From the
Recent genetic and functional evidence suggests that the amino
terminus of the retinoblastoma (Rb) protein plays an important role in
Rb-mediated growth suppression. To explore the mechanism(s) by which
this portion of Rb may regulate cell growth, we have sought to
characterize cellular proteins that associate with the Rb amino
terminus using an in vitro protein-binding assay. Here we
report that at least one such protein is a cell cycle-regulated
Rb/histone H1 kinase (RbK) whose enzymatic and/or Rb association
activity is most prevalent in G
The retinoblastoma (Rb)
Rb-1 encodes a set of 105-kDa nuclear phosphoproteins that are
distinguished from one another by their extent of post-translational
modification
(9, 17, 18, 19) . The state
of Rb phosphorylation is quite dynamic during the mammalian cell cycle;
resting, senescent, or terminally differentiated cells express largely
unphosphorylated p105-Rb, whereas in cycling cells Rb is phosphorylated
prior to S phase, as well as during S and G
Reconstitution of
Rb-negative tumor cells with a wild-type Rb gene has been shown to
block or reduce tumorgenicity and additional parameters of
transformation in some, but not all, cell systems
(31, 32, 33, 34, 35, 36, 37) .
Microinjection of the carboxyl-terminal two-thirds of p105-Rb into
certain Rb-negative tumor cells has been shown to block subsequent DNA
synthesis and suggested that Rb's growth-limiting function may be
restricted to a discrete temporal ``window'' approximately 6
h prior to the initiation of S phase
(38) . These results
suggested that Rb function may be critically important for the
management of cell cycle progression through a G
However, this
simplified view of Rb activity has been challenged by the results of
recently reported functional assays. Karantza et al. (49) reported that the induced expression of full-length p105-Rb
in synchronously growing S phase cells resulted in the arrest of cell
cycle progression in G
As a first step toward the biochemical and
functional characterization of the Rb amino terminus, we sought to
identify cellular proteins that specifically bound the amino terminus
of human and mouse Rb proteins. Here we report the detection of a novel
cell cycle-regulated serine-threonine kinase that specifically
associates with this portion of Rb and phosphorylates the Rb amino
terminus and histone H1 in vitro.
GST fusion proteins were used in in vitro protein-binding assays as described previously
(61) with
the following modifications. Mammalian cell extracts were prepared from
cells that were washed twice with phosphate-buffered saline (PBS) and
lysed by the addition of 1 ml of EBC buffer (50 m
M Tris, pH
8.0, 120 m
M NaCl, 0.5% Nonidet P-40, 100 m
M NaF, 200
µ
M sodium orthovanadate, 1 m
M
phenylmethylsulfonyl fluoride, and 10 µg/ml each of pepstatin and
leupeptin). Following incubation for 30 min on ice, cell lysates were
clarified by centrifugation at 10,000
To
prepare amino-terminal Rb reagents for in vitro protein-binding assays, Rb oligonucleotides and the PCR were used
to generate a GST fusion protein that encodes the first 380 amino acids
of human Rb. In addition, an equivalent GST-Rb fusion protein was
prepared from the mouse Rb cDNA. These fusion proteins include coding
sequence from the first 11 Rb exons and are comprised of amino acids
outside of the region of Rb required for association with viral
oncoproteins and cyclin
To determine
if excess p13
The biochemical results presented in this report support and
extend previous genetic and functional studies indicating that the
amino-terminal 380 amino acids of Rb comprise a domain of Rb function
(49, 50, 51, 52) . Using an in vitro protein-binding assay, we have determined that at least one N-RBP
is a novel p13
Our in vitro protein-binding assays have identified at least two biochemically
distinct RbKs that interact with Rb via its amino terminus. The RbKs
differ from one another with respect to their propensity to physically
associate with p13
The identities of the
RbKs are at present unknown. Our results indicate that two Rb kinases,
cdc2 and cdk2, are unlikely to be G
Perhaps the most
compelling evidence that an intact Rb amino terminus is required for
tumor-suppression derives from analyses of mutated Rb alleles in
retinoblastoma. Dryja et al. (52) described a large
kindred with low penetrance retinoblastoma in which affected members as
well as asymptomatic carriers inherited a defective Rb allele with a
precise genomic deletion of exon 4. Inheritance of this Rb allele
predisposed a fraction of individuals to unilateral or bilateral
retinoblastoma. Similarly, Hogg et al. (51) identified
an individual unrelated to the previous family with bilateral
retinoblastoma associated with a single nucleotide deletion within the
splice donor site of exon 4. Through genomic deletion or aberrant
splicing, loss of exon 4 sequences is predicted to lead to the
biogenesis of an aberrant Rb message with an in-frame fusion of exons 3
and 5. Whether the association of RbK with the Rb amino terminus is
necessary for Rb-mediated growth suppression is currently under
investigation. However, consistent with this supposition preliminary
mapping experiments indicate that amino acids contained within Rb exon
4 are required for the recovery of wild-type levels of RbK binding
activity.
Given the specific and cell cycle-regulated association of RbK
with Rb, we speculate that Rb
A mitotic (nocodozole-arrested) A549
extract was precleared with equivalent amounts of either GST-bound
glutathione-agarose or p13
We thank Drs. Joseph R. Nevins, Michael C. Ostrowski,
and Katherine I. Swenson for critically reviewing this manuscript and
members of the Horowitz laboratory for helpful discussions and support.
We also thank Katherine I. Swenson for cyclin-cdk reagents and
technical support, Charles Minkoff for help with phosphoamino acid
analyses, Dr. Nicholas Dyson for monoclonal antibodies against Rb and
p107, Dr. Mark E. Ewen for supplying a human p107 cDNA construct, Dr.
