Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.     

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.     

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.     

Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs

In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.     

Mutation of the Lck-Binding Motif of Tip Enhances Lymphoid Cell Activation by Herpesvirus Saimiri

The proline-rich SH3-binding (SH3B) motif of the tyrosine kinase-interacting protein (Tip) of herpesvirus saimiri (HVS) is required for binding to the cellular Src family kinase Lck. We constructed a mutant form of HVS in which prolines in the SH3B motif of Tip were altered to alanines. This mutant form of Tip was incapable of binding to Lck. The mutant virus, HVS/Tip mSH3B, retained its ability to immortalize common marmoset lymphocytes in culture. In fact, common marmoset lymphocytes immortalized by the HVS/Tip mSH3B mutant displayed increased expression of HLA-DR lymphocyte activation marker, an altered pattern of tyrosine phosphorylation, increased expression of the tyrosine kinase Lyn, and a shift in electrophoretic mobility of Lck compared to cells immortalized by wild-type HVS. Experimental infection of common marmosets resulted in fulminant lymphoma with both HVS/Tip mSH3B and wild-type HVS. However, HVS/Tip mSH3B produced greater infiltration of affected organs by proliferating lymphoid cells compared to wild-type HVS. These results demonstrate that Tip binding to Lck is not necessary for transformation and that abrogation of Tip binding to Lck alters the characteristics of transformed cells and the severity of the pathologic lesions.     

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.     

The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.     

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.     

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.     

Structural characterization of Lyn-SH3 domain in complex with a herpesviral protein reveals an extended recognition motif that enhances binding affinity

The Src homology 3 (SH3) domain of the Src family kinase Lyn binds to the herpesviral tyrosine kinase interacting protein (Tip) more than one order of magnitude stronger than other closely related members of the Src family. In order to identify the molecular basis for high-affinity binding, the structure of free and Tip-bound Lyn-SH3 was determined by NMR spectroscopy. Tip forms additional contacts outside its classical proline-rich recognition motif and, in particular, a strictly conserved leucine (L186) of the C-terminally adjacent sequence stretch packs into a hydrophobic pocket on the Lyn surface. Although the existence of this pocket is no unique property of Lyn-SH3, Lyn is the only Src family kinase that contains an additional aromatic residue (H41) in the n-Src loop as part of this pocket. H41 covers L186 of Tip by forming tight hydrophobic contacts, and model calculations suggest that the increase in binding affinity compared with other SH3 domains can mainly be attributed to these additional interactions. These findings indicate that this pocket can mediate specificity even between otherwise closely related SH3 domains.     

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.     

Binding, domain orientation, and dynamics of the Lck SH3-SH2 domain pair and comparison with other Src-family kinases

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.     

Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.     

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.     

Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.     

Lck couples Shc to TCR signaling

Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

Regulation and function of SKAP-55 non-canonical motif binding to the SH3c domain of adhesion and degranulation-promoting adaptor protein

The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.     

LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, directly associates with the SH3 domain of protein tyrosine kinase p56lck.

The Lck tyrosine kinase molecule plays an essential role in T cell activation and T cell development. Using the expression cloning technique, we have isolated a gene that encodes a molecule, LckBP1, able to associate with murine Lck. Analysis of full-length LckBP1 cDNA indicates at least four potentially important segments: a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif; a proline-rich region; a proline-glutamate repeat; and an SH3 domain. These four regions are very similar to the human haematopoietic-specific protein 1 (HS1). Deletion mutant analysis of LckBP1 revealed two proline-rich regions that permit association with Lck SH3. One region contains prolines conserved among HS1 and cortactin, and the other region contains a potential MAP kinase recognition site. In vivo association between Lck and LckBP1 was confirmed by immunoprecipitation of lysates from a pre-T cell line and adult thymocytes using antibodies specific for Lck and LckBP1. LckBP1 is tyrosine phosphorylated after T-cell receptor stimulation. The SH3 domain and the potential helix-turn-helix motif in LckBP1 suggest that this molecule may associate with various molecules and function as a DNA binding molecule. The data also suggest that LckBP1 mediates intracellular signalling through Lck in T cells.     

Phenotypic effects of CD3zeta targeting into glycosphingolipid-enriched membrane microdomains (GEMs) of T cells

In the present study we tested whether the forced expression of the CD3zeta chain within detergent-resistant, glycosphingolipid-enriched membrane microdomains (GEMs) will result in a constitutively activated phenotype in human T cells. To this aim, a monomeric recombinant protein (LckSH4-CD3zeta), containing the intracellular part of human CD3zeta chain fused to N-terminal double-acylation motif (SH4 domain) of protein tyrosine kinase Lck, was expressed in Jurkat human T lymphoid cell line and its Lck-negative mutant, J. CaM1.6. The Lck SH4 domain indeed predominantly targeted the chimeric protein into GEMs. In transfectants derived from wild-type Jurkat cells, but not in those derived from the Lck-deficient mutant, the LckSH4-CD3zeta protein was constitutively tyrosine-phosphorylated. Tyrosine phosphorylation of a major Jurkat cell phosphoprotein (pp85) was diminished in the transfectants. However, the transfectants did not exhibit any features of constitutively activated T cells, and their responses to anti-CD3 treatment were very similar to the wild-type Jurkat cells. Thus, the constitutive expression of this form of CD3zeta chain in GEMs is not sufficient for eliciting an activated state in the Jurkat cells.