Biomedical subjects

J S Lipsick

Publications and source records attributed to J S Lipsick.

At least 37 records · Page 2Linked to original sources

PubMed1995

Mutations in the DNA-binding and transcriptional activation domains of v-Myb cooperate in transformation.

The v-Myb protein encoded by avian myeloblastosis virus causes oncogenic transformation of monoblastic cells committed to the monocyte/macrophage lineage. v-Myb is a doubly truncated form of its normal cellular counterpart, c-Myb. In addition to its N- and C-terminal deletions, v-Myb contains a number of amino acid substitutions relative to c-Myb. We have previously shown that neither overexpression of c-Myb nor introduction of these amino acid substitutions into c-Myb is sufficient for transformation of myelomonocytic cells. However, a doubly truncated form of c-Myb which lacked these substitutions transformed myeloblastic cells that appeared to be committed to the granulocytic pathway. We demonstrate here that mutations in both the DNA-binding and transcriptional activation domains of v-Myb are required for transformation of rapidly growing monoblasts rather than more slowly growing myeloblasts. These rapidly growing monoblasts do not express mim-1, a target gene for the Gag-Myb-Ets protein of E26 leukemia virus, or C/EBP proteins which cooperate with Myb to activate mim-1 expression. Furthermore, v-Myb proteins which contain both sets of these mutations are weaker transcriptional activators relative to proteins which lack these mutations. These results support a model in which amino acid substitutions in v-Myb have been selected for their ability to activate only a subset of those genes which can be activated by a doubly truncated form of c-Myb. In particular, mim-1 appears to represent a class of genes whose expression was selected against during the development of an increasingly virulent strain of avian myeloblastosis virus by passage in animals.

Acetyltransferases↗

PubMed1994

Transformation of myelomonocytic cells by the avian myeloblastosis virus is determined by the v-myb oncogene, not by the unique long terminal repeats of the virus.

The avian myeloblastosis virus (AMV) induces acute monoblastic leukemia in chickens and transforms only myelomonocytic cells in vitro. The long terminal repeat (LTR) regulatory region of AMV is unique among the known classes of avian retrovirus LTRs. We demonstrate that the substitution of the AMV LTRs by Rous sarcoma virus LTRs did not alter the cell type specificity or the transforming ability of the virus.

Animals↗

PubMed1994

c-Myb prevents TPA-induced differentiation and cell death in v-Myb transformed monoblasts.

Myelomonocytic cells transformed by v-Myb or altered forms of c-Myb do not contain the full-length c-Myb protein found in most immature hematopoietic cells. To determine if c-Myb was a dominant inhibitor of v-Myb, we have induced the synthesis of full-length c-Myb in monoblasts transformed by v-Myb. We found that although some morphological changes occurred, the presence of both c-Myb and v-Myb was compatible with cell growth. However, the response to phorbol ester (TPA) was significantly altered by c-Myb. Monoblasts transformed by v-Myb can be induced to differentiate into macrophages by treatment with TPA. This process is accompanied by a significant amount of cell death. However, when c-Myb was made TPA-inducible in these cells, TPA-induced differentiation into macrophages was blocked and cell death was prevented. These results demonstrate a significant difference in the biological effects of v-Myb and c-Myb in transformed myelomonocytic cells.

Animals↗

PubMed1993

Dicistronic selection for nuclear proteins in living animal cells.

We present an expression/selection system designed for the purification of cell lines inducibly expressing genes coding for unselectable proteins by using dicistronic selection for the cell surface marker CD4. This system enabled us to establish and purify c-myb expressing variants of the v-myb transformed chicken monoblast cell line BM2 with high efficiency.

Animals↗

PubMed1993

Individual repeats of Drosophila Myb can function in transformation by v-Myb.

The v-Myb protein binds to specific DNA sequences and can regulate gene expression. The DNA-binding domain of v-Myb contains the second and third of the three highly conserved tandem repeats found in c-Myb. In general, the ability of mutant forms of v-Myb to transform correlates with their ability to trans activate transcription. Two mutations within the DNA-binding domain of v-Myb which preserve DNA binding in vitro but fail to trans activate or transform have been described. These results suggested that this highly conserved domain might function in specific protein-protein interactions, as well as in DNA binding. We therefore tested the ability of a related protein domain from Drosophila melanogaster to substitute functionally for the homologous region of v-Myb. We found that either the second or third repeat of Drosophila Myb, but not both, could function in trans-activation and transformation by v-Myb. The hybrid containing both the second and third repeats of Drosophila Myb bound to DNA but failed to trans activate transcription either in the context of v-Myb or as a v-Myb-VP16 fusion protein. These results demonstrate that although the protein-DNA contacts made by the Myb repeats have been conserved during the evolution of animals, the protein-protein interactions have diverged.

