Pharmacological regulation of network kinetics by protein kinase C localization

seminars in I M M U N OL OG Y, Vol 12, 2000: pp. 55]61 doi: 10.1006rsmim.2000.0207, available online at http:rrwww.idealibrary.com on

Pharmacological regulation of network kinetics by protein kinase C localization Daria Mochly-Rosen 1 and Lawrence M. Kauvar 2,U

Protein kinase C (PKC) is a conserved family of 11 seriner threonine kinases. Most cell types express multiple members of the family. Because the catalytic sites are homologous, and able to accommodate a broad range of substrates in vitro, specificity in function is dependent on subcellular localization of each isozyme in each cell type. Physiological stimulation can result in major changes in localization of individual PKC isozymes, mediated through binding to specific anchoring proteins. We describe data demonstrating that disruption of such translocations of PKC isozymes by pharmacological agents, peptides, or antibodies, causes profound effects on T cell functions. The pharmacological opportunity provided by distinct kinetic properties of complex assembly is also discussed.

likely to mediate unique biological effects, it is not surprising in retrospect that unacceptable toxicity accompanied the first generation inhibitors which targeted conserved sites within the regulatory and catalytic domains. In the past few years, new information has emerged on the role of subcellular localization as an important PKC regulatory factor. Clarification of the mechanism underlying differential localization of individual isozymes, namely activation-induced binding of PKC to anchoring proteins,3 enables a novel approach to obtaining isozyme-specific PKC inhibitors that appears more likely to overcome the toxicity encountered earlier. In parallel, the role of PKC in signal transduction in cells of the immune system has become better understood, which has suggested specific therapeutic opportunities for treatment of immune disorders.

Key words: anchoring r localization r protein kinase C ŽPKC. r RACKs Q2000 Academic Press

PKC structure Introduction

The family of 11 PKC isozymes4,5 contains four conserved ŽC1]C4. and five variable ŽV1]V5. regions, with isozymes divided into sub-families according to differences in sub-domain usage and arrangement ŽFigure 1.. The conserved regions in the regulatory domain mediate binding of the activating co-factors Ca2q ŽC2. and diacylglycerol ŽC1., the pharmacological counterpart of the latter ŽDAG. being the well known PKC activator, PMA Žphorbol myristic acid.. C1 is preceded by an autoinhibitory, pseudosubstrate sequence Ž c S.. In the resting state, c S binds the catalytic site, but cannot be phosphorylated since it lacks a phospho-acceptor amino acid.6 Upon activation by Ca2q andror lipid-derived regulators, PKC undergoes a conformational change that renders the catalytic site accessible to substrate. Other intramolecular interactions are discussed below. The C3 and C4 conserved regions, together with the small

IN THE FIRST HALF OF the 1990s, PKC had a surge of popularity as a drug target for suppressing inappropriate T-cell responses.1 Extensive pharmacological studies indicated that PKC antagonists had substantial therapeutic potential for treating allergy, asthma, transplant rejection, and a variety of autoimmune disorders. Despite the initial optimism, no drug candidates have emerged so far.2 Since multiple PKC isozymes are expressed in most cell types and each is From the 1Dept. of Molecular Pharmacology, Stanford Medical School, Stanford, CA 94305-5332, USA and 2 Trellis Bioinformatics, Inc., 1489 Webster St. a521, San Francisco, CA 94115-3769, U USA. Corresponding author. Q2000 Academic Press 1044-5323r 00 r 010055q 07 $35.00r 0

