The emerging role of protein kinase Cθ in cytoskeletal signaling

Review on the signaling of protein kinase C theta (PKCθ), focusing on signal transduction to cytoskeletal elements critical for cell-type specific responses to stimuli.

Abstract

Cytoskeletal rearrangements often occur as the result of transduction of signals from the extracellular environment. Efficient awakening of this powerful machinery requires multiple activation and deactivation steps, which usually involve phosphorylation or dephosphorylation of different signaling units by kinases and phosphatases, respectively. In this review, we discuss the signaling characteristics of one of the nPKC isoforms, PKCθ, focusing on PKCθ-mediated signal transduction to cytoskeletal elements, which results in cellular rearrangements critical for cell type-specific responses to stimuli. PKCθ is the major PKC isoform present in hematopoietic and skeletal muscle cells. PKCθ plays roles in T cell signaling through the IS, survival responses in adult T cells, and T cell FasL-mediated apoptosis, all of which involve cytoskeletal rearrangements and relocation of this enzyme. PKCθ has been linked to the regulation of cell migration, lymphoid cell motility, and insulin signaling and resistance in skeletal muscle cells. Additional roles were suggested for PKCθ in mitosis and cell-cycle regulation. Comprehensive understanding of cytoskeletal regulation and the cellular “modus operandi” of PKCθ holds promise for improving current therapeutic applications aimed at autoimmune diseases.

Introduction

The PKC family belongs to the AGC group of kinases and consists of a family of ubiquitously expressed serine/threonine phosphotransferases [1–3]. The PKC family can be divided into three main groups: cPKC (α, βI, βII, and γ), nPKC (δ, ϵ, η, θ), and aPKC (ζ and ι/λ). These groups vary in their expression pattern in tissues, in their domain types, and in their manner of activation. All PKCs share a common C-terminal catalytic domain containing an ATP-binding site (C3), and a substrate-binding domain (C4) but differ in the presence, absence, or internal positioning of the regulatory domains C1 and C2 [3]. Members of the cPKC subfamily require calcium and DAG/phorbol esters for activation. Members of the nPKC subfamily lack the typical C2 homology domain and do not require calcium for activation. Members of the aPKC subfamily lack the calcium-binding C2 domain and one-half of the DAG/phorbol ester-binding C1 homologous domain and consequently, are insensitive to DAG/phorbol esters and calcium but bind PIP3 or ceramide instead (Fig. 1; reviewed in ref. [4]).

Figure 1. Schematic representation of primary structures of three different classes of PKC.

The N-terminal regulatory module contains several domains: the C1A and C1B domains (blue) that bind DAG, as well as phorbol esters, DAG's nonhydrolyzable, nonphysiological analogues. Each of the ∼50 residue-long C1A/B tandem modules contains six Cys and two His that coordinate Zn²⁺ binding. The C2 domain binds anionic lipids in a calcium-dependent manner (green). The pseudosubstrate region (red), which is located on the N-terminal end of the C1 domain, is a sequence of amino acids that mimics a substrate; however, it lacks a critical Ser/Thr phosphor-acceptor site, which is replaced by Ala. The pseudosubstrate is involved in PKC autoregulatory mechanisms; it binds the substrate-binding motif in the catalytic domain, thereby keeping the enzyme inactive. The atypical C1 domain (found in aPKCs) does not bind phorbol ester/DAG but binds PIP3 or ceramide. The C-terminal catalytic domain contains the ATP-binding domain C3 (purple) and the substrate-binding domain C4 (orange). C-regions represent conserved domains, and V-regions represent variable domains (V1–V5).

