Running title: p16 mutants defective in Cdk interaction
phone: 617-726-5901
or 5956
fax: 617-726-6893
E-mail: brent@opal.mgh.harvard.edu
Abstract
The tumor suppressor candidate p16 is a cyclin-dependent
kinase inhibitor that inhibits cell proliferation. The p16 coding gene is
often mutated in glioblastomas, pancreatic adenocarcinomas and melanoma-prone
pedigrees, but, until recently, the significance of these allelic variants has
remained unclear. Here, we used interaction mating and coprecipitation to measure
interaction of seven p16 allelic
variants detected in melanoma-prone pedigrees with Cyclin-dependent kinases
(Cdks). We found that most variants were deficient in interaction with Cdk4
and Cdk6. One defective variant was found both in cancer prone families and in
the control population and therefore previously defined as a common polymorphism.
Another variant, which is weakly linked to familial cancer, is only slightly
affected in interaction with Cdks. These results are consistent with the idea
that p16 allelic variants that
decrease Cdk interaction predispose individuals who carry them to an increased
risk of cancer. Moreover, they suggest that determination of affinity between
p16 mutants and partner proteins may help identify functionally-significant allelic
variants not detected by classical human genetic techniques.
Introduction
Cell cycle transitions are promoted
by sequential activation of cyclin-dependent kinases, which consist of a catalytic
subunit called Cdk(s),
and activating subunits called Cyclins. Activity of these proteins is orchestrated
by transcription of specific Cyclins, specific phosphorylation and dephosphorylation
events, and association with Cdk-inhibitory proteins (Ckis) (reviewed
in Sherr, 1994; King et al., 1994; Hunter and Pines, 1994; Morgan, 1995).
One Cki, p16 (p16), specifically binds to and inhibits the activity of
Cyclin D/Cdk4 complexes, and thus regulates cellular proliferation (Serrano et
al., 1993, Serrano
et al., 1995). This small protein is composed of four ankyrin repeats,
protein motifs which have been shown to direct protein-protein interactions
between the a and b-subunits of the GA binding protein (Thompson et
al., 1991), between Cactus and Dorsal (Kidd, 1992), and between cdc10p and
sct1p (Reymond and Simanis, 1993). p16 maps to chromosome 9p21, the site of
the multiple tumor suppressor (MTS1) locus, which is rearranged, deleted or mutated
in many tumor derived cell
lines (reviewed in Kamb, 1995), suggesting that p16 might be the MTS1 product.
Consistent with this idea, p16 genes isolated from primary tumors often show
sequence differences compared to wild type. Moreover, p16 is often mutated in
members of melanoma-prone families. However, the functional significance of these
variations has not yet been assessed (reviewed in Kamb, 1995). Recently, p16
has also been shown to inactivate complexes containing Cdk6, but the significance
of this inhibition is so
far not known, (Hannon and Beach, 1994).
Here, we examined interactions
between Cdks and p16 variants isolated from melanoma-prone families. Six
of these variants are thought to be bonafide mutants linked to the development
of melanoma, and two of them "common polymorphisms", also found in the
general population (Hussussian et al., 1994). We assayed binding by both interaction-mating
and in vitro binding assays. No allelic variant
showed a broadened specificity
of interaction. All but two allelic variants were also deficient in binding Cdk6.
All but one allelic variant, including one encoded by a common polymorphism,
showed decreased affinity for Cdk4 and did not compete with cyclin D1 for Cdk4
binding.
Our results suggest that decreased affinity of p16 for
Cdk4 and Cdk6, and its resulting inability to compete with Cyclin D for binding,
contributes to tumorigenesis. Moreover, for cases in which mutants of p16
are only impaired in their binding
to Cdk4, they suggest that loss of inhibition of Cdk4 is the mechanism by which
these alleles lead to tumorigenesis. They also suggest that the p16 common
polymorphism that shows diminished binding to Cdk4 may also contribute to human
cancer, even though it is also found in the population at large. Because the
interaction mating technique used here simplifies the task of assaying binding
by different alleles, our results suggest that screening for p16-Cdk4 interactions
in yeast provides a functional
test to reveal other alleles of p16 that may contribute to cancer, and may
provide a useful supplement to more established human-genetic techniques.
