The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8+ T cells. However, IQGAP1-deficient CD8+ T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.
A productive interaction between a T cell and an APC bearing an appropriate MHC–peptide complex results in the accumulation of F-actin and polarization of the microtubule organizing center (MTOC) toward the T cell–APC contact site, known as the immunological synapse (IS) (1, 2). These cytoskeletal changes are essential for proper APC recognition, IS formation, and efficient signaling leading to T cell activation (2). Numerous signaling proteins that function downstream of the TCR are intimately involved in the molecular pathways contributing to the reorganization of the F-actin and microtubule cytoskeletons, yet the mechanisms by which many of these proteins are recruited and how they exert their actin- and microtubule-regulating activities are, for the most part, unresolved.
F-actin reorganization was shown through the use of pharmacological agents to be essential for T cell activation (1, 3, 4). MTOC polarization toward the IS also is thought to be F-actin dependent (5). Several cytoskeletal interacting proteins, including Vav1, HS1, mDia1, WAVE2, and WASP, have been identified as participants in cytoskeletal reorganization and signaling during T cell–APC interactions (2, 6–12). Despite this knowledge, there are still many unanswered questions about how the cytoskeleton modulates TCR signaling. In addition, whether other proteins participate in these processes independently of the known cytoskeletal regulators, or in concert with them, remains to be determined.
One family of adaptor proteins, the IQGAP family, has been linked to both the microtubule and the actin cytoskeletal networks in several cell types (13). The IQGAP family consists of three highly homologous isoforms (IQGAP1, IQGAP2, and IQGAP3). Whereas IQGAP1 is expressed ubiquitously, IQGAP2 and IQGAP3 have more distinct expression patterns (14). IQGAP proteins are multimodular, consisting of a calponin homology domain at the N terminus, followed by a polyproline binding WW domain, four tandem IQ motifs that bind calmodulin, a RasGAP-related domain, and a RasGAP C terminus (15, 16). The domains of IQGAP1 have been shown to mediate numerous protein–protein interactions. In fact, the WW domain was found to interact with ERK1 and ERK2, whereas the IQ domains can bind MEK1, MEK2, and B-RAF, suggesting the possibility that IQGAP1 may regulate the activation of the Ras-MAPK signaling cascade (17–19). Instead of accelerating the intrinsic GTPase activities of Cdc42 and Rac1, the GTPase-activating protein domains of IQGAP proteins keep them in their active GTP-bound states (20–22). In epithelial cells, IQGAP1 was shown to regulate E-cadherin–mediated cell–cell adhesion, polarity, motility, proliferation, and MAPK signaling (17, 18, 23–30). Thus, IQGAP1 has the capacity to integrate multiple signaling cascades downstream of cell surface receptors.
In addition to functioning as a signaling adaptor protein, IQGAP1 was shown recently to bind the barbed ends of F-actin in a calmodulin-regulated manner and to display actin-capping activity through its C terminus in vitro (31). IQGAP1 also binds to regulators of the actin cytoskeleton, including N-WASP and mDia1 (32, 33). In addition, IQ domains of IQGAP1 interact with the actin-based motor myosin via the essential L chain (34), and the RasGAP domain of IQGAP1 binds the microtubule-associated protein CLIP-170 (35). As a consequence, IQGAP1 has been suggested to function as a facilitator of the communication between the F-actin and the microtubule networks (35, 36). In fact, Stinchcombe et al. (5) demonstrated that IQGAP1 localizes to the F-actin–rich region of the cytolytic synapse formed between a CD8+ T cell and a target cell (5) and, on the basis of this cellular localization, suggested that IQGAP1 might coordinate F-actin and microtubules during cellular cytotoxicity. However, one study has suggested that these two systems can be separated (37). Therefore, the exact role of IQGAP1 in regulating the interplay between cytoskeletal systems and signaling during T cell development and activation needs to be investigated. Also, functional consequences of direct F-actin regulation by IQGAP1 have not been well characterized. So far, there is no in vivo evidence to support the recent suggestion that IQGAP1 has actin-capping activity, so its functional role in actin recruitment/stabilization at the IS also is of interest.
