Itoro Akpan

Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Enhanced T-cell signaling in cells bearing linker for activation of T-cell (LAT) molecules resistant to ubiquitylation

Linker for activation of T cells (LAT) plays a central role in T-cell activation by nucleating signaling complexes that are critical for the propagation of T-cell signals from the plasma membrane to the cellular interior. The role of phosphorylation and palmitoylation in LAT function has been well studied, but not much is known about other strategies by which the cell modulates LAT activity. We have focused on LAT ubiquitylation and have mapped the sites on which LAT is ubiquitylated. To elucidate the biological role of this process, we substituted LAT lysines with arginines. This resulted in a dramatic decrease in overall LAT ubiquitylation. Ubiquitylation-resistant mutants of LAT were internalized at rates comparable to wild-type LAT in a mechanism that required Cbl family proteins. However, these mutants displayed a defect in protein turnover rates. T-cell signaling was elevated in cells reconstituted with LAT mutants resistant to ubiquitylation, indicating that inhibition of LAT ubiquitylation enhances T-cell potency. These results support LAT ubiquitylation as a molecular checkpoint for attenuation of T-cell signaling.

Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

ABSTRACT The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.

The Ability of Sos1 to Oligomerize the Adaptor Protein LAT Is Separable from Its Guanine Nucleotide Exchange Activity in Vivo

Thymocyte development requires that Sos1 acts both as a guanine nucleotide exchange factor and as a scaffold to cluster adaptor proteins. Separating Signals Mediated by Sos Activation of the small guanosine triphosphatase Ras is central to both the development of thymocytes into T cells and the effector functions of mature T cells. Antigen-dependent activation of the T cell receptor (TCR) leads to Ras activation through Ras guanine nucleotide exchange factors (GEFs), including Sos1. Kortum et al. found that T cell–specific deletion of Sos1 in mice [Sos1(T)−/−] blocked the development of thymocytes into T cells. Furthermore, Sos1(T)−/− thymocytes exhibited reduced clustering of the adaptor protein LAT, decreased activation of the kinase extracellular signal–regulated kinase (ERK) and phospholipase C–γ1 (PLC-γ1), and reduced Ca2+ mobilization in response to TCR stimulation. Generation of Sos1(T)−/− mice expressing a GEF-defective Sos1 mutant transgene restored PLC-γ1 activation and Ca2+ mobilization, but not ERK activation, in the thymocytes, whereas a transgene expressing a Sos1 mutant unable to cluster LAT had the opposite effect. However, coexpression of both mutant Sos1 proteins in trans in the same cells was required to restore thymocyte development. These data demonstrate that the ability of Sos1 to oligomerize adaptor proteins is separate from its role as a RasGEF, suggesting that these two functions might be differentially targeted therapeutically. The activation of the small guanosine triphosphatase Ras by the guanine nucleotide exchange factor (GEF) Sos1 (Son of Sevenless 1) is a central feature of many receptor-stimulated signaling pathways. In developing T cells (thymocytes), Sos1-dependent activation of extracellular signal–regulated kinase (ERK) is required to stimulate cellular proliferation and differentiation. We showed that in addition to its GEF activity, Sos1 acted as a scaffold to nucleate oligomerization of the T cell adaptor protein LAT (linker for activation of T cells) in vivo. The scaffold function of Sos1 depended on its ability to bind to the adaptor protein Grb2. Furthermore, the GEF activity of Sos1 and the Sos1-dependent oligomerization of LAT were separable functions in vivo. Whereas the GEF activity of Sos1 was required for optimal ERK phosphorylation in response to T cell receptor (TCR) stimulation, the Sos1-dependent oligomerization of LAT was required for maximal TCR-dependent phosphorylation and activation of phospholipase C–γ1 and Ca2+ signaling. Finally, both of these Sos1 functions were required for early thymocyte proliferation. Whereas transgenic restoration of either the GEF activity or the LAT oligomerization functions of Sos1 alone failed to rescue thymocyte development in Sos1-deficient mice, simultaneous reconstitution of these two signals in the same cell restored normal T cell development. This ability of Sos1 to act both as a RasGEF and as a scaffold to nucleate Grb2-dependent adaptor oligomerization may also occur in other Grb2-dependent pathways, such as those activated by growth factor receptors.

Recruitment of calcineurin to the TCR positively regulates T cell activation

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

GTP-binding Protein-like Domain of AGAP1 Is Protein Binding Site That Allosterically Regulates ArfGAP Protein Catalytic Activity

Background: The AGAPs are a subtype of ArfGAPs with a G-protein like domain (GLD) postulated to function as an allosteric binding site. Results: The C terminus of RhoA binds to AGAP1 and stimulates GAP activity specifically for Arf1 and dependent on the GLD. Conclusion: AGAP1 is allosterically regulated by specific proteins that bind to the GLD. Significance: These results are the first example of protein-dependent allosteric regulation of an ArfGAP. AGAPs are a subtype of Arf GTPase-activating proteins (GAPs) with 11 members in humans. In addition to the Arf GAP domain, the proteins contain a G-protein-like domain (GLD) with homology to Ras superfamily proteins and a PH domain. AGAPs bind to clathrin adaptors, function in post Golgi membrane traffic, and have been implicated in glioblastoma. The regulation of AGAPs is largely unexplored. Other enzymes containing GTP binding domains are regulated by nucleotide binding. However, nucleotide binding to AGAPs has not been detected. Here, we found that neither nucleotides nor deleting the GLD of AGAP1 affected catalysis, which led us to hypothesize that the GLD is a protein binding site that regulates GAP activity. Two-hybrid screens identified RhoA, Rac1, and Cdc42 as potential binding partners. Coimmunoprecipitation confirmed that AGAP1 and AGAP2 can bind to RhoA. Binding was mediated by the C terminus of RhoA and was independent of nucleotide. RhoA and the C-terminal peptide from RhoA increased GAP activity specifically for the substrate Arf1. In contrast, a C-terminal peptide from Cdc42 neither bound nor activated AGAP1. Based on these results, we propose that AGAPs are allosterically regulated through protein binding to the GLD domain.

Development of nanoscale structure in LAT-based signaling complexes.

ABSTRACT The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation. Summary: The molecular organization of the adapter proteins LAT and SLP-76 changes during T cell activation. Upon engagement of T cell receptor, they first mix randomly. Later, SLP-76 is restricted to the edges of LAT clusters.

In vivo functional mapping of the conserved protein domains within murine Themis1

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal‐initiating receptors are lineage‐specific, most trigger ‘ubiquitous’ downstream signaling pathways. T‐lineage‐specific receptors are coupled to these signaling pathways by lymphocyte‐restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T‐cell maturation. Themis1 orthologs contain three conserved domains: a proline‐rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine‐containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1−/− progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome

Significance Activation of T cells via their antigen receptor (TCR) and the formation of a multimolecular signaling complex in or near the plasma membrane is a key initial event in the generation of an immune response. Here we find that p38, a serine/threonine kinase activated by TCR signaling, in turn phosphorylates its upstream tyrosine kinase ZAP-70. This results in a decrease in the size and longevity of the TCR signaling complex, limiting T cell effector responses. Therefore, cross-talk between an upstream and downstream kinase in the signaling complex itself generates a negative feedback loop that may limit excessive T cell responses. ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70–activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.

The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8 + T cells

Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8+ T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8+ T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8+ T cells.