Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart.

Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.

Recombinants within the tyrosine kinase region of v-abl and v-src identify a v-abl segment that confers lymphoid specificity.

The v-abl and v-src oncogenes encode protein-tyrosine kinases that possess different biological properties in spite of their high degree of amino acid conservation. To correlate functional differences with structural domains of the two oncogenes, we recombined v-abl and v-src just downstream of the lysines in their ATP-binding sites, within the kinase domain. The biological activity of the chimeric genes was studied and compared with that of v-src and v-abl. The v-src/v-abl recombinant shared with v-src and v-abl the ability to transform fibroblasts. In addition, like v-abl, it transformed lymphoid cells and relieved a hematopoietic cell line of its interleukin 3 requirement. In contrast, the reciprocal construct, v-abl/v-src, was transformation defective. Lack of biological activity correlated with formation of a stable complex between the chimeric protein and two cellular proteins and with low kinase activity. We conclude that the specificity within the kinase domain determines the particular biological behavior of protein-tyrosine kinase oncogenes.

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.

Abelson virus abrogation of interleukin-3 dependence in a lymphoid cell line.

Among several tyrosine-protein kinases, only v-abl could abrogate interleukin 3 dependence of a lymphoblastoid cell line; v-src and v-fps proteins gave partial or no interleukin 3 independence, respectively. Lymphokine independence was achieved via a nonautocrine mechanism. Direct involvement of c-myc in this process was not evident.

The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor.

Cytokines regulate cell growth and differentiation by inducing the expression of specific target genes. We have recently isolated a cytokine-inducible, immediate-early cDNA, DUB-1, that encodes a deubiquitinating enzyme. The DUB-1 mRNA was specifically induced by the receptors for interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5, suggesting a role for the beta common (betac subunit known to be shared by these receptors. In order to identify the mechanism of cytokine induction, we isolated a murine genomic clone for DUB-1 containing a functional promoter region. The DUB-1 gene contains two exons, and the nucleotide sequence of its coding region is identical to the sequence of DUB-1 cDNA. Various regions of the 5' flanking region of the DUB-1 gene were assayed for cytokine-inducible activity. An enhancer region that retains the beta c-specific inducible activity of the DUB-1 gene was identified. Enhancer activity was localized to a 112-bp fragment located 1.4 kb upstream from the ATG start codon. Gel mobility shift assays revealed two specific protein complexes that bound to this minimal enhancer region. One complex was induced by betac signaling, while the other was noninducible. Finally, the membrane-proximal region of human betac was required for DUB-1 induction. In conclusion, DUB-1 is the first example of an immediate-early gene that is induced by a specific subunit of a cytokine receptor. Further analysis of the DUB-1 enhancer element may reveal specific determinants of a betac-specific signaling pathway.

JAK2 is required for induction of the murine DUB-1 gene.

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.

The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.

Preneoplastic lesions induced by myc and src oncogenes in a heterotopic rat colon.

With the use of viral vectors harboring myc and src oncogenes, we have assessed the potential contribution of these different elements to colonic neoplasia using a transplantation technique resulting in the formation of a heterotopic colon in Wistar Furth rats. While myc alone induced atypia and some dysplasia, src induced focal dysplastic lesions throughout the colon mucosa with evidence of metaplasia. In contrast, lesions induced by myc and src acting cooperatively, were highly dysplastic with evidence of tumor formation after protracted periods. These results indicated the formation of histologically distinct preneoplastic lesions elicited by the action of a single oncogene in colon implants with the production of adenocarcinomas when such oncogenic elements act cooperatively. This model provides an opportunity for studies of the action of different oncogenes, acting singly or in combination, in the multi-step progression to colon tumor formation.

Purification and chemical characterization of melittin and acetylated derivatives.

Melittin, the main basic and hydrophobic peptide of bee venom, displays marked detergent-like properties. At high peptide concentration, and depending on salt and pH, it forms a tetramer. This is prevented by using urea. A purification procedure in presence of 4.0 M urea was developed to prepare melittin in its monomeric form, free of other venom constituents such as N alpha-formyl melittin, degradation products of peptides and phospholipase A2. NH2-residues on the melittin molecule were modified by reaction with acetic anhydride to alter the asymmetrical charge distribution supposed to confer detergent-like properties to the molecule. This gave rise to di- and mono acetyl derivatives which could be used, once isolated, to study further the melittin structure-activity relationship.

Drosophila genome-wide RNAi screens: are they delivering the promise?

