase, H. J. Winn, and R. B. Colvin. 1994. Coronary 42. Martin, G. S. 2001. The hunting of the Src. Nat. Rev. Mol. Cell Biol. 2:467. atherosclerosis in transplanted mouse hearts. II. Importance of humoral immunity. 43. Erpel, T., and S. A. Courtneidge. 1995. Src family protein tyrosine kinases and J. Immunol. 152:5135. cellular signal transduction pathways. Curr. Opin. Cell Biol. 7:176. 14. Bian, H., P. E. Harris, A. Mulder, and E. F. Reed. 1997. Anti-HLA antibody 44. Sharp, L. L., and S. M. Hedrick. 1999. Commitment to the CD4 lineage mediated ligation to HLA class I molecules expressed by endothelial cells stimulates ty- by extracellular signal-related kinase mitogen-activated protein kinase and lck rosine phosphorylation, inositol phosphate generation, and proliferation. Hum. signaling. J. Immunol. 163:6598. Immunol. 53:90. 45. Turner, C. E. 2000. Paxillin and focal adhesion signalling. Nat. Cell Biol. 2:E231. 15. Bian, H., P. E. Harris, and E. F. Reed. 1998. Ligation of HLA class I molecules 46. Hanks, S. K., and T. R. Polte. 1997. Signaling through focal adhesion kinase. http://www.jimmunol.org/ on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphory- BioEssays 19:137. lation, fibroblast growth factor receptor expression and cell proliferation. Int. 47. Xing, Z., H. C. Chen, J. K. Nowlen, S. J. Taylor, D. Shalloway, and J. L. Guan. Immunol. 10:1315. 1994. Direct interaction of v-Src with the focal adhesion kinase mediated by the 16. Bian, H., and E. F. Reed. 1999. Alloantibody-mediated class I signal transduction Src SH2 domain. Mol. Biol. Cell 5:413. in endothelial cells and smooth muscle cells: enhancement by IFN-␥ and TNF-␣. 48. Cobb, B. S., M. D. Schaller, T. H. Leu, and J. T. Parsons. 1994. Stable association J. Immunol. 163:1010. of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, 17. Bian, H., and E. F. Reed. 1999. Anti-HLA antibodies transduce proliferative pp125FAK. Mol. Cell. Biol. 14:147. signals in endothelial cells and smooth muscle cells. Transplant. Proc. 31:1924. 49. Schaller, M. D., J. D. Hildebrand, J. D. Shannon, J. W. Fox, R. R. Vines, and 18. Harris, P. E., H. Bian, and E. F. Reed. 1997. Induction of high-affinity fibroblast J. T. Parsons. 1994. Autophosphorylation of the focal adhesion kinase, pp125FAK, growth factor receptor expression and proliferation in human endothelial cells by directs SH2-dependent binding of pp60src. Mol. Cell. Biol. 14:1680. anti-HLA antibodies: a possible mechanism for transplant atherosclerosis. J. Im- 50. Eide, B. L., C. W. Turck, and J. A. Escobedo. 1995. Identification of Tyr397 as munol. 159:5697. by guest on September 27, 2021 the primary site of tyrosine phosphorylation and pp60src association in the focal 19. Skov, S., N. Odum, and M. H. Claesson. 1995. MHC class I signaling in T cells leads to adhesion kinase, pp125FAK. Mol. Cell. Biol. 15:2819. tyrosine kinase activity and PLC-␥1 phosphorylation. J. Immunol. 154:1167. 51. Schlaepfer, D. D., M. A. Broome, and T. Hunter. 1997. Fibronectin-stimulated 20. Skov, S., P. Klausen, and M. H. Claesson. 1997. Ligation of major histocompat- signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, ibility complex (MHC) class I molecules on human T cells induces cell death p130cas, and Nck adaptor proteins. Mol. Cell. Biol. 17:1702. through PI-3 kinase-induced c-Jun NH2-terminal kinase activity: a novel apopto- tic pathway distinct from Fas-induced apoptosis. J. Cell Biol. 139:1523. 52. Schaller, M. D., J. D. Hildebrand, and J. T. Parsons. 1999. Complex formation 21. Skov, S., S. Bregenholt, and M. H. Claesson. 1997. MHC class I ligation of with focal adhesion kinase: a mechanism to regulate activity and subcellular human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an al- localization of Src kinases. Mol. Biol. Cell 10:3489. ternative phenotype of the TCR/CD3 ␨-chain, and induces apoptosis. J. Immunol. 53. Salazar, E. P., and E. Rozengurt. 2001. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhe- 158:3189. 397 22. Skov, S. 1998. Intracellular signal transduction mediated by ligation of MHC sion kinase autophosphorylation at Tyr . J. Biol. Chem. 276:17788. class I molecules. Tissue Antigens 51:215. 