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[An interview with Dr. Hidesaburo Hanafusa in honor of his cultural medal].
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Production of env-deficient rous sarcoma virus (RSV) early after infection.
Production of defective virus particles during the early stage of Rous sarcoma virus (RSV) infection of chicken embryo fibroblasts (CEF) was examined. RSV harvested 2 days post infection (2pi) had 10 to 30 times lower specific infectivity (focus forming units/unit reverse transcriptase activity) than 5pi harvest. Virus particles produced on day 2 contained less env proteins than particles harvested on day 5. The amount of other viral proteins was equal in particles harvested on day 2 and day 5. Analysis of infected cells revealed that these cells synthesized less env proteins on day 2 than on day 5. RSV RNA in infected cells was spliced normally on day 2. Infection at a low multiplicity of infection (moi) prolonged the production of defective particles. When infection was initiated by a low moi (0.01), particles harvested on day 5 had the same characteristics as 2pi particles after infection with a high moi (1.0). We conclude that the low infectivity of early harvest is due to the reduced amount of env proteins in virus particles, which is a consequence of the reduced env protein synthesis.
NMR studies of the RRsrc peptide, a tyrosine kinase substrate.
The proton and carbon-13 NMR resonances for the 13-residue synthetic RRsrc peptide were completely assigned using two-dimensional NMR spectroscopy. This peptide contains a tyrosine in position 9 that can be phosphorylated by many tyrosine protein kinases. On the basis of observed nuclear Overhauser enhancements and alpha-proton and alpha-carbon chemical shifts, the peptide appears to interconvert between extended and nascent helical structures. The helical conformation found in aqueous solution is compared with the corresponding structure calculated for the tyrosine 416 site of pp60src by homology modeling to the cAMP-dependent protein kinase (PKA) and also to the conformation modelled after the bound form of a PKA-inhibitor peptide.
Signal transduction pathways regulating hematopoietic differentiation.
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Synergistic activation of the prolactin promoter by vitamin D receptor and GHF-1: role of the coactivators, CREB-binding protein and steroid hormone receptor coactivator-1 (SRC-1).
PRL gene expression is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restricted manner in cells of the anterior pituitary. In pituitary GH3 cells, vitamin D increases the levels of PRL transcripts and stimulates the PRL promoter. We have analyzed the role of GHF-1 and of the vitamin D receptor (VDR) to confer vitamin D responsiveness to the PRL promoter. For this purpose we have used nonpituitary HeLa cells, which do not express GHF-1. We found that VDR activates the PRL promoter both in a ligand-dependent and -independent manner through a sequence located between positions -45/-27 in the proximal 5'-flanking region. This sequence also confers VDR and vitamin D responsiveness to a heterologous promoter. In the context of the PRL gene, VDR requires the presence of GHF-1 to activate the promoter. Truncation of the last 12 C-terminal amino acids of VDR, which contain the ligand-dependent activation function (AF2), abolishes regulation by vitamin D, suggesting that binding of coactivators to this region mediates ligand-dependent stimulation of the PRL promoter by the receptor. Indeed, expression of the coactivators, steroid hormone receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), significantly enhances the stimulatory effect of vitamin D mediated by the wild-type VDR but not by the AF2 mutant receptor. Furthermore, CBP also increases the activation of the PRL promoter by GHF-1 and the ligand-independent activation by both wild-type and mutant VDR.
Fhit is a physiological target of the protein kinase Src.
The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that FHIT functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit in vitro and in vivo, providing insight into a biochemical pathway involved in Fhit signaling.
A Drosophila genomic sequence with homology to human epidermal growth factor receptor.
Vertebrate genomes contain an extensive family of genes possessing varying degrees of homology to the v-src oncogene. Most src-related proteins identified to date are intracellular and membrane-associated, although some are transmembrane proteins and function as receptors for peptide growth factors. Three Drosophila gene sequences related to the v-src gene have been identified, each exhibiting a high degree of homology to one or more of the src-family members encoding an intracellular protein. We have isolated a panel of cloned Drosophila sequences exhibiting weak v-src hybridization and were interested to determine whether any members of this group represented homologues of additional known src-family genes, especially those functioning as growth factor receptors. As we report here, four of these clones, representing overlapping portions of the same genomic segment, hybridized preferentially with the v-erb-B oncogene and were further characterized. The deduced amino-acid sequence from a portion of this Drosophila genomic segment is 77% homologous to the kinase domain of human epidermal growth factor (EGF) receptor, a substantially greater degree of homology than was observed with any other known src-family member. By hybridization with a human EGF receptor complementary DNA clone probe, we demonstrate that the same genomic segment showing homology with the kinase domain also contains sequences related to the extracellular domain of the EGF receptor gene.