Results
We studied a set of seven p16 mutants
isolated from melanoma-prone families. We used interaction mating, an extension
of the interaction trap two-hybrid system (Finley and Brent, 1994), to characterize
the interaction of these proteins with a panel of cyclin dependent kinases.
Here, we name
the mutations according to their distance from the ATG that begins the coding
sequence. Because the first published sequence of p16 assigned an incorrect ATG,
our numbering system differs from that used by Hussussian et al.
(1994) and Ranade et al. (1995). Four of the p16 allelic variants:
p16-N71S, -R87P, -G101W and -V126D (formerly, -N63S, -R79P, -G93W, and V118D)
are missense mutations transmitted through the germline of individuals from
affected families. Two of them,
p16-I49T and -A148T (formerly, I41T and A140T), are found in affected families,
but are also found in members of the control population, and are thus termed
"common polymorphisms" (Hussussian et al., 1994). The
last variant, p16-P81L (formerly, P73L), is a missense mutation that apparently
arose somatically in a melanoma-prone pedigree. For the interaction matings,
the reporter gene was the medium-high sensitivity pSH18-34 (8 LexA operators, Hanes
and Brent, unpublished, Finley
and Brent, 1994), which in haploid cells detects interactions between lambda
repressor C termini with Kd <10 M (Estojak, Brent, and Golemis, submitted),
but whose sensitivity is somewhat reduced in diploids (Finley and Brent, 1994;
Finley, personal communication).
The interaction mating experiments
(Figure 1a and 1b) show that, as previously reported, wild-type p16 specifically
interacts with Hs Cdk4 and Hs Cdk6, but not with
Hs Cdc2, Hs
Cdk2, Hs Cdk3 or Hs Cdk5 (Hannon and Beach,
1994; Parry et al., 1995). Moreover, they also show that p16 does
not interact with the more distantly related Hs Cdk7 or Cdks from
Drosophila and yeast (Dm Cdc2, Dm Cdc2c
and Sc CDC28; figure 1a and 1b and data not shown). As judged by
blue color on Gal Xgal interaction plates (Finley and Brent, 1994), the interaction
is stronger than the threshold
for this reporter. In addition, the fact that it is also detected on glucose
plates suggests that the LexA fusion bait is substantially occupied even when
the interacting protein (prey) is only expressed at the basal level seen on
this medium. This is consistent with an even tighter interaction.
Three allelic variants of p16, p16-P81L, -R87P and -V126D, showed decreased affinities
for Cdk4 and Cdk6 (see fig 1a and 1b). The results were similar regardless
of whether the Cdk was the
LexA fusion bait, and p16 the prey, or whether the orientation of the interacting
partners was reversed (Fig 1a and 1b). As judged by western gel analysis
of bait and prey fusions with anti-LexA and anti-hemaglutinin antibody (Gyuris
et al., 1993), all fusions were expressed to the same level (not
shown), and, as judged from repression assays (Brent and Ptashne, 1984, Golemis
and Brent, 1992) all baits occupied operator to similar extents (not shown).
None of the p16 variants showed
a relaxed specificity of interaction: none interacted with Cdks other than Cdk4
and Cdk6.
To better quantitate interactions between p16 variants
and Cdk4/Cdk6, we measured b-galactosidase activity of the EGY48/RFY206 diploid
yeast that contained appropriate bait and prey constructs. As judged by these
experiments, the amount of LexA-Cdk4 bait occupancy by the p16-P81L, -R87P,
-G101W and -V126D proteins was decreased 70 to 95% compared to wild type p16 (figure
1c). Surprisingly, p16-I49T,
previously defined as a common polymorphism, based on its occurrence in individuals
not genetically related to melanoma gene carriers, showed a similar decrease
in occupancy, showing that its affinity for Cdk4 is also impaired. By
contrast, the other allelic variant defined as a common polymorphism, p16-A148T,
showed no impairment in its interaction with Cdk4. One of the germline mutations,
p16-N71S, retained substantial affinity for Cdk4 (20% decrease in Cdk4 occupancy).
