Intermolecular multiple-quantum coherences between bulk water and a glycoprotein fragment at modest concentration (20 mM) have been experimentally produced and detected, although such coherences are inconceivable in the normal theoretical framework of nuclear magnetic resonance. A density matrix treatment explains these results by including the long-range dipolar interaction between spins and by discarding the high-temperature approximation. These results imply that peak intensities (critical for structural determinations) can be distorted in many gradient experiments, and show that magic-angle gradients provide substantial improvements with reduced gradient strengths. They also suggest methods for contrast enhancement in magnetic resonance imaging.

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The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR). The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood. Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine. Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism. The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction. By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly. Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2. We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase of the Tec family that participates in the intracellular signaling events leading to T cell activation. Tec family members contain the conserved SH3, SH2, and catalytic domains common to many kinase families, but they are distinguished by unique sequences outside of this region. The mechanism by which Itk and related Tec kinases are regulated is not well understood. Our studies indicate that Itk catalytic activity is inhibited by the peptidyl prolyl isomerase activity of cyclophilin A (CypA). NMR structural studies combined with mutational analysis show that a prolinedependent conformational switch within the Itk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. CypA and Itk form a stable complex in Jurkat T cells that is disrupted by treatment with cyclosporin A. Moreover, the phosphorylation levels of Itk and a downstream substrate of Itk, PLC␥1, are increased in Jurkat T cells that have been treated with cyclosporin A. These findings support a novel mode of tyrosine kinase regulation for a Tec family member and provide a molecular basis for understanding a cellular function of the ubiquitous peptidyl prolyl isomerase, CypA.N ormal cell growth depends on the precise control of protein tyrosine kinase activity (1). For certain families of kinases, the mechanism of catalytic regulation is well understood. Structures of Src tyrosine kinases (2, 3) reveal intramolecular interactions mediated by the Src homology 2 (SH2) and Src homology 3 (SH3) domains that control catalytic activity of the neighboring kinase domain. Specifically, distortion of the Src kinase active site is achieved in part by SH2 binding to a phosphorylated tyrosine residue in the C-terminal tail of Src (4). For other families of kinases, the mechanistic details of catalytic regulation remain elusive. In particular, the Tec family of nonreceptor tyrosine kinases (5) displays distinguishing characteristics that point to an alternative mode of regulation. The Tec family kinases modulate hematopoietic cellular responses to external stimuli (6). The T cell-specific Tec family member, interleukin-2 tyrosine kinase (Itk) (7,8), plays a role in the maturation of thymocytes, is required for intracellular signaling following T cell receptor (TCR) crosslinking, and is involved in generation of second messengers that mediate cytoskeletal reorganization (9). Itk is homologous to Src in the region spanning the SH3, SH2, and kinase domain but lacks the Src C-terminal tail that contains the regulatory tyrosine. However, activation of Itk depends on SH2-mediated interactions with phosphorylated signaling partners (9) such as Slp-76 (10) and LAT (11), suggesting a regulatory role for the Itk SH2 domain despite the absence of a Src-like C-terminal regulatory tyrosine residue.The Src regulatory mechanism highlights the role of molecular switches in controlling cellular signaling pathways. Well studied covalent modifications such as...

The Tec family tyrosine kinases regulate lymphocyte development, activation, and differentiation. In T cells, the predominant Tec kinase is Itk, which functions downstream of the T-cell receptor to regulate phospholipase C-g. This review highlights recent advances in our understanding of Itk kinase structure and enzymatic regulation, focusing on Itk protein domain interactions and mechanisms of substrate recognition. We also discuss the role of Itk in the development of conventional versus innate T-cell lineages, including both ab and gd T-cell subsets. Finally, we describe the complex role of Itk signaling in effector T-cell differentiation and the regulation of cytokine gene expression. Together, these data implicate Itk as an important modulator of T-cell signaling and function.

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.

Cyclophilin A (CypA/Ppia) is a peptidyl-prolyl isomerase (PPIase) that binds the immunosuppressive drug cyclosporine. The resulting complex blocks T cell function by inhibiting the calcium-dependent phosphatase calcineurin. To identify the native function of CypA, long suspected of regulating signal transduction, we generated mice lacking the Ppia gene. These animals develop allergic disease, with elevated IgE and tissue infiltration by mast cells and eosinophils, that is driven by CD4+ T helper type II (Th2) cytokines. Ppia(-/-) Th2 cells were hypersensitive to TCR stimulation, a phenotype consistent with increased activity of Itk, a Tec family tyrosine kinase crucial for Th2 responses. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in the SH2 domain of Itk disrupted interaction with CypA and specifically increased Th2 cytokine production from wild-type CD4+ T cells. Thus, CypA inhibits CD4+ T cell signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.

Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead. ReceiVed December 5, 2006; ReVised Manuscript ReceiVed March 7, 2007 ABSTRACT: Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in Vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in Vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.Protein kinases have evolved a variety of regulatory mechanisms that exploit the effects of site-specific phosphorylation and precise protein/domain interactions (1, 2). The largest family of nonreceptor protein tyrosine kinases consists of the eleven Src kinases (3) that each contain an N-terminal unique region, followed by an Src homology-3 (SH3 1 ) domain, Src homology-2 (SH2) domain, catalytic domain, and a C-terminal tail with a conserved regulatory tyrosine. A significant leap in our understanding of kinase regulation has been provided by the three-dimensional structures of two Src family kinases (Hck and Src) in their inactive states (4-6). These structures reveal that the SH3 and SH2 domains are located on the "back" surface of the kinase domain mediating a network of intramolecular interactions th...