Novel Protein kinase C θ: Coronin 1A complex in T lymphocytes

Protein kinase C-θ (PKCθ) plays an important role in signal transduction down-stream of the T cell receptor and T cells deficient of PKCθ show impaired NF-κB as well as NFAT/AP-1 activation resulting in strongly decreased IL-2 expression and proliferation. However, it is not yet entirely clear, how the function of PKCθ - upon T cell activation - is regulated on a molecular level. Employing a yeast two-hybrid screen and co-immunoprecipitation analyses, we here identify coronin 1A (Coro1A) as a novel PKCθ-interacting protein. We show that the NH2-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Furthermore, we confirm a physical interaction by GST-Coro1A mediated pull-down of endogenous PKCθ protein. Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells. This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively. Mechanistically, Coro1A overexpression attenuates both lipid raft and plasma membrane recruitment of PKCθ in CD3/CD28-activated T cells. Using primary CD3+ T cells, we observed that (opposite to PKCθ) Coro1A does not localize preferentially to the immunological synapse. In addition, we show that CD3+ T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation. Together, these findings both in Jurkat T cells as well as in primary T cells indicate a regulatory role of Coro1A on PKCθ recruitment and function downstream of the TCR leading to NF-κB transactivation.


Findings
An efficient adaptive immune response depends on the activation of T lymphocytes by antigen-presenting cells (APC) and the acquisition of appropriate effector T cell function. Activation of T lymphocytes occurs upon engagement of T cell receptors (TCR) and the corresponding antigen-MHC complexes together with the ligation of the co-receptor CD28 by B7 moleculesleading to the formation of the immunological synapse (IS) between T cell and APC. PKCθ, a member of the protein kinase C (PKC) family of serine/threonine kinases, is rapidly, within seconds after TCR engagement, recruited to the peripheral supra-molecular activation cluster (pSMAC) of the immunological synapse (IS) [1,2]. It has been shown that, the activation of the transcription factors NF-κB, NFAT and AP-1 downstream of the TCR critically depend on PKCθ [3][4][5], linking PKCθ function to IL-2 transcription, whose promoter activation depends on these transcription factors [6].
Activation of all PKC family members is controlled by a so-called pseudo-substrate (PS) domain in the NH 2terminus that resembles PKC substrates and forms an auto-inhibitory loop to keep the enzyme in an inactive conformation. PKCθ is released from the auto-inhibition after recruitment to the plasma membrane, where it binds to diacylglycerol (reviewed in [7]). Noteworthy, PKC mutations that disrupt this intra-molecular interaction generate constitutively active forms of PKC, which are useful tools for analysing PKC functions. Another domain involved in modulating PKCθ activity is its C2-like domain, which represents a major protein:protein interaction domain. Binding of WD40 domain-containing receptor for activated PKC proteinsso called RACK proteins -to the C2-like domain of activated PKC forms another level of regulating its enzymatic function [8]. However, so far no RACK physiologically interacting with PKCθ has been identified. In the present study, designed to discover PKCθ interacting partners, we identified coronin 1A (Coro1A) as a functional regulator of PKCθ activation.

Identification of Coro1A as physical interaction partner of PKCθ
Little is known about proteins that interact with PKCθ and regulate its function in T lymphocytes and thereby modulate activation of this immune cell subset. To contribute to this issue, we have employed a yeast twohybrid (Y2H) screen using the regulatory domain of PKCθ (PKCθ-NH 2 ) fused to the DNA-binding domain as "bait". With this approach we identify a group of clones that interact strongly with PKCθ. A detailed description of all methods used is provided in the Additional file 1 (Supplementary Methods). DNA sequencing reveals the interacting "prey" protein as the NH 2 -terminal domain of human Coro1A (Table 1). Coro1A, a member of the evolutionary conserved WDrepeat family of coronin proteins, is highly expressed in all leukocytes. Originally, Coro1A has been isolated as an actin/myosin binding protein and implicated in F-actin dynamics by negatively regulating the function of the nucleation-promoting Arp2/3 complex (reviewed in [9]). In mice and human, genetic inactivation of Coro1A results in immune deficiencies that are linked to a strong reduction of naive T cell numbers in peripheral organs [10][11][12][13][14][15]. Of note, Coro1A has been implicated in calcium mobilization after TCR triggering in naive T cells as well as TGF-β signaling in Th17 cells [11,14].
Using truncated versions of PKCθ and Coro1A ( Figure 1A), we demonstrate that the N-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Co-immunoprecipitation (Co-IP) analysis in Jurkat T cells transfected with an epitope-tagged Coro1A expression vector confirmed a complex formation between PKCθ and Coro1A in T cells ( Figure 1B). Reversely, GST-Coro1A pull-downs revealed interaction with endogenous PKCθ in mouse T cells ( Figure 1C). This PKCθ:Coro1A interaction was observed both with and without CD3/CD28 stimulation of the cells and thus being constitutive in resting cells. Of note, the Co-IP experiments show strongly increased physical association of Coro1A with the constitutively active mutant PKCθ A149E, while the binding to the dominant-negative PKCθ K409R mutant remained unaltered when compared to wild-type PKCθ ( Figure 1D). This suggests that Coro1A might function as a RACK protein regulating PKC kinase activity. Of note, based on experiments using phorbol ester as pleiotropic PKC activator, or serine/threonine protein phosphatase inhibitors, PKCs have been described as kinases phosphorylating Coro1A and thereby downregulating its binding to actin [16,17]. Itho et al. identified PKCα and PKCδ as the PKC isotypes responsible for Coro1A phosphorylation [18].