To address these issues, we have used IQGAP1-deficient mice as well as short hairpin RNA (shRNA)-mediated knockdown in the Jurkat T cell model. We found that thymocyte development was unaltered in IQGAP1 knockout mice and that IQGAP1 was surprisingly dispensable for MTOC polarization and cellular cytotoxicity. However, IQGAP1-deficient T cells showed increased cytokine production, enhanced LCK activation, and heightened phosphorylation kinetics after TCR ligation. In addition, they displayed augmented F-actin accumulation upon TCR ligation and enhanced kinetics of TCR-mediated F-actin retrograde flow. Interestingly, expression of the N terminus of IQGAP1 could partially rescue F-actin accumulation and IL-2 gene transcription, whereas increased F-actin dynamics could be fully reversed by rescue with the F-actin–capping C terminus of IQGAP1. On the basis of these results, we propose that IQGAP1 is a critical modulator of T cell activation that regulates TCR-mediated signaling and F-actin dynamics through distinct molecular mechanisms.
Materials and Methods
Reagents and plasmids
Abs against ZAP-70 and LCK have been described previously (38, 39). Anti-phospho-Src and anti-ERK2 were from Cell Signaling Technology. Anti-IQGAP1 was obtained by the immunization of rabbits with a keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to amino acids 2–25 of mouse IQGAP1 (Cocalico Biologicals, Inc.). Anti-IQGAP2 and anti-phosphotyrosine (4G10) were from Upstate Biotechnology/Millipore. Anti-γ-tubulin was from Sigma-Aldrich. Anti-human CD3 (OKT3) was purchased from the Mayo Pharmacy, and anti-human CD28 was purchased from BD Biosciences. Anti-mouse CD3 (2C11) and anti-CD28 (37.51) were purchased from Bio-X-Cell. The Mayo Peptide Synthesis Facility generated the SIINFEKL (SIN) and RAHYNIVTF (E7) peptides. The altered peptide ligands Q4R7 (SIIQFERL), Q4H7 (SIIQFEHL), and pG4 (SIIGFEKL) were a gift from Dr. Diana Gil Pages (Mayo Clinic and Elim Biopharm) (40). The shRNA suppression vectors pFRT.H1p, pCMS3.cherry.H1p, and pCMS4.eGFP.H1P have been described previously (12, 41, 42). The shIQGAP1-targeting sequence (5′-GTCCTGAACATAATCTCAC-3′) corresponds to nucleotides 1318–1336 using National Center for Biotechnology Information Genbank accession number NM_003870 (http://www.ncbi.nlm.nih.gov/genbank/). The IQGAP1 cDNA was made shRNA resistant using PCR-based site-directed mutagenesis (5′-CCgGAgCAcAATCTCAC-3′).
Cell culture and isolation
Jurkat T cells were passaged as described previously (12). The P815 cell line was described previously (43). The EL-4 mouse lymphoma cell line was cultured in 5% FBS, 5% bovine calf serum, and 1% l-glutamine. To generate primary mouse T cells, splenocytes were dissociated, and RBCs were lysed in a solution containing 155 mM ammonium chloride, 1 mM potassium bicarbonate, and 0.1 mM EDTA disodium salt. Mouse CD4+ and CD8+ T cells were negatively isolated using MACS Isolation Kit II (Miltenyi Biotec). CD4+ T cells were cultured in RPMI 1640, 1% nonessential amino acids, 1% l-glutamine, 1% sodium pyruvate, 0.05% 2-ME, and 10% FBS. CD8+ T cells were cultured in RPMI 1640, 1% nonessential amino acids, 1% l-glutamine, 1% sodium pyruvate, 0.05% 2-ME, 3% FBS, and 20 U/ml IL-2.
Jurkat T cell transfection, stimulation, and Western blot analysis
Jurkat T cells were transiently transfected using a BTX ECM 830 electroporator (315 V, 10 ms, 1 pulse). Forty micrograms of each suppression plasmid or 50–60 μg of suppression/re-expression plasmids were used in each transfection, and experiments were conducted 72 h posttransfection. Jurkat T cells were stimulated with 5–10 μg anti-CD3/CD28 and cross-linked with goat anti-mouse Ig (Cappell/MP Biomedicals).