PKCθ, a member of the nPKC subfamily, is unique among PKC isoforms with regard to its expression and cellular functions. It is the major PKC isoform present in hematopoietic [5–8] and skeletal muscle cells [9, 10], and it is also found in neuromuscular junctions [11]. PKCθ has been linked to regulation of migration [12], lymphoid cell motility [13], and insulin signaling and resistance in skeletal muscle cells [14–18]. Possible roles in mitosis and cell cycle have also been suggested [19, 20]. The most comprehensively described functions of PKCθ relate to its role in T cell activation, survival responses in adult T cells, and T cell FasL-mediated apoptosis [6, 21–26]. Intriguingly, most of these processes involve cellular relocation of PKCθ and cytoskeletal rearrangements. In this review, we discuss details of PKCθ-mediated signal transduction to cytoskeletal elements, which results in cellular rearrangements critical for cell type-specific responses to stimuli. The role of PKCθ as a modulator of cytoskeletal rearrangements critical for hematopoietic and muscle cell functions is only partially understood. Comprehensive understanding of cytoskeletal regulation and the cellular ‘modus operandi’ of PKCθ holds promise for improving current therapeutic applications aimed at autoimmune diseases.

PKCθ—CYTOSKELETON CROSS-TALK IN LYMPHOCYTES

Initial observations

The first report indicating possible involvement of PKCθ in actin polymerization-dependent processes dates to 1997, in which Tang and colleagues [27] demonstrated a requirement for PKCθ activation in cell-cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells. This report was followed by findings that PKCθ, via its interactions with various adapter and linker proteins, is also involved in maintaining T cell polarity [28, 29]. In 1997, Monks and colleagues [6] reported a unique and a highly selective translocation of PKCθ to the central region of the IS, a structure formed at the interface between an activated T cell and APC (details in section TCR clustering and the IS). This finding indicated a critical function of PKCθ in T cell activation. Subsequent observations revealed that formation of the IS required several proteins known to be critical for actin cytoskeleton regulation, including talin, vinculin, and Vav (a GEF for small Rho GTPases), which are necessary for proper signal propagation from clustered TCRs to cytoskeleton [6, 30–33]. Additional proteins known to be involved in IS formation include Rac1, a member of the Rho family of GTPases, and actin cytoskeleton regulatory proteins, Abi/WAVE [33–36].

TCR clustering and the IS

The IS is the ultimate example of coclustering of signaling components and cytoskeletal scaffold proteins [37]. The activation of T cells by APCs is spatially restricted to their site of contact, where receptors on the T cell engage their counter-receptors on the APCs. When APC interacts with TCR, a multiprotein molecular complex is formed at the contact area. This complex is called the IS or SMAC (for a detailed review, see refs. [37–41]). Formation of this multiprotein machinery is essential for the T cell immune response and is tightly regulated at signaling and spatial-cytoskeletal levels. Spatial segregation of accumulated proteins at the interface of the contact site is a multistep process, which involves formation of central SMAC, peripheral SMAC, and distal SMAC, each composed of different proteins [39, 40, 42]. The IS is considered to have three functional layers: receptor, signaling, and cytoskeletal. The receptor layer is formed from TCR, CD3, LFA-1, CD4 or CD8, LAT, CD28, and CD45. The signaling layer includes Lck, Zap-70, SLP-76, LAT, PLCγ, ITK, and PKCθ. The cytoskeletal layer is composed of actin, myosin II, tubulin, talin, vinculin, and FAK 1/2 [41].