Most variant
proteins also showed decreased interaction with Cdk6. For example, LexA-Cdk6
bait occupancy by p16-N71S, -P81L, -R87P, and -V126D, was drastically reduced
compared to wild type, while binding by the p16-I49T and -A148T polymorphisms
was almost unimpaired. However, one protein, p16-G101W, which showed decreased
interaction with Cdk4, interacted normally with Cdk6 (Figure 1d).
p16 interacts with Cdk4 in the absence of Cyclin D (Xiong et al.,
1993; Serrano et al.,
1993) and competes with Cyclin D for binding to Cdk4 in vitro
(Parry et al., 1995). The decreased affinity of the melanoma-prone
family p16 allelic variants for Cdk4 suggested that they should be impaired
in their ability to compete with cyclin D for binding to Cdk4. To test this idea,
we used an in vitro association assay. We added increasing amounts
(721 fM to 721 nM final concentration) of GST-p16 or GST-p16 variants to
mixtures of Cyclin D1 and Cdk4
made by translation in vitro, and quantitated the amount of Cdk4
that remained associated with Cyclin D1. As shown in figure 2a and 2b, immunoprecipitations
with an anti-cyclin D1 antibody show that the level of Cyclin D1-associated
Cdk4 was decreased proportionally to the amount of GST-p16 or GST-p16-A148T
"common polymorphism" added to the mixture. By comparison, it
required 50 times as much of the p16-N71S mutant, and >100 times as much of
the p16-P81L and -G101W mutants,
and of the p16-I49T common polymorphism, to compete half-maximally for Cyclin
D1. No concentration of the p16-R87P and -V126D mutants competed cyclin D1.
Because the interaction mating experiments had shown that p16-G101W
was specifically impaired in interaction with Cdk4, and not with Cdk6, we tested
its ability to associate with Cdk6 in vitro. We added increasing
amounts of GST-p16, GST-p16-R87P or GST-p16-G101W to Cyclin D1 and Cdk6 translated
in vitro, and
quantitated the ability of the fusion proteins to block cyclin D binding as
above. By contrast with wild type GST-p16, GST-p16-R87P was unable to compete
with Cyclin D1 for Cdk6 binding (Figure 3a and 3b). Competition was also achieved
by increasing amounts of GST-p16-G101W, confirming that this allelic variant
is not impaired in Cdk6 binding.
Discussion
We measured binding of allelic variants of p16 to
Cyclin Dependent Kinases. Both
interaction mating experiments and in vitro competition experiments
show that affinity for Cdk4 is strongly diminished for four allelic variants
(p16-P81L, -R87P, -G101W, and -V126D) found specifically in melanoma-prone families,
and for one allelic variant (p16-I49T), found in such families but also
in the control population, and thought to be a common polymorphism. One of these
allelic variants, p16-G101W, is not diminished in its affinity for Cdk6. Another
allelic variant, p16-N71S,
shows only a slight decrease in its affinity for Cdk4.
In summary,
both in vitro and in vivo experiments suggest that
the melanoma-prone family p16 mutants are functionally defective in their ability
to inhibit Cdk4/Cdk6 kinase activity. Their impaired affinity for Cdk4 and
Cdk6 results in a decreased capability to compete with Cyclin D for binding to
these kinases. These results support the notion that loss of p16 function may contribute
to tumorigenesis and
suggest that p16 is an excellent candidate for the MTS1 locus gene product.
