Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 18 de 18
Filter
Add more filters










Publication year range
1.
Oncogene ; 25(42): 5777-86, 2006 Sep 21.
Article in English | MEDLINE | ID: mdl-16652147

ABSTRACT

RUNX1 (AML1) is a gene that is frequently disrupted by chromosomal translocations in acute leukemia. Like its Drosophila homolog Runt, RUNX1 both activates and represses transcription. Both Runt and RUNX1 are required for gene silencing during development and a central domain of RUNX1, termed repression domain 2 (RD2), was defined as being required for transcriptional repression and for the silencing of CD4 during T-cell maturation in thymic organ cultures. Although transcriptional co-repressors are known to contact other repression domains in RUNX1, the factors that bind to RD2 had not been defined. Therefore, we tested whether RD2 contacts histone-modifying enzymes that may mediate both repression and gene silencing. We found that RD2 contacts SUV39H1, a histone methyltransferase, via two motifs and that endogenous Suv39h1 associates with a Runx1-regulated repression element in murine erythroleukemia cells. In addition, one of these SUV39H1-binding motifs is also sufficient for binding to histone deacetylases 1 and 3, and both of these domains are required for full RUNX1-mediated transcriptional repression. The association between RUNX1, histone deacetylases and SUV39H1 provides a molecular mechanism for repression and possibly gene silencing mediated by RUNX1.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Histone Deacetylases/metabolism , Methyltransferases/metabolism , Repressor Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Humans , Jurkat Cells , Transfection
2.
Cancer Chemother Pharmacol ; 48 Suppl 1: S31-4, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11587363

ABSTRACT

AML-1 is one of the most frequently translocated genes in human leukemia. AML-1 binds DNA and activates or represses transcription, while the chromosomal translocation fusion proteins in acute myeloid leukemia subvert these functions. The t(8;21) is the second most frequent translocation in acute myeloid leukemia and creates a fusion between the DNA binding domain of AML-1 and the ETO (also known as MTG8) corepressor. The t(12;21) is found in up to 25% of pediatric B cell acute lymphoblastic leukemias and fuses the ETS family transcription factor TEL to the amino terminus of AML-1. In addition, the inv(16), the most frequent translocation in acute myeloid leukemia, fuses the AML-1 cofactor CBFbeta to the smooth muscle myosin heavy chain MYH11. Both the t(8;21) and t(12;21) create transcriptional repressors that impair AML-1 target gene expression. We demonstrated that the fusion proteins encoded by these translocations contact the nuclear hormone corepressors (N-CoR/SMRT), mSin3A, and histone deacetylases. We have also found that both TEL and AML-1 interact with mSin3A. TEL also binds N-CoR and histone deacetylase-3, indicating that wild-type TEL is a transcriptional repressor. The t(12;21) fuses the mSin3A interaction domain of TEL to AML-1 to transform AML-1 from a regulated to an unregulated transcriptional repressor. The recognition that AML-1 interacts with mSin3A to repress transcription suggested that the inv(16) fusion protein might also repress the transcription of AML-1-target genes. In fact, the inv(16) encodes a protein that cooperates with AML-1 to repress transcription. The inv(16) fusion protein was found in a ternary complex with AML-1 and mSin3A, suggesting that the inv(16) also acts by recruiting transcriptional corepressors and histone deacetylases.


Subject(s)
Oncogene Proteins, Fusion/physiology , Repressor Proteins/physiology , Transcription, Genetic/physiology , 3T3 Cells , Animals , COS Cells , Chromosome Inversion , Chromosomes, Human, Pair 12 , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 21 , Core Binding Factor Alpha 2 Subunit , Humans , Mice , Nuclear Proteins/metabolism , Nuclear Receptor Co-Repressor 1 , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RUNX1 Translocation Partner 1 Protein , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiology , Translocation, Genetic
3.
Mol Cell Biol ; 21(19): 6470-83, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11533236

ABSTRACT

t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G(1) phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, Delta469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Histone Deacetylases/metabolism , Leukemia, Myelomonocytic, Acute/genetics , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Proteins , Repressor Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription Factors/physiology , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/physiology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Mice , Models, Genetic , Molecular Sequence Data , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Nuclear Receptor Co-Repressor 1 , Oncogene Proteins, Fusion/antagonists & inhibitors , Protein Structure, Tertiary , RUNX1 Translocation Partner 1 Protein , Sequence Homology, Amino Acid , Sin3 Histone Deacetylase and Corepressor Complex , Transcription Factors/antagonists & inhibitors , Transcription, Genetic , Translocation, Genetic
4.
Curr Opin Hematol ; 8(4): 197-200, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11561155