The spatial-temporal arrangement at the IS depends heavily on actin polymerization [33, 37, 38, 43] and Lck-dependent association of PKCθ with the coreceptor CD28 at the TCR site [44–46]. Antigen-induced localization of PKCθ to the IS is well recognized [6, 45, 47]; however, its detailed molecular basis remains elusive. Membrane translocation and subsequent activation of cPKCs or nPKCs require the presence of their conserved domain C1, which binds to DAG (or phorbol esters), a membrane second messenger generated upon TCR-mediated activation of PLCγ [3, 47, 48]. The activation of PLCγ1 results in the generation of IP3 and DAG. IP3 induces an increase in cytosolic calcium, whereas DAG activates PKC. The transduced signal leads to activation of AP-1 and NF-κB transcription factors controlling production of IL-2, which is essential for T cell activation (Fig. 2) [49]. PKCθ activation and recruitment to the membrane were thought to be solely dependent on PLCγ activity, until studies using PLCγ inhibitors clearly demonstrated that PKCθ recruitment to the membrane and its activation was only partially dependent on PLCγ [30]. A coexisting mechanism was postulated, in which TCR-coupled protein tyrosine kinases activate Vav, which then activates Rac and/or Cdc42, leading to polymerization of actin and TCR capping, ultimately driving translocation of PKCθ to the TCR (or cSMAC) in the IS [30, 50]. Upon TCR activation, Vav becomes phosphorylated rapidly, and this phosphorylation has been reported to be Zap-70-dependent; however, it is unlikely that Vav is a direct Zap-70 substrate [51]. Vav, via its SH2 domain, forms a stable complex with phosphorylated (by Zap-70) SLP-76, which activates effectors downstream of TCR activation, such as Rac/Cdc42, NF-κB, or NF-AT [52]. Phosphorylated SLP-76 also interacts with Nck, which is essential for the activation of WASP and WAVE [53]. The interaction of Nck with WASP seemingly brings actin polymerization machinery to the plasma membrane in the vicinity of activated TCR [54]. WASP is expressed only in hematopoietic cells and was the first identified member of the family of cytoskeletal regulatory proteins N-WASP and Scar/WAVE [55]. WASP interacts directly with Cdc42, G-actin, Arp2/3, Nck, and WIP [55]. It was shown that WASP localizes to F-actin in the IS, where it becomes activated by GTP-bound Cdc42, generated following activation of Vav [53–55]. The mechanism of dissociation of the WASP–WIP complex and IS recruitment of WASP are not well understood. It has been proposed that WIP binds to the adaptor protein CrkL, and following TCR ligation, the CrkL-WIP-WASP complex is recruited to the IS by phosphorylated (by Lck) Zap-70 (which binds CrkL); in the IS, phosphorylation of WIP by PKCθ induces dissociation of WASP [55–57]. Released WASP is then activated by binding to GTP-bound Cdc42, which induces a conformational change in WASP that allows its C-terminal domain to bind and activate the Arp2/3 complex responsible for initiation of actin polymerization. Therefore, WIP controls actin polymerization in two ways: by keeping WASP in an inactive conformation that prevents it from binding to Arp2/3 and by binding and stabilizing actin filaments during TCR activation (Fig. 3) [55–57]. Another mechanism has also been proposed, in which a complex formed by SLP-76-associated protein/Fyb, SLP-76, Nck, and WASP is recruited to the IS via the lipid-raft associated protein LAT [54]. The role of lipid rafts in TCR clustering and function remains controversial and will not be discussed here.

Figure 2. Schematic representation of the role of PKCθ in TCR activation.

Binding of antigen to the T cell antigen receptor (TCR-CD3) complex results in phosphorylation of several intracellular proteins, followed by the activation of PKCθ and by an increase in the intracellular calcium concentration, which then activates calcium-calmodulin-dependent kinases and phosphatases. Phosphorylation triggers the Zap-70/Vav pathway leading to actin rearrangements and initiates a series of coordinated signals that ensure cell survival and induce cell proliferation and the production of IL-2 through the activation of transcription factors, Oct, NF-κB, AP-1, and NF-AT. PKCθ supports the activation of Oct and in synergy with calcium-activated calcineurin, stimulates the activation of AP-1 and NFAT through the MAPK/JNK pathway. PKCθ also stimulates the translocation of NF-κB from the cytoplasm to the nucleus through activation of the IKKβ subunit. Activation of these transcription factors leads to the production of IL-2, a major cytokine critical for inflammatory responses.

Figure 3. Detailed schematic of the role of PKCθ in actin cytoskeletal reorganization in activated T cells.

Zap-70 interacts with the actin-regulatory WASP–WIP complex through SH2/SH3-containing CrkL proteins. Phosphorylation of WIP by PKCθ induces dissociation of WASP; released WASP is then activated by binding to GTP-bound Cdc42, which induces a conformational change in WASP that allows its C-terminal domain to bind and activate the Arp2/3 complex responsible for initiation of actin polymerization.