Acetyltransferases↗

PubMed1993

Oncogenic truncation of the first repeat of c-Myb decreases DNA binding in vitro and in vivo.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

Amino Acid Sequence↗

PubMed1993

Differential transcriptional activation by v-myb and c-myb in animal cells and Saccharomyces cerevisiae.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

Amino Acid Sequence↗

PubMed1993

c-Myb and v-Myb are differentially phosphorylated by p42mapk in vitro.

The product of the c-myb proto-oncogene is a highly conserved transcription factor that has been shown to function as both a transactivator and repressor. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus (AMV) encode proteins truncated at both the amino and carboxy termini, deleting portions of the DNA-binding and negative regulatory domains present in c-Myb. Similar truncations of c-Myb alter its function, suggesting that the viral proteins lack important regulatory sequences. Interestingly, eight potential sites of phosphorylation by proline-directed protein kinases conserved between the avian, murine and human Myb proteins are clustered in or near the negative regulatory domain of c-Myb. The majority of these sites are deleted in both the E26 and AMV viral proteins. In this paper we show that one proline-directed protein kinase, p42mapk, phosphorylates bacterially synthesized avian and murine c-Myb but not AMV v-Myb in vitro. We find that p42mapk phosphorylates c-Myb on serine and threonine, but not on tyrosine. Furthermore, deletion analysis indicates that the sites of phosphorylation map to the C-terminal negative regulatory domain. We speculate that the inability of v-Myb to be phosphorylated by p42mapk may contribute to its oncogenic properties.

Animals↗

PubMed1992

Carboxy-terminal elements of c-Myb negatively regulate transcriptional activation in cis and in trans.

The c-Myb protein plays a key role in normal hematopoiesis, and truncation results in its activation to a transforming protein. Truncation of the c-Myb carboxyl terminus also greatly increases its transcriptional activating activity. The role of specific carboxy-terminal domains in negative regulation was investigated using Myb and Myb fusions with GAL4, LexA, or VP16. Negative regulatory activity of the carboxyl terminus in cis resides in at least two regions. A sequence in one of these regions can also inhibit transcriptional activation by Myb, Myb-VP16, or LexA-Myb proteins in trans. Regulation in trans, or suppression, is independent of c-Myb DNA binding and, therefore, likely involves protein-protein interaction. Suppression does not require the presence of a predicted heptad leucine repeat structure on either molecule. The target of suppression is a sequence that contains part of the minimal Myb transcriptional activation domain. This sequence can confer suppressibility on fusion proteins containing heterologous DNA-binding or transcriptional activation domains.

Amino Acid Sequence↗

PubMed1992

Definition of functional domains in P135gag-myb-ets and p48v-myb proteins required to maintain the response of neuroretina cells to basic fibroblast growth factor.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

Animals↗

PubMed1992

Protein truncation is not required for c-myb proto-oncogene activity in neuroretina cells.

The v-myb oncogene of avian myeloblastosis virus (AMV) differs from its normal cellular counterpart by a truncation at both its amino and carboxyl termini and by a substitution of 11 amino acid residues. We had previously shown that v-myb-containing AMV, in the presence of basic fibroblast growth factor, transformed chicken neuroretina (CNR) cells. To understand the mechanism of c-myb activation, we have tested whether avian retroviruses that express the full-length c-Myb are also active on CNR cells. We have found that c-Myb, like v-Myb, strongly increases the basic fibroblast growth factor response of CNR cells and that these c-myb-expressing cells are able to grow in soft agar in the presence of the growth factor. We have also found that, in contrast to normal or v-myb-expressing AMV-transformed CNR cells, c-Myb-transformed cells express mim-1, a granulocyte-specific gene. However, normal v-Myb- and c-Myb-expressing CNR cells all express the pax-QNR gene, a newly described paired and homeobox-containing gene specifically expressed in the neuroretina. We conclude that, in contrast to what has been described for hematopoietic cells, overexpression of c-Myb is sufficient to activate gene expression and to induce an abnormal behavior of CNR cells.

Avian Myeloblastosis Virus↗

PubMed1992

Expression of the CD4 gene requires a Myb transcription factor.

