Identification of Itk/Tsk Src Homology 3 Domain Ligands

Abstract

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. The T cell specific tyrosine kinase Tsk/Itk/Emt (1-4) (hereafter referred to as Itk) is a member of the Tec family of nonreceptor tyrosine kinases. This family of kinases consists of Tec, Btk, 1 Itk, and Bmx (5-7), and is defined by the presence of SH3 and SH2 domains (8) adjacent to the catalytic domain and an amino-terminal region containing a pleckstrin homology (PH) domain (9), a Tec homology (TH) domain (10), and a proline-rich region (11). Btk, the best characterized member of this family, is an important component of antigen receptor signaling pathways in B cells and mast cells (12-15). The expression pattern of Itk complements that of Btk; Itk is expressed in T cells whereas Btk is expressed in most hematopoietic cells other than T cells (16). This reciprocal pattern of expression suggests that these two kinases play analogous roles in antigen receptor signaling pathways, as is the case with ZAP-70 and Syk, which are capable of functionally substituting for one another (17).Btk has been identified as the gene defective in murine X-linked immunodeficiency (xid) (18, 19) and human X-linked agammaglobulinemia (XLA) (20,21). In xid mice, B cell numbers are reduced to one-half of normal and the titers of specific immunoglobulin isotypes are significantly reduced; in addition, xid B cells are insensitive to a number of mitogenic stimuli (22, 23). For reasons which have yet to be elucidated, the human disorder is much more severe, resulting in nearly complete elimination of the B cell compartment and dramatically reduced immunoglobulin levels (24). Biochemical studies have supported multiple roles for Btk in B cell activation. Btk kinase activity and tyrosine phosphorylation are increased after crosslinking either the B cell receptor on B cells (13-15) or the high affinity IgE receptor, Fc⑀RI, on mast cells (12). Interleukin-5 (25) and interleukin-6 (26) treatment have also been shown to lead to the activation of Btk.Itk, like Btk, is tyrosine-phosphorylated upon antigen receptor cross-linking (27).2 In addition, peripheral T cells from mice lacking functional Itk are refractory to stimulation by antibodies to CD3 plus antigen presenting cells (28). These Itk-deficient T cells can be stimulated by phorbol ester and calcium ionophore, demonstrating that Itk acts in signaling pathways proximal to the TCR. However, the manner in which Itk activity is regulated by, and integrated with, other TCR-derived signals is not well understood. Unlike th...

The Tec Kinases Itk and Rlk Regulate NKT Cell Maturation, Cytokine Production, and Survival

“…Recently, these data have been extended to include the adapter protein SAP, which activates and recruits Fyn to SLAM family receptors (5, 18 -20). Interestingly, Tec family kinases, specifically Btk and Itk, have been shown to bind to the Fyn Src homology 3 domain through an N-terminal region that is conserved between these kinases (59,60). Although these findings suggest a connection between Tec family kinases and the SLAM-SAP-Fyn pathway, this possibility seems unlikely.…”

Signaling network of the Btk family kinases

“…Cbl is rapidly tyrosine-phosphorylated in response to a variety of stimuli and becomes associated with a number of intracellular signaling molecules such as protein tyrosine kinases, phosphatidylinositol 3-kinase, Crk, and 14-3-3 proteins through di erent protein-interacting modules, leading to the formation of multi-component signaling complexes. Several groups have identi®ed p120 c-Cbl as a ligand for the SH3 domain of Btk kinases in vitro (Bunnell et al, 1996;Cory et al, 1995), however, the in vivo relevance of these ®ndings remains to be established. The interaction between Itk SH3 domain and Cbl seems to be transient and is only detected within 30 min upon stimulation in Jurkat cells, while Src SH3 domain binds to Cbl constitutively.…”

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

“…1B, top panels). It should be noted that the conversion of tryptophan to lysine at amino acid position 208 has been shown to represent an inactivating mutation at the predicted binding pocket of the SH3 domain (23).…”