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. 1998 Feb;18(2):989-1002.
doi: 10.1128/MCB.18.2.989.

Molecular cloning reveals that the p160 Myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

F J Tavner  1 , R Simpson, S Tashiro, D Favier, N A Jenkins, D J Gilbert, N G Copeland, E M Macmillan, J Lutwyche, R A Keough, S Ishii, T J Gonda
Affiliations

Molecular cloning reveals that the p160 Myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

F J Tavner et al. Mol Cell Biol. 1998 Feb.

Abstract

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

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Figures

FIG. 1
FIG. 1
Molecular cloning, nucleotide sequence, and predicted amino acid sequence of p160 (see Materials and Methods for details). (A) Overlapping cDNA clones that comprise the p160 cDNA and location of p67-derived peptide sequences. The top four (open) boxes represent cDNA clones derived by PCR, library screening, or 5′-RACE PCR as indicated. The bottom box (shaded) represents the entire sequence and shows the locations of the sequences corresponding to the five p67-derived tryptic peptides (solid boxes). (B) Nucleotide sequence of p160 cDNA and predicted amino acid sequence of p160. The p67-derived tryptic peptide sequences are underlined and bold, the acidic region is boxed, and the basic repeats are italicized and bold (Note that two of these sequences contain overlapping repeat motifs, while that starting at residue 1332 is incomplete.) Potential LCD motifs are indicated by dashed overlining. The symbol ⊥⊤ indicates the carboxyl terminus of p67*.
FIG. 1
FIG. 1
Molecular cloning, nucleotide sequence, and predicted amino acid sequence of p160 (see Materials and Methods for details). (A) Overlapping cDNA clones that comprise the p160 cDNA and location of p67-derived peptide sequences. The top four (open) boxes represent cDNA clones derived by PCR, library screening, or 5′-RACE PCR as indicated. The bottom box (shaded) represents the entire sequence and shows the locations of the sequences corresponding to the five p67-derived tryptic peptides (solid boxes). (B) Nucleotide sequence of p160 cDNA and predicted amino acid sequence of p160. The p67-derived tryptic peptide sequences are underlined and bold, the acidic region is boxed, and the basic repeats are italicized and bold (Note that two of these sequences contain overlapping repeat motifs, while that starting at residue 1332 is incomplete.) Potential LCD motifs are indicated by dashed overlining. The symbol ⊥⊤ indicates the carboxyl terminus of p67*.
FIG. 2
FIG. 2
Genomic organization and chromosomal localization of P160 and the related genes, P160-rs1 and P160-rs2. (A) Southern blot analysis of murine DNA with probes corresponding to the 5′-most 1.0 kb (left) and the 3′-most 1.0 kb (right) of the p160 cDNA sequence. DNA was digested with the following restriction endonucleases: lanes 1 and 6, XbaI; lanes 2 and 7, SacI; lanes 3 and 8, PstI; lanes 4 and 9, HindIII; lanes 5 and 10, EcoRI. The positions and sizes (in kilobases) of DNA markers are shown at the left. (B) Summary of chromosomal localization of p160-related loci in the mouse genome, as determined by interspecific backcross analysis. The grids above each chromosome map represent the possible genotypes of the backcross mice, with solid squares representing the M. spretus alleles and open squares representing the M. musculus alleles; the numbers of recombinants of the indicated genotypes are shown below the grid. The number of recombinant N2 animals over the total number of N2 animals is shown at the left of each chromosome map. The recombination frequencies between each P160-related locus and the adjacent markers, expressed as genetic distance in centimorgans (±1 standard error), are also shown. The positions of corresponding loci on human chromosomes, where known, are shown at the right of each chromosome map.
FIG. 2
FIG. 2
Genomic organization and chromosomal localization of P160 and the related genes, P160-rs1 and P160-rs2. (A) Southern blot analysis of murine DNA with probes corresponding to the 5′-most 1.0 kb (left) and the 3′-most 1.0 kb (right) of the p160 cDNA sequence. DNA was digested with the following restriction endonucleases: lanes 1 and 6, XbaI; lanes 2 and 7, SacI; lanes 3 and 8, PstI; lanes 4 and 9, HindIII; lanes 5 and 10, EcoRI. The positions and sizes (in kilobases) of DNA markers are shown at the left. (B) Summary of chromosomal localization of p160-related loci in the mouse genome, as determined by interspecific backcross analysis. The grids above each chromosome map represent the possible genotypes of the backcross mice, with solid squares representing the M. spretus alleles and open squares representing the M. musculus alleles; the numbers of recombinants of the indicated genotypes are shown below the grid. The number of recombinant N2 animals over the total number of N2 animals is shown at the left of each chromosome map. The recombination frequencies between each P160-related locus and the adjacent markers, expressed as genetic distance in centimorgans (±1 standard error), are also shown. The positions of corresponding loci on human chromosomes, where known, are shown at the right of each chromosome map.
