ARL3, a small GTPase with a functionally conserved role in primary cilia and immune synapses.

The primary cilium and the immunological synapse are both specialized functional plasma membrane domains that share several similarities. Signalling output of membrane domains is regulated, spatially and temporally, by segregating and focusing lipids and proteins. ARL3, a small GTPase, plays a major role in concentrating lipid-modified proteins in both the immunological synapse and the primary cilia. Here in this review we will introduce the role of ARL3 in health and disease and its role in polarizing signalling at the primary cilia and immunological synapses.

eNOS S-nitrosylates ß-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of ß-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated ß-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of ß-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

Protein kinase C-θ clustering at immunological synapses amplifies effector responses in NK cells.

In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.

A targeted siRNA screen identifies regulators of Cdc42 activity at the natural killer cell immunological synapse.

Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.

Protein kinase C η is required for T cell activation and homeostatic proliferation.

Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη-/- mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ-/- mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.

A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.

The expression and function of ß-1,4-galactosyltransferase-I in dendritic cells.

ß-1,4-Galactosyltransferase-I (GalTI) is unusual among the galactosyltransferase family, which has two isoforms that differ only in the length of their cytoplasmic domains [1]. In this study, we found that both the long and short isoforms of GalTI were expressed in human monocyte-derived dendritic cells (MoDCs), and localized in the cytoplasm near nucleus and cytomembrane. The expression level of GalTI and cellular adhesion ability was increased when DCs continued to mature. We also demonstrated that the cellular adhesion ability of DCs was inhibited by α-lactalbumin (α-LA) via interference with cell surface GalTI function, suggesting that the adhesion ability was positively correlated with the expression of cell surface long GalTI. α-LA also could inhibit DC-T clustering and CD4(+) T cell proliferation. Collectively, the data suggests that GalTI might act as a key adhesion molecular participating in T cells-DCs contacts.

Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs.

A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.

Cutting edge: association with I kappa B kinase beta regulates the subcellular localization of Homer3.

The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.

Insufficient deactivation of the protein tyrosine kinase lck amplifies T-cell responsiveness in acute coronary syndrome.

RATIONALE: In the vulnerable atherosclerotic plaque, T cells may destabilize the tissue structure through direct cell-injurious effector functions. T cells transmit environmental signals, such as recognition of antigen, into cellular responses through regulated phosphorylation of cytoplasmic proteins, with the Src family kinase Lck (lymphocyte-specific protein tyrosine kinase) in critical membrane-proximal position of the T-cell receptor (TCR) signaling cascade. The balance between protein phosphorylation and dephosphorylation defines the signal transduction threshold and determines appropriate T-cell responses. OBJECTIVE: We have examined whether abnormal calibration of intracellular signaling pathways renders acute coronary syndrome (ACS) patients susceptible to disproportionate T-cell responses. METHODS AND RESULTS: Intracellular signaling cascades were quantified in CD4 T cells from ACS patients and control individuals after stimulation with major histocompatibility complex class II-superantigen complexes. ACS T cells mobilized more intracellular calcium and accumulated higher levels of phosphotyrosine than control T cells. Proximal steps in TCR signaling, such as recruitment of ZAP-70 and clustering of TCR complexes in the immune synapse, were abnormally enhanced in ACS T cells. Acceleration of the signaling cascade derived from a proximal defect in ACS T cells, which failed to phosphorylate Lck at Tyr505, extending activation of the Src kinase. Abnormalities in TCR signaling did not correlate with systemic inflammation as measured by C-reactive protein. CONCLUSIONS: An intrinsic abnormality in the signaling machinery of ACS T cells resulting in the accumulation of active Lck lowers the TCR threshold and renders lymphocytes hyperreactive and capable of unwanted immune responses.

Integrating receptor signal inputs that influence small Rho GTPase activation dynamics at the immunological synapse.

The Rho GTPase Cdc42 regulates cytoskeletal changes at the immunological synapse (IS) that are critical to T-cell activation. By imaging fluorescent activity biosensors (Raichu) using fluorescence lifetime imaging microscopy, Cdc42 activation was shown to display kinetics that are conditional on the specific receptor input (through two IS-associated receptors, CD3 and beta1 integrin). CD3-triggered Cdc42 activity is dependent on the cyto-2 (NPIY) motif of the beta1 integrin cytoplasmic domain. Perturbations of the ezrin-radixin-moesin (ERM) function blocked CD3- and beta1-dependent increases in Cdc42 activity. Both IS-associated receptors probably lie on a serial molecular pathway and transduce signals through the ERM-dependent machinery that is responsible for the remodeling and stabilization of the synapse. Cdc42 activity is impaired in beta1 integrin-deficient T cells that form conjugates with antigen-presenting cells but is partially restored in the context of an antigen-specific synapse. This restoration of Cdc42 activity is due, at least in part, to the recruitment and activation of beta2 integrin.

Signals and sequences that control CD28 localization to the central region of the immunological synapse.

During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-mediated signaling through PI3K results in the recruitment of protein kinase C-theta (PKCtheta) to the cSMAC, activation of NF-kappaB, and up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28 localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with the ability of CD28 to preferentially accumulate at the cSMAC. PKCtheta distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKCtheta even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-kappaB, suggesting that CD28-mediated localization of PKCtheta to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay of signals between TCR and CD28 and suggest that CD28 signaling through PCKtheta may be mediated through localization to the cSMAC region of the immunological synapse.

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8(+) but not CD4(+) CTL to lyse target cells despite having no effect of the amount of released granules by both CD8(+) and CD4(+) CTL. Consistent with this, CD4(+) CTL break their synapses more often than do CD8(+) CTL, which leads to the escape of the cytolytic molecules from the interface. CD4(+) CTL treatment with a protein kinase Ctheta inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Ctheta, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis.

T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase C theta.

Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.