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sumably serves to bring the enzyme into proximity with its proper protein substrates. The earlier view of all inactive PKCs as having a diffusely cytoplasmic localization has been superseded by more detailed studies using isozyme-specific antibodies to define specific localizations in the basal state as well as in the activated state.3 PKC localization is highly regulated, and specific inducers of cellular responses give rise to unique patterns of PKC isozyme translocations, which may differ in different cell types. The localization of each isozyme to a distinct subcellular position strongly suggested specific interactions with other proteins, designated RACKsrRICKs Žreceptors for activatedrinactivated C-kinase..3 The RACK-binding site is distinct from that for STICKs Žsubstrates that interact with C-kinase.,10 since an excess of substrate peptide that blocks STICK binding has no effect on RACK binding. The conformational change that displaces the intramolecular interaction between ␺ S and the catalytic site also exposes the RACK-binding site, presumably by displacing a pseudo-RACK sequence Ž ␺ R. that masks the RACK site in the enzyme’s basal state.11 Additional elements in the regulatory domain of PKC are also involved in the binding of each PKC to its specific anchoring proteins. These conditional interactions can account for a significant part of the unique functions of each isozyme. The role of subcellular localization in PKC function was further advanced by identifying the interaction sites on PKCs and their corresponding RACKs. Peptides corresponding to these sites have provided tools to inhibit translocation in intact cells, in an isozyme-specific manner.3,11 For example, peptide I, comprising 15 residues from the annexin I sequence, inhibits PKC binding to RACKs in vitro. Microinjection of this peptide into Xenopus oocytes inhibits insulin-induced ␤ PKC translocation and oocyte maturation.11 Similarly, peptides that block ␧ PKC translocation in cardiac myocytes inhibit adrenergic modulation of intrinsic contraction rate.3 These experiments demonstrate the feasibility of isozyme-specific pharmacological intervention.1 The phenomenon of subcellular translocation that dominates PKC physiology has parallels in several other signalling cascades.12 For example, protein kinase A ŽPKA. binds to anchoring proteins termed AKAPs via its regulatory domain, with the catalytic domain dissociating and translocating when cAMP levels rise.13 Similarly, the serinerthreonine kinase raf translocates to the plasma membrane where it associates with the small G protein ras in its GTP-

Figure 1. PKC structure. The classical group ŽcPKC. contains all four conserved amino acid regions. ␤ I and ␤ II are alternative mRNA splicing products of the same gene, differing by fewer than 50 amino acids in the V5 region. The novel group ŽnPKC. has its V1 region N-terminal to the cysteine-rich C1 region, and lacks a calcium binding C2 region, although homology of the nPKC class V1 region to C2 has been identified. The atypical group ŽaPKC. also lacks the C2 region as well as half of the C1 region. The most deviant isozyme, ␮ PKC, contains a putative membrane spanning domain at its N-terminus and a pleckstrin homology ŽPH. domain.

intervening V4 region, create a catalytic domain able to phosphorylate serinerthreonine. Although certain protein substrates show high specificity for a particular isozyme, an extensive study of consensus peptide substrates for each isozyme revealed only moderate preferences for particular sequences.7 These data suggest that other mechanisms are responsible for isozyme specificity. The most likely such mechanism is the limited access of the activated PKCs to potential substrates due to restricted subcellular localization, mediated by the PKC variable regions.3 Specific functions for individual variable region elements are not completely understood. However, structural information from X-ray crystallography and NMR of some subdomains of PKC,8 and the organization of the whole protein, for example, from analysis of two-dimensional crystals on lipid monolayers9 will help in identifying unique surfaces in different PKC isozymes that are critical for their specific functions.

Localization of PKC isozymes Translocation of PKC follows activation, which pre56

Network regulation by PKC localization

bound form Žsee review by Olson and Marais, this issue.. Therefore, inhibition of protein᎐protein interactions is likely to be a fruitful approach to identify critical members of each signal transduction event. The translocation of PKCs is regulated in a complex manner by both Ca2q and DAG. Recently, the individual domains responding to these second messengers were expressed as GFP-fusion proteins in order to dissect out the regulation and kinetic properties of PKC translocation by individual second messengers. A fusion of Green Fluorescent Protein ŽGFP. to the cysteine rich C1 domain of PKC Žthe PMA binding site. was found to move from a diffusely cytoplasmic localization to the plasma membrane following stimulation of receptors generating diacylglycerol.14 Just as a fluorescently labeled cAMP binding subunit of protein kinase A has proven effective in dissecting patterns and kinetics of signal transduction,15 so too should GFP-tagged translocating domains be useful indicators for studying the second messengers triggering PKC translocation and the time course of these events.

fluorescent antigens in a lipid bilayer as a surrogate for the antigen presenting cell.18 As discussed further below, formation of the TCR complex with other signalling enzymes is a coordinated process that occurs over a period of at least 5 min and could potentially be interrupted by a variety of pharmacological agents. Therefore, understanding the process of TCR complex formation and the manner by which interactions of individual components is controlled in normal and disease states can provide new means to block or enhance individual T cell responses.