We have analyzed the control of developmental expression of the CD4 gene, which encodes an important recognition molecule and differentiation antigen on T cells. We have determined that the CD4 promoter alone functions at high levels in the CD4+ CD8- mature T cell but not at the early CD4+ CD8+ stage of T-cell development. In addition, the CD4 promoter functions only in T lymphocytes; thus, the stage and tissue specificity of the CD4 gene is mediated in part by its promoter. We have determined that a Myb transcription factor binds to the CD4 promoter and is critical for full promoter function. Thus, Myb plays an important role in the expression of T-cell-specific developmentally regulated genes.

Animals↗

PubMed1992

The Myb DNA-binding domain is highly conserved in Dictyostelium discoideum.

The c-myb proto-oncogene encodes a protein that is highly conserved among birds and mammals. The amino-terminal domain of c-Myb contains three imperfect tandem repeats of approximately 50 amino acids each. This domain is required for DNA binding and has also been conserved to varying degrees in invertebrates, plants and yeast. Given that myb-related genes appear to control cellular differentiation in a variety of eucaryotic systems, the presence of a myb gene in the cellular slime mold Dictyostelium discoideum might provide a tractable system for studying the role of myb in differentiation. Degenerate oligonucleotide primers encoding regions that are highly conserved in the vertebrate and Drosophila Myb DNA-binding domains were used to amplify a related domain from Dictyostelium genomic DNA, which was then used to isolate a genomic clone. The putative DNA-binding domain of Dictyostelium Myb is as closely related to vertebrate c-Myb as is Drosophila Myb (65% identity), whereas the known Myb-related proteins of plants and yeast are more distantly related. The conserved domain of Dictyostelium Myb is capable of binding to the same DNA sequence as the vertebrate and Drosophila Myb proteins. The remainder of the deduced amino acid sequence of Dictyostelium Myb shows no homology to the divergent domains of the known animal, plant and yeast Myb-related proteins. Evolutionary analysis implies that the duplications that generated the repeats of the Myb DNA-binding domain began prior to the divergence of animals, plants, cellular slime molds and yeast.

Amino Acid Sequence↗

PubMed1992

A highly conserved cysteine in the v-Myb DNA-binding domain is essential for transformation and transcriptional trans-activation.

The v-Myb protein is nuclear, binds to DNA in a sequence-specific fashion, regulates the transcription of various reporter gene and transforms myelomonocytic cells. Cysteine is one of the most conserved residues during protein evolution and has been implicated in DNA binding, protein-protein interaction and redox regulation of various proteins. Therefore, we have now individually substituted each of the seven cysteines of v-Myb with a serine. All seven mutant proteins bound to DNA when they were expressed in E. coli. However, mutant C65S neither trans-activated transcription in vivo nor transformed myeloid cells, although it was transported into the nucleus. This cysteine is conserved in the Myb-related proteins of animals, plants, yeast and the cellular slime mold Dictyostelium discoideum. The C65S mutation and a nearby codon insertion mutation also abolished trans-activation by fusion proteins containing the v-Myb DNA-binding domain and the strong constitutive activation domain of herpes simplex virus (HSV) VP16. Because this domain of VP16 appears to activate transcription whenever it is bound upstream of an appropriate promoter, these results imply that C65 may be required for high-affinity DNA binding in vivo. In support of this hypothesis, we have also shown that, in contrast to wild-type v-Myb, mutant C65S is unable to block transcription from a reporter gene in which Myb binding sites overlap the initiation site.

Amino Acid Sequence↗

PubMed1991

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

Animals↗

PubMed1991

Determinants of sequence-specific DNA-binding by p48v-myb.

The v-myb oncogene of the avian myeloblastosis virus encodes a nuclear protein, p48v-myb, which binds to DNA in a sequence-specific manner. We have used wild type and mutant forms of this protein expressed in E. coli to study the protein and DNA determinants for sequence-specific DNA-binding. We have shown that only the highly conserved domain at the amino terminus of p48v-myb is required for sequence-specific DNA-binding. However, neither of the tandem 50 amino acid repeats present in this domain is alone sufficient for such binding. We have also demonstrated that p48v-myb can recognize a single consensus myb binding site and appears to interact with DNA as a protein monomer. In addition, we have shown that sequence-specific binding by p48v-myb requires nucleotides which flank the previously reported PyAACT/GG consensus.

Amino Acid Sequence↗

PubMed1990

trans activation of gene expression by v-myb.

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

Animals↗

PubMed1988

DNA-binding activity associated with the v-myb oncogene product is not sufficient for transformation.

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.

Autoradiography↗