Roger J. Davis for erk1/erk2 antisera, Dr. Nicholas K. Tonks for
purified PTP1b, and Drs. Paul Nurse and Mariano Garcia-Blanco for cdk1
antisera and GST-FH15, respectively.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/M phases of cells. In
contrast to previously characterized cyclin-dependent and Rb-associated
kinases, such as cdk1 (cdc2) and cdk2, G
/M RbK 1) is not
depleted by incubation with p13
-beads, 2) is not detected
with antisera against several Rb-associated cyclins-cdks, and 3)
associates with Rb via the Rb amino terminus, a region that is
dispensable for interaction with other Rb-associated kinases. RbK is
clearly distinct from previously characterized mitotic cdks since
cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 did not
associate with the Rb amino terminus. Coprecipitation experiments with
Rb antisera confirmed the association of Rb with a RbK-like kinase in
metaphase-arrested cells in vivo. Interestingly,
G
/M RbK did not appreciably associate with an analogous
portion of p107, a Rb-related protein. Taken together, these data
indicate that the Rb amino terminus specifically associates with a
novel cell cycle-regulated kinase in late cell cycle stages.
(
)
gene,
Rb-1, spans a 200-kilobase pair segment of the q14 region of
human chromosome 13
(1, 2, 3) . Mutational
inactivation of this gene has been clearly implicated in both sporadic
and familial forms of retinoblastoma (a rare intraocular tumor) and
secondary neoplasms of familial retinoblastoma patients such as
osteosarcoma
(4) . Functional inactivation of Rb-1 is
also associated with the genesis of a variety of human tumors in the
general population, including small cell lung, breast, and bladder
carcinomas
(5, 6, 7, 8, 9, 10, 11, 12) .
In addition to its role in human cancer, the Rb protein (p105-Rb) is
involved in another pathway to deregulated cell growth and
tumorigenesis, that being transformation induced by several families of
DNA tumor viruses. Adenovirus E1A, the large-T antigens of SV40 and
polyomavirus, and the E7 protein of papillomavirus can cause the
outgrowth of tumorigenic cells partly dependent on their formation of
stable complexes with p105-Rb
(13, 14, 15, 16) .
, and then is
progressively dephosphorylated during late stages of mitosis
(20, 21, 22, 23) . Rb is likely to be
phosphorylated by more than one kinase in vivo. Rb kinases
identified to date have proven to be members of the cyclin-dependent
kinase (cdk) family. cdk1 (cdc2), cdk2, and cdk4 phosphorylate Rb
in vitro at a subset of sites that are phosphorylated in
vivo and cdks 1 and 2 have been found in association with Rb
in vivo (19, 24, 25, 26, 27, 28) .
The cyclin component of these Rb
kinase complexes has yet to be
definitively established, although transfection of cyclins A, D, and E
can stimulate Rb phosphorylation in vivo (29, 30) . It is believed, but not as yet certain,
that Rb function is regulated by these cell cycle-dependent
phosphorylation and dephosphorylation reactions.
``checkpoint'' subsequent to which a cell is committed
to replicating DNA. Consistent with the notion that the Rb carboxyl
terminus plays an important role in mediating growth suppression, this
portion of Rb has been shown to be a ``hotspot'' for
mutations in human tumors
(5, 6, 7, 8, 9, 31, 39) .
Additionally, it is this portion of p105-Rb that is essential for
binding of Rb by viral oncoproteins and for the interaction of Rb with
cellular proteins, such as transcription factors ( e.g. E2F-1)
and cyclin
cdk complexes
(40, 41, 42, 43, 44, 45, 46) .
Precise mapping experiments with Rb mutants created in vitro have corroborated evidence from tumor cells, delineating amino
acids within exons 12 through 18 and 19 through 22 (the so called
Rb-``pocket'') as being essential for p105-Rb to complex with
these viral and cellular proteins
(47, 48) . Thus, the
carboxyl-terminal two-thirds of p105-Rb are believed to be necessary
and perhaps sufficient for at least one Rb function.
. Interestingly, an Rb cDNA carrying
a mutation within the Rb carboxyl terminus that abrogates the
association of Rb with SV40 large-T antigen also led to growth arrest
in G
suggesting that regions outside of the Rb pocket are
necessary for growth arrest in this cell cycle compartment
(49) . In addition to these observations, a variety of
amino-terminal Rb mutations have been shown by Qian et al. (50) to abrogate Rb-mediated growth suppression and to block Rb
phosphorylation in vivo despite the retention of E2F-binding
activity within the Rb pocket. More importantly, rare instances of
mutations within the Rb amino terminus have been detected in
retinoblastoma patients, supporting the supposition that lesions within
this portion of Rb result in profound loss-of-function mutations
(51, 52) . The precise function(s) of the
400 amino
acids of Rb that are linked upstream of the Rb pocket domain is(are)
unknown. This amino-terminal portion of Rb is well conserved in human,
mouse, chicken, and Xenopus Rb proteins, and results of in
vitro mutagenesis experiments suggest that this region is also
critical for Rb-mediated growth suppression
(50, 53, 54, 55) . As such, Rb appears
to be modular in structure, possessing a protein-binding domain within
the Rb carboxyl terminus and a separable upstream region of as yet
unknown function.
Cell Lines and Cell Culture
A549 and 5637 cells
were obtained from the American Type Culture Collection (ATCC,
Rockville, MD), ML-1 cells were obtained from Dr. Stephen H. Friend
(Massachusetts General Cancer Center, Charlestown, MA), and CHO cells
were obtained from the Duke Comprehensive Cancer Center shared tissue
culture facility. Cells were cultured in Dulbecco's modified
minimal essential media (DMEM) or F12 (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal bovine serum (Hyclone,
Inc., Logan, UT) and penicillin/streptomycin under 5% COin
a humidified incubator at 37 °C.
