Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.     

The phosphopeptide-binding specificity of Src family SH2 domains

BACKGROUND: Epidemiologic evidence suggests that exposure to UV radiation plays a significant role in the development of melanoma skin cancers. As early surgical removal of the melanoma is the only effective therapy, current strategies for reducing mortality from melanoma focus on prevention of the disease. Chemical sunscreens protect mice from development of skin cancers that resemble sunlight-induced human squamous cell cancers, but there appears to be a complex relationship between UV radiation exposure and development of melanoma. PURPOSE: We asked whether common sunscreens would protect mice against UV radiation-induced enhancement of melanoma incidence. METHODS: C3H mice were exposed to 4.8 kJ/m2 UVB from FS40 sunlamps twice a week for 3 weeks. Sunscreens containing 7.5% 2-ethylhexyl-p-methoxycinnamate, 8% octyl-N-dimethyl-p-aminobenzoate, 6% benzophenone-3, or the oil-in-water vehicle alone were applied to the ears and tails of the mice 20 minutes before irradiation. At various times during and after exposure, we determined UV radiation-induced inflammation by measuring ear swelling. We also examined the ears histologically for UV radiation-induced alterations. One day after the final irradiation, 2.5 x 10(4) syngeneic K1735 melanoma cells were injected into the external ears. Mice were examined weekly for tumor growth for 5-8 weeks after tumor cell injection. Control mice were treated in the identical way except for exposure to UV radiation. RESULTS: The incidence of melanomas was significantly higher in the UV-irradiated mice. All three sunscreens protected against UV radiation-induced ear swelling and clearly diminished histopathologic alterations, including sunburn cell formation, epidermal hyperplasia, and mononuclear cell infiltrate in the dermis. However, the sunscreens failed to protect against UV radiation-induced increase in melanoma incidence. The sunscreens or vehicle alone did not significantly alter tumor growth. CONCLUSIONS: Protection against sunburn does not necessarily imply protection against other possible UV radiation effects, such as enhanced melanoma growth. IMPLICATIONS: Sunscreen protection against UV radiation-induced inflammation may encourage prolonged exposure to UV radiation and thus may actually increase the risk of melanoma development. These findings suggest that further research on the ability of sunscreens to prevent melanoma is urgently needed.     

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.     

Potent inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands

To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.     

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.     

NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites

The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr.     

Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling

The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.     

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.     

Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5'-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc.SHIP complex and regulation of SHIP function during T cell receptor signaling.     

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

The ezrin-like family of tyrosine kinase substrates: receptor-specific pattern of tyrosine phosphorylation and relationship to malignant transformation

A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.     

The elusive family of imidazoline binding sites

Endopeptidase 24.11 (EP 24.11), a membrane-bound cell surface enzyme, modulates chemotactic responsiveness of neutrophils to f-Met-Leu-Phe. It is unknown if the enzyme degrades potent formylmethionyl tetrapeptides or if an enzyme with similar activities, endopeptidase 24.15 (EP 24.15), degrades formylated chemotactic peptides. In a study of five formylmethionyl tetrapeptides and f-Met-Leu-Phe, we found that EP 24.11 had high affinity for all peptides evaluated, although it did not effectively degrade f-Met-Ile-Leu-Phe. EP 24.15 had high affinity for three of the tetrapeptides, and for f-Met-Leu-Phe, although, for unclear reasons, it did not degrade f-Met-Ile-Leu-Phe or f-Met-Leu-Phe, the apparent natural products of Staphylococcus aureus and Escherichia coli, respectively.     

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.     

Solution structure of the SH3 domain from Bruton's tyrosine kinase

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.     

Cellular proteins binding to the first Src homology 3 (SH3) domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.     

Identification of serines-1035/1037 in the kinase domain of the insulin receptor as protein kinase C alpha mediated phosphorylation sites

A new site of serine phosphorylation (Ser-1035/1037) has been identified in the kinase domain of the insulin receptor. Mutant receptors missing these two serines were expressed in Chinese hamster ovary cells overexpressing protein kinase C alpha. These mutant receptors lacked a phorbol ester-stimulated phosphoserine containing tryptic peptide as demonstrated by both high percentage polyacrylamide/urea gel electrophoresis and two-dimensional tlc. Moreover, a synthetic peptide with the sequence of this tryptic peptide was phosphorylated by isolated protein kinase C alpha and co-migrated with the phosphopeptide from in vivo labeled receptor. These results indicate that serine-1035 and/or 1037 in the kinase domain of the insulin receptor are phosphorylated in response to activation of protein kinase C alpha.