The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors.

BACKGROUND: The adaptor protein Gads is a Grb2-related protein originally identified on the basis of its interaction with the tyrosine-phosphorylated form of the docking protein Shc. Gads protein expression is restricted to hematopoietic tissues and cell lines. Gads contains a Src homology 2 (SH2) domain, which has previously been shown to have a similar binding specificity to that of Grb2. Gads also possesses two SH3 domains, but these have a distinct binding specificity to those of Grb2, as Gads does not bind to known Grb2 SH3 domain targets. Here, we investigated whether Gads is involved in T-cell signaling. RESULTS: We found that Gads is highly expressed in T cells and that the SLP-76 adaptor protein is a major Gads-associated protein in vivo. The constitutive interaction between Gads and SLP-76 was mediated by the carboxy-terminal SH3 domain of Gads and a 20 amino-acid proline-rich region in SLP-76. Gads also coimmunoprecipitated the tyrosine-phosphorylated form of the linker for activated T cells (LAT) adaptor protein following cross-linking of the T-cell receptor; this interaction was mediated by the Gads SH2 domain. Overexpression of Gads and SLP-76 resulted in a synergistic augmentation of T-cell signaling, as measured by activation of nuclear factor of activated T cells (NFAT), and this cooperation required a functional Gads SH2 domain. CONCLUSIONS: These results demonstrate that Gads plays an important role in T-cell signaling via its association with SLP-76 and LAT. Gads may promote cross-talk between the LAT and SLP-76 signaling complexes, thereby coupling membrane-proximal events to downstream signaling pathways.

Neuralized functions as an E3 ubiquitin ligase during Drosophila development.

The Notch pathway is a widely studied means of intercellular signaling responsible for the determination of cell fate, cell differentiation, and boundary formation (reviewed in ). The main effectors of this pathway, Notch (N) and Delta (Dl), have been shown to function as a receptor and ligand, respectively. Genetic and phenotypic studies suggest that Neuralized (Neu), a RING finger protein, also plays a role within the N-Dl pathway, although its biochemical function is unknown. Here, we show that Neu is required at the plasma membrane for functional activity and that its RING finger domain acts as an E3 ubiquitin ligase. These data suggest that the role of Neu is to target components of the N-Dl pathway for ubiquitination, allowing for propagation and/or regulation of the signal.

Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate.

BACKGROUND: Drosophila numb was originally described as a mutation affecting binary divisions in the sensory organ precursor (SOP) lineage. The numb gene was subsequently shown to encode an asymmetrically localized protein which is required for binary cell-fate decisions during peripheral nervous system development. Part of the Drosophila NUMB protein exhibits homology to the SHC phosphotyrosine-binding (PTB) domain, suggesting a potential link to tyrosine-kinase signal transduction. RESULTS: A widely expressed mammalian homologue of Drosophila numb (dnumb) has been cloned from rat and is referred to here as mammalian Numb (mNumb). The mNUMB protein has a similar overall structure to dNUMB and 67 sequence similarity. Misexpression of mNumb in Drosophila during sensory nervous system precursor cell division causes identical cell fate transformations to those produced by ectopic dNUMB expression. In vitro, the mNUMB PTB domain binds phosphotyrosine-containing proteins, and SH3 domains of SRC-family tyrosine kinases bind to mNUMB presumably through interactions with proline-rich regions in the carboxyl terminus. Overexpression of full-length mNUMB in the multipotential neural crest stem cell line MONC-1 dramatically biases its differentiation towards neurons, whereas overexpression of the mNUMB PTB domain biases its differentiation away from neuronal fates. CONCLUSIONS: Our results demonstrate that mNUMB is an evolutionarily conserved functional homologue of dNUMB, and establish a link to tyrosine-kinase-mediated signal transduction pathways. Furthermore, our results suggest that mNUMB and dNUMB are new members of a family of signaling adapter molecules that mediate conserved cell-fate decisions during development.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.

Regulation of Numb isoform expression by activated ERK signaling.

