The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, K.
Right arrow Articles by Altman, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, K.
Right arrow Articles by Altman, A.
The Journal of Immunology, 2006, 177: 1721-1728.
Copyright © 2006 by The American Association of Immunologists

Filamin A Is Required for T Cell Activation Mediated by Protein Kinase C-{theta}1

Keitaro Hayashi and Amnon Altman2

Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Induction of T cell responses following engagement of the Ag-specific TCR depends on TCR-initiated rearrangements of the cellular actin cytoskeleton and highly coordinated and tightly regulated interactions and of diverse intracellular signaling proteins. In this study, we show that filamin A (FLNa), an actin-binding and signal mediator scaffolding protein, is required for T cell activation. Following Ag stimulation, FLNa was recruited to the T cell-APC contact area, where it colocalized with protein kinase C-{theta} (PKC{theta}). Depletion of FLNa by RNA interference did not affect TCR-induced early tyrosine phosphorylation or actin polymerization but, nevertheless, resulted in impaired IL-2 expression by human primary T cells and reduced activation of NF-{kappa}B, AP-1, and NFAT reporter genes in transfected T cells. TCR stimulation induced stable physical association of FLNa with PKC{theta}. Furthermore, the TCR/CD28-induced membrane translocation of PKC{theta} was inhibited in FLNa-depleted T cells. These results reveal novel role for FLNa in the TCR/CD28 signaling pathway leading to transcription factor activation and IL-2 production, and suggest that this role is mediated, in part, through the inducible interaction of FLNa with PKC{theta}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell recognition of specific Ag is accomplished by binding of the Ag-specific TCR to antigenic peptides associated with MHC molecules and presented by APCs. Engagement of the TCR by MHC-peptide triggers rearrangements of the T cell actin cytoskeleton, and induces the formation of a highly organized complex of receptors and intracellular signaling molecules at the interface between T cells and APCs, the so-called immunological synapse (IS)3 (1). Protein kinase C-{theta} (PKC{theta}), a member of the novel, Ca2+-independent PKC subfamily, is a major player in the early TCR/CD28-induced signaling events leading to T cell activation (2, 3, 4). The important role of PKC{theta} in mature T cell activation is evident from findings that peripheral T cells from PKC{theta}-deficient T cells display a severe impairment in IL-2 production following anti-CD3 and anti-CD28 costimulation (5, 6). A fraction of PKC{theta} is found associated with the plasma membrane and actin cytoskeleton in receptor-stimulated T cells (7, 8), and, furthermore, specific Ag stimulation induces a highly selective and sharply defined colocalization of PKC{theta} with the TCR/CD3 complex in the central supramolecular activation complex (cSMAC) within the IS (9). In contrast, other adhesion receptors and actin-binding proteins such as LFA1 and talin, respectively, are recruited into peripheral SMAC (pSMAC). Thus, the localization and proper activation of PKC{theta} appear to be closely linked to reorganization of the actin cytoskeleton. However, the precise nature of the interaction between PKC{theta} and the actin cytoskeleton is unknown. It would therefore be of interest to identify the proteins (or other molecules) that link the plasma membrane, actin, and the network intracellular signaling proteins, including PKC{theta}, in activated T cells.

Filamin was originally identified as a non-muscle actin-binding and -polymerizing protein (10, 11). In humans, three members of the filamin family, namely, filamin A (FLNa), filamin B, and filamin C, have been identified. Whereas filamin C is muscle specific (12), FLNa, but not filamin B, is expressed in T cells (13). In addition to its ability to bind actin, FLNa also associates with plasma membrane proteins and with several intracellular signaling intermediates, including enzymes and adapter proteins (14, 15). These findings indicate that filamin plays an important role as a scaffolding protein that links a wide range of intracellular signaling proteins to the actin cytoskeleton and to cell membrane receptors. Despite increasing evidences for the interaction of filamin with signaling proteins, the physiological significance of these interactions is far from being clear, because a majority of the relevant reports have used filamin-deficient cell lines. Genetic approaches have demonstrated that deletion or specific missense mutations of FLNa or B cause a wide range of developmental malformations of brain, bone, limb, and cardiovascular systems in humans (15). However, the role of filamin in other tissues, including the immune system, remains poorly understood. In this report, we show FLNa is recruited to T cell-APC contact area and plays a role in IL-2 production by TCR/CD28-costimulated T cells. In addition, FLNa colocalizes and interacts with PKC{theta} and is required for optimal activation of three PKC{theta} targets, i.e., the transcription factors NF-{kappa}B, AP-1, and NFAT. Thus, the interaction of FLNa with PKC{theta} is likely to at least partially account for its requirement for optimal T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies

The anti-human CD3 (OKT3) and anti-phosphotyrosine (pTyr; 4G10) mAbs were purified from culture supernatants as previously described (16), and the anti-human CD28 mAb was purchased from BD Pharmingen. The mouse mAb against FLNa, PM6/317, was purchased from Chemicon. A polyclonal goat anti-PKC{theta} Ab (for immunofluorescence microscopy) and a PKC{theta}-specific mAb (for immunoblotting) were from Santa Cruz Biotechnology or BD Biosciences, respectively. Rabbit anti-phospho-LAT (Y191), anti-ZAP-70, and anti-phospho-ZAP-70 (Y493) Abs were from Cell Signaling. The rabbit anti-LAT Ab was from Upstate Biotechnology, and an allophycocyanin-conjugated anti-human CD4 Ab was purchased from Biolegend. Alexa 488-, Alexa 555-, or Alexa 648-conjugated anti-goat or anti-mouse IgG Abs were from Molecular Probes.

Cells, stimulation, and IL-2 expression

Leukemic human Jurkat E6.1 T cells or Raji B cells were maintained in culture as previously described (17). Human PBMCs were prepared from healthy volunteers by Histopaque (Sigma-Aldrich) centrifugation. The study was approved by the La Jolla Institute for Allergy and Immunology Human Research Committee. For immunofluorescence analysis (see below), purified PBMCs were cultured with staphylococcal enterotoxin E (SEE; 10 µg/ml; Toxin Technology) and recombinant human IL-2 (20 U/ml) for 4 days and rested for 3 additional days in the presence of IL-2 only. These cells, or Jurkat T cells, were then mixed at a 1:1 ratio in a total volume of 150 µl with Raji B lymphoma cells, which were pulsed with SEE (10 µg/ml) for 30 min and washed, or left nonpulsed as a negative control. In some experiments, the Raji cells were prelabeled with 5-(and -6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; 4 µM; Molecular Probes) for 30 min. The cell mixture was immediately transferred onto poly-L-lysine-coated glass slides and incubated for 30 min at 37°C.

To determine IL-2 production, purified PBMCs were transfected with small interfering RNA (siRNA) (see below), stimulated with plate-bound anti-CD3 mAb and soluble anti-CD28 mAb (3 µg/ml each) plus recombinant human IL-2 (20 U/ml) for 1 day, washed, and recultured in the presence of IL-2 for 3 additional days. The cells were then restimulated with plate-bound anti-CD3 plus anti-CD28 mAbs for 14 h. IL-2-producing CD4+ T cells were enumerated using a human IL-2 secretion kit (Miltenyi Biotec) according to the manufacturer’s instructions.

RNA interference-mediated FLNa knockdown

We designed two forms of FLNa-targeted siRNA. The first one, siRNA1, consisted of a double-stranded FLNa-specific oligonucleotide having the following sequence: 5'-GUGACCGCCAAUAACGACAUU-3' (sense), and 5'-UGUCGUUAUUGGCGGUCACUU-3' (antisense), along with a control oligonucleotide: 5'-CUCUCGCCGUAAUAGCAGUUU-3' (sense), and 5'-ACUGCUAUUACGGCGAGAGUU-3' (antisense). Second, we inserted a distinct FLNa gene-specific 19-mer oligonucleotide, siRNA2 (GATCCAGCAGAACACTTTC), corresponding to nt 129–147 downstream of the transcription start site into the BglII/HindIII-digested RNA interference expression vector, pSuper.retro.neo (OligoEngine), and the empty vector was used as a transfection control. BLAST searches of the human genome database were conducted to ensure that the selected sequences did not target other gene transcripts. Jurkat cells were transfected with FLNa-specific or control siRNAs by electroporation, and purified PBMCs were transfected using the human T cell nucleofection kit (Amaxa) according to the manufacturer’s instructions. Transfection efficiency of PBMC was routinely ≥90% as assessed by the uptake of FITC-labeled control siRNA. FLNa expression in siRNA-treated cells was assessed by immunoblotting (see below).

Reporter assays

The constitutively active PKC{theta} (PKC{theta}-A/E) plasmid as well as the IL-2 promoter (IL-2p)-, NF-{kappa}B-, AP-1-, or NFAT-luciferase (Luc) reporter genes have been described (17). For reporter assays, Jurkat cells, which were transfected with FLNa-specific or control siRNA as described above, were cotransfected with Luc reporter genes driven by the proximal IL-2p, or tandem AP-1-, NF-{kappa}B-, or NFAT-binding sequences as described (17). The cells were cultured for 72 h and stimulated for 8 h with anti-CD3 plus anti-CD28 mAbs. The cells were the lysed and Luc activity was quantitated and normalized to the activity of a cotransfected beta-galactosidase reporter gene as previously described (17).