IQGAP1−/− mice (44) were obtained from Dr. Wadie Bahou (State University of New York at Stony Brook, Stony Brook, NY). Sex-matched littermates were used in all of the experiments unless indicated. In order to generate IQGAP1-deficient OT-I TCR transgenic mice, IQGAP1−/− mice were bred with homozygous OT-I TCR transgenic mice, and heterozygous progeny then were bred to generate the experimental animals. All of the mice were genotyped for the presence of the Vα2, Vβ5 TCR transgene and for the deletion of the IQGAP1 gene. Immunoblotting to demonstrate the loss of IQGAP1 and flow cytometry for the expression of Vα2 and Vβ5 were performed to confirm TCR transgene expression. All of the mice were between 5 and 40 wk of age. All of the animal work complied with Mayo Clinic’s guidelines and was approved by the Institutional Animal Care and Use Committee.
P815 cells were stained with anti-CD3/CD28 and mixed with splenic CD8+ T cells. Splenic OT-I T cells were stimulated with 1 μg/ml SIINFEKL for 48 h and allowed to rest for 48 h. EL-4 cells were pulsed with either 10 μg/ml E7 peptide, SIINFEKL peptide, or an altered peptide ligand for 1 h. Along with the peptide, these cells were loaded with 50 μCi [51Cr] per 1 × 106 cells. EL-4 and OT-I T cells were allowed to interact for 4 h and pelleted, and the supernatant was analyzed for [51Cr] release as described (45).
Splenic OT-I T cells were stimulated with 1 μg/ml SIINFEKL for 72 h and allowed to rest for 24 h. Conjugates were formed with EL-4 cells prepulsed with either E7 peptide or SIINFEKL peptide. CD8+ T cells and EL-4 cells were allowed to interact for 30 min for F-actin or 20 min for MTOC polarization at 37°C. Conjugates were spread onto poly-l-lysine–coated coverslips for 5 min at 37°C. Cells were fixed and stained for F-actin and α-tubulin as described previously (46) and imaged on a Zeiss LSM 710 confocal or Zeiss Axiovert 200M microscope. Quantification of F-actin accumulation or MTOC polarization at the IS was done as described previously (42). At least 50 conjugates were analyzed in each of three experiments. Quantification of the intensity of polarized F-actin was determined by Zen software. Pixel intensities were broken into three groups, and percentages of cells in each group are shown. At least 60 conjugates were analyzed in total. Data represent average ± SD.
All of the Abs were from BD Biosciences or eBioscience. All of the samples were run on either a BD FACSCanto II or a BD LSR II. Jurkat T cells were stimulated for 1 or 5 min with CD3/CD28. The cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 0.15% Surfact-Amps X-100 (Thermo Scientific). F-actin was labeled with either FITC- or Alexa Fluor 647-conjugated phalloidin (Invitrogen). Stimulation time points were labeled in triplicate and repeated in three different experiments. Analysis of data was performed using FlowJo (Tree Star). Statistical significance was determined using a two-tailed Student t test.
ELISA and quantitative RT-PCR
For ELISA and RT-PCR, CD4+ T cells were stimulated for 24 h with the indicated concentrations of anti-CD3 and anti-CD28. IL-2 levels in the supernatants were analyzed by ELISA (eBioscience). Quantitative analyses of mRNA expression were performed as described previously (47). In brief, T cells were stimulated with the indicated concentrations of Ab over time, and RNA was extracted subsequently using the RNeasy Mini Kit (Qiagen). One microgram of total RNA was transcribed into cDNA using the Superscript III RT-PCR Kit (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was performed using the comparative cycle threshold method with SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7900TM Sequence Detection System. Experiments were performed in triplicate using independently generated cDNAs. Gene-specific primers for mouse and human IL-2 were designed by using software from the Integrated DNA Technologies Web site (http://www.idtdna.com/scitools/Applications/RealTimePCR/) and are shown below: mouse RPLP0, forward 5′-AGATCCGCATGTCCCTTC-3′, reverse 5′-CCTTGCGCATCATGGTGTT-3′; mouse IFN-γ, forward 5′-CCTAGCTCTGAGACAATGAACG-3′, reverse 5′-TTCCACATCTATGCCACTTGAG-3′; mouse IL-2, forward 5′-TGATGGACCTACAGGAGCTCCTGA-3′, reverse 5′-GAGTCAAATCCAGAACATGCCGCAG-3′; human RPLP0, forward 5′-AGATCCGCATGTCCCTCC-3′, reverse 5′-CCTTGCGCATCATGGTGTT-3′; human IL-2, forward 5′-AACTCCTGTCTTGCATTGCAC-3′, reverse 5′-GCTCCAGTTGTAGCTGTGTTT-3′.