PKC regulation of cytokine production in T-cells Compounds that inhibit the catalytic activity of PKC have been used to demonstrate different roles for PKC in T cell responses, but cannot identify the specific isozyme that mediates them. For example, a PKC pseudosubstrate Ž ␺ S. inhibitor, which is specific for PKC compared to other protein kinases, blocks T cell activation.17 Small molecule inhibitors of the catalytic site have also been extensively studied, primarily focused on low nM staurosporine derivatives.1,2 Unlike the parent compound which inhibits many kinases, the derivatives are specific for PKC, with preferential activity on the cPKC subfamily. These compounds show good efficacy not only in human T cells stimulated under physiological conditions, but also in animal models for allergic disease.20 Of special interest for T cell responses is ␪ PKC, the PKC isozyme whose tissue distribution is most nearly restricted to T cells.17 Whereas inhibitors of the catalytic activity have implicated the PKC family in T cell activation, interference with the localization of ␪ PKC has pinpointed this particular isozyme as a key regulator of physiological T cell responses. Experiments using physiological stimulation by APC demonstrated that the early phase of a T cell response involves the specific translocation and activation of ␪ PKC.19 Thus, ␪ PKC moves from a diffuse cytoplasmic localization to a central zone in the immunological synapse between the APC and the T cell, thus placing it in close proximity to the TCR and associated proteins which cluster at this location ŽFigure 2.. Previous studies also suggested an important role for PKC, and especially ␪ PKC, in TCR-mediated responses, initially by experiments in which PKC is directly activated by PMA treatment, in the absence of antigens.16 Combined with activation of parallel pathways by a calcium ionophore, this pharmacologi-

TCR complex formation and the immunological synapse During physiological activation, an antigen-specific T cell is stimulated by specific antigenic peptide complexed with major histocompatibility proteins on the surface of an antigen presenting cell ŽAPC.. The key site for TCR signal transduction is the contact zone between the two cells, which is known as the ‘immunological synapse’ Žsee review by van der Merwe et al, this issue.. The signalling mechanism involves the regulated assembly of many transmembrane proteins, intracellular signalling proteins and cytoskeletal proteins to the contact site. Signalling at the immunological synapse is sensitive to the composition of transient quarternary complexes that cluster around the TCR.16 In addition, the temporal dynamics of assembly of these proteins into the TCR complex determine the physiological outcome arising from antigen presentation. Diverse and opposing physiological outcomes occur following TCR activation, including proliferation, apoptosis, anergy and the induction of a variety of effector functions, such as the secretion of specific cytokines.16,17 The process of assembling the mature TCR signalling complex has begun to be explored, using 57

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cation of activated ␪ PKC, but not the translocation of ␤ or ␧ isozymes, following stimulation with PMA and phytohemagglutinin.21 In these cells, Nef expression also blocks IL-2 production. Thus, suppression of IL-2 production by Nef in T cells may be due to inhibition of ␪ PKC binding to the TCR complex, perhaps explaining the suppression of IL-2 production seen after HIV infection. Antibodies have also been used to interfere with PKC localization and demonstrate a role for ␪ PKC in IL-2 receptor upregulation. ␪ PKC and ␣ PKC translocate within 10 min of T cell stimulation with an anti-CD3 antibody, OKT3, and loading T cells with antibodies to these PKCs by electroporation blocks the early phenotype of IL-2 receptor up-regulation,22 with other isozymes implicated in later events. In a more recent study, electroporation of antibodies to ␪ PKC suppressed IL-4 production in an OKT3 stimulated T cell line.23 Moreover, in the same study, a non-peptide small organic molecule inhibitor of ␪ PKC translocation was shown to selectively inhibit IL-4 secretion compared to IFN-␥ in cells secreting both ŽFigure 3.. Therefore, several approaches indicate that association of ␪ PKC with TCR complex is required for cytokine production. In addition, the 14᎐3᎐3 family of proteins has been implicated in localization of inactive ␪ PKC.17 Both ␪ PKC translocation and expression of a reporter driven by an IL-2 promoter is inhibited by over-expression of 14᎐3᎐3␶ .17 Recent data have also implicated the 14᎐3᎐3 family in cytoplasmic anchoring of other enzymes, including raf, the cell cycle phosphatase Cdc25C, and BAD, a protein that dimerizes with Bcl-XŽL. to promote apoptosis.17 Therefore, conditional co-localization of specific PKC isozymes with different subsets of signalling enzymes

