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PKC inhibitors: potential in T cell-dependent immune diseases
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PKC inhibitors: potential in T cell-dependent immune diseases Gottfried Baier1 and Ju¨rgen Wagner2 The basic mechanisms of serine/threonine protein kinase signaling networks have been elucidated in the past decade. Members of the protein kinase C (PKC) family are crucial in T cell signaling pathways. Particularly, PKC a, PKC b, and PKC u isotypes determine the nature of lymphocyte-specific in vivo effector responses. Therefore, PKC isotypes are validated drug targets in adaptive immunity. Selective PKC kinase inhibitors have been discovered and are currently in clinical development, where they may provide new therapeutic options for different immune disorders. Here we review the topic of PKC pathway activity in the regulation of T lymphocytes both in the cytokine response and adhesive capacity, and review recent results with PKC inhibitors in vitro and in vivo. Addresses 1 Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria 2 Autoimmunity, Transplantation & Inflammation, Novartis Institute for BioMedical Research, Basel, Switzerland Corresponding author: Baier, Gottfried ([email protected])
Current Opinion in Cell Biology 2009, 21:262–267 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Nikolas Tonks Available online 3rd February 2009 0955-0674/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2008.12.008
Introduction The main function of mature T cells is to recognize and respond to foreign antigens. This occurs through a complex activation process that involves activation, adhesion, and differentiation of the resting cell into a proliferating lymphoblast that actively secretes immunoregulatory lymphokines or displays targeted cytotoxicity. Ultimately, this results in the recruitment of other cell types and the initiation of an effective immune response. The activation, fate, and outcome of such induced effector functions of the clonotypic T cell are largely controlled by direct cell-to-cell contact to mediate an exchange of information via antigen and adhesion receptors as well as costimulatory molecules. The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunological (i) synapse between the membranes of the two cells. The formation of this functional signaling moiety triggers signaling pathCurrent Opinion in Cell Biology 2009, 21:262–267
ways that involve molecules such as protein kinases, protein phosphatases, and small GTPases. In T cells, this activation cycle crucially includes cytoskeletal remodeling and b2-integrin-mediated cell adhesion [1]. These events transactivate key transcription factors like NFAT (nuclear factor of activated T cells), AP-1 (activator protein-1), and NF-kB (nuclear factor of kB) during the course of sustained T cell activation [2–4]. Subsequently, immediate-early genes like cytokines, which are crucial in the regulation of T cells during clonotypic expansion [5], are expressed. Recruitment of PKC isotypes as amplifying kinases crucially modulates signal strength to overcome negative regulators of cellular signaling [6]. Sustained activation of crucial transcription factors and subsequent cytokine amplification signaling allows entry into S-phase of the cell cycle and promotes cell survival, resulting in many rounds of proliferation of the clonotypic T cells. Currently, a cohesive picture has emerged of the decisive cellular and molecular interactions that determine the ‘success’ of downstream signaling in antigen-specific lymphocytes (see for review [7]). Recently, functional analysis of PKC family members in the complex signaling pathways downstream of the T cell receptor (TCR) and CD28 co-receptor has greatly advanced our understanding of the unique aspects of T cell type-selective signaling pathways. Employing mostly PKC-isotype selective knockout mice, physiological functions within the transactivation pathways of NFkB, NFAT, AP-1 [8–11], and recently, b2-intregrin mediated T cell adhesion were delineated [12]. Given the central role of T cells in immune responses and, consistent with this concept of PKC as a central regulator of T cell fate, the first breakthrough was achieved; highly selective and potent inhibitors of PKC, like AEB071, emerged as a potential new class of immunosuppressants and were found to specifically abrogate early T cell activation in in vivo preclinical models of solid organ graft rejection [13]. Furthermore, AEB071 significantly improved symptoms of psoriasis, a T cell-dependent autoimmune disease, in a two-week proof-of-concept clinical trial [14]. Thus, the pharmacologic blockade of the PKC pathway might indeed offer an innovative mechanism-based therapeutic strategy for transplanted patients and patients suffering from T cell-dependent autoimmune pathologies. AEB071 may only be the first example of the very good overall potential of innovative therapeutic small molecule drugs targeting PKC isotypes and/or their downstream effector substrates in T cell signaling. www.sciencedirect.com
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The PKC kinases, a gene family of nine isotypes
PKC u sets the T cell activation threshold by positively regulating cytokine responses
PKC isotypes are members of the serine/threonine protein kinase family originally identified by Nishizuka and co-workers in 1977 as a cyclic nucleotide-independent protein kinase that phosphorylated histone and protamine in bovine cerebellum [15]. According to their structure and cofactor dependency, PKCs can be classified as conventional PKC isotypes a, b, and g; the novel PKCs d, e, h, and u; and the atypical PKCs z and i. Conventional PKCs require Ca2+ and diacylglycerol (DAG) for activation; novel PKCs are Ca2+-independent; and atypical PKCs require neither Ca2+ nor DAG for activation [16]. Some of the isotypes exist as different splice variants (see for review [17]). PKC function is regulated by transphosphorylation and autophosphorylation as well as by the translocation of PKC from one cellular compartment to another (see for review [18,19]), where lipid activators and proteins that bind to the activated form of the enzyme are in proximity to PKC substrates.
Published data demonstrate that PKC u, with its largely restricted T cell-expression profile [35], is essential in early T cell activation. PKC u knockout experiments reveal signaling defects in the TCR signal transduction pathways for both NFAT/AP-1 and NF-kB transactivation, and subsequently IL-2 cytokine release as well as CD25 and CD69 surface expression. A further clue was finding that PKC u also acts as a key molecule in the regulation of intrinsic T cell survival [36,37]. Mechanistically, PKC signaling to cause NFAT/AP-1 transactivation crucially involves NR2F6 inactivation, presumably by stimulating the release of NR2F6 from DNA binding sites [38]. This facilitates the binding of NFAT/AP-1 to its enhancer element in various promoters, like those of IL-2 and IL17 gene loci. Consistently, PKC u single-deficient [10] and particularly, PKC a/u double-deficient T cells have almost no remaining TCR/NFAT/AP-1 transactivation signaling (G. Baier, unpublished) and the TCR/NFAT/ AP-1 transactivation pathway is dramatically amplified in NR2F6 / CD4+ T cells [38].
