TCR signaling leads to the activation of the γ1 isoform of the enzyme phospholipase C (PLCγ1), and the products of PLCγ1-mediated hydrolysis of membrane lipids activate additional signaling events that induce specific transcription factors in T cells (Fig. 1). Soon after TCR triggering, the LAT and SLP76 adaptors are phosphorylated on tyrosine residues by ZAP70 and combine with GADS to form a com plex on the inside of the plasma membrane. At the same time PI3-kinase generates PIP3 in the inner leaflet of the plasma membrane and recruits both the TEC-family kinase ITK and PLCγ1 (both enzymes have PH domains and can thus bind to PIP3). These enzymes also contain SH2 domains that allow them to bind to specific phosphorylated tyrosine residues on the LAT/GADS/SLP76 complex, and it is here that ITK phosphorylates and activates PLCγ1. Activated PLCγ1 catalyzes the hydrolysis of the plasma membrane phospholipid PIP2, generating two breakdown products: the soluble sugar triphosphate, inositol 1,4,5-trisphosphate (IP3), and membrane-bound diacylglycerol (DAG). IP3 and DAG then activate two distinct downstream signaling pathways in T cells.
Fig1. T-cell signaling downstream of phospholipase C γ1 (PLCγ1). (A) The LAT adaptor protein that is phosphorylated on T-cell activation binds the cytosolic enzyme PLCγ1, which is phosphorylated by ITK and activated. Active PLCγ1 hydrolyzes membrane phosphatidylinositol bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3), which stimulates an increase in cytosolic calcium, and diacylglycerol (DAG), which activates the enzyme protein kinase C (PKC). (B) IP3 causes depletion of endoplasmic reticulum calcium, which is sensed by STIM1. PKC induces numerous cellular responses. (C) STIM1 induces the opening of the calcium release–activated calcium (CRAC) channel that facilitates entry of extracellular calcium into the cytosol. ORAI is a component of the CRAC channel. Increased cytosolic calcium together with PKC activate various transcription factors, leading to cellular responses. LAT, Linker for activation of T cells.
IP3 produces a rapid increase in cytosolic free calcium after T-cell activation. IP3 diffuses through the cytosol to the smooth endoplasmic reticulum (ER), where it binds to its receptor, a ligand-gated calcium channel, and stimulates release of mem brane-sequestered calcium stores. As a result, the cytosolic free calcium ion concentration increases from a resting level of about 100 nM to a peak of 600 to 1000 nM within a few minutes. The depletion of ER calcium is sensed by STIM1, an ER membrane protein, which then activates a plasma membrane ion channel called a calcium release–activated calcium (CRAC) channel. Portions of the ER membrane in the cell are often very close to the plasma membrane and this proximity facilitates the interaction between STIM1 and the CRAC channel. The activation of the CRAC channel results in an influx of extracellular calcium that sustains cytosolic levels at about 300 to 400 nM for more than 1 hour. A key component of the CRAC channel is the ORAI protein; mutations in the gene encoding this protein are the cause of a rare human immunodeficiency disease. Cytosolic free calcium acts as a signaling molecule by binding to calmodulin, a ubiquitous calcium-dependent regulatory protein. Calcium-calmodulin complexes activate several enzymes, including calcineurin, a protein serine/threonine phosphatase that is important for transcription factor activation.
DAG, the second breakdown product of PIP2, is a mem brane-bound lipid that activates the enzyme protein kinase C (PKC). Several isoforms of PKC participate in the generation of active transcription factors. The combination of elevated free cytosolic calcium and DAG activates certain isoforms of membrane-associated PKC by inducing a con formational change that makes the catalytic site of the kinase accessible to its substrates. Numerous downstream proteins are phosphorylated by PKC. The PKCθ isoform localizes to the immune synapse and is involved in the activation and nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor.
So far, we have described several signal transduction path ways initiated by ligand binding to the TCR that result in the activation of different types of enzymes: G protein–MAP kinase pathways leading to activation of kinases such as ERK and JNK; a PLCγ1-calcium–dependent pathway leading to activation of the phosphatase calcineurin; and a DAG-dependent pathway leading to activation of PKC. Each of these pathways contributes to the expression of genes encoding proteins needed for T-cell proliferation, differentiation, and effector functions. In the following section, we will describe the mechanisms by which these different signaling pathways stimulate the transcription of various genes in T cells.