Figure 2. ␪ PKC translocation. ŽA. Phase contrast image shows a T cell contacting an antigen presenting cell. ŽB. TCR, visualized with a fluorescent antibody to CD3, is clustered in the zone of contact. ŽC. ␪ PKC also becomes localized at the zone of contact, visualized with a second color fluorescent antibody.19

cal stimulus induces expression of IL-2, which then acts as an autocrine growth factor. The IL-2 promoter contains multiple transcription factor binding sites, including AP-1 Ž fos r jun., and in murine EL-4 thymoma cells, overexpression of ␪ PKC potentiates PMA-driven expression of a reporter gene coupled to an AP-1 binding site.16,17 These studies provided the early indications that ␪ PKC has a critical role in T cell responses. Further implication of ␪ PKC in T cell responses came from studies that correlated loss of T cell functions with inhibition of ␪ PKC translocation. The first study to interfere with the localization of ␪ PKC made use of the HIV protein Nef expressed in T cells. In vitro studies demonstrated that ␪ PKC is the only PKC isozyme that binds Nef.21 This binding is augmented by phosphatidylserine and diacylglycerol but is unaffected by ␺ S, suggesting that Nef interaction with PKC is not via the enzyme’s substrate binding site. In Jurkat cells, Nef expression prevents normal translo-

Figure 3. Pharmacological block of ␪ PKC translocation. Ža. Translocation of ␪ PKC from soluble to particulate fraction following stimulation by OKT3 Žantibody to CD3. is inhibited by TER14687; ␪ PKC was visualized by Western blotting. Žb. TER14687 suppresses secretion of IL-4 more than IFN-␥ , with the caveat that absolute level of IFN-␥ is 10-fold larger than IL-4 in these cells; secreted cytokines were assayed by ELISA 24 h after OKT3 stimulation.23

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PKC and kinetics of signal transduction

is likely to lead to different physiological outcomes and hence provides additional pharmacological opportunities for intervention.

The ability to respond to foreign versus self antigens by activation or suppression, respectively, of the relevant immune cells is critical to normal function. In T cells, the most informative experiments for illuminating how this is achieved have utilized altered peptide ligands of the T cell receptor.30 With these closely related peptides, the same experimental system can yield opposing functions including a productive activation, an anergic state resistant to activation by an agonist, or apoptosis.16 The property that may account for the diverse cellular responses in T cells and that distinguishes these related peptides is their off rate from the TCR, i.e. the half life of the TCR-peptide interaction.31 The importance of sustained TCR stimulation is further underscored by the more pronounced effect on T cells produced by immobilized OKT3 antibody compared to soluble OKT3.32 These data imply that in vivo self antigens, that are highly abundant but exert weak affinity for TCR are distinguishable from low abundance, high affinity foreign antigens by their off rate from the TCR.31 As mentioned earlier, recent microscopy work Žsee review by van der Merwe et al, this issue., using fluorescent antigen in a lipid bilayer, has begun to reveal the sequence of events that results in a mature immunological synapse.18 The transition from the initial stage, in which TCR and antigen first interact, to a more mature phase in which the TCRs become concentrated in a central core of the contact zone between T cell and antigen presenting cell,19 only occurs with agonist peptides with slow off-rates. Following formation of the central core of this complex, which takes approximately 5 min, there is a further phase of maturation of the synapse in which lateral mobility of the antigens is greatly reduced. This molecular anatomy of this complex is of particular interest given the observation that slow off-rate peptides favor the differentiation of naive CD4q T cells into Th1 effectors, whereas related fast off-rate peptides favor the Th2 phenotype.33 Intracellular Ca2q levels also control the time course of TCR-induced signalling and PKC activation by PMA has two counterbalancing effects on Ca2q levels.34 First, PMA reduces the Ca2q sequestered in the endoplasmic reticulum. Second, PMA potentiates the signal arising from depleted internal stores that leads to opening of SOC, a plasma membrane calcium channel activated by depletion of the intracellular stores. Importantly, agonist peptide also increases

