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.     

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.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

Identification of an Allosteric Signaling Network within Tec Family Kinases

The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the ‘regulatory spine’, which assembles and disassembles as a kinase switches between its active and inactive states, has been identified. Here, we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the Src homology 2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine, resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant, rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Bruton's tyrosine kinase R544 on the activation loop and Bruton's tyrosine kinase E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases.     

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.     

The solution structure and intramolecular associations of the Tec kinase SRC homology 3 domain.

Tec is the prototypic member of a family of intracellular tyrosine kinases that includes Txk, Bmx, Itk, and Btk. Tec family kinases share similarities in domain structure with Src family kinases, but one of the features that differentiates them is a proline-rich region (PRR) preceding their Src homology (SH) 3 domain. Evidence that the PRR of Itk can bind in an intramolecular fashion to its SH3 domain and the lack of a regulatory tyrosine in the C terminus indicates that Tec kinases must be regulated by a different set of intramolecular interactions to the Src kinases. We have determined the solution structure of the Tec SH3 domain and have investigated interactions with its PRR, which contains two SH3-binding sites. We demonstrate that in vitro, the Tec PRR can bind in an intramolecular fashion to the SH3. However, the affinity is lower than that for dimerization via reciprocal PRR-SH3 association. Using site-directed mutagenesis we show that both sites can bind the Tec SH3 domain; site 1 (155KTLPPAP161) binds intramolecularly, while site 2 (165KRRPPPPIPP174) cannot and binds in an intermolecular fashion. These distinct roles for the SH3 binding sites in Tec family kinases could be important for protein targeting and enzyme activation.     

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.     

Interaction between Btk TH and SH3 domain

Several mechanisms are involved in the regulation of cellular signaling. Bruton tyrosine kinase (Btk) of the Tec family contains in the Tec homology (TH) domain a proline-rich region (PRR) capable of interacting with several SH3 domains. The Btk has the SH3 domain adjacent to the TH domain. CD and fluorescence spectroscopy were used to study the binding of two peptides corresponding to segments in the PRR to the Btk SH3 domain. The peptide for the N-terminal half of the PRR binds specifically, whereas the other peptide had hardly any affinity. The TH domain has about four times lower affinity to the SH3 domain than the peptide, 17.0 vs 3.9 microM. The interaction was further tested with an SH3 domain construct that contained the PRR. The two peptides cannot compete for the binding to the extended protein and the TH domain has two times lower affinity to the extended SH3 domain. The intra- or intermolecular interaction between the TH and SH3 domain might have regulatory function also in the other Tec family members.     

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.     

Competing modes of self-association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization

A nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer-dimer equilibria. The Btk fragment studied contains two consecutive proline-rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino-terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy-terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino-terminal proline stretch; the other, in the carboxy-terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino-terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined K(a) values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co-localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.     

Dynamics of the Tec‐family tyrosine kinase SH3 domains

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains.     

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.     

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.     

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.     

Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

Crystallographic structure of the SH3 domain of the human c-Yes tyrosine kinase: loop flexibility and amyloid aggregation

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline-rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high-resolution crystal structure of the SH3 domain of the c-Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n-Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.     

Tec homology (TH) adjacent to the PH domain

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline-rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon-γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non-catalytic p85 subunit of PI-3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.     

A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein

The Shc (Src homology collagen-like) adaptor protein plays a crucial role in linking stimulated receptors to mitogen-activated protein kinase activation through the formation of dynamic signalling complexes. Shc comprises an N-terminal phosphotyrosine binding (PTB) domain, a C-terminal Src homology 2 (SH2) domain and a central proline-rich collagen homology 1 domain. The latter domain contains three tyrosine residues that are known to become phosphorylated. We have expressed and purified the human p52Shc isoform and characterised its binding to different ligands. CD spectra revealed that some parts of the Shc protein are not fully folded, remaining largely unaffected by the binding of ligands. The PTB domain binds peptide and Ins-1,4,5-P₃ (but not Ins-1,3,5-P₃) independently, suggesting two distinct sites of interaction. In the unphosphorylated Shc, the SH2 domain is non-functional. Ligand binding to the PTB domain does not affect this. However, phosphorylation of the three tyrosine residues promotes binding to the SH2 domain. Thus, Shc has an intrinsic phosphorylation-dependent gating mechanism where the SH2 domain adopts an open conformation only when tyrosine phosphorylation has occurred.     

Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies

Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleckstrin homology (PH) domain followed by Tec homology (TH) domain, Src homology 3 and 2 (SH3 and SH2) domains, and a C-terminal kinase domain. Mutations in the human BTK gene cause the severe immunodeficiency disease X-linked agammaglobulinemia (XLA). The structural and functional basis of several XLA-causing mutations remains unknown, since only the structures of the PH and SH3 domains of human Btk are currently available. In this study, we overexpressed and purified a protein consisting of the SH3 and SH2 domains of human Btk for biochemical and structural analysis. The purified protein was only partially soluble and had a tendency to dimerize, which made it unsuitable for further studies. To overcome the problems of low solubility and dimerization, subdomain interactions were engineered without altering the function of the protein.