Combined deficiencies of Src, Fyn, and Yes tyrosine kinases in mutant mice.
Three members of the Src family of tyrosine kinases, src, fyn, and yes, are broadly expressed throughout mouse development. Mutations in the c-src and fyn genes were shown previously to lead to restricted nonoverlapping phenotypes only in a subset of cells in which these kinases are expressed. In this work we show that a mutation in the yes gene does not lead to an overt phenotype. Except for brain, the level or distribution of related kinases is not altered in major tissues. To gain further insight into the possibility that these kinases compensate for each other, animals deficient in multiple src-kinases were generated. Whereas most of the src/fyn or src/yes double mutants die perinatally, a substantial proportion of fyn/yes double mutants are viable but undergo degenerative renal changes leading to diffuse segmental glomerulosclerosis. Taken together, these data are consistent with the hypothesis that, at least in some cells, these kinases are able to compensate for the loss of the other related kinases.
Stimulation of hTAFII68 (NTD)-mediated transactivation by v-Src.
The three genes hTAF(II)68, EWS, and TLS (called the TET family) encode related RNA binding proteins containing an RNA recognition motif and three glycine-, arginine-, and proline-rich regions in the C-terminus and a degenerated repeat containing the consensus sequence Ser-Tyr-Gly-Gln-Ser in the N-terminus. In many human cancers, the N-terminal portion of hTAF(II)68, EWS, or TLS is fused to the DNA binding domain of one of several transcription factors including Fli-1, ERG, ETV1, E1AF, WT1, ATF-1, CHOP, or TEC. We have recognized the presence of several potential tyrosine phosphorylation sites within the amino-terminal domain of hTAF(II)68 and have investigated the potential effects of cytoplasmic signaling on hTAF(II)68 function. Herein, we find that hTAF(II)68 is phosphorylated on tyrosine residue(s) by ectopic expression of v-Src protein tyrosine kinase in vitro and in vivo. The hTAF(II)68 protein can associated with the SH3 domains of several cell signaling proteins, including v-Src protein tyrosine kinase. We also document that full-length v-Src can stimulate hTAF(II)68-mediated transcriptional activation, whereas deletion mutants of v-Src are unable to exert this effect. In addition, cellular Src activity appears important for hTAF(II)68 function since hTAF(II)68-mediated transactivation is reduced in a dose-dependent fashion by ectopic overexpression of a dominant-negative mutant of Src. Taken together, our results suggest that the biological activities of hTAF(II)68 are linked to the cytoplasmic Src signal transduction pathway.
Overexpressed Drosophila src 64B is phosphorylated at its carboxy-terminal tyrosine, but is not catalytically repressed, in cultured Drosophila cells.
Little is known about the regulation of non-receptor tyrosine kinases in invertebrates. We have studied the relationship between the phosphorylation state of the Drosophila src 64B (Dsrc) gene product, p62D, and its tyrosine kinase activity in Drosophila Schneider 2 cells, using wild-type and mutated Dsrc constructs that were overexpressed by transient transfection. Phosphopeptide mapping showed that the putative regulatory C-terminal tyrosine (Tyr-547) of p62D was phosphorylated in vivo. In contrast to vertebrate src family kinases overexpressed in fibroblasts, wild-type p62D overexpressed in Schneider 2 cells was phosphorylated at additional tyrosines outside of the C-terminus. These tyrosines corresponded to the major in vitro autophosphorylation sites. Overexpression of wild-type p62D or several catalytically active p62D mutants significantly increased the phosphorylation of numerous Schneider cell proteins on tyrosine, while expression of catalytically inactive mutants of p62D had no such effect. Thus, in contrast to the repression of src family kinase activity in fibroblasts, p62D is catalytically active when overexpressed in Drosophila cells, perhaps because of substoichiometric C-terminal tyrosine phosphorylation. These results raise the possibility that fly development will be sensitive to ectopic expression of p62D.
src-related tyrosine kinases regulate transcriptional activation of the interstitial collagenase gene, MMP-1, in interleukin-1-stimulated synovial fibroblasts.
OBJECTIVE: To characterize tyrosine kinases that contribute to the transcription of interstitial collagenase. METHODS: Four thousand six hundred fourteen basepairs of the rabbit collagenase promoter region were cloned and sequenced. Plasmids containing collagenase promoter fragments linked to the luciferase reporter gene were transiently transfected into primary rabbit synovial fibroblasts. Regulation of gene activation by inflammatory mediators and tyrosine kinase inhibitors was assessed. To identify specific tyrosine kinases that contribute to collagenase gene expression, v-src was transiently expressed in rabbit synovial fibroblasts along with collagenase promoter constructs, and basal and interleukin-(IL-l)-induced collagenase transcription was assayed. RESULT: An inhibitor of src-related tyrosine kinases, herbimycin A, inhibited increases of collagenase messenger RNA in IL-1- and phorbol myristate acetate-treated fibroblasts. Transcriptional activation of collagenase by IL-1 was also inhibited by herbimycin A. Expression of v-src in synovial fibroblasts enhanced basal and IL-1-inducible transcription. CONCLUSION: Activation of collagenase transcription by inflammatory mediators involves activation of an src-related tyrosine kinase.
Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.
Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.
Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20.
A deletion of the long arm of chromosome 20 [del(20q)] is a recurring abnormality in malignant myeloid disorders. The occurrence of the del(20q) in a broad spectrum of myeloid disorders suggests that the loss of genetic material on 20q could provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor-suppressor gene. We have examined a series of patients with the del(20q) using fluorescence in situ hybridization (FISH) with unique sequence probes that map along the length of 20q, and have delineated a segment that is deleted in 95% of all patients examined (18 of 19). In addition, we have shown that the deletions are interstitial rather than terminal. This region of deletion extends from 20q11.2 to q12, and is flanked by the RPN2 (proximal) and D20S17 loci (distal). The SRC and ADA genes are located within the commonly deleted segment. Our findings emphasize the importance of FISH and other molecular mapping techniques in defining such a region. The delineation of a commonly deleted segment in 20q11.2-q12 will facilitate the identification of candidate tumor-suppressor genes on 20q.
Molecular cloning of a novel non-receptor tyrosine kinase, HYL (hematopoietic consensus tyrosine-lacking kinase).
We identified a novel non-receptor tyrosine kinase from a human megakaryoblastic cell line, UT-7, by means of a PCR-based cloning method. The HYL gene contained a SH2 and SH3 domain and a tyrosine kinase catalytic domain. The deduced amino acid sequence of the protein encoded by this gene was most homologous to CSK (c-src kinase). This gene and CSK shared some unique structural properties such as the absence of a myristylation signal and phosphorylation sites of tyrosine residues corresponding to tyrosines 416 and 527 of chicken p60c-src. Unlike CSK, the SH3 domain of HYL was unique since the ALYDY motif was absent. Northern blot analysis revealed a 2.2 kb transcript in various myeloid cell lines but not in adult tissues except for the brain and the lung, whereas CSK mRNA was ubiquitously expressed. The expression of HYL was upregulated when these myeloid cells were differentiated by induction with phorbol myristate acetate. We named this gene, hematopoietic consensus tyrosine-lacking kinase, HYL. The HYL gene was assigned to chromosome 19 at band p13. It is suggested that HYL plays a significant role in the signal transduction of hematopoietic cells.
Activity-dependent regulation of alternative splicing patterns in the rat brain.
Alternative splicing plays an important role in the expression of genetic information. Among the best understood alternative splicing factors are transformer and transformer-2, which regulate sexual differentiation in Drosophila. Like the Drosophila genes, the recently identified mammalian homologues are subject to alternative splicing. Using an antibody directed against the major human transformer-2 beta isoform, we show that it has a widespread expression in the rat brain. Pilocarpine-induced neuronal activity changes the alternative splicing pattern of the human transformer-2-beta gene in the brain. After neuronal stimulation, a variant bearing high similarity to a male-specific Drosophila tra-2179 isoform is switched off in the hippocampus and is detectable in the cortex. In addition, the ratio of another short RNA isoform (htra2-beta2) to htra2-beta1 is changed. Htra2-beta2 is not translated into protein, and probably helps to regulate the relative amounts of htra2-beta1 to beta3. We also observe activity-dependent changes in alternative splicing of the clathrin light chain B, c-src and NMDAR1 genes, indicating that the coordinated change of alternative splicing patterns might contribute to molecular plasticity in the brain.
The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.
X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.
Specific and redundant roles of Src and Fyn in organizing the cytoskeleton.
Mouse embryos lacking Csk, a negative regulator of Src family kinases, exhibit defects in neurulation and die at mid-gestation. To determine the role of activated Src family kinases in the csk- phenotype, we have introduced mutations in the src and fyn genes into the csk- mutant background. Genetic analysis reveals that src, but not fyn, is partly epistatic to the csk gene. Biochemical analysis indicates that several cytoskeletal proteins are hyperphosphorylated on tyrosine residues in csk- cells. Regulation of cortactin and tensin hyperphosphorylation is Src-dependent, whereas focal adhesion kinase and paxillin hyperphosphorylation is partly dependent on both Src and Fyn. Furthermore, the src- mutation can restore the normal distribution of cortactin and partly correct filamentous actin organization in csk-cells. Thus, Src family kinases have both specific and overlapping functions in regulation of the cytoskeleton. The disturbance of these functions may be a molecular basis for the phenotype exhibited by csk- mutants.