Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells. Engagement of the T cell antigen receptor (TCR)1 results in the activation of protein tyrosine kinases (PTK) and the subsequent tyrosine phosphorylation of numerous proteins in T cells (1). Our efforts to characterize substrates of the TCRinduced PTK activity led to the cloning of SLP-76, a tyrosinephosphorylated hematopoietic cell-specific protein that associates with the SH3 domains of Grb2 (2, 3). A possible function of SLP-76 in T cells was suggested by experiments showing that overexpression of SLP-76 augments TCR-mediated signals that lead to the induction of IL-2 gene promoter activity (4, 5). We have shown that the activity of SLP-76 requires engagement of the TCR and that overexpression of SLP-76 results in increased activation of the mitogen-activated protein kinase cascade following TCR ligation.2 Interestingly, three distinct regions of SLP-76 that are responsible for protein-protein interactions in T cells are required for its ability to augment IL-2 promoter activity when overexpressed (6, 7).2 These data suggest that SLP-76 functions as a link between proteins that regulate signals generated by TCR ligation.To investigate the function of SLP-76 in T cells further, we and others have begun to characterize SLP-76-associated proteins that may participate with SLP-76 in transducing signals from the TCR to the nucleus. These proteins include Vav, which associates with the amino-terminal acidic region of SLP-76 in a phosphotyrosine-dependent manner (5,8,9); the adapter protein Grb2, which interacts with a proline-rich motif of SLP-76 via its SH3 domains (3, 4); and two unidentified tyrosine-phosphorylated proteins of 64 and 130 kDa and a serine/threonine kinase, all of which associate with the carboxyl-terminal SH2 domain of SLP-76 (4). In this study, we report the cloning of the cDNA encoding a 130-kDa protein (SLAP-130 for SLP-76 associated phosphoprotein of 130 kDa) that as...

Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

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The mechanism by which the products of the v-rel oncogene, the corresponding c-rel proto-oncogene, and the related dorsal gene of Drosophila melanogaster exert their effects is not clear. Here we show that the v-rel, chicken c-rel, and dorsal proteins activated gene expression when fused to LexA sequences and bound to DNA upstream of target genes in Saccharomyces cerevisiae. We have defined two distinct activation regions in the c-rel protein. Region I, located in the amino-terminal half of rel and dorsal proteins, contains no stretches of glutamines, prolines, or acidic amino acids and therefore may be a novel activation domain. Lesions in the v-rel protein that diminished or abolished oncogenic transformation of avian spleen cells correspondingly affected transcription activation by region I. Region II, located in the carboxy terminus of the c-rel protein, is highly acidic. Region II is not present in the v-rel protein or in a transforming mutant derivative of the c-rel protein.Our results show that the oncogenicity of Rel proteins requires activation region I and suggest that the biological function of rel and dorsal proteins depends on transcription activation by this region.

The p105 precursor protein of NF-B1 acts as an NF-B inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor ␣ (TNF␣) and interleukin-1␣ (IL-1␣) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the IB kinase (IKK) complex is essential for these pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (AspSer 927 -Gly-Val-Glu-Thr), related to the IKK target sequence in IB␣, which is conserved between human, mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants in which serine/threonine residues within and adjacent to this motif were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression. This residue is also required for TNF␣ and IL-1␣ to stimulate p105 degradation. By using a specific anti-phosphopeptide antibody, it was confirmed that IKK2 overexpression induces serine 927 phosphorylation of co-transfected p105 and that endogenous p105 is also rapidly phosphorylated on this residue after TNF␣ or IL-1␣ stimulation. In vitro kinase assays with purified proteins demonstrated that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-B1 p105 by direct phosphorylation of serine 927 in its PEST domain.

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr -Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr -Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by BcrAbl. To investigate the contribution of Src kinases to BcrAbl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph + CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph 7 leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph + cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr -Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr -Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor ␣ (TGF-␣) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRPinduced proliferation or invasion of HNSCC cells. In addition to TGF-␣ release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-␣ and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-␣ and amphiregulin in HNSCC.

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.