(Received for publication, August 7, 1995)
From the
The retinoblastoma protein (Rb) interacts with multiple cellular
proteins that mediate its cellular function. We have identified nine
polypeptides that bind to the T-binding domains of Rb using an Rb
affinity resin. RbAp48 and RbAp46 are quantitatively the major
Rb-associated proteins purified by this approach. RbAp48 was
characterized previously and was found to be related to MSI1, a
negative regulator of Ras in the yeast Saccharomyces
cerevisiae. Here we report the cloning and characterization of
RbAp46. RbAp46 shares 89.4% amino acid identity with RbAp48. The
internal WD repeats, which are found in a growing number of eukaryotic
proteins, are conserved between RbAp46 and RbAp48. Like RbAp48, RbAp46
forms a complex with Rb both in vitro and in vivo and
suppresses the heat-shock sensitivity of the yeast RAS2 strains. We have also isolated the
murine cDNA homologs of RbAp48 and RbAp46. Although both mRNA can be
detected in all mouse tissues, their mRNA levels vary dramatically
between different tissues. No significant differences were observed in
the expression patterns of these genes in most tissues except thymus,
testis, and ovary/uterus, in which 2-fold differences were observed.
Interestingly, the mouse and human RbAp48 amino acid sequences are
completely identical, and the mouse and human RbAp46 differ only by one
conserved amino acid substitution. These results suggest that RbAp48
and RbAp46 may have shared as well as unique functions in the
regulation of cell proliferation and differentiation.
Studies of familial cancer syndromes, such as retinoblastoma, have lead to the identification of tumor suppressor genes. Loss-of-function mutations in the Rb gene are found not only in all hereditary and sporadic forms of retinoblastoma, but also in many other tumor types, including osteosarcoma, breast carcinoma, small cell lung carcinoma, bladder carcinoma, and prostate carcinoma(1) . Introduction of the wild-type Rb gene into Rb-deficient tumor cells suppresses their neoplastic phenotype(2, 3, 4, 5, 6, 7) , thus establishing the Rb gene as a tumor suppressor.
The Rb gene encodes a nuclear phosphoprotein that undergoes cyclic phosphorylation and dephosphorylation during the cell cycle(8, 9, 10) . Rb is underphosphorylated during early G1 phase, phosphorylated by members of the cyclin-dependent kinase family just before S phase, and remains phosphorylated until late mitosis. Hypophosphorylated Rb arrests cells in the G1 phase, and phosphorylation relieves this inhibition(11) . Rb protein not only plays a major role in the inhibition of G1 to S phase transition(12, 13) , but is also important for cell differentiation. In Rb-deficient mouse embryos, sensory neuronal cells fail to become post-mitotic, do not differentiate properly, and exhibit extensive apoptotic cell death. Similarly, lens epithelial cells fail to terminally differentiate and undergo programmed cell death(14, 15) .
These pleiotropic cellular functions of Rb are likely to be mediated by association with multiple cellular proteins(16) . One of the best studied Rb-associated protein is the transcriptional factor E2F, which regulates expression of several genes essential for S phase entry(17) . E2F activity appears to be essential for G1-S progression, since Drosophila E2F null mutants fail to enter S phase after initial 17 embryonic cell divisions(18) . The sequestering and concomitant inhibition of E2F by Rb has been used as a paradigm to demonstrate how Rb restrains cell cycle progression. UBF, a ribosomal transcription factor, is another well studied Rb-associated protein (19, 20, 21) . The UBF-Rb interaction results in suppression of the synthesis of ribosomal RNA by RNA polymerase I, thereby globally inhibiting cell proliferation.
RbAp48
and RbAp46 were first identified as major polypeptides from a HeLa cell
lysate that specifically bound to an Rb affinity column(22) .
The cDNA encoding RbAp48 was subsequently cloned based on partial amino
acid sequences. The predicted amino acid sequence of RbAp48 shares
significant homology with a known yeast protein, MSI1. MSI1 is
presumably a negative regulator of the Ras signal transduction pathway
in Saccharomyces cerevisiae, because overexpression of the
MSI1 gene suppresses the heat-shock sensitivity of RAS2 and ira1 mutant yeast strains
and reduces the intracellular cAMP levels in these mutants (23) . Overexpression of the human RbAp48 gene also suppresses
the heat-shock sensitivity of these yeast mutants, suggesting that the
yeast and human gene products are functionally homologous(22) .