Among the major T cell expressed PKC isotypes a, b, d, e, z, h, u, and i (for the mRNA expression profiles, see: http://symatlas.gnf.org/SymAtlas/), PKC u was identified as essential for TCR/CD3-mediated activation events [20,21]. Thus, PKC u provides a molecular basis for isotype selectivity and the non-redundant function of distinct PKC isoenzymes in T cells (see for review [22]). As shown by confocal microscopy, PKC u is indeed the only isotype that is rapidly recruited to the immunological synapse upon T cell engagement [23]. PKC u plays an important role in the activation of transcription factors as well as in T cell immune responses in vivo [8,10,11,24– 26]. Furthermore, PKC u has been shown to be crucial in the TCR-mediated activation of the b2-integrin adhesiveness of T cells [12]. Key functions in T cells have also been reported for the cPKC a and b isotypes. PKC adeficient mice have a Th1 defect and strongly reduced IFN-g production [9]. While T cell signaling appears intact in PKC b-deficient mice [27], lymphocyte function-associated antigen-1 (LFA-1) mediated outside-in signaling, which is required for T cell locomotion, is impaired [28]. Unlike other PKC family members, PKC d was mapped to a signaling pathway that is necessary for T cell attenuation [29]. By contrast, the isotypeselective functions of PKC e and PKC z appear dispensable in TCR/CD28 signaling pathways leading to IL2 secretion responses of primary CD3+ T cells [30,31]. The latter is most probably due to a functional redundancy between PKC z and the highly homologous PKC i in T cell activation responses. Nevertheless, a partial Th2 defect in PKC z knockout mice indicates a crucial function for PKC z in T cell-dependent immune responses in vivo [32]. The established role of PKC family members in B cells and other hematopoietic cell types is reviewed elsewhere [33,34]. www.sciencedirect.com
PKC u mediates the activation-dependent adhesive capacity of T cells Immune cells are migratory cells that move between tissues in response to immune defense, recognizing antigenic peptides that are presented through the major histocompatibility complex (MHC) of APC. The peptide/MHC encounter initiates a program of reciprocal signaling. As result of a stable adhesive contact, an isynapse consisting of a central zone formed by the TCRMHC-interaction and an outer ring of adhesion molecules is formed to stabilize the information flow between the T cell and the APC. Crucial adhesion molecules are the b2integrins, namely LFA-1, on T cells and the immunoglobulin-like cell adhesion molecules, namely ICAM-1, on APCs. LFA-1 is a transmembrane molecule constitutively expressed in T cells and LFA-1 avidity to ICAM-1 is regulated via control of conformation and surface distribution downstream of several signaling pathways inside the T cell, a pathway called ‘inside-out signaling’ [39]. The Rap1 pathway is essential for inside-out signaling in T cells [40]. Recently, we provided evidence of a novel PKC u/RapGEF2 complex that controls Rap-1 activation downstream of the TCR and, subsequently, b2-integrin adhesion of CD3+ T cells. PKC u interacts with and directly phosphorylates RapGEF2. These recent data demonstrate a crucial and non-redundant role for PKC u and its substrate, RapGEF2, in T cell inside-out signaling to Rap1/LFA-1 to sustain TCR engagement and enhance T cell effector functions [12].
Pharmacological inhibition of PKC; where do we stand? Since the literature suggests that PKC u is an attractive target for modulation of the adaptive immune response, Current Opinion in Cell Biology 2009, 21:262–267
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PKC u / mice were studied in several in vivo disease models for immune-related disorders. These studies showed that PKC u / mice fail to develop experimental allergic encephalomyelitis [41,42], display drastically reduced lung inflammation after induction of allergic asthma [43], and have a significantly diminished response in experimental colitis [44] and a type II collageninduced arthritis model [45]. Of note, PKC u / mice may still mount a normal protective Th1 immune response to clear viral infections [46]. Taken together, this validates PKC u as a particularly attractive target for developing ways to selectively manipulate T cell effector functions that are relevant to pathogenesis of different diseases, including asthma, rheumatoid arthritis, and multiple sclerosis. Recently, reduced alloreactivity in an allogeneic heart transplantation experiment was reported in PKC u / mice [47]. However, using our
established PKC u / mice as recipients, we found that such cardiac allograft survival times were only minimally prolonged (G. Baier, unpublished). Thus, PKC u might be necessary but insufficient to block alloimmune responses. Additional PKC isotypes might be involved in crucial signaling pathways in vivo. Together, these results suggest that PKC u inhibition alone may not be sufficient to prevent solid organ rejection in contrast with other immune-related disorders like asthma, rheumatoid arthritis, and multiple sclerosis. Consequently, it is likely that pharmacologic inhibition of multiple PKC isotypes has to be considered for effective immunomodulation after transplantation. Most protein kinase inhibitors target the adenosine triphosphate (ATP) binding site, which is well conserved even among distantly related protein kinases. Therefore,
Figure 1
PKC isotypes set the threshold for T cell activation by positively regulating both cytokine responses and the adhesive capacity of T lymphocytes. This schematic representation depicts the current knowledge of the physiological and non-redundant functions of the two crucial PKC gene products, PKC a and PKC u, in T cells. PKC u, and probably PKC a as well, are crucial in antigen receptor mediated T cell activation (abbreviations: Akt, Akt/Protein kinase B; AP-1, activating protein-1; Bcl10, B cell CLL/lymphoma 10 gene; CaN, calcineurin A; CARD11, caspase recruitment domain family, member 11; CD28 RE, CD28 response element; Cot, mitogen-activated protein kinase kinase kinase 8; CsA, cyclosporine A, IKK I-k B kinase; IL-2, interleukin2; IL-2R, interleukin-2 receptor; LFA-1, lymphocyte function-associated antigen 1; MALT1, mucosa associated lymphoid tissue lymphoma translocation gene 1; NFAT, nuclear factor of activated T cells; NF-kB, nuclear factor of kB; NR2F6, nuclear receptor subfamily 2, group F, member 6; Oct1, POU domain, class 2, transcription factor 1; PKC, protein kinase C). Current Opinion in Cell Biology 2009, 21:262–267
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PKC as a drug target Baier and Wagner 265
the design of inhibitors that selectively target even a subset of the approximately 570 related human protein kinases continues to be a daunting challenge. Early on, a number of cell-permeable inhibitors of the PKC family were developed and have been used extensively in cellbased assays to study the physiological roles of this protein kinase family. Among them are staurosporines and related bisindolylmaleimides (e.g. Go¨6976 and Ro 318220). Unfortunately, the specificity of these compounds has not been rigorously tested, and they sometimes inhibit other kinase families more potently than the targeted PKC isotypes [48,49]. Thus, conclusions drawn from the use of these inhibitors in intact cells may have been somewhat misleading. So far, the most successful of the initial PKC inhibitors is LY333531, a specific inhibitor of PKC b, which is currently being developed for diabetic complications [50]. More recently, the search for PKC inhibitors with various specificities was intensified, guided by the X-ray crystal structures of the catalytic domains of PKC b and u [51,52]. Interestingly, selective PKC u-specific inhibitors have been reported, but no clinical trials have started yet [53,54]. A major advance was achieved with AEB071, a very potent and selective low molecular weight inhibitor of both novel and classical PKCs. AEB071 has a greatly improved target selectivity profile in vitro [14]. Current biochemical and pharmacological characterization shows that AEB071 inhibits both of the crucial cytokines, IL-2 and IFN-g, effects that are reminiscent of the phenotypes of the PKC a and PKC u single and double knockout mice [9,10]. Preclinical studies have shown that AEB071 prolongs rat heterotopic heart transplant survival and cynomolgus monkey renal allograft survival when administered as an oral monotherapy or in combination with adjunct immunosuppressive agents [13]. Furthermore, AEB071 blocks T cell activation through a mechanism independent of calcineurin inhibitors, which are the current standard therapy ([13], M. Bigaud, C. Pally, Novartis, unpublished). Thus, AEB071 may lack the toxicities associated with inhibition of the calcineurin pathway. The goal of future academic research, therefore, is to define the most crucial downstream PKC substrates and their effector functions in early T cell activation and alloantigen-induced T cell responses. These effector substrates, once established, might continue to offer an innovative, mechanism-based, therapeutic strategy for transplanted patients and patients who have T cell-dependent autoimmune diseases.