Antisera, Immunoprecipitations, and Western
Blotting
Anti-Rb ascites fluid was prepared from XZ77
(56) hybridoma cells (a gift of Dr. Nicholas Dyson,
Massachusetts General Hospital Cancer Center). To generate polyclonal
antisera against human Rb protein, a full-length human Rb cDNA in
plasmid pBSKwas used as substrate for the PCR using
the exon 1 and exon 11 oligonucleotides indicated below. A resulting
1.1-kilobase pair amino-terminal Rb fragment was inserted in frame into
pGEX2TK, a bacterial-fusion protein expression vector (Pharmacia
Biotech, Inc.). This portion of the Rb cDNA encodes the amino-terminal
380 amino acids of human Rb protein. Following bacterial
transformation, fusion proteins were induced with IPTG, and a 68-kDa
GST-Rb fusion protein was purified as described previously
(57) . For immunizations, a single New Zealand White rabbit (no.
9300) was sequentially immunized with 150 µg of affinity-purified
fusion protein in Freund's complete and incomplete adjuvants.
Polyclonal rabbit anti-cyclin A (Ab-1) and PSTAIRE (Ab-1) antibodies
were obtained from Oncogene Science (Uniondale, NY), a polyclonal
rabbit anti-human cdk1 carboxyl-terminal peptide antiserum was provided
by Dr. Paul Nurse (Imperial Cancer Research Fund, Oxford, United
Kingdom), and polyclonal rabbit anti-cyclin A and E antisera were
obtained from Dr. Steven I. Reed (Scripps Institute, La Jolla, CA). A
monoclonal antibody (no. 107) that detects the MAP kinases erk1/erk2
was a kind gift of Dr. Roger J. Davis (University of Massachusetts
Medical Center, Worcester, MA). Monoclonal antibodies prepared against
p107 (SD2, SD4, SD6, SD9, and SD15) were kindly provided as hybridoma
supernatants by Dr. Nicholas Dyson (Massachusetts General Cancer
Center). A mixture of these supernatants was prepared and utilized for
immunoprecipitations of p107. For immunoprecipitations with XZ77 or
anti-p107 antiserum, whole-cell extracts were prepared and treated as
described previously
(56) . Western blots were performed as
previously described
(58) and antigen-antibody complexes were
detected using an enhanced chemiluminescent system (ECL, Amersham
Corp.) and exposure to Hyperfilm (Amersham) at ambient temperature.
Construction of GST Fusion Proteins
A bacterial
plasmid containing a human Rb cDNA, pBSK-HRbC
(59) , was
linearized with BamHI and Rb exons 1 through 11 were amplified
by the PCR using Vent polymerase (New England Biolabs, Inc., Beverly,
MA), a thermal cycler, and the following oligonucleotides as primers:
5`-GTCATGCCGCCCAAAACCCCCCGAAAA-3` (exon 1) and
5`-TAACTGGAGTGTGTGGAGGAATTATAT-3` (exon 11). Rb oligonucleotides were
synthesized with sites for BamHI cleavage at their 5` ends and
amino-terminal Rb PCR products were digested with BamHI and
ligated with BamHI-digested pGEX2TK (Pharmacia), generating
plasmid pGEX2TK-HuRb, such that the Rb reading frame is collinear with
that of GST. To generate a similar mouse GST-Rb fusion protein, a
bacterial plasmid carrying the mouse Rb cDNA, pBSK115ROX
(53) ,
was digested with NaeI and DraI to release a
1.3-kilobase pair fragment that encodes the amino-terminal 400 amino
acids of mouse Rb protein. This fragment was subsequently ligated in
frame with SmaI-digested pGEX1N (Pharmacia). A full-length
human p107 cDNA
(60) , a kind gift of Dr. Mark E. Ewen, Dana
Farber Cancer Institute, Boston, MA, was linearized with BamHI
and amplified as above using the following oligonucleotides as primers:
5`-ATGTTCGAGGACAAGCC-3`, and 5`-GGAGTAATGACTGCTTC-3`. p107
oligonucleotides were synthesized with BamHI ends, and the
amplified product was subsequently cloned in pGEX2TK (Pharmacia),
fusing the amino-terminal 386 amino acids of p107 in-frame with GST.
The nucleotide sequences at the junctions of each construction were
verified by double-stranded DNA sequencing using Sequenase (United
States Biochemicals Corp. Cleveland, OH),
[S]dATP, and GST or Rb oligonucleotide primers.
A carboxyl-terminal GST-Rb fusion protein has been previously described
(61) . A control GST fusion construct, pGEX-FH15, encoding a
Schistosome surface antigen was a gift of Dr. Mariano
Garcia-Blanco (Duke University Medical Center, Durham, NC).
Expression and Purification of GST Fusion Proteins and in
Vitro Protein Binding and Kinase Assays
BL21 bacterial cells
were transformed with GST fusion plasmids, and fusion proteins were
purified following induction with IPTG and collection on
glutathione-agarose beads (Sigma). Fusion proteins were harvested from
100 to 1000 ml of post-induction bacterial extracts with
glutathione-agarose beads that had been previously equilibrated with
NETN (20 m
M Tris, pH 8.0, 100 m
M NaCl, 1 m
M
EDTA, 0.5% Nonidet P-40) supplemented with 0.5% powdered milk. Proteins
were visualized by staining with Coomassie Brilliant Blue, and protein
concentrations were estimated by comparison with bovine serum albumin
standards.