LFA-1 signaling

PKCθ was found to mediate signal transduction from integrins to the actin cytoskeleton in processes involving LFA-1 (CD11a/CD18), the small GTPase Rap1, and its upstream regulator RapGEF2 [58–61]. LFA-1 and its counter-ligands on the APC, ICAM-1, -2, and -3, stabilize antigen-specific interactions between the T cell and APC [58]. LFA-1 plays a critical role in mediating immune cell adhesion upon engagement of the TCR; TCR clustering induces changes in LFA-1 conformation and surface distribution (relocation to the leading edge) and results in increased LFA-1 avidity to ICAMs [58, 61]. A closer look into the mechanism of immune adhesion revealed that the association of PKCθ with RapGEF2 and the subsequent phosphorylation of the latter on Ser960 facilitate activation of Rap1, which when activated, increases adhesiveness of LFA-1 to its binding partner ICAM-1, resulting in an overall increase in adhesiveness of the T cell to the endothelium and to the APC [59]. These findings indicate that among other molecules involved in the regulation of LFA-1, the PKCθ/RapGEF2 complex is an important component of TCR signaling, which positively regulates cytokine responses and adhesive capacities of T lymphocytes.

Spectrin-based membrane skeleton

PKC-dependent polarization of the spectrin-based cytoskeleton was also reported in lymphocytes [62, 63]. Spectrin is an actin cross-linking and scaffold protein that links cell membrane to the actin cytoskeleton. Evidence is emerging of its role in cell-signaling events [64]. Cellular relocation of a multiprotein polar aggregate, which included spectrin, PKCβ, and PKCθ, was induced by various chemical and physical stimuli [63, 65–68]. Within the aggregates, spectrin was found to be phosphorylated, and the aggregation process was inhibited by the PKC inhibitor, bisindolylmaleimide [67]. Similar observations were reported recently using peripheral lymphoid cells exposed to systemically administered chemotherapeutics (fludarabine/mitoxantrone/dexamethasone, the regimen for NHLs), where aggregates of PKCθ and spectrin were found in peripheral leukemic and NHL lymphoid cells, isolated 24 h after initial administration of the drugs [63, 69]. These findings suggest that physical or chemical stress may induce spectrin/PKCθ-dependent relocations and changes within the membrane skeleton, and these changes could be related to early stages of apoptosis. These observations also point to a correlation between spectrin aggregation and signals transduced by PKCθ and indicate a possible link between PKCθ signaling and the spectrin-based membrane skeleton. The nature of this association and whether spectrin is a substrate for PKCθ remain to be determined; however, these observations may suggest that cytoskeletal rearrangements could play an active role in the process of apoptosis, and the cytoskeletal scaffold may be used for assembly of apoptotic protein complexes, especially in the early stages of this process.

MTOC and cell polarity

PKCθ has also been shown to be involved in the establishment of T cell polarity. The formation of the IS is associated with the polarization of the MTOC adjacent to the IS, thereby establishing the axis of polarization and facilitating directional release of cytokines and cytolytic factors toward the APC [70]. Actin polymerization stabilizes the IS network of adhesive contacts and receptor-ligand interactions, whereas the microtubule cytoskeleton positions MTOC just beneath the IS. The second messenger DAG is the major link between TCR signaling and MTOC polarization and was shown to be crucial for proper orientation of the MTOC downstream of TCR [71, 72]. The mechanisms that couple DAG to the MTOC are not known; however, its role in the IS and MTOC assembly suggests that proteins containing C1 domains are involved in the process. Indeed, it was recently shown that PKCϵ, -η, and -θ were recruited by DAG to the IS in a two-step process, in which PKCϵ and -η arrive first and promote subsequent recruitment of PKCθ. Both steps are required for proper MTOC reorientation, and it was proposed that broad accumulation of PKCϵ and -η controls early polarization steps, whereas PKCθ recruitment refines positioning of MTOC in the proximity to IS at later stages [72].