Coro1A modulates PKCθ-mediated functions
After having observed a complex formation between PKCθ and Coro1A, we next asked the question about the functional relevance of this interaction. Therefore, it was analysed whether Coro1A does influence the transcriptional activation of genes that are established downstream targets of PKCθ such as IL-2 and Cyclin D1. In functional analyses using IL-2 promoter luciferase reporter assays, overexpression of wild-type Coro1A but not the COOHdeletion mutant, lacking the actin-binding domain, negatively interferes with PKCθ-dependent IL-2 transactivation in Jurkat T cells ( Figure 2A). Thus, even though the actinbinding function of Coro1A is not necessary for its interaction with PKCθ ( Figure 1), it appears to be of relevance for Coro1A modulating PKCθ function. In these experiments, Jurkat T cells co-transfected with the constitutively active mutant PKCθ A149E and wild-type or truncated Coro1A, were stimulated with the calcium ionophore, ionomycin. Co-transfection with the dominantnegative PKCθ K409R mutant or the dominant-negative mutant of Rac1, Rac1 N17, which leads to inhibition of IL-2 reporter transcription via actin polymerization defects served as positive controls. Those findings suggest that actin is part of a functional PKCθ:Coro1A axis identified in the Jurkat T cell line. In addition, wild-type but not the deletion mutant of Coro1A repressed the induction of an NF-κB-dependent promoter luciferase reporter ( Figure 2B). This effect could be phenocopied both by cell-permeable pharmacological inhibitors of actin polymerisation and PKC function, respectively ( Figure 2C). Similarly, Cyclin D1 promoter reporter activation (that was PKC isotypeselectively dependent on PKCθ function) was attenuated by wild-type Coro1A co-expression ( Figure 2D).     NF-κB activation independent of PKCθ, based on the experiments described above, we conclude that Coro1A, which is in a complex with PKCθ, modulates PKCθ functionally.
Taken together, Coro1A likely may act as a safeguard for stochastic membrane recruitment/IS translocation of PKCtheta upon transient T cell activation signals, e.g. by low affinity antigens.

Coro1A is involved in NF-κB signaling in primary T lymphocytes
Next, we investigated the subcellular localization of Coro1A and PKCθ upon T cell activation. For this purpose human T cell blasts from immunized donors were incubated with APCs loaded or not with the corresponding peptide and analysed by confocal microscopy for the localization of PKCθ and Coro1A with regard to the IS (stained by antibodies against (p)tyrosine). Of note, while as already published, activation-induced PKCθ recruitment to the IS was consistently observed by confocal microscopy [19], Coro1A was not recruited to the IS. Coro1A was rather excluded from the IS in approximately 65% of antigen:APC-stimulated T cell blasts ( Figure 4A/B), suggesting a role as negative regulator in TCR signaling.
Results on the molecular mechanism of Coro1A in T cell signaling are controversial in part due to the diverse results obtained with the different conventional knockout mice strains established in several laboratories [10,11,20]. In particular, Mueller et al. described a physical interaction between Coro1A and PLC-γ1 promoting calcium mobilization from intracellular stores upon activation of naive T cells [11], while no defect in other pathways downstream of the TCR was detected. In contrast, Föger et al. did not observe any impairment of T cell activation at all when analysing T cell function using their knockout strain [10]. Using our Coro1a knockout mice [14], we addressed the potential involvement of Coro1A in the NF-κB signaling pathway, known to be regulated by PKCθ, in primary mouse T cells. The results revealed reduced levels of phosphorylated inhibitor of NF-κB (I-κBα) in T cells isolated from Coro1a-deficient mice upon stimulation with anti-CD3 and anti-CD28 ( Figure 4C). Furthermore, NF-κB:DNA binding upon anti-CD3/CD28 treatment was strongly reduced in Coro1a-deficient T cells when analysed by electrophoretic mobility shift assay (EMSA) ( Figure 4D).
Using a combination of phorbol ester and ionomycin, which bypass early activation events downstream of the TCR by directly activating PKC isotypes and inducing calcium influx, respectively, only partially restored I-κBα phosphorylation and NF-κB:DNA binding, pointing to an important role of Coro1A for PKC activation processes.
Taken together, the present results provide evidence that Coro1A is a functional interaction partner of PKCθ in the established PKCθ/IKK/NF-κB/IL-2 transactivation pathway in CD3 + T cells.  Figure 4 Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830-843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254-268; GDLLGILESRGIKAR) were used with autologous Epstein-Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3 + T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3 + T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.
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