g for 10 min at
4 °C. Aliquots of cell extracts (0.5 ml, 5
10
to 1
10
cell equivalents) were precleared of
proteins with affinity for the GST moiety by incubation with gentle
rocking for 1 h at 4 °C with at least 5 µg of GST protein bound
to glutathione-agarose beads. GST protein-bound beads were pelleted by
brief centrifugation and discarded. Precleared cell extracts were then
incubated with 5 µg of bead-bound GST fusion proteins with gentle
rocking for 1 h at 4 °C. Following exhaustive washes with NETN,
protein-bound beads were resuspended in 30 µl of 50 m
M
HEPES, pH 7.2, containing 10 or 20 m
M MgCl
, 1
m
M dithiothreitol, 1 m
M phenylmethylsulfonyl
fluoride, 10 µCi of [
P]ATP, and 25
µ
M unlabeled ATP. In some experiments 0.1 mg/ml histone H1
(Sigma) or 1 µg of various GST fusion proteins or purified cellular
proteins were included as kinase substrates. GST-E2F-1 and purified
phosphotyrosine phosphatase 1B (PTP1B) were kind gifts of Drs. Joseph
R. Nevins (Duke University Medical Center) and Nicholas K. Tonks (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY). After incubation for
10-20 min at room temperature, samples were boiled in Laemmli
sample buffer and resolved on SDS-polyacrylamide gels. Radiolabeled
kinase substrates were visualized following exposure to Hyperfilm for 5
h to overnight at -80 °C. Phosphoamino acid analyses were
performed as described previously
(19, 62) .
Protein-binding and in vitro kinase assays that employed
extracts from vaccinia virus-infected cells
(63, 64) utilized unlabeled whole cell extracts prepared from cells
multiply infected with recombinant viruses carrying epitope-tagged
cyclin A-cdc2 and cyclin A-cdk2 or cyclin B-cdc2 and cyclin B-cdk2
cDNAs. Each cyclin cDNA was epitope tagged at its amino terminus with a
nine amino acid moiety (NH
-EEEEYMPME-CO
H)
derived from the medium T antigen of polyoma virus to which a
monoclonal antibody was available.
p13
BL21 cells were transformed with a bacterial
p13Purification and Extract
Depletion
expression plasmid, pRKDSUC (a gift of Dr. Katherine
I. Swenson, Duke University Medical Center), and p13
expression was induced by the addition of IPTG to cultures at an
OD
of 0.6. After 4 h of growth at 37 °C, cells were
harvested, resuspended in p13
lysis buffer (50
m
M Tris pH 8.0, 5 m
M EDTA, 10% glycerol, 1
m
M phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and
pepstatin), sonicated, and clarified by centrifugation at 17,400
g for 30 min. Recovered supernatants (6 ml) were
applied to a Sephacryl S-200 column (100 cm
2.6 cm)
equilibrated in 0.1
M NaHCO
, pH 8.3, and 0.5
M NaCl. Aliquots of column fractions were resolved on 13%
SDS-polyacrylamide gels, stained with Coomassie Brilliant Blue, and
fractions containing pure p13
protein were pooled and
coupled to CNBR-Sepharose (5 mg of p13
protein/ml of
Sepharose) according to the instructions of the manufacturer
(Pharmacia). To preclear cell extracts of p13
-associated
kinases, 0.5 ml of unlabeled cell extracts were incubated with 100
µl of p13
-Sepharose beads (containing 500 µg of
p13
protein) with gentle agitation for 60 min at 4
°C. Precleared cell extracts were then clarified by centrifugation
at 4 °C and supernatants examined for in vitro Rb binding
activity and/or histone H1 kinase activity.
Cell Cycle Synchronization and Growth
Arrest
Exponentially growing A549 cells were arrested in early
Gby incubation in methionine-free media supplemented with
10% fetal bovine serum for 48 h. Cells were arrested at the
G
/S boundary by incubation in DMEM supplemented with 5
µg/ml aphidicolin (Sigma) and 0.5 m
M hydroxyurea (Sigma)
for 48 h. To obtain an S phase cell population,
G
/S-arrested cells were washed with serum-free DMEM, refed
with complete DMEM and harvested 6 h later. Populations of cells
arrested in mitotic metaphase were prepared by incubation of cells in
0.4 µg/ml nocodazole (Sigma) for 16 h. CHO cells were synchronized
as described by Seth et al. (65) , and cell cycle
status was monitored by propidium iodide staining and flow-cytometric
analysis. Approximately 2
10
cells were collected
by trypsinization or gentle agitation (metaphase-arrested cells),
washed with PBS, and fixed in methanol for 10 min at -20 °C.
Fixed cells were washed with PBS, resuspended in 10 µg/ml
DNase-free RNase (Sigma), and stained with a solution containing 50
µg/ml propidium iodide for 30 min at room temperature. The timing
of DNA synthesis was determined by monitoring the incorporation of
[
H]thymidine into mammalian cell DNA. Briefly,
cells were incubated with 5 µCi/ml
[
H]thymidine in serum-free media, trypsinized,
washed with PBS, and precipitated by the addition of 10%
trichloroacetic acid. Incorporation of
[
H]thymidine into cell precipitates was
quantified by scintillation counting.