Eight-well Lab-Tek II chambered cover glasses (Nunc) were prepared and coated with OKT3 (Biolegend), as described previously (48). Immediately before being imaged, wells were covered with 400 μl of imaging medium (phenol red-free RPMI 1640 with 25 μM HEPES) and equilibrated to 37°C on the microscope stage within a Solent environmental chamber. Cultured cells were harvested and resuspended in imaging medium at a concentration of 2 × 106 cells per milliliter. Spreading was initiated by adding 10 μl of cell suspension to the well. Time-lapse series of randomly selected cells were collected on a Zeiss Axiovert 200 equipped with a ×63 objective and a PerkinElmer ERS6 Ultraview spinning disk confocal system. The 0.5-μm-thick Z stacks of two planes were collected using an Orca ER camera (Hamamatsu) at 3-s intervals for 1–5 min.
Kymography and statistical analysis
Kymographs were generated along the radii of spread T cells using Volocity 6.0 software (PerkinElmer) and analyzed for F-actin features. Velocity was determined based on the deflection angles of diagonal trajectories. Four kymographs were generated per IS, and one velocity measurement was made per kymograph. Statistical analysis was performed using Microsoft Excel, with statistical significance determined using a two-tailed Student t test for unpaired samples with equal variances.
Thymocyte development and peripheral T cell numbers are normal in IQGAP1-deficient mice
The role of IQGAP1 has been studied extensively in epithelial cells and cancer. However, the functional role of IQGAP1 in T cells remains unclear. We began our analysis by verifying that the IQGAP1 protein was absent in T cells from IQGAP1−/− mice (44, 49). These mice have a neomycin cassette inserted in the IQGAP1 gene locus replacing exon 27. Using an Ab against the IQGAP1 N terminus, we found that IQGAP1 was completely absent from total thymocytes and splenic CD4+ T cells (Fig. 1A), and a smaller truncated protein product was never observed (Supplemental Fig. 1A). Thus, although it was reported that IQGAP1 mRNA was found in IQGAP1−/− T cells (50), we could not detect intact or partial IQGAP1 protein in either thymocytes or purified splenic CD4+/CD8+ T cells. We conclude that any remaining RNA likely is degraded by nonsense RNA-mediated decay.
We next analyzed T cell development in the absence of IQGAP1. We found that double-negative (DN), double-positive (DP), and single-positive (SP) thymocyte populations were unaltered in the absence of IQGAP1 (Fig. 1B, 1C). Early thymocyte progenitors, DN2, DN3, and DN4, also were unchanged compared with wild-type T cells (J.A. Gorman and D.D. Billadeau, unpublished observations). In addition, there were no differences in the number of splenic or lymphatic CD4+ or CD8+ T cells (Fig. 1D). Before testing the functionality of the knockout T cells, we confirmed that TCR and CD28 surface levels were normal (Fig. 1E). Taken together, these data indicate that the loss of IQGAP1 does not alter thymocyte development, the ability of naive T cells to migrate into peripheral lymphoid organs, or TCR levels.