FIG. 3
FIG. 3
Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A)+ RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.
FIG. 4
FIG. 4
Proteins detected by antisera raised to the N and C termini of p160. Cytoplasmic (Cyt) and nuclear (Nuc) extracts from FDC-P1 and NIH 3T3 cells, as indicated, were fractionated by SDS-PAGE and immunoblotted with antisera against the N-terminal (anti-160N) (A) or the C-terminal (anti-160C) (B) regions of p160. The positions of p160 and p67 are indicated by arrows, and those of size standards (in kilodaltons) are indicated at the left.
FIG. 5
FIG. 5
Proteolysis of p160 by cell extracts. In vitro-translated p160 labelled with [35S]methionine was incubated with extracts from FDC-P1 or NIH 3T3 cells for the indicated times (in minutes), after which the products were analyzed by SDS-PAGE. The positions and molecular masses of size standards are indicated at the right, and the bands corresponding to p160 and the 67-kDa product (p67) are indicated at the left.
FIG. 6
FIG. 6
Expression and binding properties of p160FLAG and p67*FLAG following transfection into 293T cells. In each panel, proteins were detected by immunoblotting with anti-FLAG monoclonal antibody M2. (A) SDS-PAGE of extracts from cells transfected with pact expression vectors containing p160FLAG, p67*FLAG constructs, or mock-transfected cells, as indicated. (B) Analysis of bound and unbound p160FLAG and p67*FLAG following incubation of the cell extracts shown in panel A with Sepharose beads carrying the GST-NRD2 (lanes M), GST-L3,4P (lanes M′), or GST-Jun (lanes J) fusion proteins. Note that only 50% of each unbound fraction was loaded.
FIG. 7
FIG. 7
Coimmunoprecipitation of Myb and p160. Extracts from 293T cells cotransfected with expression vectors encoding p160FLAG, wild-type Myb (Myb), L3,4P mutant Myb (L3,4PMyb), or combinations of these as indicated were subject to immunoprecipitation with anti-FLAG antibodies. The resultant immunoprecipitates were then analyzed by SDS-PAGE and immunoblotting with anti-Myb monoclonal antibody 1.1. The immunoblots were then stripped and reprobed with M2 anti-FLAG antibodies. Also shown is a direct immunoblot of a portion of each cell lysate used for immunoprecipitation (Cell Lysate).
FIG. 8
FIG. 8
Effect of p160 and p67* on transactivation by wild-type (WT) and L3,4P mutant (Mut) Myb. (A) CAT activity (measured by thin-layer chromatography) in CV-1 cells transfected with the pc-myc-CAT reporter plasmid, pact-β-galactosidase, and pact expression vectors encoding Myb, p160FLAG, or p67*FLAG, as indicated at the bottom of the chromatograms. Where the p160FLAG or p67*FLAG vector was included, the amount of plasmid used (in micrograms) is shown. The degree of activation, expressed as the ratio of CAT activity obtained in each transfection compared to that in the control transfection with no Myb vector (leftmost lane), is shown above each lane; these values are normalized with respect to β-galactosidase activity to correct for differences in transfection efficiency. See Materials and Methods for details. (B) Representation of the data from panel A to indicate the degree of inhibition of Myb-stimulated transactivation by p160 and p67*, which is taken as 100% for each form of Myb (WT or MUT, as indicated). Error bars represent the standard error as determined from triplicate transfections.
FIG. 9
FIG. 9
Immunofluorescence analysis of the subcellular localization of p160. Each panel shows phase-contrast (left) and fluorescence (right) images of the same field of cells. The cells shown in panels A to C are NIH 3T3 fibroblasts, and those shown in panel D are NIH 3T3 fibroblasts expressing p160FLAG (see the text for details). These were stained with pre-immune rabbit serum (A), 160C rabbit antiserum (B), and anti-FLAG monoclonal antibody M2 (C and D).
See this image and copyright information in PMC

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