Here we describe the cloning and characterization of RbAp46. RbAp46
and RbAp48 are not only highly homologous at the amino acid sequence
level, but also appear to be functionally homologous with each other
and with S. cerevisiae MSI1, since overexpression of RbAp46
suppresses the heat-shock sensitivity of RAS2 mutant yeast strains. We have also isolated mouse cDNAs encoding
RbAp48 and RbAp46 and shown that they are evolutionarily conserved.
Figure 1: Anti-p48 antibodies recognize RbAp46. As described previously(22) , Rb protein-containing matrices were incubated with a mixture of radiolabeled and unlabeled HeLa cell lysates. Bound proteins were separated by two-dimensional gel electrophoresis and visualized by immunoblotting with anti-p48 antibodies (right), followed by fluorography (left).
Figure 2: Epitope mapping of anti-p48 monoclonal antibodies. A, a series of COOH-terminal deletions of GST-RbAp48 were generated as described under ``Materials and Methods.'' Bacterial cell lysates were separated by SDS-PAGE and visualized by Coomassie Blue staining. B, 13 anti-p48 monoclonal antibodies were mapped using Western blotting analysis. Five of them cross-reacted with RbAp46.
Figure 3: Sequence of RbAp46 and sequence comparison. A, cDNA sequence and deduced amino acid sequence of RbAp46. The numbers correspond to the nucleotide (upper line in each pair) or amino acid (lower line in each pair). An open reading frame of 425 amino acid residues is shown. Peptide sequences identical to that obtained from purified protein are underlined. B, comparison of aligned amino acid sequences of RbAp48 and RbAp46. The complete sequences of the two proteins are shown. Identical amino acids are shown by the solid vertical line; conservative amino acid substitutions are shown by the colons (these amino acids are grouped as ILMV, AS, TS, KR, DE, DN, QN, and QE). The dashes indicate the placement of gaps to maximize alignment between the two sequences.
Figure 4: Southern blot analysis of RbAp46 and RbAp48. 10 µg of genomic DNA isolated from HeLa or Molt-4 cell lines were digested with EcoRI or HindIII, electrophoresed in a 0.8% agarose gel, transferred to Hybond paper (Amersham Corp.), and hybridized with cDNA probes as indicated.
Figure 5:
Interaction of Rb and RbAp46 in vivo and in vitro. A, Rb forms complexes with RbAp46
and RbAp48 in vivo. Equal amounts of total HeLa cell lysates
(40 mg) were used for immunoprecipitation using anti-Rb antibody 0.495,
rabbit anti-nm23 antibody 2669 (E. Y.-H. P. Lee, unpublished), or
normal rabbit serum (NRS), and the immunoprecipitates were
separated by SDS-PAGE and analyzed by Western blotting with a
combination of anti-p48 antibodies (19H9, 15B9, 19D9, 12B1, and 15G12). B, Rb directly interacts with RbAp46 and RbAp48. Left, purified p56, RbAp48, RbAp46, and different
combinations of proteins were separated by native polyacrylamide gel
electrophoresis and visualized by silver staining. Right, the
new band seen in the p56
and RbAp46 mixture was excised.
Polypeptides within the band were separated by electrophoresis through
a 10% SDS-polyacrylamide gel and visualized by silver
staining.
Figure 6:
Effect of expressing RbAp46 on the
heat-shock sensitivity of strains containing RAS2. Heat-shock experiments were performed
as described under ``Materials and Methods.'' The yeast
strains used in this experiment are wild-type strain SP1 (MATa leu2
ura3 trp1 his3 ade8 can1) (1) and the RAS2
strain TK161-R2V (MATa
RAS2
leu2 ura3 trp1 his3 ade8 can1) (2-7). The plasmids used here are pd3 (3),
pd3::48K (4), pYCp48 (5), pYCp46 (6), and
pYC-DE2 (7).
Figure 7: Sequencing comparison of RbAp48 and RbAp46 between human and mouse. The predicted amino acid sequence of human RbAp48 is shown. Amino acid residues of mouse RbAp48 and human and mouse RbAp46 that are identical to those of human RbAp48 are shown by asterisks. Gaps (dashed lines) were inserted to optimize the alignment.
Figure 8: Expression of RbAp46 and RbAp48 genes in mouse tissues. A, a 10-µg sample of total RNA from 2-month-old mouse tissues was subjected to RNase protection assays to detect the presence of RbAp48 and RbAp46 mRNA. B, the same amount (10 µg) of total RNA samples was run on a 1.2% formaldehyde agarose gel and visualized by ethidium bromide staining. C, the abundance of RbAp48 and RbAp46 mRNA in A was densitometrically quantitated and normalized to the 28 and 18 S in B. The amount of RbAp48 in the brain was arbitrarily adjusted to 1. The RNase protection experiment was repeated three times, with similar results.