IQGAP1 does not regulate MTOC polarization or cytotoxicity
Because IQGAP1 was shown to localize to the cytolytic synapse (5) and was hypothesized to regulate T cell MTOC polarity in line with its role in epithelial cells (25), we examined T cell-mediated cytotoxicity and MTOC polarization in the IQGAP1 knockout. We used IQGAP1+/+ and IQGAP1−/− OT-I TCR transgenic CD8+ T cells to investigate MTOC polarization to the IS and cell-mediated killing using control (E7) or Ag-loaded (SIINFEKL) EL4 cells. Consistent with our findings in nontransgenic mice, thymocyte development was normal in the OT-I mice (unpublished observations). Interestingly, TCR transgenic IQGAP1−/− lymphocytes were able to polarize their MTOC toward the IS (Fig. 2A, 2B) and efficiently killed Ag-loaded EL4 cells (Fig. 2C). To confirm this result, we used an alternate system to measure cytotoxicity. Using CD8+ T cells isolated from non-TCR transgenic mice, we assessed IQGAP1−/− T cells’ ability to kill 2C11-pulsed P815 cells. Again, we saw no difference between IQGAP1−/− and IQGAP1+/+ killing abilities (Supplemental Fig. 1B). To determine whether we were driving cytotoxicity as a result of overstimulating the system with a high concentration of peptide, we tested both lower concentrations and different strengths of agonists. Surprisingly, but consistent with the notion that IQGAP1 was not involved in TCR-driven cell-mediated cytotoxicity, we find that there is no significant difference in killing between wild-type and IQGAP1-deficient OT-I T cells regardless of SIN concentration or agonist strength (Fig. 2D). Thus, the loss of IQGAP1 does not affect T cell signaling leading to either MTOC polarization or cellular cytotoxicity.
IQGAP1 deficiency increases cytokine gene expression and signaling
Because IQGAP1 was shown to regulate the MAPK pathway (17–19), we next examined the effect of depleting IQGAP1 on T cell signaling and activation. Interestingly, IQGAP1−/− OT-I CD8+ T cells displayed increased basal tyrosine phosphorylation levels (Fig. 3A, compare 0 time points) and demonstrated enhanced global tyrosine phosphorylation after TCR cross-linking (Fig. 3A). Consistent with heightened tyrosine phosphorylation in IQGAP1−/− OT-I T cells, we also observed augmented IL-2 and IFN-γ mRNA levels by quantitative RT-PCR (Fig. 3B, 3C). Consistent with this result, IQGAP1-deficient T cells secreted more IL-2 and IFN-γ compared with control cells (Fig. 3D, 3E). Furthermore, IQGAP1−/− CD4+ T cells and IQGAP1-suppressed Jurkat T cells show increased IL-2 mRNA and secretion of IL-2 (Fig. 4A, Supplemental Fig. 2A, 2B), which is consistent with IQGAP1 regulating TCR signaling leading to elevated cytokine production in both mouse T cell subsets as well as in human T cells. Taken together, these data indicate that IQGAP1 likely functions as a negative regulator or modulator of T cell signaling leading to IL-2 and IFN-γ gene transcription.
We next sought to examine at which point in the signaling cascade IQGAP1 was exerting its effect. We began our investigation using IQGAP1-suppressed Jurkat T cells, because they phenocopy IQGAP1−/− T cells at the level of increased IL-2 gene transcription. Interestingly, IQGAP1-suppressed Jurkat T cells displayed increased global tyrosine phosphorylation levels after TCR cross-linking when compared with control cells (Fig. 4B). Consistent with the mouse model, we routinely observed that IQGAP1 depletion increased basal tyrosine phosphorylation (Figs. 3A, 4B, compare 0 time points), suggesting that IQGAP1 may suppress proximal kinase activation. We therefore analyzed TCR-induced LCK phosphorylation at Tyr394 within the kinase activation loop. Indeed, IQGAP1-suppressed Jurkat and CD8+ knockout T cells showed increased TCR-mediated LCK phosphorylation at Tyr394 (Figs. 3A, 4B). Because this Ab also can cross-react with FYN, we also immunoprecipitated LCK and assessed Tyr394 phosphorylation. Again, we found that the kinetics and extent of phosphorylation were increased in IQGAP1-depleted Jurkat cells (Fig. 4C). Taken together, these data indicate that IQGAP1 regulates LCK activation kinetics after TCR ligation.
