Phosphorylation of proteins and lipids plays a central role in the transduction of signals from the TCR complex and co receptors. Even before TCR ligation by relatively high-affinity MHC-peptide ligands that drive T-cell activation, there is some basal tyrosine phosphorylation of ITAM tyrosines and some recruitment of ZAP70, described later, to these phosphorylated ITAMs, since T cells are kept alive in part by interactions with low-affinity self MHC-peptide ligands. Within seconds of TCR ligation, LCK phosphorylates the ITAMs of the CD3 and ζ chains (Fig. 1).
Fig1. Early tyrosine phosphorylation events in T-cell activation. On antigen recognition, there is clustering of TCR complexes with coreceptors (CD4, in this case). CD4-associated LCK phosphorylates tyrosines in the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3 and ζ chains (A). ZAP70 binds to the phosphotyrosines of the ζ chains and is itself phosphorylated and activated. (The illustration shows one ZAP70 molecule binding to two phosphotyrosines of one ITAM in the ζ chain, but it is likely that initiation of a T-cell response requires the assembly of multiple ZAP70 molecules on ITAMs of the two ζ chains as well as on CD3 chains.) Active ZAP70 then phosphorylates tyrosines on various adaptor molecules, such as LAT, SLP76, GRB2, and GADS (B). The adaptors become docking sites for cellular enzymes such as PLCγ1 and guanosine diphosphate–guanosine triphosphate exchange factors such as SOS, which associate with adaptors including GRB2 that activate RAS and other small G proteins upstream of mitogen-activated protein kinases (MAPKs) (C), and these enzymes activate various cellular responses. PIP3 in the membrane recruits many proteins including PLCγ1 and ITK which contribute to downstream activation of calcium signaling. APC, Antigen-presenting cell; LAT, linker for activation of T cells; NFAT, nuclear factor of activated T cells; PIP3, phosphatidylinositol trisphosphate; PLCγ1, phospholipase Cγ1.
The tyrosine-phosphorylated ITAMs in the ζ chain are docking sites for the SYK family tyrosine kinase called ZAP70 (ζ-associated protein of 70 kD). ZAP70 contains two SH2 domains that can bind to ITAM phosphotyrosines. As dis cussed earlier, each ITAM has two tyrosine residues, and both of these must be phosphorylated to provide a docking site for one ZAP70 molecule. The bound ZAP70 becomes a substrate for the adjacent LCK after TCR recognition of antigen, and LCK phosphorylates specific tyrosine residues of ZAP70. As a result, ZAP70 acquires its own tyrosine kinase activity and is then able to phosphorylate a number of other cytoplasmic signaling molecules. A critical threshold of ZAP70 activity may be needed before downstream signaling events will proceed; this threshold is achieved by the recruitment of multiple ZAP70 molecules to the phosphorylated ITAMs on the ζ chains and on CD3 tails.
Another signaling pathway in T cells involves the activation of PI3-kinase (Fig. 2). This enzyme is recruited to adaptor proteins associated with the TCR complex and phosphorylates the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), located in the inner leaflet of the plasma membrane, to generate phosphatidylinositol 3, 4, 5-trisphosphate (PIP3). Certain signaling proteins in the cytosol have specialized pleckstrin homology (PH) domains that have an affinity for PIP3, and as a result, PH domain containing proteins can bind to the inside of the cell membrane only when PIP3 is generated. Examples of PH domain–containing enzymes that are activated when they bind to PIP3 include TEC-family tyrosine kinases such as ITK in T cells and BTK in B cells, and phospholipase Cγ1 (PLCγ1), a key enzyme in the calcium signaling pathway in T cells. Another important PIP3 dependent enzyme is PDK1, a serine/threonine kinase that is required for the phosphorylation and activation of an important downstream kinase called AKT. Activated AKT phosphorylates crucial targets and contributes to both increased protein synthesis and metabolism, leading to cell growth (enlargement), and survival. AKT activates another serine/threonine kinase, called mammalian target of rapamycin (mTOR, also called mechanistic or molecular target of rapamycin), that regulates protein syn thesis and cell metabolism. AKT also induces cell survival in a number of ways, including increased production and activation of antiapoptotic proteins of the BCL2 family, such as BCL2.
Fig2. Role of PI3-kinase in TCRs. Membrane PIP3, generated by PI3K, activates PDK1, which phosphorylates and activates the Akt kinase, which in turn phosphorylates downstream targets that are involved in cell survival and cell growth. mTOR, Mammalian target of rapamycin; PDK1, 3-phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate.