Immunofluorescence microscopy

Cells stimulated and plated on glass slides as described above were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-FLNa or -PKC{theta} Abs for 45 min at room temperature. The cells were then incubated with the relevant Alexa-labeled secondary Abs for 30 min and washed. Immunofluorescence images were recorded using a Marianas digital fluorescence microscopy system (Intelligent Imaging Innovations). The imaging system included an Axiocam MRm charge-coupled device camera (Zeiss), an Axiovert 200M microscope (Zeiss), and excitation and emission filter wheels (Sutter Instrument) with narrow-band optical filters (Chroma Technology). All of these components were controlled by SlideBook software (Intelligent Imaging Innovations). SlideBook software was used for image capture and analysis. To assess actin polymerization, stimulated Jurkat T cells were fixed with 4% (v/v) paraformaldehyde and washed. The cells were incubated for 30 min with FITC-labeled phalloidin (Sigma-Aldrich), and phalloidin staining was analyzed using a FACS.

Actin polymerization assay

Jurkat T cells were fixed with 4% (v/v) paraformaldehyde and washed. The cells were incubated for 30 min with FITC-labeled phalloidin (Sigma-Aldrich), and phalloidin staining was analyzed using a FACS.

Subcellular fractionation, immunoprecipitation, and immunoblotting

Subcellular fractionation of cell lysates into cytosolic, membrane, and detergent-insoluble (cytoskeletal) fractions was performed as described (8, 17). For immunoprecipitation, cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM sodium phosphate, 5 mM EDTA, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM NaF, 3 mM beta-glycerophosphate plus 1% Nonidet P-40, and 10 µg/ml each of aprotinin, leupeptin, and PMSF). After centrifugation at 16,000 x g (5 min, 4°C), the supernatants were incubated with optimal concentrations of Abs for 2 h at 4°C, followed by 30 µl of protein A/G-agarose (Santa Cruz Biotechnology) for an additional hour at 4°C. Samples were washed with lysis buffer, and precipitates were resolved by SDS-PAGE. Electrophoresed samples were processed for immunoblot analysis as previously described (8, 16, 17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Localization of FLNa to the T cell-APC contact site

Because actin cytoskeleton reorganization plays a critical role in the process of IS formation (18), and FLNa is an actin-binding protein as well as a scaffold for several signaling proteins, we wanted to determine initially whether FLNa undergoes relocalization during Ag stimulation and IS formation. Therefore, we stimulated Jurkat T cells or preactivated human peripheral blood T cells with SEE-pulsed Raji cells, allowed T-APC to form, and analyzed the localization of endogenous FLNa in the T cells by immunofluorescence and confocal imaging. In SEE-stimulated cells, FLNa was clearly concentrated at the T-APC contact site, i.e., the IS; in sharp contrast, we observed a fairly uniform cytoplasmic localization of FLNa in control unstimulated T cells (Fig. 1A). To assess the extent of FLNa concentration in the IS in a more quantitative manner, T cells were mixed with CMTMR-prelabeled, SEE-pulsed Raji cells, and the distribution of FLNa vs CMTMR along an imaginary axis intersecting the T-APC conjugate through the IS was analyzed (Fig. 1B). This analysis confirmed the marked enrichment of FLNa in the T cell contact area with the Raji APCs. These results indicate that recognition of SEE-pulsed APCs by T cells induces the recruitment of FLNa to the IS in primary or transformed human T cells.


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 1. Localization of FLNa at T-APC in the IS. A, Jurkat cells or SEE-preactivated human peripheral blood cells were incubated with nonpulsed or SEE-pulsed Raji B for 15 min and fixed. Localization of FLNa was analyzed by immunofluorescence and confocal imaging using an anti-FLNa Ab followed by a secondary, Alexa 488-coupled anti-mouse Ig Ab. The image is representative of 12 conjugates analyzed in each group. B, Jurkat cells were incubated with CMTMR-labeled, SEE-pulsed Raji cells. Cells were fixed and stained as in A. Analysis of green (FLNa) vs red (CMTMR, Raji cell) fluorescence intensity (in relative units) as a function of the distance (pixel number) along a line dissecting a T-APC conjugate through the IS. These curves are representative of three conjugates analyzed in each group.