The Amino Terminus of Rb Specifically Associates with a
Histone H1/Rb Kinase That Phosphorylates Rb in Vitro
Since
previous reports have suggested that the Rb amino terminus is
functionally important, we sought to identify and characterize cellular
proteins that specifically interact with this portion of Rb. We
reasoned that cellular proteins (N-RBPs) that associate with Rb via its
amino terminus in vivo might form stable complexes with Rb
in vitro and thus might be detected by affinity
chromatography. Furthermore, we speculated that one or more N-RBPs
might be a cell cycle-regulated protein kinase since Rb is
phosphorylated in vivo in a cell cycle-dependent manner.
cdk complexes (the Rb pocket). Following
induction and purification from bacteria, GST-Rb fusion proteins were
bound to glutathione-agarose beads and used as affinity reagents in an
in vitro protein-binding assay followed by an in vitro kinase assay. A549 human adenocarcinoma (Rb-positive) cells were
utilized to prepare unlabeled, cell cycle-staged extracts following
growth arrest by methionine deprivation, treatment with
hydroxyurea-aphidicolin, or nocodazole. Extracts were incubated with
glutathione-agarose beads that were bound to either GST-Rb or a
heterologous GST fusion protein (GST-FH15), and beads were exhaustively
washed and subsequently examined for kinase activity following the
addition of [
-
P]ATP and histone H1 as
exogenous substrate. As shown in Fig. 1 A, extracts incubated
with human GST-Rb contained histone H1 kinase activity that was not
detected with glutathione-agarose beads bound to GST-FH15. Histone H1
kinase activity (termed RbK) was noted to be low in A549 extracts
prepared from cells in G
, G
/S, and S phases,
and maximal in mitotic extracts. Identical results were obtained with
an analogous GST-Rb fusion prepared from a mouse Rb cDNA (data not
shown). Moreover, as shown in Fig. 1 B the GST-Rb fusion
protein utilized as an affinity reagent to recover RbK activity is also
an in vitro substrate of mitotic RbK. In contrast to
RbK-mediated phosphorylation of histone H1, efficient in vitro phosphorylation of GST-Rb was noted to require a somewhat lower
Mg
concentration (Fig. 1 B).
Significantly, in the absence of exogenous histone H1, GST-Rb was the
only phosphorylated protein detected in in vitro kinase assays
following incubation of GST-Rb with mitotic extracts. Neither RbK
itself nor additional exogenous substrates we have examined, including
Sp1, E2F-1, PTP1B, or GST , were appreciably phosphorylated by
RbK in vitro, suggesting that the phosphorylation of GST-Rb
and histone H1 by RbK is specific (Fig. 1 B and data not
shown). We have also observed that RbK phosphorylation of Rb is largely
confined to the amino terminus of human and mouse Rb proteins; GST-Rb
fusion proteins carrying the carboxyl-terminal 600 amino acids of Rb
are not appreciably phosphorylated by mitotic RbK in vitro (data not shown). To identify the amino acids phosphorylated by
RbK, phosphoamino acid analyses of radiolabeled histone H1 and GST-Rb
proteins were performed. Results of these experiments showed that
serine -threonine residues are substrates of mitotic RbK (data
not shown).
Figure 1:In vitro protein binding and
kinase assays. Panel A, in vitro histone H1 kinase
assay. Whole-cell extracts were prepared from human A549 adenocarcinoma
cells arrested in early Gby methionine deprivation
(G
), at the G
/S transition by treatment with
hydroxyurea/aphidicolin (G
/S), or cells arrested at
metaphase by nocodazole addition ( M). In addition, a
synchronously growing population of S phase cells prepared 6 h
following release from hydroxyurea arrest ( S) was included for
analysis. Following preclearing of cell extracts with GST-protein bound
to glutathione-agarose, extracts were incubated with GST fusion
proteins, and in vitro kinase assays were performed subsequent
to the addition of [
-
P]ATP and histone H1
protein in 20 m
M Mg
. Radiolabeled proteins
were resolved on a SDS-polyacrylamide gel and visualized by
autoradiography. Left, histone H1 kinase activity recovered
following incubation of cell extracts with human GST-Rb;
right, histone H1 kinase activity recovered following
incubation of cell extracts with GST-FH15, a heterologous GST fusion
protein prepared from a Schistosome surface antigen. Histone
H1 is indicated with an arrow on the right, and molecular
weight markers are indicated on the left. Panel B, in
vitro kinase assay in the presence and absence of histone H1.
Metaphase cell extracts were prepared from nocodazole-arrested A549
cells, incubated with human GST-Rb, and analyzed as in panel A except that the in vitro kinase buffer was adjusted to 10
m
M Mg
. GST-Rb-bound kinase activity was
assessed in the presence ( left lane) or absence ( right
lane) of exogenous histone H1. Arrows on the right
indicate radiolabeled GST-Rb and histone H1
proteins.
Biochemical and Immunochemical Characterization of
Mitotic RbK Activity
Since cyclin-cdk activity is cell cycle
dependent and members of this kinase family associate with Rb in
vivo and in vitro, we reasoned that these were likely RbK
candidate proteins. To determine whether previously identified
cyclins-cdks account for mitotic RbK activity, three experiments were
performed. 1) We assessed whether RbK activity could be depleted by
prior incubation of mitotic extracts with an excess of
p13-bound Sepharose beads, a regulatory protein that
binds with high affinity to a variety of cyclin
cdk complexes. 2)
We examined mitotic N-RBP eluants for reactivity with antisera to human
cyclins A and E, cdk1 (cdc2), or PSTAIRE, a conserved sequence motif
present in several cdks. 3) We prepared cell extracts overexpressing
cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 and
assessed whether these mitotic kinases could associate with and/or
phosphorylate the Rb amino terminus in vitro.
protein could deplete RbK activity in
mitotic extracts, we quantified RbK activity in GST-Rb eluants that had
been precleared with p13
beads. To ensure that
p13
beads were employed in excess, cdk1 abundance was
examined by Western blotting with cdk1 antisera before and after
incubation of mitotic extracts with p13
beads. As shown
in Fig. 2 A, preincubation of mitotic extracts with an excess
of p13
beads effectively depleted cdk1 protein from
mitotic extracts ( left panel). Consistent with previously
published evidence
(19, 24, 25, 26, 27, 28) that Rb associates with cyclin
cdk complexes in
vivo, Western blots of p13
bead-bound proteins using
anti-Rb antisera demonstrated that preincubation of mitotic extracts
with an excess of p13
beads also resulted in the
precipitation of Rb-associated, p13
-sensitive
cyclin
cdk complexes (Fig. 2 A, right
panel). Yet, despite the depletion of p13
-sensitive
cyclins-cdks, preincubation of mitotic extracts with p13
beads had no effect on the recovery of RbK activity (Table I).
