Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.     

Itk phosphorylation sites are required for functional activity in primary T cells

The Tec family kinase Itk plays a critical role in signal transduction downstream of the T cell antigen receptor and has been implicated in the activation of phospholipase C-gamma1, a key regulator of calcium mobilization and extracellular signal-regulated kinase (ERK) activation. We have shown previously that Itk is regulated by an activating transphosphorylation event in which Tyr-511 in the kinase domain is phosphorylated by Lck (Heyeck, S. D., Wilcox, H. M., Bunnell, S. C., and Berg, L. J. (1997) J. Biol. Chem. 272, 25401-25408). In this study, we present evidence for another mode of regulation for Itk, the autophosphorylation of Tyr-180 in the Src homology 3 (SH3) domain. To investigate the role of Itk trans- and autophosphorylation in T cell signaling, a retroviral transduction system was used to introduce different versions of Itk into Itk-deficient primary T cells. We report that Itk mutated at either the trans- or the autophosphorylation site is unable to fully restore cytokine production and ERK activation in the Itk-deficient cells; Itk-Y511F is severely defective, whereas Itk-Y180F has partial activity. Because phosphorylation at Tyr-180 is predicted to interfere with ligand binding by the SH3 domain, an SH3 point mutant that cannot bind ligand was also examined and found to be unable to restore function to the Itk-/- cells. These data provide new insights into the complex regulation of Itk in primary T cells.     

A specific intermolecular association between the regulatory domains of a Tec family kinase

Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. We have examined a novel intermolecular interaction between the SH3 and SH2 domains of Itk. In addition to the interaction between the isolated domains, we have found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that we describe provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, we note a striking correlation between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agamaglobulinemia.     

Mechanism and functional significance of Itk autophosphorylation

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full-length Itk mutant containing phenylalanine in place of the autophosphorylated tyrosine has been studied in Itk-deficient primary T cells. These studies revealed that the non-phosphorylated enzyme restores Itk mediated signaling only partially. In spite of these insights, the precise role of the Tec kinase autophosphorylation site is unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here, we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay, we show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand-binding capability of the Itk SH3 domain in a ligand-dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.     

TCR/CD3-Induced activation and binding of Emt/Itk to linker of activated T cell complexes: requirement for the Src homology 2 domain

Expressed in mast and T cells/inducible T cell tyrosine kinase (Emt/Itk), a Tec family protein tyrosine kinase, is critical for the development and activation of T lymphocytes. The mechanism through which Emt/Itk mediates its effector functions is poorly understood. In this study, we show that the Emt/Itk Src homology 2 (SH2) domain is critical for the transphosphorylation and activation of Emt/Itk catalytic activity that is mediated by TCR/CD3 engagement. Furthermore, we find that the Emt/Itk SH2 domain is essential for the formation of TCR/CD3-inducible Emt/Itk-LAT complexes, whereas the SH3 domain and catalytic activity are not required. The Emt/Itk-linker of activated T cells (LAT) complexes are biologically important because Jurkat T cells with deficient LAT expression (JCaM2) fail to increase Emt/Itk tyrosine phosphorylation upon TCR/CD3 stimulation. Confocal microscopy reveals that in activated cells, LAT complexes colocalize with TCR/CD3. The present data suggest that upon TCR/CD3 engagement, the Emt/Itk SH2 domain mediates the formation of a molecular complex containing Emt/Itk, LAT, and TCR/CD3; this complex is essential for Emt/Itk activation and function.     

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.     

Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade

Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.     

Tec kinase Itk forms membrane clusters specifically in the vicinity of recruiting receptors

The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 A of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.     

SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.     

A remote substrate docking mechanism for the tec family tyrosine kinases

During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.     

Controlling the activity of the Tec kinase Itk by mutation of the phenylalanine gatekeeper residue

The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.     

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.     

Binding of the proline-rich segment of myelin basic protein to SH3 domains: spectroscopic, microarray, and modeling studies of ligand conformation and effects of posttranslational modifications

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.     

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.     

Emt/Itk associates with activated TCR complexes: role of the pleckstrin homology domain

Expressed in mast and T-cells/inducible T cell tyrosine kinase (Emt/Itk) is a protein tyrosine kinase required for T cell Ag receptor (TCR)-induced activation and development. A physical interaction between Emt/Itk and TCR has not been described previously. Here, we have utilized laser scanning confocal microscopy to demonstrate that Ab-mediated engagement of the CD3epsilon chain induces the membrane colocalization of Emt/Itk with TCR/CD3. Removal of the Emt/Itk pleckstrin homology domain (DeltaPH-Emt/Itk) abrogates the association of the kinase with the cell membrane, as well as its activation-induced colocalization with the TCR complex and subsequent tyrosine phosphorylation. The addition of a membrane localization sequence to DeltaPH-Emt/Itk from Lck restores all of these deficiencies except the activation-induced tyrosine phosphorylation. Our data suggest that the PH domain of Emt/Itk can be replaced with another membrane localization signal without affecting the membrane targeting and activation-induced colocalization of the kinase with the TCR. However, the PH domain is indispensable for the activation-induced tyrosine phosphorylation of the kinase.     

Interaction between the N-terminal SH3 domain of Nck-alpha and CD3-epsilon-derived peptides: non-canonical and canonical recognition motifs

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.     

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.     

The linker between SH2 and kinase domains positively regulates catalysis of the Tec family kinases

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.