 
FLNa interacts and colocalizes with PKC{theta}

Because PKC{theta} is the only PKC family member, which selectively localizes to the IS (19), and a fraction of it is also associated with the cytoskeleton in Ag-stimulated T cells (7, 8), we were interested to find out whether FLNa associates with PKC{theta}. Consistent with this notion, an earlier study demonstrated an association between FLNa and another member of the PKC family, PKC{alpha}, in HeLa cells (20). We first addressed this possibility by coimmunoprecipitation. When FLNa was immunoprecipitated from unstimulated or anti-CD3/CD28-costimulated Jurkat cells and the immunoprecipitates were analyzed for the presence of PKC{theta} by immunoblotting, we found that although no association between the two proteins was detected in unstimulated cells, costimulation induced a physical interaction between FLNa and PKC{theta}. This interaction was first detected after 1 min of stimulation, reached a peak after 3 min, and declined at later times although it was still prominent after 30 min of stimulation (Fig. 2). An isotype-matched control Ab did not immunoprecipitate detectable amounts of either FLNa or PKC{theta}.


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 2. Interaction of FLNa with PKC{theta}. Jurkat T cells were left unstimulated or stimulated for the indicated times with anti-CD3/CD28 mAbs. Immunoprecipitates were prepared from cell lysates with an anti-FLNa mAb or an isotype-matched control IgG. Immunoprecipitates or aliquots of total cell lysates were analyzed by immunoblotting using anti-PKC{theta} or -FLNa Abs.

 
We also analyzed the relative localization of FLNa and PKC{theta} by immunofluoresence microscopy. Fig. 3 clearly demonstrates colocalization of FLNa and PKC{theta} at the contact site between primary human T cells or Jurkat cells and SEE-presenting Raji APCs. However, very little, if any, colocalization can be seen in unstimulated T cells. Taken together, these results indicate that FLNa physically associates and colocalizes with PKC{theta} in response to TCR/CD28 costimulation.


Figure 3
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 3. Colocalization of PKC{theta} and FLNa at the IS. Jurkat cells or SEE-preactivated human peripheral blood cells were incubated with unpulsed or SEE-pulsed Raji B cells. Cells were fixed and stained with anti-FLNa and anti-PKC{theta} Abs followed by secondary Alexa-coupled Abs. The images are representative of six conjugates analyzed in each group.

 
FLNa is required for translocation of PKC{theta} from cytosol to cell membrane

The association of FLNa with PKC{theta} and that there is colocalization in stimulated T cells raised the possibility that FLNa, via its association with actin, may play a role in inducing the translocation of PKC{theta} from the cytosol to the membrane fraction upon T cell stimulation. Therefore, we analyzed the effect of siRNA-mediated FLNa knockdown on anti-CD3/CD28-induced PKC{theta} translocation by subcellular fractionation of siRNA-transfected Jurkat T cells. Preliminary experiments indicated that transfection of these cells with FLNa-specific siRNA1 for 3 days was required to achieve maximal reduction of endogenous FLNa expression (data not shown). Under these conditions, the specific siRNA, but not a control siRNA, reduced the expression of FLNa by ≥80% (Figs. 4, 5, and 7). In cells transfected with a control siRNA, anti-CD3/CD28 costimulation induced the translocation of a substantial fraction of PKC{theta} from the cytosol to the membrane fraction (Fig. 4). However, no increase in this membrane translocation was observed in stimulated cells expressing the FLNa-specific siRNA. As a specificity control, transfection with FLNa-specific siRNA did not affect the relative distribution of Vav1, a potential upstream regulator of PKC{theta}, localization and activation (17). In accordance with our earlier findings (21), anti-CD3/CD28 costimulation did not affect the distribution of Vav1. These results suggest that FLNa may be required for optimal TCR/CD28-induced PKC{theta} translocation from the cytoplasm to the membrane.


Figure 4
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of FLNa knockdown on anti-CD3/CD8-induced PKC{theta} translocation. Jurkat T cells were transfected with FLNa-specific or control siRNA for 72 h and left unstimulated or stimulated with anti-CD3/CD28 mAbs for 8 min. Cytosol (C), membrane (M), and detergent-insoluble (I) fractions were prepared and resolved by SDS-PAGE, and the expression of PKC{theta} or Vav1 in each fraction was determined by immunoblotting. Bottom panel shows the expression of FLNa and Vav1 (as a loading control) in cells transfected with control or FLNa-specific siRNA.

 

Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of FLNa knockdown on various TCR/CD28 signaling events. A, Jurkat cells were cotransfected with FLNa-specific or control siRNA plus an IL-2p-Luc reporter gene and cultured for 72 h. The cells were then left unstimulated ({square}) or stimulated with anti-CD3/CD28 mAbs ({blacksquare}) for 8 h. Luc activity was measured and normalized to the activity of a cotransfected beta-galactosidase reporter gene. The expression of FLNa or actin in cells transfected with control or FLNa-specific siRNA is shown in the right panel. B, Jurkat cells were transfected with FLNa-specific or control siRNA as in A and were left unstimulated or stimulated for the indicated times with anti-CD3/CD28 mAbs. Cells lysates were resolved by SDS-PAGE and analyzed by immunoblotting with Abs specific for phospho-ZAP-70, ZAP-70, phospho-LAT, LAT, or pTyr. C, siRNA-transfected Jurkat cells were left unstimulated (filled histograms) or stimulated (empty histograms) for 15 min as in B. The cells were fixed and the relative F-actin content was measured by staining with FITC-labeled phalloidin and FACS analysis.

