Identification and characterization of the protein encoded by the human c-myb proto-oncogene.

Klempnauer, Karl‐Heinz; Bonifer, Constanze; Sippel, Albrecht E.

doi:10.1002/j.1460-2075.1986.tb04443.x

Cited by 68 publications

(41 citation statements)

References 38 publications

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“…After fractionation of nuclei isolated from cells under hypotonic conditions, the association of p48v-m'b with an insoluble nuclear fraction is dependent on the temperature at which nuclease digestion occurs, as was previously reported (8,12,14). In contrast, the association between p48v-mYb and an insoluble subnuclear structure generally defined as the nuclear matrix/lamina complex (1,19) is independent of the temperature if the nuclei are isolated and treated under isotonic conditions.…”

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confidence: 67%

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Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

Boyle

Baluda

1987

Mol. Cell. Biol.

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The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-.Yb from the nuclear matrix.The product of the oncogene v-myb of avian myeloblastosis virus (AMV) in AMV-transformed myeloblasts is a 48,000-Mr nuclear protein, p48V--myb (4,13). To help define the molecular role of p48v-myb in leukemogenesis, several investigators have sought to identify and define its nuclear interactions (1,2,8,12,14,15). The association between p48v-myb and nuclear elements has been analyzed in fractionated nuclei isolated from AMV-transformed cells (BM2) by two different strategies (1,8). One isolation procedure has been previously described (1, 3), in which nuclei are obtained from cells lysed under isotonic conditions in buffer containing 5 mM NaH2PO4 (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol (DTT), 1 mM MgCl2, 0.5 mM CaCl2, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Kyro EOB detergent (isotonic nuclei). After extraction of these isotonic nuclei with isotonic Nonidet P-40 (NP-40) buffer (0.5% NP-40, 10 mM NaH2PO4, pH 7.4, 120 mM NaCl, and 0.1 mM PMSF) (1), brief digestion with micrococcal nuclease at 37°C, and then extraction is 2.0 M NaCl (high salt), p48v-myb was removed from the nucleus in varying amounts in three distinct fractions: approximately 29% was contained in nucleoplasm, 7% was in nuclease-sensitive chromatin, and 64% was associated with the nuclear matrix/lamina complex (1). The other procedure we used to isolate nuclei relies on osmotic and shear force to separate the plasma membrane by disrupting cells which had been preincubated in hypotonic buffer (RSB: 10 mM NaCl, 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4, 1.5 mM MgCl2, 0.1% sodium deoxycholate [DOC], and 0.1 mM PMSF) with a Dounce homogenizer as described by Evan and Hancock (8).After fractionation of these nuclei (hypotonic nuclei) by digestion with DNase I at low temperature (4°C) followed by extraction in high salt, most or all of p48v-myb was eluted from nuclei along with soluble nucleoplasm and chromatin (Fig. 1A) as previously described (8). However, when these hypotonic nuclei were transiently exposed to temperatures near 37°C during nuclease digestion, most or all of p48v-myb remained associated with the resulting insoluble nuclear complex (Fig. 1A) 92138-9216. isolated by this hypotonic procedure is an important factor in nuclear subfractionation. These results could suggest that localization of p48V-myb within the nuclear matrix fraction is an artifact induced by a temperature-dependent insolubilization of nuclear proteins.Alternatively, the isolation procedure alone, not the subsequent method of subnuclear fractionation, may affect the associations of some nuclear proteins, including p48vmYb. In nuclei isolated under hypotonic conditio...

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supporting

confidence: 67%

“…To help define the molecular role of p48v-myb in leukemogenesis, several investigators have sought to identify and define its nuclear interactions (1,2,8,12,14,15). The association between p48v-myb and nuclear elements has been analyzed in fractionated nuclei isolated from AMV-transformed cells (BM2) by two different strategies (1,8).…”

mentioning

confidence: 99%

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

Boyle

Baluda

1987

Mol. Cell. Biol.

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“…This result suggests that at least part of the difference in electrophoretic mobility between the in vivo-and in vitro-produced proteins was due to phosphorylation. The remaining difference may have been due to phosphorylation that was resistant to alkaline phosphatase treatment or to some other forms of posttranslational mod- (15,19). N-terminal truncation of the proteins, as in MML7 cells, did not alter the rate of turnover (data not shown).…”

Section: Resultsmentioning

confidence: 88%

“…The c-myb proto-oncogene encodes a predominantly nuclear protein (c-Myb) with an apparent molecular weight of 75 to 80 kilodaltons in avian, murine, and human cells (5,15,16,18). Translation of murine c-Myb is thought to initiate at the first ATG in c-myb (located in exon UE3) and to terminate in a 3' exon downstream of vE7 (1, 10; Fig.…”

mentioning

confidence: 99%

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

1989

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The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein.Here we report the identification of an 85-kilodalton (p85C-mYb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c mYb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-mYb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z pre-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

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“…It is possible that this conservative amino acid change decreases CI DNA binding; however, the Cl-I protein (with Glu^°*) is believed to bind DNA (cited in Paz-Ares et al 1990). This Asp residue is conserved evolutionarily between the various members of the myb oncogene family (Bergmann et al 1981;Klempnauer et al 1982Klempnauer et al , 1986Gonda et al 1985;Boyle et al 1986;Shen-Ong et al 1986;Peter et al 1987) and may occupy a position in the DNA-recognition helix (Ohlendorf et al 1982;Lehming et al 1987;Aggarwal et al 1988;Jordan and Pabo 1988;Otting et al 1988). This conservation suggests that an analogous change in any of the various mammalian or insect myb-homologous domains could also decrease the transcriptional activation function of these proteins dramatically.…”

Section: Discussionmentioning

confidence: 99%

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

1991

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Genes encoding fusions between the maize regulatory protein CI and the yeast transcriptional activator GAL4 and mutant CI proteins were assayed for their ability to trans-activate anthocyanin biosynthetic genes in intact maize tissues. The putative DNA-binding region of CI fused to the transcriptional activation domain of GAL4 activated transcription of anthocyanin structural gene promoters in cl aleurones, cl Rscm2 embryos, and cl r embryogenic callus. Cells receiving these constructs accumulated purple anthocyanin pigments. The Cl acidic region fused to the GAL4 DNA-binding domain activated transcription of a GAL4-regulated promoter. An internal deletion of Cl also induced pigmentation; however, frameshifts in either the amino-terminal basic or carboxy-terminal acidic region blocked tra/is-activation, and the latter generated a dominant inhibitory protein. Fusion constructs between the wild-type Cl cDNA and the dominant inhibitor allele Cl-I cDNA were used to identify the amino acid changes in Cl responsible for the Cl-I inhibitory phenotype. Results from these studies establish that amino acids within the myb-homologous domain are critical for transcriptional activation.

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Identification and characterization of the protein encoded by the human c-myb proto-oncogene.

Cited by 68 publications

References 38 publications

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins.

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