The emergence of RNA interference (RNAi) on the heels of the successful completion of the Drosophila genome project was seen by many as the ace in functional genomics: Its application would quickly assign a function to all genes in this organism and help delineate the complex web of interactions or networks linking them at the systemic level. A few years wiser and a number of genome-wide Drosophila RNAi screens later, we reflect on the state of high-throughput RNAi screens in Drosophila and ask whether the initial promise was fulfilled. We review the impact that this approach has had in the field of Drosophila research and chart out strategies to extract maximal benefit from the application of RNAi to gene discovery and pursuit of systems biology.

Specific transforming potential of oncogenes encoding protein-tyrosine kinases.

Several chimeric murine retroviruses were constructed to test whether the gag sequence of Abelson murine leukemia virus (A-MuLV) could influence the in vitro specificity of two sarcoma-inducing oncogenes: src of Rous sarcoma virus and fps of Fujinami sarcoma virus. Although the src- or fps- containing chimerae could transform fibroblasts, they were unable to mimic the action of A-MuLV in causing lymphoid transformation in vitro. A-MuLV-derived gag sequences could, however, functionally replace the 5' end of src and restore the transformation potential of a 5'-truncated src gene. To investigate this functional similarity, we replaced the gag sequence of an A-MuLV virus with the 5' end of src. This recombinant virus behaved like the A-MuLV virus from which it was derived: it transformed both fibroblasts and lymphoid cells in vitro. Taken together, these results suggest that lymphoid transformation in vitro is a specific property of abl and not of src or fps. Furthermore, it shows that a functional homology exists between the gag sequence of A-MuLV and the 5' end of src.

Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity.

One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery.

Interleukin-3-induced activation of the JAK/STAT pathway is prolonged by proteasome inhibitors.

One facet of cytokine receptor signaling involves the activation of signal transducers and activators of transcription (STATs). STATs are rapidly activated via tyrosine phosphorylation by Janus kinase (JAK) family members and subsequently inactivated within a short period. We investigated the effect of proteasome inhibition on interleukin-3 (IL-3) activation of the JAK/STAT pathway following stimulation of Ba/F3 cells. Treatment of Ba/F3 cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-3 receptor, beta common (betac), and STAT5 following stimulation. The effects of LLnL were not restricted to the JAK/STAT pathway, as Shc and mitogen-activated protein kinase (MAPK) phosphorylation were also prolonged in LLnL-treated cells. Further investigation showed these stable phosphorylation events were the result of prolonged activation of JAK2 and JAK1. These observations were confirmed using pharmacologic inhibitors. In the presence of LLnL, stable phosphorylation of STAT5 and betac was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on STAT5 phosphorylation could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting phosphorylated STAT5 could be stabilized by phosphatase, but not by proteasome inhibition per se. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the JAK/STAT pathway by regulating the deactivation of JAK.

Rapid selection of tetracycline-controlled inducible cell lines using a green fluorescent-transactivator fusion protein.

We describe a modification of the tetracycline-controlled expression system that facilitates the rapid identification of tetracycline-sensitive clones. The TetR/VP16 transactivator protein was tagged with the green fluorescent protein (GFP) at its N-terminus. This results in a functional transactivator which allows cells expressing high levels of the modified transactivator to be selected by fluorescent-activated cell sorting. After expansion, single cell clones that maintain a high level of GFP fluorescence can be tested for their ability to transactivate a luciferase gene under control of the Tet operator, leading to the rapid identification of clones with strong inducibility.

A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein.

We obtained a regressing-tumor antiserum specific for the unique sequence of the transforming protein P140 of Fujinami sarcoma virus by injecting Fischer rats with syngeneic embryo cells transformed with Fujinami sarcoma virus. This serum is capable of immunoprecipitating a protein of 98,000 daltons from cell extracts of normal, uninfected chicken bone marrow cells. This normal cellular protein (NCP98) was shown to be structurally related to P140, sharing the majority of 35S-methionine-labeled tryptic peptides with the viral gene product P140. NCP98 is a phosphoprotein in vivo, with an associated in vitro protein kinase activity, capable of phosphorylating specifically at tyrosine residues of NCP98 itself and alpha-casein, an externally added substrate. This kinase activity is biochemically indistinguishable from the kinase activity associated with P140 by all criteria tested. Moreover, in vitro-phosphorylated NCP98 and P140 shared the same phosphopeptides. The expression of NCP98 is tissue-specific. It is readily detectable in bone marrow cells and detectable to a lesser extent in liver and lung cells from 6--18 day old chickens.

Revertants and partial transformants of rat fibroblasts infected with Fujinami sarcoma virus.