The endocytic adaptor protein Numb has a major role in development as an intrinsic regulator of cell fate determination and inhibitor of the Notch signaling pathway. In vertebrates, four protein isoforms of Numb are produced through alternative splicing (AS) of two cassette exons (exons 3 and 9). AS of coding exon 9 (E9) produces E9-included (p72/p71) and -excluded (p66/p65) protein products. Expression of Numb isoforms is developmentally regulated and E9-included products are expressed in progenitors, whereas E9-excluded isoforms are dominantly expressed in differentiated cells. Analyses of AS events in multiple cancers previously identified a switch in Numb transcript and protein expression from the E9-excluded to the E9-included isoform, suggesting that misregulation of the mechanisms that control E9 inclusion may have a role in tumorigenesis. Here we identify splicing factors ASF/SF2 and PTBP1 as regulators of E9 splicing and show that activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway promotes E9 inclusion in cancer cells. Our evidence supports a mechanism by which Numb AS is regulated in response to oncogenic signaling pathways, and contributes to activation of downstream pathways to promote tumorigenesis.

Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc.

Shc proteins are important substrates of receptor and cytoplasmic tyrosine kinases that couple activated receptors to downstream signaling enzymes. Phosphorylation of Shc tyrosine residues 239 and 317 leads to recruitment of the Grb2-Sos complex, thus linking Shc phosphorylation to Ras activation. We have used phosphorylated peptides corresponding to the regions spanning tyrosine 239/240 and 317 of Shc in an expression library screen to identify additional downstream targets of Shc. Here we report the identification of Gads, a novel adaptor protein most similar to Grb2 and Grap that contains amino and carboxy terminal SH3 domains flanking a central SH2 domain and a 120 amino acid unique region. Gads is most highly expressed in the thymus and spleen of adult animals and in human leukemic cell lines. The binding specificity of the Gads SH2 domain is similar to Grb2 and mediates the interaction of Gads with Shc, Bcr-Abl and c-kit. Gads does not interact with Sos, Cbl or Sam68, although the isolated carboxy terminal Gads SH3 domain is able to bind these molecules in vitro. Our results suggest that the unique structure of Gads regulates its interaction with downstream SH3 domain-binding proteins and that Gads may function to couple tyrosine-phosphorylated proteins such as Shc, Bcr-Abl and activated receptor tyrosine kinases to downstream effectors distinct from Sos and Ras.

Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells.

Shc adaptor proteins play a role in linking activated cell surface receptors to the Ras signaling pathway in response to receptor mediated tyrosine kinase activation. While the function of Shc in the activation of the Ras pathway via binding to Grb2 has been well characterized, it is becoming increasingly apparent that Shc participates in additional signaling pathways through interactions with other cytoplasmic proteins. Using the yeast two-hybrid system, we have identified a unique Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes) with no similarity to other known proteins. mPAL binds specifically to the Shc SH2 domain and unlike previously described Shc SH2 domain-protein interactions, the association of mPAL and Shc is phosphotyrosine-independent. Both mPAL RNA and protein expression are restricted to tissues containing actively dividing cells and proliferating cells in culture. mPAL expression is induced upon growth factor stimulation and is down-regulated upon growth inhibition. This pattern, and timing of mPAL expression and its association with the Shc adaptor molecule suggests a role for this protein in signaling pathways governing cell cycle progression.

The role of Gads in hematopoietic cell signalling.

Gads is a member of the family of SH2 and SH3 domain containing adaptor proteins that is expressed specifically in hematopoietic cells and functions in the coordination of tyrosine kinase mediated signal transduction. Gads plays a critical role in signalling from the T cell receptor by promoting the formation of a complex between SLP-76 and LAT. This complex couples the T cell receptor to Ras through a novel pathway involving PLC-gamma1, Tec family kinases, and RasGRP. Studies with Gads-deficient mice have highlighted its importance for thymocyte proliferation during T cell maturation. Emerging evidence suggests that Gads may also play additional roles in antigen-receptor signalling and receptor tyrosine kinase mediated signalling in other hematopoietic lineages. Gads is a unique member of the Grb2 adaptor family, because its activity can be regulated by caspase cleavage. Gads nucleates multi-protein complexes that are required for tyrosine kinase-dependent signalling in immune cells and may also represent a point of modulation for these pathways through the activation of caspase-dependent signalling events.

Caspase-dependent cleavage of the hematopoietic specific adaptor protein Gads alters signalling from the T cell receptor.

