Modulation of Lck function through multisite docking to T cell-specific adapter protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Genetic analysis of sorting nexins 1 and 2 reveals a redundant and essential function in mice

Sorting nexins 1 (Snx1) and 2 (Snx2) are homologues of the yeast gene VPS5 that is required for proper endosome-to-Golgi trafficking. The prevailing thought is that Vps5p is a component of a retrograde trafficking complex called the retromer. Genetic and biochemical evidence suggest mammals may have similar complexes, but their biological role is unknown. Furthermore, if SNX1 and SNX2 belong to such complexes, it is not known whether they act together or separately. Herein, we show that mice lacking SNX1 or SNX2 are viable and fertile, whereas embryos deficient in both proteins arrest at midgestation. These results demonstrate that SNX1 and SNX2 have a highly redundant and necessary function in the mouse. The phenotype of Snx1(-/-);Snx2(-/-) embryos is very similar to that of embryos lacking another retromer homologue, Hbeta58. This finding suggests that SNX1/SNX2 and Hbeta58 function in the same genetic pathway, providing additional evidence for the existence of mammalian complexes that are structurally similar to the yeast retromer. Furthermore, the viability of Snx1(-/-) and Snx2(-/-) mice demonstrates that it is not necessary for SNX1 and SNX2 to act together. Electron microscopy indicates morphological alterations of apical intracellular compartments in the Snx1(-/-);Snx2(-/-) yolk-sac visceral endoderm, suggesting SNX1 and SNX2 may be required for proper cellular trafficking. However, tetraploid aggregation experiments suggest that yolk sac defects cannot fully account for Snx1(-/-); Snx2(-/-) embryonic lethality. Furthermore, endocytosis of transferrin and low-density lipoprotein is unaffected in mutant primary embryonic fibroblasts, indicating that SNX1 and SNX2 are not essential for endocytosis in all cells. Although the two proteins demonstrate functional redundancy, Snx1(+/-);Snx2(-/-) mice display abnormalities not observed in Snx1(-/-);Snx2(+/-) mice, revealing that SNX1 and SNX2, or their genetic regulation, are not equivalent. Significantly, these studies represent the first mutations in the mammalian sorting nexin gene family and indicate that sorting nexins perform essential functions in mammals.     

Characterization of bacteriophage T4 and D RNA, a low-molecular-weight RNA of unknown function

The nucleotide sequence of T4 band D RNA, a stable RNA species encoded by bacteriophage T4, has been deduced from analysis of the ³²P-labeled RNA and comparison with the DNA sequence of the T4 genome in the region encoding the RNA. The sequence is: pA-U-G-A-G-A-A-A-C-C-G-G-G-U-C-G-C-U-A-C-C-G-G-U-A-A-G-U-C-G-U-C-G-G-A-C-U-G-A-U-G-G-U-U-C-C-C-U-G-A-G-U-A-A-G-G-A-A-U-U-G-C-G-U-U-A-A-U-A-A -U-C-U-U-U-G-C-G-U-U-U-A-U-U-G-A-U-G-C-C-C-U-C-U-U-A-C-A-U-C-A-C-A-G-C-A-G-A-A-A-C-G-G-C-G-C-A-C-C-A_OH. Band D RNA is 120 nucleotides long, and contains no modified nucleotides. The sequence can be arranged in a secondary structure consistent with the results of limited digestion with nuclease S1, but shows no striking similarities to tRNAs. While a biological function for band D RNA is unknown, similar molecules are encoded by bacteriophages T2 and T6, indicating that the molecule has been preserved during evolution. This retention may reflect a significant function for the RNA.     

The related adaptors, adaptor in lymphocytes of unknown function X and Rlk/Itk-binding protein, have nonredundant functions in lymphocytes

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.     

Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.     

Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.     

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.     

SETA is a multifunctional adapter protein with three SH3 domains that binds Grb2, Cbl, and the novel SB1 proteins

Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.     

AP-4, a novel protein complex related to clathrin adaptors

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.     

IL-2 receptor signaling through the Shb adapter protein in T and NK cells

We have investigated the effect of hypoxia on the excitatory synaptic transmission in the substantia gelatinosa neurons using perforated-patch-clamp configuration. Brief periods of hypoxia induced a depression in the evoked excitatory postsynaptic current (eEPSC) amplitude. The hypoxia-induced depression of eEPSC was not observed in the presence of theophylline, a nonselective adenosine receptor antagonist, and DPCPX, a selective adenosine receptor A1 antagonist. Application of adenosine (100 microM) also depressed eEPSC in a similar way as with hypoxia. This adenosine-induced depression of eEPSC was inhibited by DPCPX. Hypoxia and exogenous adenosine decreased the frequency of the spontaneous excitatory postsynaptic current (sEPSC) but not the amplitude of sEPSC and increased the paired-pulse ratio. From these results, it is suggested that acute hypoxia depresses the excitatory synaptic transmission by activating the presynaptic adenosine A1 receptor.     

Genetic analysis of structure and function in phage T4 tRNASer

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.     

Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.     

Lad, an adapter protein interacting with the SH2 domain of p56lck, is required for T cell activation.

T cell-specific Src family tyrosine kinase, p56lck, plays crucial roles in T cell differentiation, activation, and proliferation. These multiple functions of p56lck are believed to be conducted through the protein-protein interactions with various cellular signaling proteins. To clarify the mechanisms through which p56lck contributes to T cell signaling, we identified the proteins binding to the Src homology 2 (SH2) domain of p56lck through a tyrosine phosphorylation-dependent yeast two-hybrid screening. Subsequent characterization of positive clones revealed the presence of a protein of 366 aa named Lad (Lck-associated adapter protein), which is a potential murine homologue of previously reported TSAd, a T cell-specific adapter protein. Lad contains several protein-protein interaction domains including a zinc-finger motif, an SH2 domain, a proline-rich SH3 binding motif, and several phosphotyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56lck in vivo and redistributed from cytoplasm to the plasma membrane in a T cell activation-dependent manner. Moreover in T cells, IL-2 promoter activity was enhanced upon coexpression of Lad but was inhibited by the coexpression of antisense Lad RNA. These characteristics of Lad suggest that Lad play an essential role as an adapter protein in p56lck-mediated T cell signaling.     

DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells

Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells.     

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.