 

Figure 7
View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 7. Silencing of FLNa inhibits IL-2 production in human T cells. Freshly isolated human peripheral blood cells were transfected with FLNa-specific or control siRNA, stimulated, and rested in the presence of IL-2 for 4 days. The cells were then left unstimulated or restimulated with plate-bound anti-CD3 plus -CD28 mAbs for 14 h. IL-2-producing CD4+ T cells were enumerated as described in Materials and Methods. The bottom panel shows the expression of FLNa or actin in cells transfected with control or FLNa-specific siRNA.

 
FLNa is required for optimal IL-2p activation but not for proximal TCR signaling

Because PKC{theta} is required for TCR/CD28-induced IL-2 production by mature T cells (5, 6), and we observed an association between PKC{theta} and FLNa, we next determined the effect of knocking down FLNa expression on the activation of an IL-2p reporter gene. Costimulation of Jurkat T cells cotransfected with an IL-2p-Luc construct plus a control siRNA plasmid induced a 4- to 5-fold stimulation of the IL-2p in comparison with unstimulated cells (Fig. 5A). However, this level of stimulation was inhibited by ~60% when the cells were cotransfected with a FLNa-specific siRNA, which reduced the expression of endogenous FLNa by ~80%. This result suggests that FLNa plays a role in inducing optimal IL-2 expression in response to TCR/CD28 costimulation.

TCR triggering is followed by early TCR-proximal signaling events, which include sequential activation of Lck and ZAP-70 tyrosine kinase and tyrosine phosphorylation of receptor subunits such as the TCR-associated {zeta} chain and adaptor proteins, e.g., LAT. Tyrosine-phosphorylated LAT functions to recruit other adaptors and enzymes, which trigger downstream signaling pathways leading to productive T cell activation. Given the inhibition of IL-2p activation in FLNa-depleted cells (Fig. 5A), we asked whether FLNa knockdown would affect more proximal signaling events, i.e., phosphorylation of ZAP-70 or LAT. Control siRNA- and FLNa siRNA-transfected cells displayed a similar pattern of ZAP-70 and LAT phosphorylation (Fig. 5B, top two panels). In addition, the profile of inducible total tyrosine phosphorylation as determined by immunoblotting of cell lysates with an anti-pTyr mAb was also very similar in the two groups of transfected T cells (Fig. 5B, bottom panel). Lastly, FLNa depletion also did not have any detectable effect on the TCR/CD28-stimulated increase in F-actin content (Fig. 5C) or activation of mitogen-activated protein kinases ERK and JNK (data not shown). The latter finding is consistent with previous reports that PKC{theta}–/– peripheral T cells display intact ERK and JNK activation in response to anti-CD3/CD28 costimulation (5, 6). These results suggest that TCR-proximal signals and actin polymerization most likely are independent of FLNa.

FLNa knockdown inhibits activation of PKC{theta}-dependent transcription factors

Induction of IL-2 gene expression depends on activation and binding of several critical transcription factors to their cognate sequences in the IL-2p. Among these transcription factors, AP-1, NF-{kappa}B, and NFAT have clearly been shown to be targets of PKC{theta} (5, 6, 7, 22, 23). To understand in more detail the basis for inhibition of IL-2p activation, which was observed in FLNa-depleted T cells, we further analyzed the effect of transfecting cells with a FLNa-specific siRNA on the PKC{theta}-induced activation of cotransfected NF-{kappa}B, AP-1, and NFAT reporter genes. Transfection of Jurkat T cells with constitutively active PKC{theta} mutant (PKC{theta}-A/E) induced a ~4-, ~8-, and ~15- to 20-fold activation of NF-{kappa}B, AP-1, or NFAT, respectively, in cells cotransfected with a control siRNA. However, expression of a FLNa-specific siRNA (siRNA1) reduced the activation of these reporter genes by ~50% (Fig. 6, A–C). The possibility that the FLNa-specific siRNA1 nonspecifically targeted other genes besides FLNa was essentially ruled out by demonstrating that a second siRNA (siRNA2) targeted to a different FLNa nucleotide sequence, which effectively reduced FLNa expression, also substantially reduced activation of the NFAT reporter gene (Fig. 6D). These results indicate that FLNa is important for optimal PKC{theta}-dependent activation of NF-{kappa}B, AP-1, and NFAT.