Conclusion PKC isotypes, particularly PKC u and PKC a, set the threshold for T cell activation by positively regulating both cytokine responses and the adhesive capacity of T lymphocytes (Figure 1). Genetic evidence and pharmawww.sciencedirect.com
cological studies reviewed here strongly suggest that PKC isotypes have essential functions in promoting both early T cell activation and sustained T cell adhesion. Thus, substances like AEB071 may well represent novel treatment options for patients who have T cell-dependent immune disorders, even though the PKC isotype selectivity profile required for in vivo efficacy may differ between subsets of diseases. The validity of this hypothesis and its possible clinical implications remain to be seen.
Acknowledgements This work was supported by grants from the FWF Austrian Science Fund (SFB-021 and P19505-B05), the Tyrolian Science Fund (TWF-2008-1-563), the Jubila¨umsfonds of the OeNB Austrian National Bank (Project 12196), and the European Community Seventh Framework Programme under grant agreement n8HEALTH-F4-2008-201106. We apologize to those colleagues whose work could not be cited because of space restrictions.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Krawczyk C, Penninger JM: Molecular controls of antigen receptor clustering and autoimmunity. Trends Cell Biol 2001, 11:212-220.
2.
Crabtree GR, Olson EN: NFAT signaling: choreographing the social lives of cells. Cell 2002, 109(Suppl.):S67-S79.
3.
Macian F, Lopez-Rodriguez C, Rao A: Partners in transcription: NFAT and AP-1. Oncogene 2001, 20:2476-2489.
4.
Schmitz ML, Krappmann D: Controlling NF-kB activation in T cells by costimulatory receptors. Cell Death Differ 2006, 13:834-842.
5.
Weaver CT, Hatton RD, Mangan PR, Harrington LE: IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol 2007, 25:821-852.
6.
Zheng Y, Zha Y, Gajewski TF: Molecular regulation of T cell anergy. EMBO Rep 2008, 9:50-55.
7.
Marsland BJ, Kopf M: T cell fate and function: PKC u and beyond. Trends Immunol 2008, 29:179-185.
8.
Sun Z, Arendt CW, Ellmeier W, Schaeffer EM, Sunshine MJ, Gandhi L, Annes J, Petrzilka D, Kupfer A, Schwartzberg PL et al.: PKC u is required for TCR-induced NF-kB activation in mature but not immature T lymphocytes. Nature 2000, 404:402-407.
9.
Pfeifhofer C, Gruber T, Letschka T, Thuille N, Lutz-Nicoladoni C, Hermann-Kleiter N, Braun U, Leitges M, Baier G: Defective IgG2a/2b class switching in PKCaS/S mice. J Immunol 2006, 176:6004-6011.
10. Pfeifhofer C, Kofler K, Gruber T, Tabrizi NG, Lutz C, Maly K, Leitges M, Baier G: Protein kinase C u affects Ca2+ mobilization and NFAT activation in primary mouse T cells. J Exp Med 2003, 197:1525-1535. 11. Altman A, Kaminski S, Busuttil V, Droin N, Hu J, Tadevosyan Y, Hipskind RA, Villalba M: Positive feedback regulation of PLCg1/ Ca2+ signaling by PKC u in restimulated T cells via a Tec kinase-dependent pathway. Eur J Immunol 2004, 34:2001-2011. 12. Letschka T, Kollmann V, Pfeifhofer-Obermair C, Lutz Nicoladoni C, Obermair GJ, Fresser F, Leitges M, HermannKleiter N, Kaminski S, Baier G: PKC u selectively controls the adhesion-stimulating molecule Rap1. Blood 2008, 112:4617-4627. Using molecular cell biology approaches as well as PKC isotype-deficient T cells, this study for the first time implicates PKCu in TCR-induced Rap1 activation and LFA-1 mediated T cell adhesiveness. Current Opinion in Cell Biology 2009, 21:262–267
266 Cell regulation
13. Vincenti F, Kirk AD: What’s next in the pipeline. Am J Transplant 2008, 8:1972-1981. 14. Skvara H, Dawid M, Kleyn E, Wolff B, Meingassner JG, Knight H, Dumortier T, Kopp T, Fallahi N, Stary G et al.: The PKC inhibitor AEB071 may be a therapeutic option for psoriasis. J Clin Invest 2008, 118:3151-3159. This is the first study that demonstrates that treatment with a PKC inhibitor may improve the symptoms of patients suffering from severe psoriasis. Long-term studies will be required as a follow-up to this twoweek proof-of-concept study to fully assess safety and efficacy of AEB071. 15. Takai Y, Kishimoto A, Inoue M, Nishizuka Y: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem 1977, 252:7603-7609. 16. Steinberg SF: Structural basis of protein kinase C isoform function. Physiol Rev 2008, 88:1341-1378. 17. Kofler K, Erdel M, Utermann G, Baier G: Molecular genetics and structural genomics of the human protein kinase C gene module. Genome Biol 2002:3. 18. Newton AC: Lipid activation of protein kinases. J Lipid Res 2008 Nov 24. [Epub ahead of print] PMID: 19033211. 19. Gallegos LL, Newton AC: Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm. IUBMB Life 2008, 60:782-789. 20. Baier G, Telford D, Giampa L, Coggeshall KM, Baier-Bitterlich G, Isakov N, Altman A: Molecular cloning and characterization of PKC u, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. J Biol Chem 1993, 268:4997-5004. 21. Baier-Bitterlich G, Uberall F, Bauer B, Fresser F, Wachter H, Grunicke H, Utermann G, Altman A, Baier G: Protein kinase C u isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol Cell Biol 1996, 16:1842-1850. 22. Altman A, Isakov N, Baier G: Protein kinase C u: a new essential superstar on the T cell stage. Immunol Today 2000, 21:567-573. 23. Monks CR, Kupfer H, Tamir I, Barlow A, Kupfer A: Selective modulation of PKC u during T cell activation. Nature 1997, 385:83-86. 24. Hermann-Kleiter N, Thuille N, Pfeifhofer C, Gruber T, Schafer M, Zitt C, Hatzelmann A, Schudt C, Leitges M, Baier G: PKCu and PKA are antagonistic partners in the NFAT transactivation pathway of primary mouse CD3+ T lymphocytes. Blood 2006, 107:4841-4848. 25. Thuille N, Heit I, Fresser F, Krumbock N, Bauer B, Leuthaeusser S, Dammeier S, Graham C, Copeland TD, Shaw S et al.: Critical role of novel Thr-219 autophosphorylation for the cellular function of PKC u in T lymphocytes. EMBO J 2005, 24:3869-3880. 26. Gruber T, Freeley M, Thuille N, Heit I, Shaw S, Long A, Baier G: Comment on ‘PDK1 nucleates T cell receptor-induced signaling complex for NF-kB activation’. Science 2006, 312:55. 27. Thuille N, Gruber T, Bock G, Leitges M, Baier G: Protein kinase C b is dispensable for TCR-signaling. Mol Immunol 2004, 41:385-390. 28. Volkov Y, Long A, McGrath S, Ni Eidhin D, Kelleher D: Crucial importance of PKC b(I) in LFA-1-mediated locomotion of activated T cells. Nat Immunol 2001, 2:508-514. 29. Gruber T, Barsig J, Pfeifhofer C, Ghaffari-Tabrizi N, Tinhofer I, Leitges M, Baier G: PKC d is involved in signal attenuation in CD3+ T cells. Immunol Lett 2005, 96:291-293. 30. Gruber T, Fresser F, Jenny M, Uberall F, Leitges M, Baier G: PKC u cooperates with atypical PKC z and PKC i in NF-kB transactivation of T lymphocytes. Mol Immunol 2007. 31. Gruber T, Thuille N, Hermann-Kleiter N, Leitges M, Baier G: Protein kinase C e is dispensable for TCR/CD3-signaling. Mol Immunol 2005, 42:305-310. Current Opinion in Cell Biology 2009, 21:262–267