ABSTRACT

The t(8;21), t(16;21), inv(16), and t(12;21) are some of the most frequent chromosomal translocations found in acute myeloid and acute lymphoblastic leukemia. The fusion proteins created by these chromosomal translocations are transcriptional repressors. A full understanding of the types of proteins that these fusion proteins recruit to repress transcription will not only clarify understanding of the molecular mechanism of action of these fusion proteins but also provide further targets for therapeutic intervention.


Subject(s)
Leukemia, Myeloid/genetics , Oncogene Proteins, Fusion/physiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Acute Disease , Core Binding Factor Alpha 2 Subunit , Gene Expression Regulation, Neoplastic , Humans , RUNX1 Translocation Partner 1 Protein , Repressor Proteins/physiology , Transcription Factors/physiology , Transcription, Genetic , Translocation, Genetic
5.
Oncogene ; 19(42): 4886-95, 2000 Oct 05.
Article in English | MEDLINE | ID: mdl-11039906

ABSTRACT

The myc family of genes plays an important role in several cellular processes including proliferation, apoptosis, differentiation, and transformation. B-myc, a relatively new and largely unstudied member of the myc family, encodes a protein that is highly homologous to the N-terminal transcriptional regulatory domain of c-Myc. Here, we show that high level B-myc expression is restricted to specific mouse tissues, primarily hormonally-controlled tissues, with the highest level of expression in the epididymis. We also report the identification of the endogenous B-Myc protein from mouse tissues. Like other Myc family proteins, B-Myc is a short-lived nuclear protein which is phosphorylated on residues Ser-60 and Ser-68. Rapid proteolysis of B-Myc occurs via the ubiquitin-proteasome pathway. Finally, we found that overexpression of B-Myc significantly slows the growth of Rat la fibroblasts and COS cells suggesting B-Myc functions as an inhibitor of cellular proliferation.


Subject(s)
Cell Division/genetics , Epididymis/metabolism , Gene Expression Regulation , Genes, myc , Hormones/physiology , Proto-Oncogene Proteins c-myc/biosynthesis , Adrenal Glands/metabolism , Animals , Brain/metabolism , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Cysteine Endopeptidases/metabolism , DNA, Complementary/genetics , Female , Fibroblasts , Growth Inhibitors/biosynthesis , Growth Inhibitors/genetics , Growth Inhibitors/physiology , Male , Mammary Glands, Animal/metabolism , Mice , Multienzyme Complexes/metabolism , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Organ Specificity , Ovary/metabolism , Phosphorylation , Pituitary Gland/metabolism , Prostate/metabolism , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-myc/genetics , Rats , Recombinant Fusion Proteins/biosynthesis , Transfection , Ubiquitins/metabolism , Uterus/metabolism
6.
Gene ; 245(2): 223-35, 2000 Mar 21.
Article in English | MEDLINE | ID: mdl-10717473

ABSTRACT

Chromosomal translocations affecting the AML-1 gene are among the most frequent aberrations found in acute leukemia. Because the AML-1 transcription factor is a critical regulator of hematopoeitic cell development, normal homeostasis is disrupted in cells containing these translocations. In this review we describe the mechanisms of transcriptional activation and repression by AML-1 and how this transcriptional control is disrupted by the chromosomal translocations that affect AML-1. Finally, we discuss how the mechanism of transcriptional repression by these chromosomal translocation fusion proteins is a possible target of therapeutic intervention in acute leukemia.


Subject(s)
DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/cytology , Leukemia, Myeloid/genetics , Proto-Oncogene Proteins , Transcription Factors/genetics , Acute Disease , Cell Differentiation , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Myeloid/physiopathology , Transcription Factors/physiology , Translocation, Genetic
7.
J Biol Chem ; 275(5): 3438-45, 2000 Feb 04.
Article in English | MEDLINE | ID: mdl-10652337

ABSTRACT

The AML-1-encoded transcription factor, AML-1B, regulates numerous hematopoietic-specific genes. Inappropriate expression of AML-1-family proteins is oncogenic in cell culture systems and in mice. To understand the oncogenic functions of AML-1, we established cell lines expressing AML-1B to examine the role of AML-1 in the cell cycle. DNA content analysis and bromodeoxyuridine pulse-chase studies indicated that entry into the S phase of the cell cycle was accelerated by up to 4 h in AML-1B-expressing 32D.3 myeloid progenitor cells as compared with control cells or cells expressing E2F-1. However, AML-1B was not able to induce continued cell cycle progression in the absence of growth factors. The DNA binding and transactivation domains of AML-1B were required for altering the cell cycle. Thus, AML-1B is the first transcription factor that affects the timing of the mammalian cell cycle.