Gads is a SH2 and SH3 domain-containing, hematopoietic-specific adaptor protein that functions in signalling from the T cell receptor. Gads acts by linking SLP-76, bound by the carboxy-terminal Gads SH3 domain, to tyrosine phosphorylated LAT which contains binding sites for the Gads SH2 domain. Gads is distinguished from Grb2 and the closely related Grap protein by the presence of a 120 amino acid unique region between the SH2 domain and the carboxy terminal SH3 domain. Here we demonstrate that the unique region of Gads contains a capase cleavage site. Induction of apoptosis in lymphocytes results in detectable Gads cleavage by 60 min. Gads cleavage is blocked in vivo by treating cells with a caspase 3 inhibitor. A putative caspase 3 cleavage site was identified within the unique region and mutation of this site prevented Gads cleavage in vitro, and in vivo. The Gads cleavage products retained the predicted binding specificity for SLP-76 and LAT. Expression of the Gads cleavage products in Jurkat T cells inhibited NFAT activation following TCR cross linking. These findings indicate that cleavage of Gads in vivo could function to alter signalling downstream of the T cell receptor by disrupting cross talk between SLP-76 and LAT.

HACS1 encodes a novel SH3-SAM adaptor protein differentially expressed in normal and malignant hematopoietic cells.

SH3 and SAM domains are protein interaction motifs that are predominantly seen in signaling molecules, adaptors, and scaffold proteins. We have identified a novel family of putative adaptor genes that includes HACS1. HACS1 encodes a 441 amino acid protein that is differentially expressed in hematopoietic cells and has restricted expression in human tissues. Its SH3 domain is most similar to the same motif in Crk and its SAM domain shares homology with a family of uncharacterized putative scaffold and adaptor proteins. HACS1 maps to human chromosome 21q11.2 in a region that is frequently disrupted by translocation events in hematopoietic malignancies. Polyclonal antibodies against HACS1 recognized a 49.5 kDa protein whose mRNA is expressed in human immune tissues, bone marrow, heart, lung, placenta and brain. Cell lines and primary cells from acute myeloid leukemias and multiple myeloma patients express HACS1. Immunostaining and cellular fractionation studies localized the HACS1 protein predominantly to the cytoplasm.

Gads (Grb2-related adaptor downstream of Shc) is required for BCR-ABL-mediated lymphoid leukemia.

Philadelphia chromosome-positive leukemias, including chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (B-ALL), are driven by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice receiving whole bone marrow transduced with BCR-ABL. Y177 of BCR-ABL is critical to the development of myeloid disease, and phosphorylation of Y177 has been shown to induce GRB2 binding to BCR-ABL, followed by activation of the Ras and phosphoinositide 3 kinase signaling pathways. We show that the GRB2-related adapter protein, GADS, also associates with BCR-ABL, specifically through Y177 and demonstrate that BCR-ABL-driven lymphoid disease requires Gads. BCR-ABL transduction of Gads(-/-) bone marrow results in short latency myeloid disease within 3-4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates.

Cloning and chromosomal localization of a new member of the bHLH/PAS transcription factor family.

Here we report the cloning and primary characterization of TIC, a new member of the bHLH/PAS domain family of transcription factors. Northern blot analysis indicates that the 3.3-kb Tic mRNA is widely expressed. A polyclonal antibody against TIC identifies multiple protein species in most cell lines and tissues tested, suggesting that alternative splicing may result in the production of protein isoforms. Interspecies backcross and FISH mapping have been used to localize the Tic gene to mouse Chromosome (Chr) 7 F2-F3, given the locus name Arntl. FISH mapping was also used to localize the human gene to Chr 11p15.

Mapping of a phosphorylation site in the 176R (19 kDa) early region 1B protein of human adenovirus type 5.

The 176-residue (176R) early region 1B (E1B) protein of human adenovirus type 5 (Ad5) was shown to be phosphorylated at serine in lytically infected KB cells at a level estimated to be about one phosphate group per 28 176R molecules. Through the analysis of peptides generated by cleavage with cyanogen bromide and Staphylococcus aureus V-8 protease the phosphorylation site was mapped to Ser-164. Using site-directed mutagenesis, a mutant was produced in which the codon for Ser-164 was changed to that of asparagine while leaving the coding sequence for the overlapping 496R protein unchanged. This virus, which replicated well on human KB cells, produced normal levels of 176R, but in an unphosphorylated form. The mutant transformed baby rat kidney cells in cooperation with E1A at an efficiency about one-half that obtained with wt E1B. These data therefore gave little indication that phosphorylation is essential for the function of 176R.