32. Martin P, Villares R, Rodriguez-Mascarenhas S, Zaballos A, Leitges M, Kovac J, Sizing I, Rennert P, Marquez G, Martinez AC et al.: Control of Th2 cell function and allergic airway inflammation by PKC z. Proc Natl Acad Sci U S A 2005, 102:9866-9871. 33. Guo B, Su TT, Rawlings DJ: Protein kinase C family functions in B cell activation. Curr Opin Immunol 2004, 16:367-373. 34. Tan SL, Parker PJ: Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem J 2003, 376:545-552. 35. Wilda M, Ghaffari-Tabrizi N, Reisert I, Utermann G, Baier G, Hameister H: Protein kinase C isoenzymes: selective expression pattern of protein kinase C u; during mouse development. Mech Dev 2001, 103:197-200. 36. Hayashi K, Altman A: Protein kinase C u (PKCu): a key player in T cell life and death. Pharmacol Res 2007, 55:537-544. 37. Saibil SD, Jones RG, Deenick EK, Liadis N, Elford AR, Vainberg MG, Baerg H, Woodgett JR, Gerondakis S, Ohashi PS: CD4+ and CD8+ T cell survival is regulated differentially by protein kinase C u, c-Rel, and protein kinase B. J Immunol 2007, 178:2932-2939. 38. Hermann-Kleiter N, Gruber T, Lutz-Nicoladoni C, Thuille N, Fresser F, Labi V, Schiefermeier N, Warnecke M, Huber L, Villunger A et al.: The nuclear orphan receptor NR2F6 suppresses lymphocyte activation and T helper 17-dependent autoimmunity. Immunity 2008, 29:205-216. In this study orphan receptor NR2F6, a direct substrate and effector of PKC, is established as transcriptional repressor to NFAT/AP-1 and subsequently IL-17 transcription and Nr2f6-deficient mice have hyper-reactive lymphocytes and develop autoimmune pathology. 39. Burbach BJ, Medeiros RB, Mueller KL, Shimizu Y: T cell receptor signaling to integrins. Immunol Rev 2007, 218:65-81. 40. Menasche G, Kliche S, Bezman N, Schraven B: Regulation of T cell antigen receptor-mediated inside-out signaling by cytosolic adapter proteins and Rap1 effector molecules. Immunol Rev 2007, 218:82-91. 41. Salek-Ardakani S, So T, Halteman BS, Altman A, Croft M: Protein kinase C u controls Th1 cells in experimental autoimmune encephalomyelitis. J Immunol 2005, 175:7635-7641. 42. Tan SL, Zhao J, Bi C, Chen XC, Hepburn DL, Wang J, Sedgwick JD, Chintalacharuvu SR, Na S: Resistance to experimental autoimmune encephalomyelitis and impaired IL17 production in protein kinase C u-deficient mice. J Immunol 2006, 176:2872-2879. 43. Salek-Ardakani S, So T, Halteman BS, Altman A, Croft M: Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase C u. J Immunol 2004, 173:6440-6447. 44. Nagahama K, Ogawa A, Shirane K, Shimomura Y, Sugimoto K, Mizoguchi A: Protein kinase C u plays a fundamental role in different types of chronic colitis. Gastroenterology 2008, 134:459-469. This is the first study showing a role of PKC u in experimental colitis, a mouse model similar to inflammatory bowel disease. 45. Healy AM, Izmailova E, Fitzgerald M, Walker R, Hattersley M, Silva M, Siebert E, Terkelsen J, Picarella D, Pickard MD et al.: PKC u-deficient mice are protected from Th1-dependent antigeninduced arthritis. J Immunol 2006, 177:1886-1893. 46. Giannoni F, Lyon AB, Wareing MD, Dias PB, Sarawar SR: Protein kinase C u is not essential for T cell-mediated clearance of murine g–herpesvirus 68. J Virol 2005, 79:6808-6813. 47. Manicassamy S, Yin D, Zhang Z, Molinero LL, Alegre ML, Sun Z: A critical role for protein kinase C u-mediated T cell survival in cardiac allograft rejection. J Immunol 2008, 181:513-520. This is the first study showing a role of PKC u in alloreactivity and graft survival of experimental organ transplants. www.sciencedirect.com
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48. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P: The selectivity of protein kinase inhibitors: a further update. Biochem J 2007, 408:297-315. 49. Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT et al.: A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008, 26:127-132. The most comprehensive study of kinase selectivity to date including >50% of the predicted human protein kinome. The interaction maps of 38 kinase inhibitors that are marketed or in late stage developed reveal very diverse interaction patterns. 50. Donnelly R, Idris I, Forrester JV: Protein kinase C inhibition and diabetic retinopathy: a shot in the dark at translational research. Br J Ophthalmol 2004, 88:145-151. 51. Xu ZB, Chaudhary D, Olland S, Wolfrom S, Czerwinski R, Malakian K, Lin L, Stahl ML, Joseph-McCarthy D, Benander C et al.: Catalytic domain crystal structure of PKC u. J Biol Chem 2004, 279:50401-50409.
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52. Grodsky N, Li Y, Bouzida D, Love R, Jensen J, Nodes B, Nonomiya J, Grant S: Structure of the catalytic domain of human protein kinase C b II complexed with a bisindolylmaleimide inhibitor. Biochemistry 2006, 45:13970-13981. 53. Cywin CL, Dahmann G, Prokopowicz AS 3rd, Young ER, Magolda RL, Cardozo MG, Cogan DA, Disalvo D, Ginn JD, Kashem MA et al.: Discovery of potent and selective PKC u inhibitors. Bioorg Med Chem Lett 2007, 17:225-230. 54. Cole DC, Asselin M, Brennan A, Czerwinski R, Ellingboe JW, Fitz L, Greco R, Huang X, Joseph-McCarthy D, Kelly MF et al.: Identification, characterization and initial hit-to-lead optimization of a series of 4-arylamino-3-pyridinecarbonitrile as protein kinase C u inhibitors. J Med Chem 2008, 51:5958-5963. In this study, the discovery of a novel class of PKC inhibitors is described. The optimized compounds are selective for PKC u over the other isotypes and a range of serine/threonine or tyrosine kinases.