Subject(s)
Cell Cycle/genetics , DNA-Binding Proteins , G1 Phase/genetics , Gene Expression Regulation , Transcription Factors/genetics , Animals , Cell Line , Core Binding Factor Alpha 2 Subunit , Flow Cytometry , Humans , Mice , Proto-Oncogene Proteins/genetics , Transfection
8.
Mol Cell Biol ; 20(6): 2075-86, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10688654

ABSTRACT

The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.


Subject(s)
Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid/genetics , Proto-Oncogene Proteins , Transcription Factors/genetics , Translocation, Genetic , Acute Disease , Animals , COS Cells , Humans , Kruppel-Like Transcription Factors , Promyelocytic Leukemia Zinc Finger Protein , RUNX1 Translocation Partner 1 Protein , Transfection , Zinc Fingers
9.
J Biol Chem ; 275(1): 651-6, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10617663

ABSTRACT

AML1 is one of the most frequently translocated genes in human leukemia. Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1). Unexpectedly, this repression did not require interactions with the Groucho co-repressor. To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1. We demonstrate that AML-1 interacts with the mSin3 co-repressors. Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells. A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription. The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.


Subject(s)
Cyclins/genetics , Leukemia, Myeloid/genetics , Neoplasm Proteins , Proto-Oncogene Proteins , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Translocation, Genetic , Acute Disease , Binding Sites , Core Binding Factor Alpha 2 Subunit , Core Binding Factor alpha Subunits , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/metabolism , Histone Deacetylases , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/metabolism
10.
Proc Natl Acad Sci U S A ; 96(22): 12822-7, 1999 Oct 26.
Article in English | MEDLINE | ID: mdl-10536006

ABSTRACT

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). The inv(16) fusion protein acts by dominantly interfering with AML-1/core binding factor beta-dependent transcriptional regulation. Here we demonstrate that the inv(16) fusion protein cooperates with AML-1B to repress transcription. This cooperativity requires the ability of the translocation fusion protein to bind to AML-1B. Mutational analysis and cell fractionation experiments indicated that the inv(16) fusion protein acts in the nucleus and that repression occurs when the complex is bound to DNA. We also found that the inv(16) fusion protein binds to AML-1B when it is associated with the mSin3A corepressor. An AML-1B mutant that fails to bind mSin3A was impaired in cooperative repression, suggesting that the inv(16) fusion protein acts through mSin3 and possibly other corepressors. Finally, we demonstrate that the C-terminal portion of the inv(16) fusion protein contains a repression domain, suggesting a molecular mechanism for AML-1-mediated repression.


Subject(s)
Chromosome Inversion , Proto-Oncogene Proteins , Repressor Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic , Animals , COS Cells , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/metabolism
11.
Mol Cell Biol ; 19(10): 6566-74, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10490596

ABSTRACT

t(12;21) is the most frequent translocation found in pediatric B-cell acute lymphoblastic leukemias. This translocation fuses a putative repressor domain from the TEL DNA-binding protein to nearly all of the AML-1B transcription factor. Here, we demonstrate that fusion of the TEL pointed domain to the GAL4 DNA-binding domain resulted in sequence-specific transcriptional repression, indicating that the pointed domain is a portable repression motif. The TEL pointed domain functioned equally well when the GAL4 DNA-binding sites were moved 600 bp from the promoter, suggesting an active mechanism of repression. This lead us to demonstrate that wild-type TEL and the t(12;21) fusion protein bind the mSin3A corepressor. In the fusion protein, both TEL and AML-1B contribute mSin3 interaction domains. Deletion mutagenesis indicated that both the TEL and AML-1B mSin3-binding domains contribute to repression by the fusion protein. While both TEL and AML-1B associate with mSin3A, TEL/AML-1B appears to bind this corepressor much more stably than either wild-type protein, suggesting a mode of action for the t(12;21) fusion protein.