Moreover, Western blots of mitotic proteins released from GST-Rb beads
did not react with anti-cyclin A, anti-cyclin E, anti-cdk1,
anti-PSTAIRE, or anti-MAP kinase (erk1/erk2) antisera (data not shown).
Finally, to determine if known mitotic cdks could associate with and/or
phosphorylate the Rb amino terminus we prepared extracts from cells
infected with recombinant vaccinia viruses that overexpress the
following epitope-tagged cyclins-cdks: cyclin A-cdc2, cyclin A-cdk2,
cyclin B-cdc2, and cyclin B-cdk2. Levels of cyclin
cdk complexes in
such extracts have previously been estimated to be 100-1000-fold higher
than in extracts prepared from uninfected cells
(63, 64) . As shown in Fig. 2 B, each extract
contained abundant amounts of cyclins A and B ( lanes 4 and
8) and histone H1 kinase activity ( lanes 12 and
15). However, neither cyclin A nor cyclin B kinase complexes
bound the Rb amino terminus in vitro ( lanes 6 and
10) and each only marginally phosphorylated GST-Rb ( lanes
13 and 16). Consistent with a previous report
(29) , cyclin A and cyclin B kinase complexes also did not
associate with GST or a GST-Rb pocket fusion protein in vitro (Fig. 2 B). Taken together, our results indicate
that mitotic RbK activity is not due to kinase complexes containing
cyclin A
cdc2 or cyclin A
cdk2 (two previously analyzed
Rb-associated kinases), cyclin B
cdc2 or cyclin B
cdk2, closely
related cyclins
cdks, or erk1-erk2 and therefore suggest that RbK
is a novel amino-terminal Rb-associated mitotic kinase activity. Strong
support for this conclusion is also provided by recent protein-binding
experiments that indicate some cyclin
cdk complexes interact with
Rb via the carboxyl-terminal 600 amino acids of Rb, a region not
contained within our amino-terminal GST-Rb fusion proteins
(25, 29, 66) . We have not as yet determined
whether the N-RBP kinase(s) that we detect in extracts prepared from
early cell cycle stages (G
through S phase)
is(are) antigenically related to cdks; however, at least a portion of
this activity appears to be sensitive to depletion with p13
beads (see Fig. 5below).
Figure 2:
Biochemical and immunochemical
characterization of mitotic RbK kinase. Panel A, anti-cdk1 and
anti-Rb Western blots of mitotic A549 extracts that were precleared
with an excess of p13-Sepharose beads. Left,
mitotic A549 extracts were precleared with p13
-Sepharose,
and supernatants were subsequently equilibrated with GST-Rb.
p13
-Sepharose and GST-Rb-bound proteins were eluted by
boiling in Laemmli sample buffer and assayed for cdc2 immunoreactivity
in a Western blot using a polyclonal rabbit antibody prepared against a
carboxyl-terminal peptide from human cdc2. S, GST-Rb-bound
proteins recovered from mitotic extracts that had been precleared with
an excess of p13
-Sepharose; P,
p13
-Sepharose-bound proteins prepared from mitotic
extracts. cdc2 is indicated by an arrow on the right,
and molecular weight markers are indicated on the left.
Right, p13
-Sepharose-bound proteins were
prepared from mitotic extracts as in the left panel and
assayed for Rb immunoreactivity in a Western blot with a polyclonal
anti-Rb antibody (9300, 56). Panel B, in vitro protein-binding and kinase assays using recombinant vaccinia
virus-infected cell extracts. Top, Coomassie-stained proteins
(2 µg/lane) used in binding assays, lane 1, GST; lane
2, GST-Rb amino terminus; lane 3, GST-Rb carboxyl
terminus. Center, Western blot analysis of epitope-tagged
cyclin A and cyclin B in infected cell extracts ( lanes 4 and
8, respectively) and in eluants from GST beads ( lanes 5 and 9), amino-terminal Rb beads ( lanes 6 and
10), and carboxyl-terminal Rb beads ( lanes 7 and
11). Each protein binding reaction employed extracts from 3
10
cells multiply infected with recombinant
vaccinia viruses carrying either cyclin A-cdc2 and cyclin A-cdk2 or
cyclin B-cdc2 and cyclin B-cdk2. Arrows indicate recombinant
cyclin proteins. Bottom, in vitro kinase assay of
virus-infected cell extracts. 20 µl (1.5
10
cell equivalents) of uninfected ( lane 18) or
virus-infected extracts ( lanes 12-17) were employed in
each in vitro kinase assay using histone H1 and amino-terminal
GST-Rb (indicated by arrows) as substrates as in Fig.
1 B. Whole cell extracts ( lanes 12 and 15),
extracts following incubation with amino-terminal GST-Rb ( lanes 13 and 16), or carboxyl-terminal GST-Rb ( lanes 14 and 17) are shown. Levels of histone H1 phosphorylation
in each lane were directly quantified with a PhosphorImager as:
uninfected cells, 8
10
counts/min; infected cells,
9.8
10
-2.1
10
counts/min.
Figure 5:
Cell cycle dependence of recovered RbK
activity in a synchronously growing population of CHO cells. CHO cells
were synchronized as described previously (65), and unlabeled extracts
were prepared at the indicated times. CHO cells were arrested with
hydroxyurea ( G/ S) and released from
growth arrest following exhaustive cell washes. Log phase CHO extracts
were analyzed in parallel for comparison. The abundance of RbK activity
was assayed with (+) and without (-) prior incubation of
extracts with p13
-Sepharose using equivalent amounts of
CHO proteins and purified GST-Rb in each lane.