To confirm that the heighted levels of phosphorylation and IL-2 mRNA expression were a result of the loss of IQGAP1, we used a suppression/rescue system to reconstitute IQGAP1-suppressed Jurkat T cells with a shRNA-resistant IQGAP1 cDNA (12). Importantly, re-expression of wild-type IQGAP1 restored the kinetics of TCR-stimulated tyrosine phosphorylation and LCK phosphorylation to that of control transfected cells (Fig. 5A, 5B). We subsequently used deletion mutants (Fig. 5A) to examine which domains of IQGAP1 modulate TCR signaling. TCR-stimulated IL-2 mRNA levels were fully restored to that of control transfected cells after reconstitution with either wild-type IQGAP1 or a N-terminal fragment of IQGAP1 encompassing amino acids 1–735 (Fig. 5C). In contrast to the N-terminal portion of IQGAP1, the C-terminal half of the molecule (amino acids 744–1657) did not rescue IL-2 mRNA levels. Thus, the N terminus of IQGAP1 modulates TCR-mediated activation leading to IL-2 production, whereas the C terminus, which has been implicated in F-actin capping (31), does not. To further investigate the role of the IQGAP1’s N terminus, we examined the phosphorylation kinetics after TCR ligation (Supplemental Fig. 2C). Interestingly, we found that neither the N-terminal nor the C-terminal fragments rescued tyrosine phosphorylation kinetics. Taken together, the N terminus of IQGAP1 appears to modulate TCR-driven IL-2 gene transcription independent of its ability to regulate TCR-dependent proximal signaling (see 17Discussion).
IQGAP1 negatively regulates F-actin accumulation at the IS
In a recent report, IQGAP1 was shown to cap F-actin in vitro (31). To investigate the role of IQGAP1 in modulating F-actin dynamics in T cells, we first formed conjugates with TCR transgenic OT-I wild-type or IQGAP1−/− T cells and SIINFEKL-loaded EL-4 cells. Although the overall proportion of IQGAP1−/− OT-I T cells that accumulated F-actin at the IS was similar to that of the control (Fig. 6A), the amount of F-actin that accumulated at the IS was increased dramatically without IQGAP1 (Fig. 6B). In fact, measuring pixel intensity at the IS, we found that there were more IQGAP1−/− cells with increased F-actin content at the IS compared with control cells (Fig. 6C). To further analyze stimulated F-actin content, we examined IQGAP1−/− OT-I T cells spread on 2C11-coated coverslips (Fig. 6D, Supplemental Fig. 3). Indeed, knockout T cells had augmented TCR-stimulated F-actin. Furthermore, the IQGAP1−/− T cells showed faster F-actin kinetics and prolonged spreading. Moreover, using three-dimensional reconstruction of Z stacks at 5 min poststimulation, we observed that the enhanced F-actin in IQGAP1−/− cells accumulated at the base of the cell (Fig. 6E).
We next used a flow cytometry-based assay to quantify the amount of TCR-stimulated F-actin. Consistent with the qualitative and quantitative microscopic analyses of the knockout cells, IQGAP1-suppresssed Jurkat T cells showed increased F-actin after TCR ligation (Fig. 7A), whereas unstimulated baseline F-actin content was not affected (J.A. Gorman and D.D. Billadeau, unpublished observations). Having observed more TCR-stimulated F-actin accumulation at the IS, we hypothesized that IQGAP1 might regulate F-actin retrograde flow during TCR signaling. To evaluate the effects of IQGAP1 knockdown on F-actin dynamics, we used Jurkat T cells stably expressing low levels of GFP-actin (12). T cells then were imaged while spreading on an anti-CD3–coated cover glass, as described previously (51). Confocal imaging of the IS confirmed our observations of increased F-actin enrichment in the lamellipodia of T cells suppressed for IQGAP1 compared with control transfected cells (Fig. 7B, Supplemental Video 1). IQGAP1-deficient T cells also spread to a greater extent, and spreading persisted longer relative to that of nonsuppressed cells (A. Babich and J.K. Burkhardt, unpublished observations). Furthermore, kymography showed that T cells lacking IQGAP1 had faster F-actin retrograde flow relative to that of the control cells (Fig. 7C, 7D). The lamellipodia also occupied a larger portion of the synapse area than those in control counterparts. Taken together, these data suggest that IQGAP1 regulates the extent of F-actin accumulation and the velocity of F-actin movement at the IS.