Subject(s)
DNA-Binding Proteins/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins , Repressor Proteins/genetics , Transcription Factors/genetics , Translocation, Genetic , Burkitt Lymphoma/genetics , Child , Chromosomes, Human, Pair 12 , Chromosomes, Human, Pair 21 , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/metabolism , Humans , Models, Genetic , Neoplasm Proteins , Oncogene Proteins, Fusion/metabolism , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-ets , RUNX1 Translocation Partner 1 Protein , Repressor Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex , Transcription Factors/metabolism , ETS Translocation Variant 6 Protein
12.
J Cell Biochem ; 72(4): 483-91, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10022608

ABSTRACT

We have isolated and characterized cellular kinases which associate with the transactivation domain of c-Myc and phosphorylate Ser-62. We demonstrate that cellular Map kinases associate with c-Myc under stringent conditions and phosphorylate Ser-62. We also find that TPA stimulates the activity of the Myc-associated Map kinase to phosphorylate Ser-62. However, we do not observe an increase in Ser-62 phosphorylation in endogenous c-Myc after TPA treatment of cells. Since the regulation of the c-Myc-associated Map kinases does not correlate with the in vivo regulation of Ser-62 phosphorylation in c-Myc, we conclude that Map kinases are not the in vivo kinases for Ser-62. Although Ser-62 phosphorylation was not affected by TPA, phosphorylation at a different serine residue was significantly upregulated by TPA.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Protein Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , 3T3 Cells , Animals , COS Cells , Mice , Phosphopeptides/analysis , Phosphorylation , Phosphoserine/analysis , Tetradecanoylphorbol Acetate/pharmacology
13.
Mol Cell Biol ; 18(12): 7176-84, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9819404

ABSTRACT

t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.


Subject(s)
Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , DNA-Binding Proteins/genetics , Leukemia, Myeloid/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins , Transcription Factors/genetics , Translocation, Genetic/genetics , Cell Line , Core Binding Factor Alpha 2 Subunit , Histone Deacetylases/genetics , Humans , Nuclear Receptor Co-Repressor 1 , Precipitin Tests , RUNX1 Translocation Partner 1 Protein , Recombinant Fusion Proteins/genetics
14.
Mol Cell Biol ; 18(6): 3604-11, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9584201

ABSTRACT

Chromosomal translocations in acute leukemia that affect the AML-1/CBFbeta transcription factor complex create dominant inhibitory proteins. However, the mechanisms by which these proteins act remain obscure. Here we demonstrate that the multidrug resistance 1 (MDR-1) promoter is a target for AML/ETO transcriptional repression. This repression is of basal, not activated, expression from the MDR-1 promoter and thus represents a new mechanism for AML/ETO function. We have defined two domains in AML/ETO that are required for repression of basal transcription from the MDR-1 promoter: a hydrophobic heptad repeat (HHR) motif and a conserved zinc finger (ZnF) domain termed the MYND domain. The HHR mediates formation of AML/ETO homodimers and AML/ETO-ETO heterodimers. Single serine substitutions at conserved cysteine residues within the predicted ZnFs also abrogate transcriptional repression. Finally, we observe that AML/ETO can also inhibit Ets-1 activation of the MDR-1 promoter, indicating that AML/ETO can disrupt both basal and Ets-1-dependent transcription. The fortuitous inhibition of MDR-1 expression in t(8;21)-containing leukemias may contribute to the favorable response of these patients to chemotherapeutic drugs.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Oncogene Proteins, Fusion , Promoter Regions, Genetic , Repressor Proteins/metabolism , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/metabolism , Dimerization , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Myeloid/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Point Mutation , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , RUNX1 Translocation Partner 1 Protein , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Transcription Factors/genetics , Transcription Factors/metabolism , Translocation, Genetic , Tumor Cells, Cultured , Zinc Fingers/genetics
15.
Oncogene ; 14(8): 967-75, 1997 Feb 27.
Article in English | MEDLINE | ID: mdl-9050996