Current Opinion in Cell Biology 2009, 21:262–267
PKC inhibitors: potential in T cell-dependent immune diseases Gottfried Baier1 and Ju¨rgen Wagner2 The basic mechanisms of serine/threonine protein kinase signaling networks have been elucidated in the past decade. Members of the protein kinase C (PKC) family are crucial in T cell signaling pathways. Particularly, PKC a, PKC b, and PKC u isotypes determine the nature of lymphocyte-specific in vivo effector responses. Therefore, PKC isotypes are validated drug targets in adaptive immunity. Selective PKC kinase inhibitors have been discovered and are currently in clinical development, where they may provide new therapeutic options for different immune disorders. Here we review the topic of PKC pathway activity in the regulation of T lymphocytes both in the cytokine response and adhesive capacity, and review recent results with PKC inhibitors in vitro and in vivo. Addresses 1 Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria 2 Autoimmunity, Transplantation & Inflammation, Novartis Institute for BioMedical Research, Basel, Switzerland Corresponding author: Baier, Gottfried ([email protected])
Current Opinion in Cell Biology 2009, 21:262–267 This review comes from a themed issue on Cell regulation Edited by Brian Hemmings and Nikolas Tonks Available online 3rd February 2009 0955-0674/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2008.12.008
Introduction The main function of mature T cells is to recognize and respond to foreign antigens. This occurs through a complex activation process that involves activation, adhesion, and differentiation of the resting cell into a proliferating lymphoblast that actively secretes immunoregulatory lymphokines or displays targeted cytotoxicity. Ultimately, this results in the recruitment of other cell types and the initiation of an effective immune response. The activation, fate, and outcome of such induced effector functions of the clonotypic T cell are largely controlled by direct cell-to-cell contact to mediate an exchange of information via antigen and adhesion receptors as well as costimulatory molecules. The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunological (i) synapse between the membranes of the two cells. The formation of this functional signaling moiety triggers signaling pathCurrent Opinion in Cell Biology 2009, 21:262–267
ways that involve molecules such as protein kinases, protein phosphatases, and small GTPases. In T cells, this activation cycle crucially includes cytoskeletal remodeling and b2-integrin-mediated cell adhesion [1]. These events transactivate key transcription factors like NFAT (nuclear factor of activated T cells), AP-1 (activator protein-1), and NF-kB (nuclear factor of kB) during the course of sustained T cell activation [2–4]. Subsequently, immediate-early genes like cytokines, which are crucial in the regulation of T cells during clonotypic expansion [5], are expressed. Recruitment of PKC isotypes as amplifying kinases crucially modulates signal strength to overcome negative regulators of cellular signaling [6]. Sustained activation of crucial transcription factors and subsequent cytokine amplification signaling allows entry into S-phase of the cell cycle and promotes cell survival, resulting in many rounds of proliferation of the clonotypic T cells. Currently, a cohesive picture has emerged of the decisive cellular and molecular interactions that determine the ‘success’ of downstream signaling in antigen-specific lymphocytes (see for review [7]). Recently, functional analysis of PKC family members in the complex signaling pathways downstream of the T cell receptor (TCR) and CD28 co-receptor has greatly advanced our understanding of the unique aspects of T cell type-selective signaling pathways. Employing mostly PKC-isotype selective knockout mice, physiological functions within the transactivation pathways of NFkB, NFAT, AP-1 [8–11], and recently, b2-intregrin mediated T cell adhesion were delineated [12]. Given the central role of T cells in immune responses and, consistent with this concept of PKC as a central regulator of T cell fate, the first breakthrough was achieved; highly selective and potent inhibitors of PKC, like AEB071, emerged as a potential new class of immunosuppressants and were found to specifically abrogate early T cell activation in in vivo preclinical models of solid organ graft rejection [13]. Furthermore, AEB071 significantly improved symptoms of psoriasis, a T cell-dependent autoimmune disease, in a two-week proof-of-concept clinical trial [14]. Thus, the pharmacologic blockade of the PKC pathway might indeed offer an innovative mechanism-based therapeutic strategy for transplanted patients and patients suffering from T cell-dependent autoimmune pathologies. AEB071 may only be the first example of the very good overall potential of innovative therapeutic small molecule drugs targeting PKC isotypes and/or their downstream effector substrates in T cell signaling. www.sciencedirect.com
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The PKC kinases, a gene family of nine isotypes
PKC u sets the T cell activation threshold by positively regulating cytokine responses
PKC isotypes are members of the serine/threonine protein kinase family originally identified by Nishizuka and co-workers in 1977 as a cyclic nucleotide-independent protein kinase that phosphorylated histone and protamine in bovine cerebellum [15]. According to their structure and cofactor dependency, PKCs can be classified as conventional PKC isotypes a, b, and g; the novel PKCs d, e, h, and u; and the atypical PKCs z and i. Conventional PKCs require Ca2+ and diacylglycerol (DAG) for activation; novel PKCs are Ca2+-independent; and atypical PKCs require neither Ca2+ nor DAG for activation [16]. Some of the isotypes exist as different splice variants (see for review [17]). PKC function is regulated by transphosphorylation and autophosphorylation as well as by the translocation of PKC from one cellular compartment to another (see for review [18,19]), where lipid activators and proteins that bind to the activated form of the enzyme are in proximity to PKC substrates.
Published data demonstrate that PKC u, with its largely restricted T cell-expression profile [35], is essential in early T cell activation. PKC u knockout experiments reveal signaling defects in the TCR signal transduction pathways for both NFAT/AP-1 and NF-kB transactivation, and subsequently IL-2 cytokine release as well as CD25 and CD69 surface expression. A further clue was finding that PKC u also acts as a key molecule in the regulation of intrinsic T cell survival [36,37]. Mechanistically, PKC signaling to cause NFAT/AP-1 transactivation crucially involves NR2F6 inactivation, presumably by stimulating the release of NR2F6 from DNA binding sites [38]. This facilitates the binding of NFAT/AP-1 to its enhancer element in various promoters, like those of IL-2 and IL17 gene loci. Consistently, PKC u single-deficient [10] and particularly, PKC a/u double-deficient T cells have almost no remaining TCR/NFAT/AP-1 transactivation signaling (G. Baier, unpublished) and the TCR/NFAT/ AP-1 transactivation pathway is dramatically amplified in NR2F6 / CD4+ T cells [38].