PKCθ was also shown to be involved in the regulation of formation of DPC, a rigid membrane projection with cytoskeletal components that is formed during the process of T cell–APC recognition and is located distal to IS [73]. The highly homologous ezrin, radixin, and moesin, which are critical for the proper formation of DPC, are specific linkers between membrane proteins and actin cytoskeleton [73]. In response to TCR engagement, ezrin and moesin are phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localize to DPC [74]. PKCθ was shown to phosphorylate one of the ERM proteins—moesin—on Thr558 within the ⁵⁵⁵KYKpTLRQIR⁵⁶² sequence located in a conserved, putative, actin-binding domain [28]. Further studies indicated that this phosphorylated sequence is homologous to the PKC pseudosubstrate sequence, and Thr558 phosphorylation is essential for interaction of ERM proteins with actin [29]. It must be noted that these studies were performed under TCR activation conditions in a leukemic cell line; therefore, the broad biological role of moesin phosphorylation in the context of T cell activation is uncertain, but it seems probable that this process is involved in remodeling of actin cytoskeleton, which occurs following the interaction of T cells with an APC [29].

PKCθ SIGNALING TO CYTOSKELETON IN MYOGENESIS

PKCθ is known to be involved in the regulation of cytoskeletal rearrangements during myogenesis. PKCθ expression was detected in skeletal muscle and neuromuscular junction, with a four fold increase in expression occurring exclusively in the membrane fraction during Postnatal Days 3–21 in skeletal muscle and appearing in neuromuscular junctions as early as Postnatal Day 4 [11, 75]. Myogenesis involves a substantial reorganization of cytoskeletal actin to ensure appropriate function of enzymatic and contractile apparatuses, both of which are required for fetal and postnatal muscle development [76, 77]. Fetal muscle development-specific genes, such as MCK and β-enolase, are controlled by a transcription factor, Nfix [78], which is thought to act as a switch from embryonic to fetal myogenesis. It was shown that Nfix forms a complex with PKCθ and MEF2A and acts as a bridge between MEF2A and PKCθ, likely facilitating MEF2A phosphorylation by PKCθ. The Nfix/MEF2A/PKCθ complex controls transcription of MCK and β-enolase, both of which are critical components in fetal myogenesis [78]. Beside complexing with Nfix in myoblasts, PKCθ also triggers phosphorylation of MARCKS, a myristoylated, membrane-bound protein that anchors the actin cytoskeleton to the plasma membrane [79–81]. The membrane-associated state of MARCKS was shown to be dependent, not only on its myristoylation state but also on direct interactions between membrane phospholipids and basic residues present in the MARCKS catalytic domain [82]. As the PKCθ consensus phosphorylation sites are located within these groups of basic amino acids, it has become apparent that PKCθ has the ability to modulate interactions between MARCKS and the membrane and indirectly, to affect the arrangement of the undermembrane actin cytoskeleton. Indeed, it was shown that MARCKS translocates from the cytosol to the plasma membrane in one of the critical processes of skeletal muscle cell development, in which mononucleated myoblasts fuse into multinucleated myotubes. The level of MARCKS in the cytosol decreased with a concomitant increase in its level in the plasma membrane as cell fusion proceeded. The expression of PKCθ, which is responsible for the phosphorylation of MARCKS, was down-regulated during the process of fusion and myotube formation [80]. These observations showed the cooperation of PKCθ and MARCKS during the course of myogenesis and confirmed the importance of PKCθ for myocyte development.

PKCθ AND THE GENERAL CONCEPT OF “PARTICIPATION BY TRANSLOCATION”

The traditional model of PKC activation and translocation derives from early studies of PKCα, which localizes to cytosol in resting cells and weakly associates with their membranes in the absence of DAG/calcium. When the membrane-restricted product of phosphoinositide hydrolysis, DAG, is produced, PKCα translocates to the membrane, undergoes a conformational change, and becomes activated [83]. Cellular relocalization of PKCθ upon activation was initially observed in platelets by Wang and colleagues [84]. Upon stimulation, a rapid relocation of PKCθ from the cytosol to the membrane was observed, and increased membrane-bound PKC activity and decreased cytosolic PKC activity were noted. These studies were shortly followed by the analysis of the cellular localization of six isoforms of PKC in APC-activated T cells, which revealed that only PKCθ translocated to the site of cell–cell contact [6]. In addition, a local increase in the activity of PKCθ in immunoprecipitates from T cell–APC conjugates indicated that the translocation of PKCθ to the membranes coincides with its activation [6]. It is now widely accepted that PKCθ activation is associated with its membrane and cytoskeletal translocations, as well as its colocalization with the TCR at the contact site between antigen-specific T cells and APCs (for a detailed review, see refs. [37–41, 45, 85]).