[
H]Thymidine incorporation and flow cytometry
were performed as described (65). The fraction of cells in
G
/M phases and counts/minute of
[
H]thymidine incorporated into high molecular
weight DNA are indicated at the bottom. Phosphorylated GST-Rb
is indicated by an arrow at the
right.
As one further test of the
specificity of Rb-RbK association, we prepared a GST fusion protein
from an analogous region of the amino terminus of p107
(60) , a
Rb-related protein, and utilized this construction in in vitro protein binding and kinase assays. p107 has previously been shown
to interact with many of the same viral and cellular proteins as Rb and
shares several regions of sequence homology with Rb in their respective
amino termini
(60, 67, 68) . As shown in Fig. 3,
RbK kinase activity is readily harvested from mitotic extracts depleted
with p13beads using GST-Rb. In contrast, only marginal
amounts of H1 kinase activity were detected with a GST-p107
amino-terminal fusion protein. We conclude that despite the structural
and functional homology between Rb and p107, the differential
association of RbK suggests that their amino termini are biochemically
distinct.
A RbK-like Histone H1 Kinase Is Physically Associated
with Rb in Metaphase-arrested Cells
The data presented thus far
indicate that a potent Rb-histone H1 kinase activity may be found in
association with the Rb amino terminus following incubation of mitotic
extracts with Rb in vitro. It became, therefore, important to
determine whether a mitotic RbK-like activity is associated with Rb
in vivo. Extracts were prepared from nocodazole-arrested
Rb-positive (A549 and ML-1; 11, 19) and Rb-negative (5637; 5) cells and
were again exhaustively precleared with p13beads,
immunoprecipitated with a Rb monoclonal antibody, XZ77
(56) ,
and immunoprecipitates were assayed for Rb abundance by Western
blotting and coprecipitating histone H1 kinase activity in an in
vitro kinase assay. XZ77 immunoprecipitates of Rb-positive
extracts contained abundant amounts of hyperphosphorylated Rb protein
when analyzed in Western blots using a polyclonal anti-Rb antiserum
(no. 9300) prepared against the human Rb amino terminus (data not
shown). As shown in Fig. 4, these immunoprecipitates also contained
abundant amounts of coprecipitating histone H1 kinase activity. As
would be predicted, Rb-negative mitotic extracts similarly treated
showed little or no XZ77-associated histone H1 kinase activity
(Fig. 4). Consistent with our in vitro results, only
marginal amounts of coprecipitating histone H1 kinase activity were
recovered in similar immunoprecipitates prepared with antisera against
p107 (data not shown). Thus, wild-type Rb protein may be found in
association with at least one mitotic p13
-insensitive
histone H1 kinase in vivo. Moreover, the association of this
Rb
kinase complex is likely to be compatible with Rb
phosphorylation, since phosphorylated Rb predominates in mammalian
mitotic extracts. Interestingly, similar p13
-depleted
immunoprecipitates of mitotic extracts that were prepared using an
anti-Rb monoclonal antibody directed against the Rb amino terminus
(C36; 13) did not contain significant amounts of histone H1 kinase
activity nor Rb protein (data not shown). Since we and others have
shown that C36 precipitates abundant amounts of Rb protein from log
phase extracts or mitotic extracts that have not been precleared with
p13
beads, we presume that the epitope recognized by C36
is masked by one or more N-RBPs complexed with a fraction of Rb
molecules in metaphase cells.
Figure 4:
Coprecipitation of Rb and RbK-like histone
H1 kinase activity from mitotic Rb-positive cell extracts. Mitotic
extracts prepared from nocodazole-arrested Rb-positive ( A549 and ML-1; 11, 19) and Rb-negative ( 5637; 5)
cells were precleared with p13-Sepharose as in Fig. 2.
Supernatants were immunoprecipitated with anti-Rb antisera (XZ77; 56)
and assayed for H1 kinase activity as in Fig. 1 B. An arrow on the right indicates histone H1, and molecular weight
markers are indicated on the left.
RbK Enzymatic and/or Rb Association Activity Is Maximal
in G
The data presented above suggest that RbK enzymatic and/or
Rb association activity is(are) most abundant in extracts prepared from
metaphase cells (Fig. 1 A). To more precisely determine
the abundance of RbK activity in late cell cycle stages and to ensure
that the recovery of mitotic RbK activity is not induced by nocodazole
arrest, we examined the cell cycle dependence of RbK activity in
extracts prepared from a synchronously growing cell population. Thus,
CHO cells were arrested at the G/M Phases of Synchronously Growing Populations of
Cells
/S boundary with
hydroxyurea and then released to yield a synchronously dividing cell
population. Cell cycle position of synchronously growing CHO cells was
monitored by [
H]thymidine incorporation and by
flow-cytometric analysis following staining with propidium iodide (Fig.
5). To ensure that we quantified the kinetics of recovered RbK activity
and not a p13
-sensitive cdk, RbK activity was assessed
prior to and following preincubation with an excess of p13
beads. Consistent with previous results using growth-arrested
A549 cell extracts (Fig. 1 A), a low level of kinase
activity is detected in G
thru S phases ( Fig. 5and
Table II; G
/S boundary thru 6 h post-release). The recovery
of a portion of this kinase activity (approximately 50-75%)
appears to be sensitive to prior incubation with p13
beads (). At the transition from a population
containing predominantly S phase cells to one containing predominantly
G
/M cells (9 h post-release from hydroxyurea arrest), the
abundance of p13
-insensitive kinase activity, as
quantified by Rb phosphorylation in vitro, increased nearly
3-fold ( Fig. 5and ). Identical temporal results
were obtained in parallel in vitro kinase assays that utilized
histone H1 as exogenous substrate (data not shown). We conclude that
the Rb amino terminus interacts with one or more Rb-histone H1 kinases
that are most prevalent and/or active in G
/M phases and
that RbK activity is not artificially induced by mitotic drug arrest.