Among the major T cell expressed PKC isotypes a, b, d, e, z, h, u, and i (for the mRNA expression profiles, see: http://symatlas.gnf.org/SymAtlas/), PKC u was identified as essential for TCR/CD3-mediated activation events [20,21]. Thus, PKC u provides a molecular basis for isotype selectivity and the non-redundant function of distinct PKC isoenzymes in T cells (see for review [22]). As shown by confocal microscopy, PKC u is indeed the only isotype that is rapidly recruited to the immunological synapse upon T cell engagement [23]. PKC u plays an important role in the activation of transcription factors as well as in T cell immune responses in vivo [8,10,11,24– 26]. Furthermore, PKC u has been shown to be crucial in the TCR-mediated activation of the b2-integrin adhesiveness of T cells [12]. Key functions in T cells have also been reported for the cPKC a and b isotypes. PKC adeficient mice have a Th1 defect and strongly reduced IFN-g production [9]. While T cell signaling appears intact in PKC b-deficient mice [27], lymphocyte function-associated antigen-1 (LFA-1) mediated outside-in signaling, which is required for T cell locomotion, is impaired [28]. Unlike other PKC family members, PKC d was mapped to a signaling pathway that is necessary for T cell attenuation [29]. By contrast, the isotypeselective functions of PKC e and PKC z appear dispensable in TCR/CD28 signaling pathways leading to IL2 secretion responses of primary CD3+ T cells [30,31]. The latter is most probably due to a functional redundancy between PKC z and the highly homologous PKC i in T cell activation responses. Nevertheless, a partial Th2 defect in PKC z knockout mice indicates a crucial function for PKC z in T cell-dependent immune responses in vivo [32]. The established role of PKC family members in B cells and other hematopoietic cell types is reviewed elsewhere [33,34]. www.sciencedirect.com
PKC u mediates the activation-dependent adhesive capacity of T cells Immune cells are migratory cells that move between tissues in response to immune defense, recognizing antigenic peptides that are presented through the major histocompatibility complex (MHC) of APC. The peptide/MHC encounter initiates a program of reciprocal signaling. As result of a stable adhesive contact, an isynapse consisting of a central zone formed by the TCRMHC-interaction and an outer ring of adhesion molecules is formed to stabilize the information flow between the T cell and the APC. Crucial adhesion molecules are the b2integrins, namely LFA-1, on T cells and the immunoglobulin-like cell adhesion molecules, namely ICAM-1, on APCs. LFA-1 is a transmembrane molecule constitutively expressed in T cells and LFA-1 avidity to ICAM-1 is regulated via control of conformation and surface distribution downstream of several signaling pathways inside the T cell, a pathway called ‘inside-out signaling’ [39]. The Rap1 pathway is essential for inside-out signaling in T cells [40]. Recently, we provided evidence of a novel PKC u/RapGEF2 complex that controls Rap-1 activation downstream of the TCR and, subsequently, b2-integrin adhesion of CD3+ T cells. PKC u interacts with and directly phosphorylates RapGEF2. These recent data demonstrate a crucial and non-redundant role for PKC u and its substrate, RapGEF2, in T cell inside-out signaling to Rap1/LFA-1 to sustain TCR engagement and enhance T cell effector functions [12].
Pharmacological inhibition of PKC; where do we stand? Since the literature suggests that PKC u is an attractive target for modulation of the adaptive immune response, Current Opinion in Cell Biology 2009, 21:262–267
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PKC u / mice were studied in several in vivo disease models for immune-related disorders. These studies showed that PKC u / mice fail to develop experimental allergic encephalomyelitis [41,42], display drastically reduced lung inflammation after induction of allergic asthma [43], and have a significantly diminished response in experimental colitis [44] and a type II collageninduced arthritis model [45]. Of note, PKC u / mice may still mount a normal protective Th1 immune response to clear viral infections [46]. Taken together, this validates PKC u as a particularly attractive target for developing ways to selectively manipulate T cell effector functions that are relevant to pathogenesis of different diseases, including asthma, rheumatoid arthritis, and multiple sclerosis. Recently, reduced alloreactivity in an allogeneic heart transplantation experiment was reported in PKC u / mice [47]. However, using our
established PKC u / mice as recipients, we found that such cardiac allograft survival times were only minimally prolonged (G. Baier, unpublished). Thus, PKC u might be necessary but insufficient to block alloimmune responses. Additional PKC isotypes might be involved in crucial signaling pathways in vivo. Together, these results suggest that PKC u inhibition alone may not be sufficient to prevent solid organ rejection in contrast with other immune-related disorders like asthma, rheumatoid arthritis, and multiple sclerosis. Consequently, it is likely that pharmacologic inhibition of multiple PKC isotypes has to be considered for effective immunomodulation after transplantation. Most protein kinase inhibitors target the adenosine triphosphate (ATP) binding site, which is well conserved even among distantly related protein kinases. Therefore,
Figure 1
PKC isotypes set the threshold for T cell activation by positively regulating both cytokine responses and the adhesive capacity of T lymphocytes. This schematic representation depicts the current knowledge of the physiological and non-redundant functions of the two crucial PKC gene products, PKC a and PKC u, in T cells. PKC u, and probably PKC a as well, are crucial in antigen receptor mediated T cell activation (abbreviations: Akt, Akt/Protein kinase B; AP-1, activating protein-1; Bcl10, B cell CLL/lymphoma 10 gene; CaN, calcineurin A; CARD11, caspase recruitment domain family, member 11; CD28 RE, CD28 response element; Cot, mitogen-activated protein kinase kinase kinase 8; CsA, cyclosporine A, IKK I-k B kinase; IL-2, interleukin2; IL-2R, interleukin-2 receptor; LFA-1, lymphocyte function-associated antigen 1; MALT1, mucosa associated lymphoid tissue lymphoma translocation gene 1; NFAT, nuclear factor of activated T cells; NF-kB, nuclear factor of kB; NR2F6, nuclear receptor subfamily 2, group F, member 6; Oct1, POU domain, class 2, transcription factor 1; PKC, protein kinase C). Current Opinion in Cell Biology 2009, 21:262–267
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the design of inhibitors that selectively target even a subset of the approximately 570 related human protein kinases continues to be a daunting challenge. Early on, a number of cell-permeable inhibitors of the PKC family were developed and have been used extensively in cellbased assays to study the physiological roles of this protein kinase family. Among them are staurosporines and related bisindolylmaleimides (e.g. Go¨6976 and Ro 318220). Unfortunately, the specificity of these compounds has not been rigorously tested, and they sometimes inhibit other kinase families more potently than the targeted PKC isotypes [48,49]. Thus, conclusions drawn from the use of these inhibitors in intact cells may have been somewhat misleading. So far, the most successful of the initial PKC inhibitors is LY333531, a specific inhibitor of PKC b, which is currently being developed for diabetic complications [50]. More recently, the search for PKC inhibitors with various specificities was intensified, guided by the X-ray crystal structures of the catalytic domains of PKC b and u [51,52]. Interestingly, selective PKC u-specific inhibitors have been reported, but no clinical trials have started yet [53,54]. A major advance was achieved with AEB071, a very potent and selective low molecular weight inhibitor of both novel and classical PKCs. AEB071 has a greatly improved target selectivity profile in vitro [14]. Current biochemical and pharmacological characterization shows that AEB071 inhibits both of the crucial cytokines, IL-2 and IFN-g, effects that are reminiscent of the phenotypes of the PKC a and PKC u single and double knockout mice [9,10]. Preclinical studies have shown that AEB071 prolongs rat heterotopic heart transplant survival and cynomolgus monkey renal allograft survival when administered as an oral monotherapy or in combination with adjunct immunosuppressive agents [13]. Furthermore, AEB071 blocks T cell activation through a mechanism independent of calcineurin inhibitors, which are the current standard therapy ([13], M. Bigaud, C. Pally, Novartis, unpublished). Thus, AEB071 may lack the toxicities associated with inhibition of the calcineurin pathway. The goal of future academic research, therefore, is to define the most crucial downstream PKC substrates and their effector functions in early T cell activation and alloantigen-induced T cell responses. These effector substrates, once established, might continue to offer an innovative, mechanism-based, therapeutic strategy for transplanted patients and patients who have T cell-dependent autoimmune diseases.