Examples cited here clearly indicate that the communication between PKCθ and undermembrane actin/spectrin/myosin-based cytoskeleton often involves shuttling of PKCθ between the cytoplasm and the membrane, which is reflected physiologically by the relocalization of the kinase, often accompanied by the translocation of the cytoskeletal proteins, in response to different stimulatory events [30, 69, 86–88]. These rearrangements are associated not only with activation of cells and proliferation but also with developmental differentiation and possibly apoptosis, indicating that such rearrangements are universal, and raise further questions regarding their regulation. It is possible that PKCθ associates with cytoskeletal scaffold proteins and phosphorylates some of them only when they are in a specific conformational/spatial arrangement; alternatively, these proteins may serve as substrates of PKCθ, and their specific conformational/spatial arrangements are dictated by their phosphorylation status. Interestingly, there is evidence that the capacity of PKCθ to mediate its own relocalization remains under autoregulatory control. PKCθ displays an autoregulatory potential, which appears to involve autophosphorylation of Thr219, located between the tandem C1 domains of the regulatory fragment of PKCθ [89, 90]. A T219A mutation was shown to abrogate the capacity of PKCθ to mediate signaling cascades leading to activation of NF-κB, NF-AT, and AP-1. Furthermore, this mutation prevented proper recruitment of PKCθ to the membrane in activated T cells, suggesting that the mechanism of PKCθ relocalization might be autoregulated [90]. It seems likely that phosphorylation-dependent and nonphosphorylation-dependent associations between PKCθ and the cytoskeletal components, as well as kinase autoregulatory mechanisms, are all required for full effectiveness of relocation-dependent cellular responses mediated by this enzyme.

PHARMACOLOGICAL CONTROL

It is reasonable to ask how we might pharmacologically manipulate and control PKCθ relocalization or the “participation-by-relocation” mode for therapeutic benefit. The crystal structure of the catalytic domain of staurosporine-complexed PKCθ resolved by Xu and colleagues provided crucial information for the development of specific inhibitors [91]. Staurosporine, which has a core indolo[2,3-a]carbazole ring, is a PKC inhibitor with phorbol ester agonistic properties. Staurosporine is known to cause the translocation of PKC isoenzymes ϵ, δ, and θ from the cytosol to the membrane; a similar effect is also caused by the phorbol esters [92]. The bisindolylmaleimides (LY333531, LY317615, and AEB071) were found to induce similar effects on PKCα, -β, -γ, -ϵ, -ζ, and -η [92, 93]. Paradoxically, PKCθ transmembrane relocation is observed upon treatment with inhibitors (e.g., staurosporine or bisindolylmaleimide) or activators (e.g., phorbol esters). Recent studies by Seco and colleagues [2] provided crucial insight into the PKCθ activation process and shed light on the paradoxical activation of PKCθ by its inhibitors, referred to as “ligand priming” or “inhibitor hijacking” (notably, a similar phenomenon is observed for PKC and Akt) [94, 95]. It was recently shown that PKCθ activation during TCR signaling is achieved upon phosphorylation (at Thr538) of its AL by GLK (also named MAPK4K3) [96]. In addition phosphorylation, on Ser676 and Ser695, (on turn and hydrophobic motifs respectively), may positively regulate T cell activation during TCR signaling [97, 98]. Phosphorylation of these sites is crucial for reaching a complete activation state of PKCθ, whereas Thr538 monophosphorylated PKCθ displays lower activity [2]. Seco and colleagues showed that conformational changes occurring in unphosphorylated PKCθ, upon binding of some inhibitors and activators, may result in enhanced exposure of the AL and ATP-binding sites to phosphorylation and may facilitate activation of the enzyme [2]. This unique transmission mechanism may explain activation of PKCs by their inhibitors [2].