Last, we used suppression/re-expression studies to identify which region of IQGAP1 impacts F-actin accumulation. Significantly, TCR-stimulated F-actin levels were reduced to control levels after reconstitution with either wild-type IQGAP1 or the C-terminal fragment (744–1657) that has F-actin–capping potential (Fig. 7E). Interestingly, the region that rescued the modulation of IL-2 gene transcription, IQGAP1 1–735, was capable of at least partial rescue of F-actin accumulation in this assay.
IQGAP1 regulates signaling cascades and the cytoskeleton through its scaffolding functions (13, 52). However, in this study, we show that the loss of IQGAP1 does not affect MTOC polarization or cytotoxicity. Alternatively, we demonstrate that IQGAP1 normally attenuates signaling events and F-actin dynamics after TCR ligation. Specifically, we find that IQGAP1 modulates proximal TCR-mediated LCK activation leading to increased production of IL-2 and IFN-γ. Furthermore, we show that IQGAP1-deficient T cells display enhanced F-actin accumulation and increased velocities of retrograde flow at the IS. Altogether, our data suggest that IQGAP1 is a critical negative modulator of T cell activation.
IQGAP1 has been suggested to integrate cross talk between the cytoskeletal systems, leading to MTOC polarization and cytotoxicity (5). In fact, a recent study examining the role of IQGAP1 in NK cells demonstrated a critical role for IQGAP1 in NK cell-mediated cytotoxicity (53). These results conflict with our analysis of IQGAP1-deficient CD8+ T cells, which show no obvious defect in either MTOC polarization to the cytotoxic synapse or cell-mediated killing. The differences might be attributable to either the use of shRNA versus a knockout model or the differences in the cell types being analyzed. In fact, it has been suggested in a previous study that the requirements for granule secretion in NK cells are different (54). The delivery of lytic granules to the NK IS is through small areas devoid of F-actin; however, there is still a need for myosin IIA (54, 55). On the contrary, previous work has suggested that actin is not a barrier for granule secretion in cytotoxic T lymphocytes (5, 56). These differences could explain the conflicting observed phenotypes. Our results suggest that neither MTOC polarization nor cytotoxicity is affected in CD8+ T cells completely lacking IQGAP1.
The T cell cytoskeleton acts as a crucial platform for T cell signaling and ultimately T cell activation. Previous studies have shown that both F-actin and the microtubule cytoskeleton are needed for proper IS formation, TCR signaling, and T cell activation (57). However, the exact functions of F-actin in TCR signaling remain unclear. Recent reports suggest that F-actin dynamics may modulate TCR function by exerting physical forces on signaling complexes or microcluster biogenesis (57, 58) (A. Babich and J.K. Burkhardt, unpublished observations). Although this is not the only possible explanation for the functional effects of IQGAP1 suppression, this paradigm implicates IQGAP1 in the mechanical aspects of TCR signaling. However, evidence from our rescue experiments suggests that the IQGAP1 C terminus rescues F-actin levels completely but does not rescue the augmented activation phenotype or TCR signaling. This observation suggests that the negative regulation of signaling by IQGAP1 is independent of its F-actin regulation.