Conclusion PKC isotypes, particularly PKC u and PKC a, set the threshold for T cell activation by positively regulating both cytokine responses and the adhesive capacity of T lymphocytes (Figure 1). Genetic evidence and pharmawww.sciencedirect.com
cological studies reviewed here strongly suggest that PKC isotypes have essential functions in promoting both early T cell activation and sustained T cell adhesion. Thus, substances like AEB071 may well represent novel treatment options for patients who have T cell-dependent immune disorders, even though the PKC isotype selectivity profile required for in vivo efficacy may differ between subsets of diseases. The validity of this hypothesis and its possible clinical implications remain to be seen.
Acknowledgements This work was supported by grants from the FWF Austrian Science Fund (SFB-021 and P19505-B05), the Tyrolian Science Fund (TWF-2008-1-563), the Jubila¨umsfonds of the OeNB Austrian National Bank (Project 12196), and the European Community Seventh Framework Programme under grant agreement n8HEALTH-F4-2008-201106. We apologize to those colleagues whose work could not be cited because of space restrictions.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Krawczyk C, Penninger JM: Molecular controls of antigen receptor clustering and autoimmunity. Trends Cell Biol 2001, 11:212-220.
2.
Crabtree GR, Olson EN: NFAT signaling: choreographing the social lives of cells. Cell 2002, 109(Suppl.):S67-S79.
3.
Macian F, Lopez-Rodriguez C, Rao A: Partners in transcription: NFAT and AP-1. Oncogene 2001, 20:2476-2489.
4.
Schmitz ML, Krappmann D: Controlling NF-kB activation in T cells by costimulatory receptors. Cell Death Differ 2006, 13:834-842.
5.
Weaver CT, Hatton RD, Mangan PR, Harrington LE: IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol 2007, 25:821-852.
6.
Zheng Y, Zha Y, Gajewski TF: Molecular regulation of T cell anergy. EMBO Rep 2008, 9:50-55.
7.
Marsland BJ, Kopf M: T cell fate and function: PKC u and beyond. Trends Immunol 2008, 29:179-185.
8.
Sun Z, Arendt CW, Ellmeier W, Schaeffer EM, Sunshine MJ, Gandhi L, Annes J, Petrzilka D, Kupfer A, Schwartzberg PL et al.: PKC u is required for TCR-induced NF-kB activation in mature but not immature T lymphocytes. Nature 2000, 404:402-407.
9.
Pfeifhofer C, Gruber T, Letschka T, Thuille N, Lutz-Nicoladoni C, Hermann-Kleiter N, Braun U, Leitges M, Baier G: Defective IgG2a/2b class switching in PKCaS/S mice. J Immunol 2006, 176:6004-6011.
10. Pfeifhofer C, Kofler K, Gruber T, Tabrizi NG, Lutz C, Maly K, Leitges M, Baier G: Protein kinase C u affects Ca2+ mobilization and NFAT activation in primary mouse T cells. J Exp Med 2003, 197:1525-1535. 11. Altman A, Kaminski S, Busuttil V, Droin N, Hu J, Tadevosyan Y, Hipskind RA, Villalba M: Positive feedback regulation of PLCg1/ Ca2+ signaling by PKC u in restimulated T cells via a Tec kinase-dependent pathway. Eur J Immunol 2004, 34:2001-2011. 12. Letschka T, Kollmann V, Pfeifhofer-Obermair C, Lutz Nicoladoni C, Obermair GJ, Fresser F, Leitges M, HermannKleiter N, Kaminski S, Baier G: PKC u selectively controls the adhesion-stimulating molecule Rap1. Blood 2008, 112:4617-4627. Using molecular cell biology approaches as well as PKC isotype-deficient T cells, this study for the first time implicates PKCu in TCR-induced Rap1 activation and LFA-1 mediated T cell adhesiveness. Current Opinion in Cell Biology 2009, 21:262–267
266 Cell regulation
13. Vincenti F, Kirk AD: What’s next in the pipeline. Am J Transplant 2008, 8:1972-1981. 14. Skvara H, Dawid M, Kleyn E, Wolff B, Meingassner JG, Knight H, Dumortier T, Kopp T, Fallahi N, Stary G et al.: The PKC inhibitor AEB071 may be a therapeutic option for psoriasis. J Clin Invest 2008, 118:3151-3159. This is the first study that demonstrates that treatment with a PKC inhibitor may improve the symptoms of patients suffering from severe psoriasis. Long-term studies will be required as a follow-up to this twoweek proof-of-concept study to fully assess safety and efficacy of AEB071. 15. Takai Y, Kishimoto A, Inoue M, Nishizuka Y: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem 1977, 252:7603-7609. 16. Steinberg SF: Structural basis of protein kinase C isoform function. Physiol Rev 2008, 88:1341-1378. 17. Kofler K, Erdel M, Utermann G, Baier G: Molecular genetics and structural genomics of the human protein kinase C gene module. Genome Biol 2002:3. 18. Newton AC: Lipid activation of protein kinases. J Lipid Res 2008 Nov 24. [Epub ahead of print] PMID: 19033211. 19. Gallegos LL, Newton AC: Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm. IUBMB Life 2008, 60:782-789. 20. Baier G, Telford D, Giampa L, Coggeshall KM, Baier-Bitterlich G, Isakov N, Altman A: Molecular cloning and characterization of PKC u, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. J Biol Chem 1993, 268:4997-5004. 21. Baier-Bitterlich G, Uberall F, Bauer B, Fresser F, Wachter H, Grunicke H, Utermann G, Altman A, Baier G: Protein kinase C u isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol Cell Biol 1996, 16:1842-1850. 22. Altman A, Isakov N, Baier G: Protein kinase C u: a new essential superstar on the T cell stage. Immunol Today 2000, 21:567-573. 23. Monks CR, Kupfer H, Tamir I, Barlow A, Kupfer A: Selective modulation of PKC u during T cell activation. Nature 1997, 385:83-86. 24. Hermann-Kleiter N, Thuille N, Pfeifhofer C, Gruber T, Schafer M, Zitt C, Hatzelmann A, Schudt C, Leitges M, Baier G: PKCu and PKA are antagonistic partners in the NFAT transactivation pathway of primary mouse CD3+ T lymphocytes. Blood 2006, 107:4841-4848. 25. Thuille N, Heit I, Fresser F, Krumbock N, Bauer B, Leuthaeusser S, Dammeier S, Graham C, Copeland TD, Shaw S et al.: Critical role of novel Thr-219 autophosphorylation for the cellular function of PKC u in T lymphocytes. EMBO J 2005, 24:3869-3880. 26. Gruber T, Freeley M, Thuille N, Heit I, Shaw S, Long A, Baier G: Comment on ‘PDK1 nucleates T cell receptor-induced signaling complex for NF-kB activation’. Science 2006, 312:55. 27. Thuille N, Gruber T, Bock G, Leitges M, Baier G: Protein kinase C b is dispensable for TCR-signaling. Mol Immunol 2004, 41:385-390. 28. Volkov Y, Long A, McGrath S, Ni Eidhin D, Kelleher D: Crucial importance of PKC b(I) in LFA-1-mediated locomotion of activated T cells. Nat Immunol 2001, 2:508-514. 29. Gruber T, Barsig J, Pfeifhofer C, Ghaffari-Tabrizi N, Tinhofer I, Leitges M, Baier G: PKC d is involved in signal attenuation in CD3+ T cells. Immunol Lett 2005, 96:291-293. 30. Gruber T, Fresser F, Jenny M, Uberall F, Leitges M, Baier G: PKC u cooperates with atypical PKC z and PKC i in NF-kB transactivation of T lymphocytes. Mol Immunol 2007. 31. Gruber T, Thuille N, Hermann-Kleiter N, Leitges M, Baier G: Protein kinase C e is dispensable for TCR/CD3-signaling. Mol Immunol 2005, 42:305-310. Current Opinion in Cell Biology 2009, 21:262–267