In a high-throughput screening campaign, 2,4-diamino-5-nitropyrimidines [99] and 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile [100] were identified as new, relatively specific PKCθ inhibitors. Upon optimization, 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile was shown to be selective for inhibition of nPKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the cPKC and aPKC, PKCβ and PKCζ, respectively [100]. The most recent comprehensive summary of PKCθ inhibitors can be found in a review by Boschelli [93]. The broadly overlapping substrate specificity among PKC family members and the high level of homology within the group of nPKC isoforms (PKCθ and PKCδ display nearly 70% homology at the nucleotide and amino acid levels [5]) present a major challenge for development of specific PKCθ inhibitors. Indeed, no inhibitor of PKCθ is currently in clinical development except AEB071, which has strong activity on PKCθ, PKCα, and PKCβ and lesser activity on PKCδ, PKCϵ, and PKCη, indicating its rather broad specificity (clinical trials, ID #NCT01402440, #NCT01430416, and #NCT00416546; reference available from http://www.clinicaltrials.gov, October 23, 2012). No PKCθ crystal structure with any compound other than staurosporine (GenomeNet; Database: PDB; Entry: 1XJD; http://www.genome.jp/dbget-bin/www_bget?pdb:1XJD) has been reported to date [91]. The need for a clinically available PKCθ inhibitor is apparent, as PKCθ-deficient mouse models of various diseases clearly indicate potential therapeutic benefits of PKCθ inhibition. Antigen-induced arthritis, experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis), allergic asthma, cardiac allograft rejection, and inflammatory bowel disease have all shown less severity in PKCθ(−/−) animals [101–104]. The benefits appear to be mainly the results of decreased production of cytokines and decreased proliferation, as observed for T cells from PKCθ knockout mice [105, 106].

CONCLUDING REMARKS

PKCθ is involved in numerous intracellular signal transduction pathways. It is required for the activation, proliferation, and control of apoptosis in T cells, and it regulates several transcription factors involved in these processes. Importantly, PKCθ is also crucial for cytoskeletal rearrangements associated with activation, proliferation, and apoptosis of lymphoid cells. Components of the cytoplasmic or undermembrane cytoskeleton have been identified as substrates for PKCθ, and their functions are dependent on their phosphorylation status (summarized in Table 1). This implies that PKCθ may serve as a versatile regulator of spatial and temporal signal transduction, not only to the immediate regulatory molecules but also to the end-point effector cytoskeletal components. Although details regarding physiological functions of PKCθ continue to emerge, we still lack a comprehensive understanding of its induced transduction pathways and cellular targets. Most of the cellular phenotypic data are provided by relatively recently developed genetic knockouts, knockdowns, or PKCθ-deficient cellular systems. These approaches have defined the end-point targets of PKCθ signaling and will continue to deepen our knowledge of its intermediate players.

Table 1. Role of PKCθ in Cytoskeleton Signaling.

Immunological synapse	Polarity	Myogenesis	Motility
Rac/Vav/actin polymerization-dependent recruitment of PKCθ to activated TCR site [50]	PKCθ recruitment to the membrane is critical for proper orientation of the MTOC and cytotoxic response in T cells [71, 72].	PKCθ phosphorylates membrane-bound MARCKS and modulates arrangement of the under-membrane actin cytoskeleton in skeletal muscle cell development [80].	PKCθ regulates stromal cell-derived factor 1-induced chemotaxis [13].
Zap-70/Slp-76/Vav signaling mediates activation of PKCθ [52].	PKCθ phosphorylates moesin on Thr558 within its actin-binding domain, modulating actin cytoskeleton-dependent cell polarization [28].		Overexpression of PKCθ expression-dependent θ-associated protein causes a decrease of focal adhesion formation and enhances migratory properties of endothelial cells [12].
PKCθ-dependent phosphorylation of WASP inhibitory WIP leads to activation of Cdc42 and initiates actin polymerization at the TCR site [55].
PKCθ-mediated phosphorylation of RapGEF2 on Ser960 facilitates activation of small GTPase Rap1, leading to increased adhesiveness of T cells to APCs [59].
Spectrin and PKCθ are found in polar aggregates in early apoptotic lymphoid cells [63, 69].

AUTHORSHIP

All authors wrote the paper.

DISCLOSURES

The authors declare no conflicts of interest.