Fifteen revertants were isolated from three independent clones of rat fibroblasts transformed by Fujinami sarcoma virus (FSV). Three revertant clones resulted from the deletion of the one copy of the FSV provirus, and one encoded an enzymatically inactive, transformation-defective protein. The remaining revertant clones were characterized by a transcriptional block of the provirus. Digestion of chromosomal DNA with MspI and HpaII revealed that the FSV provirus was hypermethylated in these revertants, whereas proviral DNA of their spontaneous retransformants was hypomethylated. Furthermore, the revertants had lost DNase I-hypersensitive sites in and around the FSV provirus. The effect of transcriptional regulation of the FSV provirus was further analyzed in clones showing various degrees of phenotypic transformation. We quantitated v-fps mRNA levels in these cells by liquid hybridization and found that increasing levels of viral RNA correlated with a more pronounced transformed phenotype. These results suggest that transcription of FSV proviral DNA is under both viral and cellular control and that transformation by FSV is a function of the dosage of v-fps mRNA.

Differentiation domains of the erythropoietin receptor.

Ectopic expression of the erythropoietin receptor (Epo-R) in Ba/F3, an interleukin-3 (IL-3)-dependent progenitor cell line, confers both Epo-dependent cell growth and Epo-dependent induction of beta-globin mRNA. We have used this system of limited erythroid differentiation to characterize the role of the Epo-R in differentiation. In particular, we have been interested in identifying a differentiation domain of the Epo-R. We have studied three chimeras encoding regions of the extracellular region of the Epo-R and the intracellular region of the IL-3R beta IL-3. After transfection into Ba/F3 cells, all three chimeras conferred Epo-dependent growth and induced the expression of beta-globin, suggesting that the extracellular region of the Epo-R plays a critical role in differentiation. However, a truncated Epo-R containing only the extracellular region of the Epo-R and a 15 amino acid cytoplasmic tail does not induced beta-globin expression, although it is processed to the cell surface and binds Epo. These experiments show that the extracytoplasmic region of the Epo-R is necessary but not sufficient to induce erythroid-specific differentiation in this system.

Multiple proteins interact with the nuclear inhibitory protein repressor element in the human interleukin-3 promoter.

T cell expression of interleukin 3 (IL-3) is directed by positive and negative cis-acting DNA elements clustered within 300 base pairs of the transcriptional start site. A strong repressor element, termed nuclear inhibitory protein (NIP), was previously mapped to a segment of the IL-3 promoter between nucleotides -271 and -250. Functional characterization of this element demonstrates that it can mediate repression when linked in cis to a heterologous promoter. DNA binding experiments were carried out to characterize the repressor activity. Using varying conditions, three distinct complexes were shown to interact specifically with the NIP region, although only one correlates with repressor activity. Complex 1 results from binding of a ubiquitous polypeptide that recognizes the 3' portion of this sequence and is not required for repression. Complex 2 corresponds to binding of transcription factor (upstream stimulatory factor) to an E-box motif in the 5' portion of the NIP region. DNA binding specificity of complex 3 overlaps with that of upstream stimulatory factor but is clearly distinct. To determine which of the latter two complexes represents NIP activity, we incorporated small alterations into the NIP site of an IL-3 promoter-linked reporter construct and examined their effects on NIP-mediated repression. Functional specificity for repression matches the DNA binding specificity of complex 3; both repressor activity and complex 3 binding require the consensus sequence CTCACNTNC.

Cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3.

Tissue-specific expression of interleukin-3 (IL-3) is mediated via cis-acting elements located within 315 base pairs of the transcription start. This is achieved in part through the positive activities of the AP-1 and Elf-1 sites in the IL-3 promoter. The contribution to T cell-specific expression by other promoter sites was assessed in a transient expression assay with IL-3 promoter constructs linked to a luciferase gene, focusing initially on the core binding factor (CBF) site, which is footprinted in vivo upon T cell activation. Activity of the CBF site is shown to be critically dependent on the adjacent activator site Act-1. Together the Act-1 and CBF sites form a functional unit (AC unit) with dual activity. The AC unit is demonstrated to enhance basal activity of promoters both in fibroblasts and T cells. This activity is further inducible in activated T cells, but not in fibroblasts. In addition to the already identified NIP repressor site, evidence is presented for a second repressor region that restricts promoter activity in fibroblasts. Finally, a novel positive regulatory element has been mapped in the IL-3 promoter between nucleotide -180 and -210 that leads to increased expression in T cells. Together these results demonstrate that T cell expression of IL-3 is not specified by the activity of a single tissue-specific element, but instead involves multiple interacting elements that provide both specific positive regulation in T cells and specific negative regulation in fibroblasts.