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2+ Intracellular Ca Protein Kinase C and the Increase in Pyk2: Opposing T Cell Activation Up-Regulates the Expression of the Focal Adhesion Kinase Pyk2: Opposing Roles for the Activation of Protein Kinase C and the Increase in This information is current as Intracellular Ca2+ of September 27, 2021. Masahiro Tsuchida, Eric R. Manthei, Tausif Alam, Stuart J. Knechtle and Majed M. Hamawy J Immunol 1999; 163:6640-6650; ; http://www.jimmunol.org/content/163/12/6640 Downloaded from References This article cites 62 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/163/12/6640.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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. T Cell Activation Up-Regulates the Expression of the Focal Adhesion Kinase Pyk2: Opposing Roles for the Activation of Protein Kinase C and the Increase in Intracellular Ca21 Masahiro Tsuchida, Eric R. Manthei, Tausif Alam, Stuart J. Knechtle, and Majed M. Hamawy1 T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase Downloaded from (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca21/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca21 ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca21 has opposing effects on IL-2 and Pyk2 21 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca ionophore-mediated inhibition of Pyk2 expression, impli- http://www.jimmunol.org/ cating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells. The Journal of Immunology, 1999, 163: 6640–6650. cell receptor engagement initiates an array of intracellular scriptional activity of transcription factors such as cAMP response signals, leading to an increase in the transcriptional ac- element binding protein, which, in turn, up-regulates the activity of T tivity of cytokine genes, cell adhesion, proliferation, and c-Fos (11). Stimulated calcineurin dephosphorylates the transcrip- differentiation. Cytokines released from activated T cells stimulate tion factor c-NF-AT, leading to its activation and translocation to Ab production, induce maturation of cytotoxic effector cells, and the nucleus (5, 12). Calcineurin also regulates the activity of other by guest on September 27, 2021 activate various accessory cells, such as phagocytes. TCR-trig- transcription factors, including Elk-1, Oct-1, and NF-kB (13–15). Ac- gered signals include the stimulation of protein tyrosine kinases tivated transcription factors bind to promoters of genes, leading to the (PTKs),2 the generation of inositol triphosphate and diacylglyc- transcription of genes that encode cytokines, cytokine receptors, and erol, the activation of protein kinase C (PKC), the increase in protooncogenes, such as c-myc,c-fos, and src family PTKs. intracellular Ca21, the activation of the Ras and Rho family of PTKs are involved in the signal transduction pathways of var- GTPases, and the activation of several mitogen-activated protein ious receptors and are critical for controlling cell growth and dif- kinases (MAPKs) (1, 2). Stimulated MAPKs translocate from the ferentiation (1, 2, 16–18). The earliest detectable biochemical cytoplasm to the nucleus, where they phosphorylate and, in turn, event following TCR engagement is the activation of several activate transcription factors such as c-Fos, c-Jun, Elk-1, and c- PTKs, resulting in transient tyrosine phosphorylation of numerous 1 Myc (3, 4). The increase in intracellular Ca2 following TCR li- intracellular substrates. Among the PTKs that become rapidly 1 gation stimulates various enzymes, such as the Ca2 /calmodulin- phosphorylated on tyrosine following TCR ligation are PTKs that dependent kinases (CaMKs) and the serine/threonine phosphatase belong to the Src family, Syk/Zap-70 family, Csk family, and the calcineurin, key enzymes in the T cell signal transduction cascade recently reported focal adhesion kinase family. In contrast to the (5–10). Activated CaMKs phosphorylate and up-regulate the tran- extensive data available on the role of the Src, Csk, and the Syk/ Zap-70 PTKs in T cell activation, little is known about the in- volvement of the focal adhesion PTKs Pyk2 and Fak in T cell Department of Surgery, Division of Transplantation, University of Wisconsin, Mad- processes. The focal adhesion kinase family consists of two cyto- ison, WI 53792 solic PTKs, Pyk2 and Fak (;65% similarity at the amino acid Received for publication July 13, 1999. Accepted for publication October 6, 1999. sequence level) (19–25). Pyk2 and Fak share a similar structural The costs of publication of this article were defrayed in part by the payment of page organization: a central tyrosine kinase flanked by noncatalytic do- charges. This article must therefore be hereby marked advertisement in accordance mains at both the N- and C-termini. Both PTKs lack SH2 and SH3 with 18 U.S.C. Section 1734 solely to indicate this fact. domains and therefore rely on their tyrosine-phosphorylated resi- 1 Address correspondence and reprint requests to Dr. Majed M. Hamawy, H4/749, Department of Surgery, 600 Highland Avenue, Madison, WI 53792. E-mail address: dues for interacting with SH2-containing signaling molecules. Fak [email protected] is expressed in all tissues, whereas Pyk2 is primarily expressed in 2 Abbreviations used in this paper: PTK, protein tyrosine kinase; Act-D, actinomycin the nervous system and in cells of hemopoietic lineage. Although D; CaMKs, Ca21/calmodulin-dependent kinases; CsA, cyclosporin A; Jurkat T cells, in adherent cells Fak is localized to focal adhesion sites, Pyk2 is acute human leukemia T cells; MAPK, mitogen-activated protein kinase; ERK, ex- tracellular signal-related kinase; MEK, MAP/ERK kinase; PKC, protein kinase C; mainly diffused throughout the cytoplasm. The differential distri- WCL, whole cell lysates; Fak, focal adhesion kinase. bution of these PTKs in tissues and their localization to different Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 6641 sites within the cell suggest that these PTKs have different cellular tivated FCS, 4 mM L-glutamine, and antibiotic-antimycotic mixture) at functions. 37°C in 5% CO2 and were subcultured three times per week. Receptor-mediated activation of T cells has been shown to in- Cell activation and preparation of cell lysates duce tyrosine phosphorylation of the focal adhesion PTKs (26– 31). Notably, although both Pyk2 and Fak become tyrosine phos- T cell activation was performed as reported previously (27, 32–34). For stimulating T cells in suspension, Jurkat T cells or purified normal human phorylated following TCR ligation, only Pyk2 appears to become peripheral blood T cells were washed with RPMI-5% FCS and suspended tyrosine phosphorylated upon CD28 ligation (26, 27, 29, 30). To in the same medium in polyethylene tubes (8 3 105 cells in 400 mlof further examine the involvement of Pyk2 and Fak in T cell pro- medium). The cells were then incubated with 100 ml of RPMI-5% FCS cesses, we examined the effect of T cell stimulation on the expres- containing the indicated concentrations of the stimuli with or without in- sion of Pyk2 and Fak in T cells. We found that the activation of hibitors for 16 h at 37°C. After incubation, the cells were washed once with RPMI 1640 containing 0.001% BSA and immediately solubilized with Jurkat T cells and human normal T cells by ligating TCR or by boiling SDS-PAGE sample buffer. For stimulating T cells with immobi- treatment with PMA increased Pyk2 expression. In contrast, TCR lized
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