54. Turner, C. E. 2000. Paxillin interactions. J. Cell Sci. 113:4139. 23. Skov, S., M. Nielsen, S. Bregenholt, N. Odum, and M. H. Claesson. 1998. Activation 55. Gur, H., F. el-Zaatari, T. D. Geppert, M. C. Wacholtz, J. D. Taurog, and P. E. Lipsky. of Stat-3 is involved in the induction of apoptosis after ligation of major histocom- 1990. Analysis of T cell signaling by class I MHC molecules: the cytoplasmic do- patibility complex class I molecules on human Jurkat T cells. Blood 91:3566. main is not required for signal transduction. J. Exp. Med. 172:1267. 24. Dasgupta, J. D., E. Egea, V. Relias, A. Iglesias, P. Gladstone, and E. J. Yunis. 56. Due, C., M. Simonsen, and L. Olsson. 1986. The major histocompatibility com- 1990. Involvement of major histocompatibility complex class I antigens in T cell plex class I heavy chain as a structural subunit of the human cell membrane activation. Eur. J. Immunol. 20:1553. insulin receptor: implications for the range of biological functions of histocom- 25. Smith, J. D., C. Lawson, M. H. Yacoub, and M. L. Rose. 2000. Activation of patibility antigens. Proc. Natl. Acad. Sci. USA 83:6007. NF-␬B in human endothelial cells induced by monoclonal and allospecific HLA 57. Phillips, M. L., M. L. Moule, T. L. Delovitch, and C. C. Yip. 1986. Class I antibodies. Int. Immunol. 12:563. histocompatibility antigens and insulin receptors: evidence for interactions. Proc. 26. Wacholtz, M. C., S. S. Patel, and P. E. Lipsky. 1989. Patterns of costimulation of Natl. Acad. Sci. USA 83:3474. T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules. 58. Schreiber, A. B., J. Schlessinger, and M. Edidin. 1984. Interaction between major J. Immunol. 142:4201. histocompatibility complex antigens and epidermal growth factor receptors on 27. Geppert, T. D., M. C. Wacholtz, L. S. Davis, and P. E. Lipsky. 1988. Activation of human cells. J. Cell Biol. 98:725. human T4 cells by cross-linking class I MHC molecules. J. Immunol. 140:2155. 59. Danen, E. H., and K. M. Yamada. 2001. Fibronectin, integrins, and growth con- 28. Roosnek, E., A. Tunnacliffe, and A. Lanzavecchia. 1990. T cell activation by a trol. J. Cell. Physiol. 189:1. bispecific anti-CD3/anti-major histocompatibility complex class I antibody. Eur. 60. Mason, I. J. 1994. The ins and outs of fibroblast growth factors. Cell 78:547. J. Immunol. 20:1393. 61. Karin, M., and T. Hunter. 1995. Transcriptional control by protein phosphoryla- 29. Geppert, T. D., L. S. Davis, H. Gur, M. C. Wacholtz, and P. E. Lipsky. 1990. tion: signal transmission from the cell surface to the nucleus. Curr. Biol. 5:747. Accessory cell signals involved in T-cell activation. Immunol. Rev. 117:5. 62. Bonnet, H., O. Filhol, I. Truchet, P. Brethenou, C. Cochet, F. Amalric, and 30. Geppert, T. D., and P. E. Lipsky. 1988. Activation of T lymphocytes by immo- G. Bouche. 1996. Fibroblast growth factor-2 binds to the regulatory ␤ subunit of CK2 bilized monoclonal antibodies to CD3: regulatory influences of monoclonal an- and directly stimulates CK2 activity toward nucleolin. J. Biol. Chem. 271:24781. tibodies to additional T cell surface determinants. J. Clin. Invest. 81:1497. 63. Maher, P. A. 1996. Nuclear translocation of fibroblast growth factor (FGF) re- 31. Hansen, N. Q., T. Tscherning, and M. H. Claesson. 1991. T-cell activation. IV. ceptors in response to FGF-2. J. Cell Biol. 134:529. Evidence for a functional linkage between MHC class I, interleukin-2 receptor, 64. Keresztes, M., and J. Boonstra. 1999. Import(ance) of growth factors in(to) the and interleukin-4 receptor molecules. Cytokine 3:35. nucleus. J. Cell Biol. 145:421. 32. Dissing, S., C. Geisler, B. Rubin, T. Plesner, and M. H. Claesson. 1990. T cell 65. Nath, N., H. Bian, E. F. Reed, and S. P. Chellappan. 1999. HLA class I-mediated activation. II. Activation of human T lymphoma cells by cross-linking of their induction of cell proliferation involves cyclin E-mediated inactivation of Rb func- MHC class I antigens. Cell. Immunol. 126:196. tion and induction of E2F activity. J. Immunol. 162:5351.