These data also support our earlier observation that at least one
additional kinase associates with the Rb amino terminus. The enzymatic
and Rb association activities of this additional kinase are apparent
prior to S phase, and the recovery of this kinase activity is
diminished by preincubation with p13
beads.
-insensitive Rb-histone H1 kinase (RbK)
whose enzymatic and/or Rb association activity is maximal in extracts
prepared from post-S phase cells. Moreover, using a monoclonal antibody
prepared against a carboxyl-terminal Rb epitope, we have shown that
endogenous p105-Rb associates with a histone H1 kinase with identical
biochemical properties in vivo.
beads and in their temporal abundance
and/or Rb association activity. The most predominant RbK, in terms of
Rb-histone H1 kinase activity, is a p13
-insensitive
kinase that associates with Rb in vivo and in vitro.
The abundance, enzymatic, and/or Rb association activity of this RbK is
maximal in extracts prepared from post-S-synchronized cells. That this
G
/M RbK is likely to be a novel Rb-associated kinase is
indicated by the following observations. 1) The abundance of
G
/M RbK kinase activity in cell extracts is insensitive to
prior incubation with an excess of p13
beads. 2) Using
appropriate antisera, G
/M RbK-containing protein eluants of
GST-Rb beads are devoid of cyclin-cdk proteins previously shown to
interact with Rb. 3) RbK associates with Rb via a region of Rb
previously shown to be dispensible for interaction with several
cyclin
cdk complexes. 4) We have shown that previously
characterized mitotic cdks, cyclin A-cdc2, cyclin A-cdk2, cyclin
B-cdc2, and cyclin B-cdk2, do not associate with and only marginally
phosphorylate the Rb amino terminus in vitro. Taken together,
these data strongly support the conclusion that G
/M RbK
activity is not due to previously studied Rb-associated kinase
complexes. In addition, we have also provided evidence for a distinct
p13
-sensitive kinase activity that associates with the Rb
amino terminus in earlier cell cycle stages.
/M RbKs, and one
additional Rb kinase, cdk4, has been shown not to phosphorylate histone
H1. Additionally, based on our results from Western blotting
experiments the G
/M RbK kinase(s) appears not to be
antigenically related to the MAP kinases erk1/erk2. Since Rb is subject
to successive waves of phosphorylation as cells progress from late
G
to mitosis
(18) and only a subset of these sites
of phosphorylation coincide with sites phosphorylated by cdk kinases
in vitro (19, 27) , RbKs may be responsible for
additional Rb phosphorylations in vivo.
(
)
In addition to analyses of naturally
occurring Rb mutations in tumor cells, in vitro mutagenesis
has been effectively exploited to probe Rb-mediated growth suppression.
Using a series of internally deleted Rb cDNAs, Qian et al. (50) have previously shown that subtle deletions within the Rb
amino terminus often yield proteins that are unphosphorylated in
vivo and are functionally defective in an in vitro growth
suppression assay. Importantly, such mutated Rb proteins retain the
ability to physically associate with at least one target of Rb
function, transcription factor E2F. Consistent with our observation
that Rb amino acids comprising exon 4 are required for the recovery of
wild-type levels of RbK activity, several mutated Rb cDNAs with
internal deletions proximal to exon 4 have also been shown to perturb
the recovery of G
/M RbK activity in vitro.
RbK complexes are functional and are
required for the regulation of cell cycle events subsequent to the
G
/S transition. This hypothesis is supported by recent
evidence that the conditional expression of full-length Rb in
synchronously growing cells can trigger growth arrest in G
(49) . Interestingly, the conditional expression of a cDNA
suffering a Rb pocket mutation was as active as wild-type Rb at
eliciting G
growth arrest. This latter observation is
consistent with the notion that interactions of Rb with proteins
distinct from those that bind the Rb pocket, such as RbK, may be
required for growth arrest in G
. Although we can only
speculate as to the precise functional role of Rb in mediating cell
cycle events in G
, it is likely that Rb participates in
transcriptional regulation perhaps involving protein targets distinct
from those in G
. However, it is also conceivable that Rb
performs functions in G
that are distinct from those in
earlier cell cycle stages. For example, in conjunction with RbK, Rb may
function to prevent the reinitiation of DNA replication in G
or perhaps participate in a checkpoint leading to the
establishment of post-mitotic or apoptotic cells. Finally, our
observation that a p13
-insensitive histone H1 kinase
activity similar to that of RbK does not appreciably associate with the
p107 amino terminus suggests that the amino termini of Rb and p107 may
perform dissimilar functions. The cloning and characterization of the
RbKs and cellular RbK substrates will undoubtedly provide insight into
the viability of these and other possibilities.
Table:
Abundance of p13-sensitive and
-insensitive RbK activity in extracts prepared from A549
metaphase-arrested cells
-Sepharose, and supernatants
were subsequently equilibrated with GST-Rb-bound glutathione-agarose
beads. All beads were then assayed for bound histone H1 kinase activity
as in Fig. 1 B. Following autoradiography, radiolabeled histone
H1 was excised from a SDS-polyacrylamide gel and quantified by
scintillation counting.
Table:
Abundance
of p13-sensitive and -insensitive kinase activity in
synchronously growing populations of CHO cells
-
D-galactopyranoside; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; RbK, histone H1
kinase; cdk, cyclin-dependent kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.