Biochemical Interactions Integrating Itk with the T Cell Receptor-initiated Signaling Cascade

Abstract

The T cell receptor (TCR), 1 B cell receptor, and mast cell receptor (Fc⑀RI) are composed of multiple subunits, enabling the coupling of a variable antigen-recognition subunit to conserved signaling modules (1-3). Collectively, these receptors direct the antigen-specific responses of T cells, B cells, and mast cells. Although these antigen receptors do not contain intrinsic tyrosine kinases, the early events downstream of these antigen receptors are controlled by a relatively conserved series of tyrosine kinase-dependent events. In each case, Src family kinases initiate the signaling cascade by phosphorylating immunoreceptor tyrosine-based activation motifs present in the cytoplasmic tails of the signal-transducing receptor subunits. Complete immunoreceptor tyrosine-based activation motifs phosphorylation results in the recruitment of Syk/ZAP-70 family tyrosine kinases to the receptor. These kinases are subsequently activated by Src kinase-mediated transphosphorylation. The substrates of the Syk kinases include internal phosphorylation sites as well as transphosphorylation sites in LAT, SLP-76, and BLNK, a SLP-76-related adapter protein (1, 4 -7). These Syk kinase-dependent phosphorylations direct the recruitment of additional effector and adapter molecules to LAT, SLP-76, BLNK, and the Syk kinases themselves (7-13). In T cells LAT, which is recruited into lipid rafts by dual palmitoylation, nucleates a raft-associated complex containing multiple effectors, including Grb2 and Sos, Cbl, Vav, PLC␥1, and PI3K (5,14,15). The perturbation of this complex by the disruption of Lck, ZAP-70, LAT, or SLP-76 results in profound defects in the activation of calcium influxes and mitogen-activated protein kinases (MAPKs) downstream of the TCR (16 -20). Similar complexes may form in both B cells and mast cells.The Tec family kinases (Tec, Itk, Btk, Bmx, and Txk (21,22)) also participate in signal transduction pathways downstream of antigen receptors (3,(23)(24)(25)(26)(27)(28)(29)(30)(31). In particular, Tec kinases have been observed to contribute to the PI3K-dependent phosphorylation and activation of PLC␥ isoforms, the concomitant induction of sustained calcium influxes (32), and the activation of MAPKs (33-35). However, the mechanisms that integrate the Tec kinases into antigen receptor-dependent signaling pathways are not yet well understood. Structurally, these kinases share the conserved arrangement of SH3, SH2, and kinase domains found in Src kinases, but are distinguished by the absence of amino-terminal acylations, the absence of the carboxyl-terminal regulatory tyrosine, and by the presence of a conserved amino-terminal region containing a pleckstrin homology (PH) domain, a Btk homology motif, and a proline-rich region (PRR). Although the functions of the protein-binding domains of Tec kinases remain unclear, the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding pocket present in Tec kinase PH domains is clearly required for the cooperative activation of PLC␥ isoforms by PI3K and Tec kinases (32)...

TCR/CD3-Induced Activation and Binding of Emt/Itk to Linker of Activated T Cell Complexes: Requirement for the Src Homology 2 Domain

“…A fusion protein encompassing the proline-rich region of Emt/Itk, but excluding the SH3 and SH2 domains, was able to interact with LAT, but not with SLP-76. Because LAT does not contain a complementary SH3 domain to bind to the proline-rich region of Emt/Itk, we suggest that this interaction is due to an intermediate protein such as Grb2, which contains both SH3 and SH2 domains, with the latter known to interact with LAT (28).…”

“…SLP-76 is an important adapter protein that is phosphorylated by ZAP-70 and is required for PLC␥1 activation, intracellular Ca 2ϩ mobilization, IL-2 production, and development of T lymphocytes (29 -33). Bunnell et al (28) showed that GST-SH2 fusion proteins interacted only with phosphorylated SLP-76, whereas GST-SH3 constructs reacted with SLP-76 regardless of its phosphorylation status. Because of the inability of full-length Emt/Itk to interact with unphosphorylated SLP-76, these authors suggested that the SH3 domain is sequestered in the intact Emt/Itk molecule and, furthermore, that the finding that SLP-76 possesses adjacent binding sites for the Emt/Itk SH2 and SH3 domains suggested that the interaction of SLP-76 with Emt/ Itk may represent a synergistic effect between these two domains (28).…”

“…Bunnell et al (28) showed that GST-SH2 fusion proteins interacted only with phosphorylated SLP-76, whereas GST-SH3 constructs reacted with SLP-76 regardless of its phosphorylation status. Because of the inability of full-length Emt/Itk to interact with unphosphorylated SLP-76, these authors suggested that the SH3 domain is sequestered in the intact Emt/Itk molecule and, furthermore, that the finding that SLP-76 possesses adjacent binding sites for the Emt/Itk SH2 and SH3 domains suggested that the interaction of SLP-76 with Emt/ Itk may represent a synergistic effect between these two domains (28). A fusion protein encompassing the proline-rich region of Emt/Itk, but excluding the SH3 and SH2 domains, was able to interact with LAT, but not with SLP-76.…”

“…Recently Bunnell et al (28), utilizing GST fusion proteins representing various domains of Emt/Itk, were able to capture another adapter protein, SLP-76. SLP-76 is an important adapter protein that is phosphorylated by ZAP-70 and is required for PLC␥1 activation, intracellular Ca 2ϩ mobilization, IL-2 production, and development of T lymphocytes (29 -33).…”

In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

“…The N terminus harbors three tyrosines that become phos- phorylated upon TCR engagement [5,6] and have been reported to serve as docking sites for the guanine nucleotide exchange factor Vav, the adaptor protein Nck, the Tec family kinase Itk, and the p85 subunit of PI3K [7][8][9][10][11][12][13][14][15][16]. A single SH2 domain resides in the C-terminal portion of SLP-76 and links it to the adhesion and degranulation-promoting adaptor protein (ADAP) and to the hematopoietic progenitor kinase 1 [17][18][19].…”