Individual adenovirus E1B proteins induce transformation independently but by additive pathways.

The specific contributions of human adenovirus type 5 early region 1B (E1B) proteins were examined using mutants which synthesize these products individually. In cooperation with E1A, transformation of primary baby rat kidney cells was achieved with either the 176R protein or 496R protein alone, albeit at an efficiency considerably less than that observed when both were present. These results indicate that transformation mediated by either E1B product can proceed independently, but that the processes involved are additive.

Phosphotyrosine-independent binding of SHC to the NPLH sequence of murine protein-tyrosine phosphatase-PEST. Evidence for extended phosphotyrosine binding/phosphotyrosine interaction domain recognition specificity.

The phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domain of the proto-oncoprotein p52SHC binds to an NPXpY consensus sequence found in several growth factor receptors (Kavanaugh, W. M., Turck, C. W., and Williams, L. T. (1994) Science 268, 1177-1179). The amino-terminal region of p52SHC, which includes the PTB/PI domain, has been previously shown to associate with protein-tyrosine phosphatase-PEST (PTP-PEST) in vivo (Habib, T. , Herrera, R., and Decker, S. J. (1994) J. Biol. Chem. 269, 25243-25246). We report here the detailed mapping of this interaction in a murine context using glutathione S-transferase fusion protein binding studies and peptide competition assays. We show that the interaction between murine SHC and murine PTP-PEST is mediated through the PTB/PI domain of murine SHC and an NPLH sequence found in the carboxyl terminus of murine PTP-PEST. Since this interaction is not dependent on the presence of a tyrosine-phosphorylated residue in the target sequence, this reveals that the PTB/PI domain of SHC can recognize both tyrosine-phosphorylated sequences and non-tyrosine-based recognition motifs.

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.

Identification of the phosphorylation sites in early region 1A proteins of adenovirus type 5 by amino acid sequencing of peptide fragments.

We have mapped the positions of three of the phosphorylation sites on the 289 and 243 residue (289R and 243R) early region 1A (E1A) proteins of human adenovirus type 5 (Ad5). These proteins, which play roles in both transcriptional control and oncogenic transformation, have identical sequences except for the presence in 289R of 46 additional internal amino acids. Phosphorylation was detected exclusively at serine residues. E1A proteins purified from [35S]methionine- or [32P]orthophosphate-labeled Ad5-infected cells were digested with trypsin, and two phosphopeptides were isolated by reverse-phase chromatography and subjected to automated Edman degradation. The major species was shown to contain a single phosphorylation site at Ser-219. The second phosphopeptide was shown to contain at least one phosphorylation site at Ser-231. A third phosphorylated tryptic peptide could not be eluted from the column but was isolated using an E1A-specific rat monoclonal antibody. Following subcleavage by Staphylococcus aureus V-8 protease, this peptide was shown to contain at least one phosphorylation site at Ser-89. The present data indicate that both the 289R and 243R E1A proteins are phosphorylated at the same sites, at least one in the amino terminal half of the molecule, and at least two toward the carboxyl terminus.

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.

The adaptor protein Gads (Grb2-related adaptor downstream of Shc) is implicated in coupling hemopoietic progenitor kinase-1 to the activated TCR.

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.

The mammalian numb phosphotyrosine-binding domain. Characterization of binding specificity and identification of a novel PDZ domain-containing numb binding protein, LNX.

Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during development. A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for Numb function. Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain. Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and 70 kDa (LNX-b). LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains. The unique amino-terminal region of LNX includes a RING finger domain. The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY, found in both protein isoforms. Mutational analysis of LNX and peptide competition experiments showed that phosphorylation of the tyrosine residue within this motif was not required for binding to the Numb PTB domain. Finally, we also provide evidence that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent binding of other PTB domain-containing proteins such as SHC. We speculate that LNX may be important for clustering PTB-containing proteins with functionally related transmembrane proteins in specific membrane compartments.