Figure 6
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of FLNa knockdown on PKC{theta}-induced reporter gene activation. Jurkat cells were cotransfected with PKC{theta}-A/E plus FLNa-specific siRNA1 or control siRNA (A–C), or with siRNA2-expressing pSuper.retro.neo vector (or empty vector as a control) (D) plus an NF-{kappa}B-Luc (A), AP-1-Luc (B), or NFAT-Luc (C and D) reporter genes and cultured for 72 h. Luc activity was measured and normalized to the activity of a cotransfected beta-galactosidase reporter gene. D, Bottom panels show the expression of FLNa and actin (as a loading control) in cells transfected with control or FLNa-specific siRNA2.

 
FLNa is necessary for activation of primary human T cells

To extend our findings and confirm them in a biologically relevant context, we also evaluated the effect of siRNA-mediated FLNa knockdown on receptor stimulation IL-2 production by preactivated and restimulated peripheral human blood lymphocytes. Reduction of FLNa expression by >80% resulted in a marked decrease in the proportion of CD4-gated IL-2 secreting cells from 10% (in control siRNA-transfected cells) to 3.8%, i.e., a 62% inhibition (Fig. 7). Similar results were also obtained when IL-2 expression was analyzed by intracellular cytokine staining (data not shown). Thus, FLNa is required for maximal IL-2 production by TCR/CD28-costimulated primary T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Filamins link cell mechanics and motility to cellular signal transduction events by binding simultaneously plasma membrane receptors, various intracellular signaling molecules, and actin (14, 15). The importance of filamins is evident from findings that filamin gene mutations lead to severe developmental defects in humans (15). However, a clear role for filamins has not been previously established in immune system cells, where FLNa is the predominant form, although it is known that FLNa is physically associated with the cytoplasmic tails of integrins, and that increased FLNa binding to beta1 integrin in Jurkat T cells inhibits their chemokine-stimulated migration (13, 24, 25). In this report, we began to analyze the role of FLNa in T cell activation using imaging, coimmunoprecipitation, and siRNA approaches. We demonstrate that FLNa 1) localizes to the IS in response to Ag stimulation, 2) colocalizes and coimmunoprecipitates with PKC{theta} in stimulated T cells, and 3) is required for optimal IL-2 expression and activation of the transcription factors NF-{kappa}B, AP-1, and NFAT in T cells. Thus, the major novel aspect of our study is the demonstration of a functional role for FLNa in receptor-induced T cell activation and IL-2 production.

The TCR/CD28-induced recruitment of PKC{theta} to specific sites, i.e., the IS (9, 19) and membrane lipid rafts (26) represents an important mechanism that regulates its unique functions and access to substrates in T cells. Polymerized actin is known to concentrate in the contact area between T cells and APCs (18), and a fraction of PKC{theta} is found in the detergent-insoluble cytoskeleton fraction of Ag-stimulated T cells. Although binding of the C1 domain of classical and novel PKC enzymes to membrane-localized diacylglycerol represents a general mechanism for the nonselective association of these PKC family members with the inner leaflet of the plasma membrane, an additional mechanism that selectively recruits PKC{theta} to specific membrane subdomains must exist. However, the molecular details of this mechanism are unknown. Therefore, identification of factors, which regulate the recruitment of PKC{theta} to the IS and lipid rafts, is critical for our understanding of the pathways that regulate the localization and function of this enzyme in T cells. Our previous findings demonstrated that Vav1 as well as PI3K are involved in the selective localization of PKC{theta} induced by TCR stimulation (17, 27). Our present findings extend these reports and demonstrate that PKC{theta} colocalizes and associates with FLNa, and that activating TCR/CD28 signals increase this association. Thus, FLNa could play a role in the selective localization of PKC{theta} in the IS via its simultaneous association with PKC{theta} and actin. Alternatively (or in addition), FLNa may function as a bridge to attach PKC{theta} to plasma membrane lipids given the demonstration that FLNa can also directly bind lipid membranes (28). Thus, it is possible that FLNa acts as a scaffold to promote the association of PKC{theta} with specific membrane subdomains or with other proteins. This notion is consistent with our findings, including the colocalization and association between the two proteins and the increased association following T cell stimulation, and the decreased membrane translocation of PKC{theta} in T cells treated with FLNa-specific siRNA. Nevertheless, we could not detect impaired localization of PKC{theta} in the IS in FLNa siRNA-treated cells (data not shown), perhaps reflecting the fact that a sufficient amount of FLNa was still present in the cells. Furthermore, although PKC{theta} is known to specifically localize in the cSMAC of the IS (9, 19), the localization of FLNa appears to be less selective because we find it localized across the whole T cell-APC contact area (data not shown). Therefore, other entities (proteins? lipids?) besides FLNa likely play additional roles in the selective localization and function of PKC{theta}.