In this study, we have cloned and characterized RbAp46, an Rb-associated protein which shares high homology (89.4% identity) with the previously characterized RbAp48(22) . The RbAp48 and RbAp46 polypeptides share many common features; both are nuclear proteins and bind Rb protein in vitro and in vivo; both contain the same internal WD (Trp-Asp) repeats; both share amino acid sequence homology with yeast protein MSI1 and mimic the function of MSI1 in yeast; both share similar tissue expression patterns, except thymus, testis, and ovary/uterus in which 2-fold differences were observed.
RbAp48 and RbAp46 are evolutionarily conserved. The predicted amino
acid sequences of RbAp48 for human and mouse are completely identical,
and the predicted protein sequences of RbAp46 for human and mouse are
identical, except for one conserved amino acid substitution. Homologs
of RbAp48 and RbAp46 have been cloned from tomato and Arabidopsis
thaliana as well, sharing 65% amino acid sequence identity to the
human counterparts. ()The high degree of conservation of
RbAp48 and RbAp46 genes during the course of evolution is indicative of
their functional importance.
The presence of WD repeats in both
RbAp48 and RbAp46 is of interest. The WD repeat was first found in the
-subunit of heterotrimeric GTP-binding proteins (G proteins) which
transduce signals across the plasma membrane(29) . It has been
called the
-transducin repeat (30) , the WD-40
repeat(29, 31) , or the GH-WD repeat(32) . WD
repeat proteins are made up of highly conserved repeating units usually
ending with Trp-Asp (WD) and have been found in all eukaryotes but not
in prokaryotes. These proteins appear to perform regulatory functions
in diverse cellular processes, such as cell division, cell fate
determination, gene transcription, transmembrane signaling, mRNA
modification, and vesicle fusion(33) . In mammalian cells, only
few nuclear WD repeat proteins have been characterized. Recently, the
p60 subunit of chromatin assembly factor I has been cloned and shown to
contain WD repeats(34) . However, it is unclear whether the WD
repeats are required for chromatin assembly factor I function. On the
other hand, several WD proteins have been shown to form multiprotein
complexes, interacting with other proteins through the WD repeat region (31, 35, 36, 37, 38) .
Preliminary data indicate that the WD repeat region of RbAp48 is not
required for its interaction with Rb. (
)It will be of
interest to test whether, besides Rb, RbAp48 interacts with other
cellular proteins.
Although homologs of RbAp48 and RbAp46 genes have been found in plants and animals, it is not clear whether yeast has other genes homologous to MSI1. Gene disruption experiments indicated that the MSI1 gene is not essential for the growth of yeast cells. Yeast cells carrying the MSI1 null mutation were indistinguishable from wild-type cells with regard to growth rate, sporulation efficiency, and heat-shock sensitivity(23) . It is possible that there is a second gene in yeast that can complement at least some of the functions of MSI1. Although it has been speculated that MSI1 negatively regulates Ras activities, nothing is known about the mechanisms involved(23) . The central role of Ras proteins in growth control and differentiation in many species and, specifically, in neoplasia in humans has been well documented (39, 40, 41) . Many aspects of Ras signaling are similar in yeasts and mammals. Mammalian RAS genes are functional in yeast, and mutated yeast RAS genes efficiently transform mouse fibroblast cell lines. Although in lower and higher eukaryotes, Ras regulates different biochemical pathways, Raf1 and mitogen-activated protein kinase in mammals (42) and adenylyl cyclase in yeasts(43, 44) , there is some evidence indicating that the effects of Ras activity on gene expression are similar in yeasts and in mammals. For example, both in yeasts and in mammals, Ras activates AP-1 transcription factors in response to UV irradiation (45) and controls transcription of similar heat-shock genes in response to heat shock(46) . It could be that many effects of Ras on gene expression are crucial for cellular responses to external signals and, therefore, are highly conserved in evolution. However, the pathways that transmit the signal from Ras to the transcription factors are not conserved. Since both RbAp48 and RbAp46 are functionally equivalent to MSI1 in the suppression of the Ras mutant phenotype, it could be that RbAp48 and RbAp46 in mammals and MSI1 in yeasts regulate similar effects of Ras on gene expression in response to external signals. The high degree of conservation of RbAp48 and RbAp46 genes during the course of evolution and the similar but distinguished tissue expression patterns might indicate that they have shared as well as unique functions.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35141 [GenBank](mRbAp48), U35142 [GenBank](mRbAp46), and U35143 [GenBank](RbAp46).