ABSTRACT

Using an extensive series of deletion and site-specific mutation constructs, we have identified five new phosphorylation sites in c-Myc in the N-terminal transactivation domain and near the C-terminal DNA binding/heterodimerization domain. We have also found that Thr-58 phosphorylation is regulated by specific cellular events. When c-Myc is overexpressed in cells Thr-58 phosphorylation was greatly enhanced in the overexpressed, exogenous c-Myc as compared with the endogenous protein. In contrast, an inhibition of Thr-58 phosphorylation and an enhancement of Serine 62 phosphorylation was observed in c-Myc from immortalized cells compared with primary cells. No significant changes in c-Myc phosphorylation were found when transformed and nontransformed cells were compared. Finally, mutations at these phosphorylation sites, either individually or in combination with previously described sites, did not affect the ability of c-Myc to transactivate through the CACGTG Myc/Max DNA binding sites. These results further suggest that either the molecular role for c-Myc phosphorylation does not involve modulating transcriptional activity of c-Myc or that the CACGTG site does not represent a physiological promoter element.


Subject(s)
Cell Transformation, Neoplastic , Proto-Oncogene Proteins c-myc/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Survival , Mice , Molecular Sequence Data , Peptide Mapping , Phosphorylation , Phosphoserine/metabolism , Phosphothreonine/metabolism , Sequence Deletion , Structure-Activity Relationship , Transcriptional Activation , Tumor Cells, Cultured/metabolism
16.
Mol Cell Biol ; 15(8): 4031-42, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7623799

ABSTRACT

The c-Myc protein is a transcription factor with an N-terminal transcriptional regulatory domain and C-terminal oligomerization and DNA-binding motifs. Previous studies have demonstrated that p107, a protein related to the retinoblastoma protein, binds to the c-Myc transcriptional activation domain and suppresses its activity. We sought to characterize the transforming activity and transcriptional properties of lymphoma-derived mutant MYC alleles. Alleles encoding c-Myc proteins with missense mutations in the transcriptional regulatory domain were more potent than wild-type c-Myc in transforming rodent fibroblasts. Although the mutant c-Myc proteins retained their binding to p107 in in vitro and in vivo assays, p107 failed to suppress their transcriptional activation activities. Many of the lymphoma-derived MYC alleles contain missense mutations that result in substitution for the threonine at codon 58 or affect sequences flanking this amino acid. We observed that in vivo phosphorylation of Thr-58 was absent in a lymphoma cell line with a mutant MYC allele containing a missense mutation flanking codon 58. Our in vitro studies suggest that phosphorylation of Thr-58 in wild-type c-Myc was dependent on cyclin A and required prior phosphorylation of Ser-62 by a p107-cyclin A-CDK complex. In contrast, Thr-58 remained unphosphorylated in two representative mutant c-Myc transactivation domains in vitro. Our studies suggest that missense mutations in MYC may be selected for during lymphomagenesis, because the mutant MYC proteins have altered functional interactions with p107 protein complexes and fail to be phosphorylated at Thr-58.


Subject(s)
Burkitt Lymphoma/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Genes, myc/genetics , Proto-Oncogene Proteins c-myc/genetics , Alleles , Amino Acid Sequence , Base Sequence , Cyclins/metabolism , DNA-Binding Proteins/metabolism , Humans , Models, Genetic , Molecular Sequence Data , Mutation , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-myc/metabolism , Retinoblastoma-Like Protein p107 , Structure-Activity Relationship , Suppression, Genetic , TATA-Box Binding Protein , Threonine/metabolism , Transcription Factors/metabolism , Transcriptional Activation
17.
Mol Cell Biol ; 14(8): 5510-22, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8035827

ABSTRACT

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.


Subject(s)
Mitogens/pharmacology , Mitosis , Proto-Oncogene Proteins c-myc/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Nucleus/metabolism , Chick Embryo , Coturnix , Cytoplasm/metabolism , Molecular Sequence Data , Phosphorylation , Phosphoserine/metabolism , Phosphothreonine/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcriptional Activation
18.
Bioessays ; 13(8): 413-7, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1953703

ABSTRACT

During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms. It is widely accepted that this undertaking must include the elucidation of the genetic blueprint - the genome sequence - of a number of model organisms. As a prelude to the determination of these sequences, clone-based physical maps of the genomes of a number of multicellular animals and plants are being constructed. Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps. The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone 'islands' to be linked together in an efficient manner. The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility. Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large.


Subject(s)
Caenorhabditis/genetics , Chromosome Mapping/methods , Chromosomes, Fungal , Genome , Saccharomyces cerevisiae/genetics , Animals , Cloning, Molecular/methods , Cosmids , Genetic Vectors , Restriction Mapping
SELECTION OF CITATIONS
SEARCH DETAIL
...