In addition to reducing the membrane fraction of PKC{theta} in stimulated T cells, siRNA-mediated reduction of FLNa expression also had clear functional effects on T cells. Thus, FLNa-depleted Jurkat T cells displayed significantly (albeit incompletely) reduced activation of IL-2, NF-{kappa}B, AP-1, and NFAT reporter genes. More importantly, siRNA-mediated reduction of FLNa expression in human peripheral blood T cells consistently reduced the fraction of IL-2-producing T cells by ≥60%, indicating that FLNa is required for optimal IL-2 production in a biologically relevant context of primary T cells. The reduction of FLNa expression might inhibit transcription factor activation and IL-2 production by interfering with the proper translocation of PKC{theta} from the cytoplasm to the plasma membrane and its subsequent activation. Indeed, the finding that FLNa knockdown reduces activation of three distinct transcription factors known to be controlled by PKC{theta} (5, 6) is consistent with the notion that FLNa functions at a PKC{theta}-proximal step to regulate different PKC{theta}-dependent downstream signals.

The nature of the association between FLNa and PKC{theta}, as well as the basis for its increase following TCR/CD28 costimulation, is unknown. Interestingly, the tyrosine kinase, Lck, has been reported to phosphorylate FLNa on tyrosine, a modification that resulted in regulation of its association with integrins and actin filaments (29). Thus, one possibility is that phosphorylation of FLNa by Lck (or another kinase) following TCR stimulation enhances its interaction with PKC{theta}. Conversely, the Lck-induced phosphorylation of PKC{theta} in its regulatory domain (30) may also promote its association with FLNa. In summary, our findings document a novel stimulus-regulated association between FLNa and PKC{theta} in the physiologically relevant context of the T cell IS and, more importantly, reveal that FLNa is required for optimal T cell activation. These results open the way for future studies aimed at precisely defining the molecular basis for the FLNa-PKC{theta} association and its significance in T cell responses.


   Acknowledgments
 
We thank Dr. T. Mustelin for the use of the Amaxa nucleofection apparatus.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant CA35299 (to A.A.). Back

2 Address correspondence and reprint requests to Dr. Amnon Altman, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: amnon{at}liai.org Back

3 Abbreviations used in this paper: IS, immunological synapse; PKC{theta}, protein kinase C-{theta}; SMAC, supramolecular activation cluster; FLNa, filamin A; SEE, staphylococcal enterotoxin E; CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; siRNA, small interfering RNA; pTyr, phosphotyrosine; IL-2p, IL-2 promoter; Luc, luciferase. Back