32. Martin P, Villares R, Rodriguez-Mascarenhas S, Zaballos A, Leitges M, Kovac J, Sizing I, Rennert P, Marquez G, Martinez AC et al.: Control of Th2 cell function and allergic airway inflammation by PKC z. Proc Natl Acad Sci U S A 2005, 102:9866-9871. 33. Guo B, Su TT, Rawlings DJ: Protein kinase C family functions in B cell activation. Curr Opin Immunol 2004, 16:367-373. 34. Tan SL, Parker PJ: Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem J 2003, 376:545-552. 35. Wilda M, Ghaffari-Tabrizi N, Reisert I, Utermann G, Baier G, Hameister H: Protein kinase C isoenzymes: selective expression pattern of protein kinase C u; during mouse development. Mech Dev 2001, 103:197-200. 36. Hayashi K, Altman A: Protein kinase C u (PKCu): a key player in T cell life and death. Pharmacol Res 2007, 55:537-544. 37. Saibil SD, Jones RG, Deenick EK, Liadis N, Elford AR, Vainberg MG, Baerg H, Woodgett JR, Gerondakis S, Ohashi PS: CD4+ and CD8+ T cell survival is regulated differentially by protein kinase C u, c-Rel, and protein kinase B. J Immunol 2007, 178:2932-2939. 38. Hermann-Kleiter N, Gruber T, Lutz-Nicoladoni C, Thuille N, Fresser F, Labi V, Schiefermeier N, Warnecke M, Huber L, Villunger A et al.: The nuclear orphan receptor NR2F6 suppresses lymphocyte activation and T helper 17-dependent autoimmunity. Immunity 2008, 29:205-216. In this study orphan receptor NR2F6, a direct substrate and effector of PKC, is established as transcriptional repressor to NFAT/AP-1 and subsequently IL-17 transcription and Nr2f6-deficient mice have hyper-reactive lymphocytes and develop autoimmune pathology. 39. Burbach BJ, Medeiros RB, Mueller KL, Shimizu Y: T cell receptor signaling to integrins. Immunol Rev 2007, 218:65-81. 40. Menasche G, Kliche S, Bezman N, Schraven B: Regulation of T cell antigen receptor-mediated inside-out signaling by cytosolic adapter proteins and Rap1 effector molecules. Immunol Rev 2007, 218:82-91. 41. Salek-Ardakani S, So T, Halteman BS, Altman A, Croft M: Protein kinase C u controls Th1 cells in experimental autoimmune encephalomyelitis. J Immunol 2005, 175:7635-7641. 42. Tan SL, Zhao J, Bi C, Chen XC, Hepburn DL, Wang J, Sedgwick JD, Chintalacharuvu SR, Na S: Resistance to experimental autoimmune encephalomyelitis and impaired IL17 production in protein kinase C u-deficient mice. J Immunol 2006, 176:2872-2879. 43. Salek-Ardakani S, So T, Halteman BS, Altman A, Croft M: Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase C u. J Immunol 2004, 173:6440-6447. 44. Nagahama K, Ogawa A, Shirane K, Shimomura Y, Sugimoto K, Mizoguchi A: Protein kinase C u plays a fundamental role in different types of chronic colitis. Gastroenterology 2008, 134:459-469. This is the first study showing a role of PKC u in experimental colitis, a mouse model similar to inflammatory bowel disease. 45. Healy AM, Izmailova E, Fitzgerald M, Walker R, Hattersley M, Silva M, Siebert E, Terkelsen J, Picarella D, Pickard MD et al.: PKC u-deficient mice are protected from Th1-dependent antigeninduced arthritis. J Immunol 2006, 177:1886-1893. 46. Giannoni F, Lyon AB, Wareing MD, Dias PB, Sarawar SR: Protein kinase C u is not essential for T cell-mediated clearance of murine g–herpesvirus 68. J Virol 2005, 79:6808-6813. 47. Manicassamy S, Yin D, Zhang Z, Molinero LL, Alegre ML, Sun Z: A critical role for protein kinase C u-mediated T cell survival in cardiac allograft rejection. J Immunol 2008, 181:513-520. This is the first study showing a role of PKC u in alloreactivity and graft survival of experimental organ transplants. www.sciencedirect.com
PKC as a drug target Baier and Wagner 267
48. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P: The selectivity of protein kinase inhibitors: a further update. Biochem J 2007, 408:297-315. 49. Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT et al.: A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008, 26:127-132. The most comprehensive study of kinase selectivity to date including >50% of the predicted human protein kinome. The interaction maps of 38 kinase inhibitors that are marketed or in late stage developed reveal very diverse interaction patterns. 50. Donnelly R, Idris I, Forrester JV: Protein kinase C inhibition and diabetic retinopathy: a shot in the dark at translational research. Br J Ophthalmol 2004, 88:145-151. 51. Xu ZB, Chaudhary D, Olland S, Wolfrom S, Czerwinski R, Malakian K, Lin L, Stahl ML, Joseph-McCarthy D, Benander C et al.: Catalytic domain crystal structure of PKC u. J Biol Chem 2004, 279:50401-50409.
www.sciencedirect.com
52. Grodsky N, Li Y, Bouzida D, Love R, Jensen J, Nodes B, Nonomiya J, Grant S: Structure of the catalytic domain of human protein kinase C b II complexed with a bisindolylmaleimide inhibitor. Biochemistry 2006, 45:13970-13981. 53. Cywin CL, Dahmann G, Prokopowicz AS 3rd, Young ER, Magolda RL, Cardozo MG, Cogan DA, Disalvo D, Ginn JD, Kashem MA et al.: Discovery of potent and selective PKC u inhibitors. Bioorg Med Chem Lett 2007, 17:225-230. 54. Cole DC, Asselin M, Brennan A, Czerwinski R, Ellingboe JW, Fitz L, Greco R, Huang X, Joseph-McCarthy D, Kelly MF et al.: Identification, characterization and initial hit-to-lead optimization of a series of 4-arylamino-3-pyridinecarbonitrile as protein kinase C u inhibitors. J Med Chem 2008, 51:5958-5963. In this study, the discovery of a novel class of PKC inhibitors is described. The optimized compounds are selective for PKC u over the other isotypes and a range of serine/threonine or tyrosine kinases.
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