Our results differ in two regards from a recent study (Ranade et
al., 1995). First, Ranade et al. deemed the allelic variant
p16-I49T a common polymorphism, whereas we showed that it has an impaired ability
to bind Cdk4 and compete with cyclin D1. However, it is worth noting that
our interaction results (figure 1c) and the kinase assays of Ranade et
al. in fact gave very similar
results: we show that p16-I49T retains 43% of the binding activity of p16
(figure 1c) while Ranade et al. show that p16-I49T retains about
~50% of the kinase inhibitory activity of p16-WT, consistent with a diminution
in its binding function. Consistent with this idea, our coimmunoprecipitation
experiments (see figure 2b) also show that the allelic variant p16-I49T is affected
in binding. Second, our experiments (figure 1d, and figure 3) clearly show
that p16-G101W retains binding
activity to Cdk6, while Ranade et al. show that purified p16-G101W
apparently does not inhibit Cdk6/cyclin D1 kinase activity in vitro.
If the p16-G101W used by Ranade et al. was active, this result
suggests that this allelic variant can bind Cdk6, and compete for cyclin D1
binding without inbiting its kinase activity, a possibility we are investigating.
On the other hand, our findings correlate well with existing human
genetic data. Consistent
with our interaction data, possession of p16-R87P, -G101W or -V126D alleles is
highly correlated with melanoma in familial pedigrees, whereas possession of p16-N71S
is only weakly correlated (Hussussian et al., 1994). Moreover,
because our results show that the p16-G101W protein is specifically affected
in binding to Cdk4, but not Cdk6, our results strongly suggest impaired interaction
with Cdk4 is the mechanism by which this mutant contributes to cancer.
However, there is one case in
which our results differ from those predicted by the human-genetic studies. Our
results show that the p16-I49T allele, which occurs in both melanoma-prone pedigrees
and in control populations (Hussussian et al., 1994), is deficient
in interaction with Cdk4. This result suggests that p16-I49T may well
dispose individuals who carry it to increased cancer risk. We suggest that the
interaction-mating assay used in this study will facilitate demonstration of
loss-of-interaction for allelic
variants of p16 and of other proteins. It is thus possible that interaction mating
assays may augment existing human genetic strategies by providing alternative
means to detect weakly penetrant mutations, and those that are not confined
to genetically-distinct family cohorts.
Materials
and Methods
Plasmids. LexA- and B42- containing plasmids
are derivatives of pEG202 and pJG4-5 plasmids respectively (Gyuris et al.,
1993; Zervos et
al., 1993). All were constructed by standard techniques (Ausubel et al.,
1987-1995). All sequences generated by PCR were confirmed by direct DNA sequencing.
LexA derivatives (baits). pLexA-p16: EcoRI/XhoI
ended fragments that contained the entire coding regions for
p16 or p16 allelic variants were introduced into the EcoRI/SalI
site of pEG202AAT, a modified version of the plasmid pEG202, which
adds one nucleotide upstream
of the EcoRI restriction site and thus directs the synthesis of fused
moieties in a different frame (E.Golemis, unpublished, Gyuris et al.,
1993). pLexA-Cdks: EcoRI/SalI, EcoRI/XhoI
or MunI/SalI fragments DNAs that
contained the coding regions for Cdks were cloned in the EcoRI/SalI
site of pEG202 (Gyuris et al., 1993; Finley and
Brent, 1994 and this work).
B42
derivatives (preys). pB42-p16: EcoRI/XhoI
ended coding regions for p16 or p16 allelic variants, as above, were cloned into
pJG4-5AAT, a modified version of the plasmid pJG4-5, which expresses fused moieties
in a different frame due to the fact that it carries an additional A upstream
of the EcoRI restriction site ( E.Golemis, unpublished, Gyuris et
al., 1993). pB42-Cdks: coding regions for Cdks were cloned into EcoRI/XhoI
pJG4-5,
as above (Gyuris et al., 1993 and this work).
Yeast
manipulations. Standard microbiological techniques and media were used throughout
(Guthrie and Fink, 1991; Ausubel et al., 1987-1995). Yeast minimal
dropout media are designated by the nutrients component(s) left out. They
contain, in addition to the dropout mix, either 2% glucose or 2% galactose/1% raffinose
as a carbon source. X-Gal minimal drop out plates contained X-Gal and
phosphate buffer at pH 7.0. (Ausubel
et al., 1987-1995). Yeast transformation was carried out as
described by Gietz et al. (1992). Interaction mating assays were
performed using the yeast strains RFY206 (Mata his3Æ200 leu2-3
lys2Æ201 ura3-52 trp1Æ::hisG) and EGY48 (Mata his3
leu2::3 lexAop-LEU2 ura3 trp1 LYS2) that contained the pSH18-34 reporter
plasmid as described in Finley and Brent (1994).
Expression and purification
of p16 proteins. Coding
regions for p16 or p16 allelic variants were cloned in frame into pGEX4T (Pharmacia).
E.coli BL21 carrying the plasmids were grown to an OD600
of 0.5 at 37C, IPTG was added to the media to a final concentration of 0.33 mM
and the culture was grown 10h at 20C. Cells were harvested by centrifugation,
frozen and thawed twice by immersing the tubes in dry ice and a 37o waterbath ,
and resuspended in 1/50 of the starting culture volume of PBS pH7.2, 5 mM EDTA,
1 mM DTT, 2 mg/ml aprotinin,
2 mg/ml leupeptin, 2 mg/ml pepstatin A, 1mM PMSF and 2mg/ml lysozyme (Sigma). After
30 min on ice, Triton X-100 was added to a final concentration of 1% and the
cells were sonicated until the viscosity was reduced. The supernatant was collected
by centrifugation and the protein was bound to 100 ml of Glutathione-sepharose
resin (Pharmacia) for 60 min. The resin was washed three times with washing
buffer (WB: PBS, 1% Triton X-100, 5 mM EDTA, 1 mM DTT, 2 mg/ml aprotinin,
2 mg/ml leupeptin, 2 mg/ml pepstatin
A, 1 mM PMSF), three times with WB containing 250 mM KCl and equilibrated
by two washes with 50 mM Tris-HCl pH8.0, 25% glycerol. GST fusion proteins were
eluted in 50 mM Tris-HCl pH8.0, 10mM glutathione, 25% glycerol, aliquoted and
stored at -80C. Protein quantitation was done by the Bradford Coomassie dye-binding
method according to the manufacturer's instructions (BioRad)
In
vitro binding assays. Fragments that carried the coding sequence for
human Cdk4, Cdk6 and Cyclin
D1 were subcloned into pGEM derived plasmids (Promega) to allow transcription with
the T7 RNA polymerase. Proteins were synthesized in vitro using
the TNT coupled transcription/translation system and L-[S]methionine according
to the manufacturer's instructions (Promega). Samples were incubated for 30 min
at 30C. Equal amounts of in vitro translated Cyclin D1 and Cdk4
were mixed with GST-p16 fusions, diluted in 3 ml of 20 mM Tris-HCl pH8.0, 150
mM NaCl and incubated for 30 min
at 30C. Volume was then adjusted to 500 ml with ice cold 20mM Tris-HCl pH8.0,
150 mM NaCl, and 0.25% Nonidet P40 (Sigma). After centrifugation the supernatant
was immunoprecipitated with 5 mg of a rabbit anti-human cyclin D1 antibody
(UBI). The immune complexes were collected with protein A-Sepharose as described,
run on 10 or 12% SDS acrylamide gels (Protogel, National Diagnostics), dried,
and visualized and quantitated using a Phosphoimager (Molecular Dynamics).
Acknowledgments
We
thank N. C. Dracopoli, C. J. Hussussiana and
M. Meyerson for plasmid DNA, members of the Brent laboratory for helpful discussions
and reagents, and B. Cohen, P. Colas, R. L.Finley, F. J. Slack and W. Xu
for critical reading of the manuscript. This work was supported by a Swiss Cancer
League Fellowship to A. R., and a Pew Scholar's Award and an American Cancer
Society Faculty Research Award to R.B.
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Legends
to figures:
Figure 1.
p16 melanoma-prone family mutants show decreased affinity for Cdk4 and Cdk6.
A:
Interaction-mating assays between strains carrying Cdks (baits) and melanoma-prone
families p16 mutants (preys). Bait strains containing plasmids that expressed
LexA fusions to Homo sapiens Cdc2, Cdk2, Cdk3, Cdk4, Cdk6,
Cdk7 and Max and Saccharomyces
cerevisiae CDC28 were mated to EGY48 derivatives that contained
B42 fusions to p16 or p16 allelic variants. Plates are -ura -his -trp X-gal and
contain either glucose or galactose/raffinose.
B: Interaction-mating assays
between strains carrying melanoma-prone families p16 mutants (baits) and Cdk (preys).
Bait strains containing plasmids that expressed LexA fusions to p16 or
p16 allelic variants were mated to EGY48 derivatives that contained B42 fusions
to Homo sapiens
Cdc2, Cdk3, Cdk4, Cdk6, Saccharomyces cerevisiae CDC28 and Drosophila
melanogaster Cdc2c. Plates are as in panel A.
C: b-galactosidase
activity induced by interactions between Cdk4 and p16 WT or p16 mutants.
Activities are normalized to the b-galactosidase activity induced by the Cdk4-p16
wild type interaction.
D: b-galactosidase activity induced by interactions
between Cdk6 and p16 WT or p16 mutants. Activities are normalized to the
b-galactosidase activity induced
by the Cdk6-p16 WT interaction.
Figure 2: p16 melanoma-prone family mutants
do not compete with Cyclin D1 for binding to Cdk4 in vitro.
Sequential dilutions of bacterially expressed GST-p16 WT or GST-p16 mutants (1pg-1mg,
corresponding to final concentrations of 721fM-721nM) were added to a fixed
amount of labeled, in vitro translated, Cyclin D1 and Cdk4. After
incubation, the proteins were immunoprecipitated using cyclin D1 antiserum.
Immunoprecipitates were separated
on 12% SDS acrylamide gels and exposed to X-ray film.
A: Coimmunoprecipitated
Cdk4 (open circles) and Cyclin D1 (closed circles) competed with GST-p16-WT,
-P81L, -R87P, -V126D. Lanes 1-7 show competition with 1pg (721fM), 10pg
(7pM), 100pg (72pM), 1ng (721pM), 10ng (7.2nM), 100ng (72nM) and 1mg (721nM)
of the p16 competitor. Figure shows p16 allelic variants that compete with Cyclin
D1 (p16-WT), that compete only at high concentrations (p16-P81L), and that
do not compete (p16-R87P, p16-V126D).
B:
Coimmunoprecipitated Cdk4 was quantitated by a PhosphoImager
(Molecular Dynamics) and plotted against the amount of competitor protein added
to the reaction.
Figure 3: p16-G101W competes with Cyclin D1 for binding
to Cdk6 in vitro. Sequential dilutions of bacterially expressed
GST-p16 and allelic variants -R87P and -G101W (1pg-1mg, corresponding to 721fM-721nM)
were added to a fixed amount of labeled, in vitro translated,
Cyclin D1 and Cdk6. After
incubation, the proteins were immunoprecipitated using Cyclin D1 antiserum. The
immunoprecipitates were separated on a 10% SDS-PAGE and exposed to X-ray film.
A:
autoradiographs of coimmunoprecipitated Cdk6 (open square) and Cyclin
D1 (closed circle) competed with GST-p16-WT, -R87P or -G101W. Lanes 1-7 correspond
to competition with 1pg (721fM), 10pg (7pM), 100pg (72pM), 1ng (721pM), 10ng
(7.2nM), 100ng (72nM) and 1mg (721nM) of competitor protein, respectively.
B:
Coimmunoprecipitated
Cdk6 was quantitated by a PhosphoImager (Molecular Dynamics) and plotted against
the amount of competitor protein added to the reaction.