Received for publication October 11, 2005. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221-227. [Abstract/Free Full Text]
  2. Altman, A., N. Isakov, G. Baier. 2000. Protein kinase C{theta}: a new essential superstar on the T-cell stage. Immunol. Today 21: 567-573. [Medline]
  3. Isakov, N., A. Altman. 2002. Protein kinase C{theta} in T cell activation. Annu. Rev. Immunol. 20: 761-794. [Medline]
  4. Arendt, C. W., B. Albrecht, T. J. Soos, D. R. Littman. 2002. Protein kinase C-{theta}: signaling from the center of the T-cell synapse. Curr. Opin. Immunol. 14: 323-330. [Medline]
  5. Sun, Z., C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi, J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, D. R. Littman. 2000. PKC-{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature T lymphocytes. Nature 404: 402-407. [Medline]
  6. Pfeifhofer, C., K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges, G. Baier. 2003. Protein kinase C {theta} affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197: 1525-1535. [Abstract/Free Full Text]
  7. Coudronniere, N., M. Villalba, N. Englund, A. Altman. 2000. NF-{kappa}B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-{theta}. Proc. Natl. Acad. Sci. USA 97: 3394-3399. [Abstract/Free Full Text]
  8. Villalba, M., K. Bi, F. Rodriguez, Y. Tanaka, S. Schoenberger, A. Altman. 2001. Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells. J. Cell Biol. 155: 331-338. [Abstract/Free Full Text]
  9. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395: 82-86. [Medline]
  10. Hartwig, J. H., T. P. Stossel. 1975. Isolation and properties of actin, myosin, and a new actin binding protein in rabbit alveolar macrophages. J. Biol. Chem. 250: 5696-5705. [Abstract/Free Full Text]
  11. Wang, K., J. F. Ash, S. J. Singer. 1975. Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells. Proc. Natl. Acad. Sci. USA 72: 4483-4486. [Abstract/Free Full Text]
  12. Van der Flier, A., A. Sonnenberg. 2001. Structural and functional aspects of filamins. Biochim. Biophys. Acta 1538: 99-117. [Medline]
  13. Calderwood, D. A., A. Huttenlocher, W. B. Kiosses, D. M. Rose, D. G. Woodside, M. A. Schwartz, M. H. Ginsberg. 2001. Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration. Nat. Cell Biol. 3: 1060-1068. [Medline]
  14. Stossel, T. P., J. Condeelis, L. Cooley, J. H. Hartwig, A. Noegel, M. Schleicher, S. S. Shapiro. 2001. Filamins as integrators of cell mechanics and signalling. Nat. Rev. Mol. Cell Biol. 2: 138-145. [Medline]
  15. Feng, Y., C. A. Walsh. 2004. The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat. Cell Biol. 6: 1034-1038. [Medline]
  16. Villalba, M., S. Kasibhatla, L. Genestier, A. Mahboubi, D. R. Green, A. Altman. 1999. Protein kinase C{theta} cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death. J. Immunol. 163: 5813-5819. [Abstract/Free Full Text]
  17. Villalba, M., N. Coudronniere, M. Deckert, E. Teixeiro, P. Mas, A. Altman. 2000. A novel functional interaction between Vav and PKC{theta} is required for TCR-induced T cell activation. Immunity 12: 151-160. [Medline]
  18. Wulfing, C., M. M. Davis. 1998. A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282: 2266-2269. [Abstract/Free Full Text]
  19. Monks, C. R., H. Kupfer, I. Tamir, A. Barlow, A. Kupfer. 1997. Selective modulation of protein kinase C-{theta} during T-cell activation. Nature 385: 83-86. [Medline]
  20. Tigges, U., B. Koch, J. Wissing, B. M. Jockusch, W. H. Ziegler. 2003. The F-actin cross-linking and focal adhesion protein FLNa is a ligand and in vivo substrate for protein kinase C{alpha}. J. Biol. Chem. 278: 23561-23569. [Abstract/Free Full Text]
  21. Charvet, C., A. J. Canonigo, D. D. Billadeau, A. Altman. 2005. Membrane localization and function of Vav3 in T cells depend on its association with the adapter SLP-76. J. Biol. Chem. 280: 15289-15299. [Abstract/Free Full Text]
  22. Baier-Bitterlich, G., F. Uberall, B. Bauer, F. Fresser, H. Wachter, H. Grunicke, G. Utermann, A. Altman, G. Baier. 1996. Protein kinase C-{theta} isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol. Cell. Biol. 16: 1842-1850. [Abstract/Free Full Text]
  23. Lin, X., A. O’Mahony, Y. Mu, R. Geleziunas, W. C. Greene. 2000. Protein kinase C-{theta} participates in NF-{kappa}B activation induced by CD3-CD28 costimulation through selective activation of I{kappa}B kinase beta. Mol. Cell. Biol. 20: 2933-2940. [Abstract/Free Full Text]
  24. Sharma, C. P., R. M. Ezzell, M. A. Arnaout. 1995. Direct interaction of filamin (ABP-280) with the beta2-integrin subunit CD18. J. Immunol. 154: 3461-3470. [Abstract]
  25. Loo, D. T., S. B. Kanner, A. Aruffo. 1998. Filamin binds to the cytoplasmic domain of the beta1-integrin: identification of amino acids responsible for this interaction. J. Biol. Chem. 273: 23304-23312. [Abstract/Free Full Text]
  26. Bi, K., Y. Tanaka, N. Coudronniere, K. Sugie, S. Hong, M. J. B. van Stipdonk, A. Altman. Antigen-induced translocation of PKC-{theta} to membrane rafts is required for T cell activation. Nat. Immunol. 2: 556-563.
  27. Villalba, M., K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham, A. Altman. 2002. Translocation of PKC{theta} in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J. Cell Biol. 157: 253-263. [Abstract/Free Full Text]
  28. Goldmann, W. H., J. M. Teodoridis, C. P. Sharma, B. Hu, G. Isenberg. 1999. Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers. Biochem. Biophys. Res. Commun. 259: 108-112. [Medline]
  29. Sharma, C. P., W. H. Goldmann. 2004. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking. Cell Biol. Int. 28: 935-941. [Medline]
  30. Liu, Y., S. Witte, Y. C. Liu, M. Doyle, C. Elly, A. Altman. 2000. Regulation of protein kinase C{theta} function during T cell activation by Lck-mediated tyrosine phosphorylation. J. Biol. Chem. 275: 3603-3609. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Sanchez-Lockhart, B. Graf, and J. Miller
Signals and Sequences That Control CD28 Localization to the Central Region of the Immunological Synapse
J. Immunol., December 1, 2008; 181(11): 7639 - 7648.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hayashi, K.
Right arrow Articles by Altman, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hayashi, K.
Right arrow Articles by Altman, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS