(Received for publication, July 5, 1995)
From the
The retinoblastoma protein, Rb, is detected in extracts of
monkey CV-1 cells complexed with Pur, a sequence-specific
single-stranded DNA-binding protein implicated in control of gene
transcription and DNA replication. These complexes can be
immunoextracted from cell lysates using monoclonal antibodies to either
Pur
or Rb. The Pur
Rb complexes contain a form of
Pur
with extensive post-synthetic modification, as demonstrated
following expression of Pur
cDNA fused to a 9-amino acid epitope
tag. Human Pur
, expressed as a glutathione S-transferase
fusion protein, specifically binds to the hypophosphorylated form of Rb
with an affinity as high as that of SV40 large T-antigen. In the
absence of DNA, glutathione S-transferase-Pur
binds to
p56
, an NH
-terminal-truncated Rb protein
purified from Escherichia coli, containing the T-antigen
binding domain, to form multimeric complexes. The single-stranded DNA
Pur
recognition element disrupts these complexes. Conversely, high
concentrations of p56
prevent Pur
binding to DNA.
Through use of a series of deletion mutants, the DNA binding activity
of Pur
is localized to a series of modular amino acid repeats. Rb
binding involves a Pur
region with limited homology to the
Rb-binding region of SV40 large T-antigen. Binding of Pur
to
p56
, the COOH-terminal portion of Rb, is inhibited by a
synthetic peptide containing the T-antigen Rb-binding motif.
The retinoblastoma protein, Rb, ()product of the rb tumor suppressor gene, is modified by phosphorylation in
late G1 phase of the cell cycle(1, 2) . This
phosphorylation alters the activity of Rb, causing it to release
several cellular proteins with which it is associated and allowing the
cell to progress from G1 to S-phase. (
)Through such
associations Rb controls a link between DNA synthesis, required for
cell proliferation, and expression of specific genes, required for cell
differentiation. Hypophosphorylated Rb associates with members of the D
cyclin family(4, 5) . It has been reported that at
least one member of this family, cyclin D1, together with cyclin E,
participates in the phosphorylation of Rb.
In
non-transformed cells the D cyclins reportedly form complexes which
include the DNA polymerase
processivity factor proliferating cell
nuclear antigen(6) . Hypophosphorylated Rb also binds to the
SV40 large T-antigen(7, 8) , the adenoviral protein
E1A(9) , and human papilloma virus protein E7(10) ,
each of which plays a role in both viral gene transcription and DNA
replication. Hypophosphorylated Rb also associates directly with
cellular transcription factor E2F, originally identified as a protein
binding to sites in the adenovirus E2A promoter (11) . E2F,
which binds the same region of Rb as do the D cyclins, T-antigen, and
E1A, activates many genes, including several involved in aspects of DNA
replication(12, 13, 14, 15, 16) .
The mammalian cellular protein Pur
has recently been implicated in
control of JC viral DNA gene transcription and DNA replication. Because
of homologies between Pur
and certain Rb-binding proteins, we have
examined the interaction of Pur
with the Rb protein.
Pur
binds both single-stranded and double-stranded DNA in a
sequence-specific fashion, but it has a 10-fold preference for the
purine-rich single strand of its recognition element, which consists of
repeats containing the sequence (GGN)
, where N is
not G(17) . Two members of a Pur protein family have been
identified, Pur
and Pur
, and the complete sequence and
DNA-binding properties of Pur
have been reported(18) . The
protein consists of 322 amino acids in human and 321 in
mouse(19) . Human and mouse Pur
proteins differ by only 2
amino acids, a very high degree of conservation. Pur
mRNA is
detected in every mammalian tissue thus far examined (17) .
Evidence from a variety of sources indicates that Pur is a
transcriptional transactivator. Pur
has been implicated in control
of transcription of genes activated by two different retroviruses. In
avian fibroblasts infected with Rous sarcoma virus v-src, a PUR element functions as an enhancer for the clusterin gene.
Positive regulation of clusterin gene transcription is mediated by an
avian Pur protein closely related to human Pur
(20) . In
human glial cells Pur
mediates activation of the late promoter of
neurotropic virus JC by the HIV-1 Tat
protein(21, 22) .
Pur
interacts
with the cellular protein YB-1, which binds to the opposite strand of
the JCV Pur
recognition element. The binding of Pur
to its
element is altered by association of YB-1 with the JC viral T-antigen,
possibly playing a role in the shift from early to late viral gene
transcription(22) . Pur
, encoded by a transfected
expression vector, stimulates transcription of a CAT reporter gene
linked to the myelin basic protein gene promoter, and a Pur
recognition site in this promoter is required for stimulation.
Transactivation may be mediated by a glutamine-rich domain,
including Q
, located near the COOH terminus of
Pur
(18) .
Pur binds a series of recognition
elements, located approximately 1.6 kilobases upstream of the P1
promoter of the human c-myc gene near the center of a recently
mapped zone of initiation of DNA replication(23) . The Pur
binding repeats are positioned at an intrinsic bend in the DNA near an
extensive AT-rich region(17) . Both DNA bending and AT-rich
elements are correlated with origins of
replication(24, 25) . The position of PUR elements in the c-myc locus, and in other reported
replication initiation
zones(24, 26, 27, 28) , is
reminiscent of their position in JCV. In the viral DNA PUR elements in the late-region promoter overlap the origin of
replication and are juxtaposed with AT-rich regions. Pur
recognition elements in such a configuration are present in two 98-base
pair repeats located on the late side of the central palindrome of the
core origin, and these elements are essential for T-antigen-mediated
replication(29) . These PUR elements are necessary for
helical alterations in the adjacent AT-rich region effected by
T-antigen helicase activity(30) . While Pur
and JCV
T-antigen may not directly associate they mutually interact with
cellular protein YB-1(22) . In this paper we show that Pur
and SV40 T-antigen mutually interact with Rb.
Pur has a region
of limited homology to T-antigen which may be involved in
protein-protein interaction(19) . The homology of Pur
to
SV40 large T-antigen involves a region in each protein beginning with
PTY and ending in SEEM (19) (aa's 251-278 in
Pur
). Allowing for a 2-amino acid gap, there are 9 identities in
26 residues. Although this homology is limited, it includes several
residues conserved not only in initiator proteins of DNA tumor viruses,
but also in certain cellular proteins potentially involved in
initiation, including MCM2 of yeast and BM28 of human
cells(19) . BM28 is a human homolog of MCM2 reportedly required
for entry of cultured cells into S phase of the cell
cycle(31) . Most intriguingly, the homology of Pur
to
T-antigen spans a region necessary for binding of T-antigen to Rb, the
product of the human Rb tumor suppressor
gene(7, 32) . In this regard it became of interest to
determine whether or not Pur
binds the Rb protein and whether or
not this motif is involved. We show here that this motif is included in
the region of Pur
involved in binding to the hypophosphorylated
form of Rb. Furthermore, Rb modulates the binding of Pur
to its
single-stranded recognition element, and presence of this element
affects the binding of Rb to Pur
.
CTAGC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GCG
G TAC ATG GGT ATG CTA CAA GGT CTA ATG CGA CGC CTG
M Y P Y D V P D Y A A
The resulting fragment was then ligated into the NheI and EcoRI sites of eukaryotic expression vector pBK-CMV
(Stratagene) downstream of the cytomegalovirus (CMV) promoter. The
resulting HA-Pur expression vector is termed pHAPur1. This vector
was transfected into cells using a modified calcium phosphate procedure (40) either alone or together with plasmid pCEP4-Rb,
expressing a full-length Rb protein under control of the CMV promoter.
5 µg of each vector were transfected per monolayer dish of 10
cells. Cells were harvested, lysed as described above for WR2E3
cells, and subjected to immunoextraction procedures as described in the
legends to the figures. Monoclonal antibody 12CA5 (38) was used
for immunodetection of the HA epitope.
Figure 1:
Pur binds the hypophosphorylated
form of p110
. GST-Pur
and GST-T, a mutant form of
SV40 large T-antigen, were synthesized in E. coli and bound to
glutathione-agarose beads as described under ``Experimental
Procedures.'' Unfused GST was prepared in the same manner for use
as a control. Beads containing equivalent amounts of each protein were
used for each lane. The amounts used represent approximately 40 pmol of
GST-Pur
or GST-T. Beads were collected, washed, and bound proteins
subjected to electrophoresis on 7.5% polyacrylamide SDS gels as
described previously(35) . After electrophoresis, proteins were
blotted to Immobilon membranes and probed with anti-Rb monoclonal
antibody 11D7 followed by alkaline phosphatase-conjugated secondary
antibody, and Rb protein bands visualized as described under
``Experimental Procedures.'' IPP represents Rb
immunoprecipitated from WR2E3 cells using rabbit polyclonal anti-Rb
antibody 0.47.
Figure 2:
Immunoprecipitation of a PurRb
complex from lysates of monkey CV-1 cells using monoclonal antibodies
to Pur
. Monkey CV-1 cells, not transfected with any vector, were
cultured as monolayers, pulsed with [
S]Met and
lysed as described under ``Experimental Procedures.''
Monoclonal antibodies were added to aliquots of lysate, and
antibody-antigen complexes extracted with protein A-agarose beads as
described. Lane labels refer to the precipitating antibody as follows. prot A, protein A-agarose beads alone were added to the lysate
with no prior antibody; mab's 16A2, 12A4, 9C12, 5B11, and 2B3, anti-Pur
monoclonal antibodies; anti-HA, monoclonal antibody 12CA5, to influenza virus coat
protein hemagglutinin (irrelevant antibody). Immunoprecipitates were
washed, proteins eluted and subjected to SDS-polyacrylamide gel
electrophoresis on a 10% gel. Bottomleftpanel, [
S]Met incorporation into
immunoprecipitated proteins as revealed by autoradiography of the gel.
Exposure was 72 h. Markers, not visible, are the Sigma high-MW
prestained standards. Topleftpanel,
anti-Rb Western blot of the gel autoradiographed at bottom. The gel was
electroblotted and probed with anti-Rb monoclonal antibody 11D7 using
the Renaissance system (DuPont NEN). Segments of the lanes
corresponding to 110 kDa are shown. Exposure of the filter was for 1.5
h. Bands from the first antibody, detected by the Renaissance second
antibody, are below 80 kDa and are not shown. As a control for possible
110-kDa bands generated by the first antibody, mab 5B11 was run alone
in the lane at right of this panel. Rightlane, specificity of reaction of anti-Rb mab 11D7 with a
CV-1 cell lysate. A single lane containing the cell lysate was run,
blotted, excised from the filter and subjected to Western blot analysis
with anti-Rb mab 11D7 as described for the top panel. Additional controls for the specificity of anti-Pur
mab and
the position of the Pur
bands in cell lysates are presented in Fig. 3.
Figure 3:
Immunoprecipitation, using an anti-Rb
monoclonal antibody, of a PurRb complex from lysates of CV-1
cells transfected to overexpress Pur
and Rb. CV-1, HeLa, and mouse
NIH-3T3 cells were transfected with vector pHAPur1, and G418-resistant
clones were selected as described under ``Experimental
Procedures.'' pHAPur 1 expresses Pur
mRNA, from the CMV
promoter, fused to a sequence encoding the 9-amino acid HA epitope tag (38) . Monolayers of selected clones were then transiently
transfected with vector pCEP4, which expresses a full-length Rb mRNA
from the CMV promoter. Lysates of cells were prepared as described and
subjected to immunoprecipitation using various mouse antibodies
followed by Dynal magnetic beads coupled to sheep anti-mouse antibody
as described under ``Experimental Procedures.''
Immunoprecipitates were washed, proteins eluted and subjected to
SDS-polyacrylamide gel electrophoresis on a 10% gel. Following blotting
of the gel to a membrane filter, the filter was cut so that parallel
lanes could be reacted with one of two different antibodies. Western
blots were performed according to the Renaissance protocol supplied by
DuPont NEN. Left panel, Western blot with anti-Pur
mouse
monoclonal antibody 12A4 followed by peroxidase-conjugated goat
anti-mouse antibody (Sigma). Lysate lanes indicate total lysates from
CV-1, HeLa, or NIH-3T3 cells as described. Imm. ext. lanes
indicate immunoextractions prepared with the following antibodies. No 1st ab, magnetic beads alone with no first antibody; Anti-Rb 11D7, monoclonal anti-Rb antibody 11D7. GST-Pur
thrombin is a lane with bacterially expressed GST-Pur
treated with the protease thrombin, releasing Pur
from GST, to
demonstrate that the antibody reacts with Pur
but not with GST
(27.5 kDa). Right panel, Western blot from parallel lanes to
those at left, but reacted with anti-HA rabbit polyclonal antibody
(BabCo) followed by peroxidase-conjugated donkey anti-rabbit antibody
(Amersham). No 1st ab, immunoprecipitation of the transfected
CV-1 lysate with magnetic beads but no first antibody; Anti-Rb
11D7, immunprecipitation of the lysate with anti-Rb mouse
monoclonal antibody 11D7 and magnetic beads; mab 9C12, mouse
anti-Pur
antibody 9C12, 20 ng; Rabbit ab 0.47, anti-Rb
rabbit polyclonal antibody 0.47, 20 ng.
Figure 4:
Formation of complexes between
GST-Pur and p56
is dependent on p56
concentration and is altered by single-stranded Pur
recognition element. Proteins were allowed to interact, subjected to
nondenaturing gel electrophoresis, blotted to a nitrocellulose
membrane, and the membrane incubated with labeled MFO677
single-stranded oligonucleotide as described under ``Experimental
Procedures.'' An autoradiograph is shown. p56Rb, purified
p56
, used at 320 ng in lane1 in absence
of other proteins or DNA; GST-Pur, purified
GST-Pur
, used at 80 ng in absence of other proteins or DNA (lane2) or in the presence of 100 ng unlabeled
oligonucleotide MFO677 (lane8); P + R1, etc., 80 ng of GST-Pur
incubated with a 1-, 5-, 20-,
50-, or 100-fold molar excess of p56
, respectively.
Samples for the seven lanes at the left were incubated and
loaded for electrophoresis in the absence of any DNA. For the six lanes
at the right (+Pur R.E.), proteins were
incubated and loaded for electrophoresis in the presence of 100 ng of
unlabeled single-stranded Pur
recognition element, MFO677. After
electrophoresis, gel lanes were electroblotted to a nitrocellulose
membrane, and the membrane was incubated with
P-labeled
single-stranded MFO677, washed, and subjected to autoradiography as
described under ``Experimental
Procedures.''
Figure 5:
Mutational analysis of specific
single-stranded DNA-binding regions of the Pur protein. Deletion
mutants of the Pur
cDNA, cloned as a fusion gene in vector
pGEX-1
T, were constructed as described under ``Experimental
Procedures'' and subjected to standard gel band-shift analysis (17) using labeled single-stranded 24-mer oligonucleotide
MFO677, representing the c-myc PUR element. A, depiction of deletion mutants with respect to structural features
of the Pur
protein. The NH
-terminal GST portion of
each fusion protein is omitted from this diagram. The * after 1-314 is
to note that whereas 8 amino acids have been deleted, 14 other amino
acids have been added. B, autoradiograph of a gel band-shift
analysis carried out on a 6% polyacrylamide gel(17) . -
and + lanes refer to the absence or presence of a 30-fold excess
of unlabeled competitor MFO677 DNA. GST-Pur lanes refer to a
binding reaction carried out with full-length GST-Pur
. Other lanes
refer to GST-Pur
deletion mutants described in panelA.
Figure 6:
Binding to p110 requires a
region of the Pur
protein comprising amino acids 216-274.
Binding of Rb in extracts of WR2E3 cells to affinity columns containing
GST, GST-T, GST-Pur
, or GST-Pur
deletion mutants was carried
out exactly as described for Fig. 1. A, description of
GST-Pur
deletion mutants employed in panel B. Refer to Fig. 5A for structural features of Pur
. B, proteins bound to columns were subjected to SDS-gel
electrophoresis, transferred to an Immobilon membrane, reacted with
anti-Rb monoclonal antibody 11D7, and visualized as described for Fig. 1. IPP refers to an immunoprecipitate of Rb
protein from WR2E3 cells. Each other lane consists of protein bound to
the particular affinity column indicated.
Figure 7:
Binding of Pur to p56
and inhibition by a synthetic peptide containing the Rb-binding
motif of SV40 large T-antigen. The mutant p56
was
incubated with either Sepharose-GST beads (GST; 5-µl bead
volume; Pharmacia) or Sepharose-GST-Pur
beads (GST-Pur;
5-µl bead volume) in lysis 150 buffer with bovine serum albumin
(1.0 mg/ml), with or without 33 pmol of p56
, in the
presence or absence of synthetic peptide (T-Ag pept), 2X, 10X,
or 100X molar excess over p56
. The sequence of the peptide
is: KKENLFCSEEMPSSDDEATA, all but the first two lysines were derived
from the T-antigen sequence, with the LXCXE motif
printed in bold. After incubation at 22 °C for 45 min,
beads were collected by centrifugation, washed, and proteins eluted
with SDS sample buffer and subjected to SDS-polyacrylamide gel
electrophoresis on a 10% gel. Production of Sepharose-GST-Pur
beads was as described for agarose-GST-Pur
beads, and washing was
conducted with lysis 150 buffer as described under ``Experimental
Procedures.'' Proteins were blotted to an Immobilon membrane and
probed with anti-Rb monoclonal antibody 11D7 using the Renaissance
system as described in the legend to Fig. 3. The lane at the right (CV-1 lysate) contains 50 µg of
total protein from a CV-1 cell lysate to indicate the position of
full-length Rb at approximately 110 kDa. Markers, not visible, were the
Sigma high-MW prestained markers.
Several cellular and viral proteins have now been identified
which bind to Rb. For certain of these proteins interaction has been
demonstrated by co-immunoprecipitation from cell extracts or through
use of yeast hybrid-protein interaction systems, evidence taken by many
investigators to indicate in vivo interaction (see Goodrich
and Lee (45) for a review). The results of this study
demonstrate that Pur binds specifically to the hypophosphorylated
form of the Rb protein, p110
( Fig. 1and Fig. 6), and that the Pur
Rb complex is present in
CV-1 cell extracts ( Fig. 2and Fig. 3). Although several
different proteins bind specifically to the hypophosphorylated form of
Rb, a variety of binding mechanisms are implicated. Hypophosphorylated
Rb binds to three known viral proteins, SV40 large
T-antigen(7, 8) , adenoviral protein E1A(9) ,
and human papilloma viral protein E7(10) . Each of these
proteins possesses a region of 23-26 aa's, with limited
homology, containing the motif LxCxE(19) . The three viral
proteins each bind to a T/E1A domain located in the COOH-terminal
portion of Rb. Most naturally occurring mutations in rb in
cancers are found in this T/E1A domain(46, 47) . In
mammalian cells the Ets-related transcription factor Elf-1 (32) and the cell cycle regulatory proteins, cyclins D1, D2,
and D3(4, 5) , also possess the LxCxE motif in their
regions of Rb binding. In contrast, the Rb-binding domain of the
transcriptional regulatory protein E2F possesses neither the viral
protein homology nor the LxCxE motif, but E2F also binds the
hypophosphorylated form of
Rb(12, 13, 14, 15) . A domain near
the COOH terminus of E2F containing approximately 33% acidic residues
may be involved in Rb binding(13, 14, 15) .
Although E2F does not bind Rb through an LxCxE motif, E2F does bind to
the COOH-terminal portion of Rb, as do the viral proteins. Recently a
nuclear matrix Rb-binding protein, p84, has been identified using the
yeast two-hybrid system(44) . This protein binds specifically
to the amino-terminal portion of Rb. As do the other proteins cited
here, p84 binds to the hypophosphorylated form of Rb. Thus evidence
points to a complex array of protein interaction mechanisms involving
Rb, all subject to control by phosphorylation. The region of Pur
essential for Rb-binding, delineated in Fig. 6, includes a
region of 28 aa's, the psycho motif, with limited homology to the
T-antigen Rb-binding motif but lacking the LxCxE
configuration(19) . At the COOH terminus of Pur
lies a
segment of 18 aa's with 39% Asp and Glu residues. Disruption of
this region strongly inhibits Rb binding but does not eliminate it.
This mutant was not included in Fig. 6because the disruption
was not technically a deletion mutant but a combination of 8-aa
deletion and 14-aa insertion. Binding of Pur
to Rb may thus be
complex in that disparate Pur
domains influence the interaction.
Nonetheless, the region of Rb bound by Pur
includes or overlaps
the region bound by SV40 large T-antigen. This binding domain is
retained in p56
, and its binding to Pur
is blocked by
a 20-aa peptide containing the T-antigen Rb-binding motif (Fig. 7).
The interaction of Pur and Rb in cells is
highly modulated. Pur
binds only to a hypophosphorylated form of
Rb, and Rb binds to a post-synthetically altered form of Pur
. Both
of these proteins are capable of forming large multi-protein
aggregates. Rb is a nuclear protein, and Pur
is a nuclear protein
which can also be extracted from cytoplasm. Little is known about the
cellular compartmentalization of modified forms of either Rb or
Pur
. It is conceivable that within a particular cell compartment, e.g. the nucleus, a hypophosphorylated form of Rb could
sequester, through binding and aggregation, most or all Pur
molecules even though the latter may be present in molar excess. Such
sequestering would be subject to additional modulation if only a
specifically modified form of Pur
were the target of Rb binding.
It is equally likely that Pur
could bind, and potentially alter
activity, of most or all hypophosphorylated Rb molecules within a given
cell compartment. Studies on the cellular distribution of modified
forms of Pur
and Rb will help elucidate the functional
significance of their interaction at particular cell locations.
Association of Pur and Rb affects the interaction of Pur
with its single-strand DNA recognition element in a dynamic fashion.
When Rb is hypophosphorylated, as it is in early G1, it forms
filamentous aggregates which are tethered to the inner regions of the
nuclear membrane, possibly through associations with lamins A and C (48) or to the nuclear matrix(44) . In this state Rb is
associated with Pur
in cells, and this would limit access of
Pur
to many, perhaps all, of its recognition elements in
chromosomes in a manner yet to be detailed. We show here that Pur
also forms multimeric aggregates and that this formation is promoted by
Rb (Fig. 4). The Pur
Rb complex is disrupted by Rb
phosphorylation ( Fig. 1and Fig. 6) and by the presence
of the single-stranded DNA recognition element (Fig. 4). Rb
phosphorylation begins prior to entry of cells into S-phase. The
presence of single-stranded DNA at origins of replication could provide
a reinforcing mechanism for desequestering Pur
from Rb since
Pur
has a 10-fold preference for binding to the purine-rich single
strand of its recognition element. While it is intriguing to speculate
a role for Pur
in creating such single-stranded regions, there is
presently no evidence for this in cells. For most Rb-binding proteins
the functional consequences of the interaction are not known. In the
present case we have established the nature of Rb binding to Pur
in vivo and in vitro. Studies of association of
Pur
and Rb in the cell cycle, beyond the scope of the present
publication, will help to detail functional aspects of the interaction.
Rb phosphorylation occurs primarily on Ser and Thr residues and is
modulated during the cell cycle, Rb phosphorylation beginning in late
G1(1, 2) . Each of the three Rb-binding viral proteins
described above plays a role in cell transformation, and, in the case
of T-antigen, in the initiation of viral DNA replication. E2F is
involved in regulation of transcription of several genes, most if not
all of which are themselves involved indirectly in regulation of DNA
synthesis. For example, the dhfr gene, regulated by
E2F(16) , controls a step in the generation of nucleotides
necessary for DNA synthesis. Certain D-type cyclins which associate
with the Rb protein also associate with the DNA polymerase
processivity factor proliferating cell nuclear antigen(6) .
Nonetheless, gene knockout experiments call into question a universal
role for Rb in the cell cycle control of DNA replication. Homozygous rb
fetal mice develop apparently normally
until day 12-14 of embryogenesis, at which point they
die(49, 50) . At death, defects are found in
development of both the hematopoietic system and the hindbrain, two
systems which require a cessation of proliferation for terminal
differentiation. Since DNA replication clearly occurs in the early
embryonic cells of rb
mice, Rb cannot be
essential for initiation at all origins of replication at that stage.
However, it is also clear that an aberrant lack of cessation of
proliferation occurs in the blood and brain tissues. It is conceivable
that this loss of control occurs at the level of initiation of DNA
replication, particularly if certain Rb-binding proteins, possibly
including Pur
, influence replication initiated at only a specific
subset of chromosonal loci. It is also conceivable that Rb itself does
not play a primary role in cell cycle control of proliferation, but
that other Rb-like proteins, such as p107 or p130(51, 52, 53, 54, 55, 56) are
more directly involved. The p107 protein in human U937 cells is found
in a complex with cyclin A and the cdc2 kinase p33, two proteins
thought to be involved in entry of cells into
S-phase(57, 58) . The p130 protein associates with
CDK2 and cyclins A, D
, D
, D
, and
E(53, 54) . It is not presently known whether Pur
associates with p107 or p130, although both of these proteins possess
homology to the Rb T-antigen-binding domain.
It is of interest
regarding Pur that rb
mice are
arrested in development with defects in the hematopoietic system. The
human pur
gene has recently been localized to chromosome
band 5q31(59) . Partial deletion of 5q is commonly observed in
myelodysplasias and acute nonlymphocytic leukemias, causing clinical
and hematological symptoms termed the 5q
syndrome.
Recent observations indicate that 5q31 is the most common deleted locus
among 5q
individuals(3, 60) . It is
conceivable that Pur
and Rb participate in a common regulatory
pathway that can be altered aberrantly in different ways to cause
defects in development or cancer.