We find that IQGAP1-deficient T cells displayed augmented TCR signaling events as receptor-proximal as LCK phosphorylation, resulting in increased IL-2 and IFN-γ production in CD8+ T cells. A recent study has shown that CD3/CD28-stimulated, IQGAP1-depleted CD8+ T cells produced more IFN-γ when compared with wild-type cells (50). In this study, the authors demonstrated that IQGAP1 exists in a large cytoplasmic RNA–protein complex containing NFAT, a long, intergenic noncoding RNA repressor of NFAT, and several kinases that regulate NFAT subcellular localization. Importantly, they demonstrated that either depletion of noncoding RNA repressor of NFAT or IQGAP1 led to an increase in the expression of NFAT-dependent cytokines after stimulation. Although most IQGAP1 binding partners have been shown to interact with the C-terminal fragment, the N-terminal fragment interacts with ERK1/2 through its WW repeat region (17). Although ERK1 and ERK2 are downstream of TCR signaling, these kinases provide a positive feedback loop on TCR signaling through interference with the interaction of LCK and SHP1 (59). Despite this, we found that the expression of the N-terminal fragment of IQGAP1 rescues IL-2 production to wild-type levels, whereas TCR-stimulated signaling remained elevated similar to that seen in IQGAP1-suppressed cells or those expressing the C-terminal fragment of IQGAP1. It remains possible that the N-terminal region assembles the large RNA–protein complex regulating NFAT nuclear shuttling and enhanced NFAT-mediated transcription but is incapable of modulating proximal signaling at the level of LCK activation. Clearly, further experiments aimed at elucidating the mechanism by which IQGAP1 integrates TCR signaling through its dynamic interactions are needed.
We provide evidence that IQGAP1-deficient T cells generate significantly more TCR-stimulated F-actin at the IS and exhibit altered T cell spreading with lamellipodia that are consistently larger than those in control T cells. Recent in vitro studies from Pelikan-Conchaudron et al. (31) have demonstrated that the C terminus of IQGAP1 harbors a calmodulin-regulated F-actin–capping region and as such may control F-actin kinetics during cellular activation. Our results showing that the C terminus of IQGAP1 is able to rescue the increased F-actin phenotype are consistent with these in vitro findings, such that, in the absence of IQGAP1, F-actin filaments continue to grow and generate an outward force on the cell membrane. Furthermore, F-actin networks of T cells spreading on CD3-coated coverslips, acutely treated with jasplakinolide, a F-actin–stabilizing agent, collapsed inward toward the cSMAC (A. Babich and J.K. Burkhardt, unpublished observations); thus F-actin polymerization is the driving force on cell membranes. This is consistent with the idea that heightened F-actin levels could generate an increased outward force on the cell membrane, thus causing the larger lamellipodia. Under normal circumstances, it is possible that IQGAP1 regulation of GTPase activity or its cooperation with myosin motor proteins may be critical for maintaining balance during TCR-mediated F-actin remodeling (34, 52, 60). In addition, the IQGAP1 C terminus interacts with VASP (61), which can positively regulate F-actin nucleation by blocking capping proteins from binding to the barbed ends of actin filaments (62), as well as Dia1 (32), which can protect barbed ends from capping proteins (63). Thus, it is possible that the C terminus of IQGAP1 could sequester these actin regulatory proteins permitting efficient capping. In addition to the C terminus of IQGAP1, the N terminus also partially rescued actin dynamics. This might be explained by either the ability of the N terminus to bind and bundle F-actin via its CH domain (64, 65) or other unknown binding partners. In future studies, it will be important to understand how these various proteins cooperate with IQGAP1 to efficiently regulate lamellipodial dynamics during TCR stimulation.
Taken together, we demonstrate that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms. Our study also highlights the complexity of IQGAP1 function in T cells, underscoring the importance of future research aimed at further characterizing the cellular activities of IQGAP proteins, which are likely to illuminate the exact mechanisms by which IQGAP1 can distinctively modulate T cell activation and F-actin dynamics at the IS.
This work was supported in part by an Immunology Predoctoral Training Grant from the National Institutes of Health (T32-AI07425 to J.A.G.), an Allergic Diseases Training Grant from the National Institutes of Health (T32-AI07047 to T.S.G.), and National Institutes of Health Grant R01-AI065474 (to D.D.B.). D.D.B. is a Leukemia and Lymphoma Society Scholar.
The online version of this article contains supplemental material.
Abbreviations used in this article:
microtubule organizing center
short hairpin RNA
The authors have no financial conflicts of interest.