PKC and other regulatory networks in immune cells TCR ligation triggers the activation of multiple downstream signalling proteins, which are part of a complex regulatory network. PKC activators and inhibitors influence the phosphorylation of several of these proteins, including fos kinase, I ␬ B, and c-raf-1.20 However, it is not clear at this point which of these are direct substrates of PKC. By transfecting Jurkat cells with PKC isozymes Žwild-type, kinase dead and constitutively active mutants., it has also been established that ␪ PKC, but neither ␣ PKC nor ␧ PKC, participates in JNK activation, acting synergistically with calcineurin; no such effects were seen on the parallel ERK pathway.24 Moreover, the same proteins have only additive effects on JNK activation in HeLa cells, implying that some additional T-cell specific factor is required to achieve this synergistic effect. The emerging understanding of Fas-mediated apoptosis in T cells further implies a role for PKC in responses to stress stimuli. In a murine CD4q cell line, circumstantial evidence implicates ␣ and ␤ I isozymes25 in this response. In that system, PKC inhibitors block the apoptotic response to concurrent TCR and Fas activation, but only partially block expression of Fas ligands. In light of these results, it is surprising that the same PKC inhibitorŽs. applied during autoantigen stimulation facilitated apoptosis, thereby preventing the development of symptoms in two rat models of T cell mediated autoimmune diseases.26 Duration of stimulation of TCR may account for this discrepancy, as discussed further below. Lymphocyte activation triggers additional effector functions besides cytokine production and mitogenesis. ␤ I and ␦ PKC, for example, translocate to microtubule cytoskeleton elements where they regulate lymphocyte crawling.27 Likewise, exocytosis of cytolytic granules from cytotoxic T lymphocytes is regulated by PKC via microtubule reorganization.28 Even the surface density of TCR, which presumably modulates responsiveness to antigen, is controlled in part by PKC-dependent phosphorylation of CD3␥ .29 Therefore, a variety of phosphorylation events, mediated by different PKC isozymes, influence multiple responses of T cells. 59

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intracellular Ca2q in two phases, corresponding to inositol triphosphate ŽIP3 . induced mobilization of intracellular stores, followed by opening of SOC.16 In contrast, a partial agonist induces the second phase of Ca2q rise, but not the first. As illustrated in Figure 4, genistein, a protein tyrosine kinase inhibitor, markedly inhibits both phases.35 By contrast, genistein has no effect on Ca2q mobilized by a partial agonist. Inhibition of the partial agonist effect can be achieved, however, by GF109203X, a staurosporinederived PKC inhibitor more selective for the calcium activated cPKC subfamily. To complicate matters, GF109203X has no effect on the agonist peptide response. Finally, an antagonist peptide does not cause an early phase of calcium rise but shows high frequency oscillations in the second phase, that is inhibitable by GF109203X. Together, these data indicate a composite role for different PKC isozymes ŽCa2q-sensitive and Ca2q-insensitive. as well as tyrosine kinases in orchestrating the intricate kinetics that govern T cell responses. More detailed mapping of the time course of translocation of each PKC isozyme at different stages of the T cell response may

reveal critical periods in which the composition of the TCR associated complex results in responses as different as anergy and T cell activation. In summary, the deeper appreciation of the PKC family’s complexity gained over the past decade has set the stage for a renewed effort to target selective PKC isozymes for therapeutic purposes. Ameliorating or reversing each of the multiple disease states of the immune system may require targeting different signalling steps. Detailed in-cell microscopy studies can play an important role, complementary to biochemistry, in identifying which translocation event provides the best therapeutic intervention point for a particular syndrome. Feasibility of small molecule intervention in these processes has been established. In addition to the findings in T cells described above,23 another recent report demonstrated the covalent binding of a small molecule to the RACKbinding site of ␣ PKC following UV activation, which may account for the compound’s anti-tumor effects seen in adenocarcinoma cells.36 Translocation inhibitors of PKC, that block or slow down signalling complex assembly, thus represent credible candidates for

Figure 4. PKC influence on calcium mobilization. T cells were immobilized for microscopy and calcium concentration visualized with a fluorescent chelator. Ža. Intracellular calcium time course following activation with full agonist, partial agonist, and antagonist peptides. Effects of Žb. genistein Ža tyrosine kinase inhibitor. and Žc. GF109203X Ža staurosporine based PKC inhibitor..35

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therapeutics to treat diseases associated with the immune system.

19. 20.

Acknowledgements

21.

Figure 2 is reproduced by kind permission from Dr Abraham Kupfer’s web site, - http:rrwww.njc.orgrResearchr LabsrMonkrstruct.html) , where a rotating three-dimensional image of the localization of these proteins in an activated T-cell is also presented. We thank Drs Amnon Altman and Yasuharu Nishimura for careful reading of the manuscript. DM-R’s work is supported by NIH grants HL52141 and AA11147.

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23.

24.

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