Download Structure/Function Analysis of ras Using Random Mutagenesis

OVERVIEW
Structure/Function Analysis of ras
Using Random Mutagenesis Coupled
with Functional Screening Assays*
Larry A. Feig, Michael Corbley, Bin-Tao Pan, Thomas M. Roberts,
and Geoffrey M. Cooper
Dana-Farber Cancer Institute
and Department of Pathology
Harvard Medical School
Boston, Massachusetts 02115
We review the use of functional assays for the ras
protein, p21, that have allowed us to screen for
mutant ras genes encoding proteins defective in
either interactions with guanine nucleotides or transforming activity. GTP binding and GTP-dependent
autokinase activities were assayed directly on lysed
bacterial colonies expressing p21. Mutants encoding ras proteins deficient in these activities were
isolated after randomly mutagenizing a v-rasH
expression vector. Transformation defective mutants were isolated by randomly mutagenizing a vrasH retroviral shuttle vector. NIH cells were then
infected with a stock of nonreplicating mutagenized
retroviruses and nontransformed infected colonies
were isolated. The mutant ras genes were then
rescued from these cells for analysis. Characterization of these mutants defines domains of p21
involved in both biochemical and biological activities
and addresses the role of guanine nucleotide binding in p21 function. (Molecular Endocrinology 1:
127-136,1987)
All three cellular ras genes encode proteins (designated p21s) of 189 amino acids which differ primarily in
a region of 20 amino acids near the carboxy-terminus
(5-7). The viral rasH and rasK gene products differ from
their normal cellular homologs by three and six amino
substitutions, respectively (8, 9). Overexpression of
normal cellular rasH is sufficient to induce cell transformation (10). However, activation of ras genes in tumors
and viruses commonly involves structural alterations in
the form of single amino acid substitutions which increase ras transforming potential. In human tumors,
amino acid substitutions at either position 12,13, or 61
have been identified as activating mutations (11-15). In
Harvey and Kirsten sarcoma viruses, ras activation is
similarly associated with amino acid substitutions at
position 12 (8, 9). The substitution of threonine for
alanine at position 59 is a second activating mutation
in the viral rasH and rasK genes (8, 9,16).
Both the viral and cellular ras proteins are localized
to the inner face of the plasma membrane (17, 18).
They are posttranslationally modified by palmitylation
of cysteine-186, which is required for both membrane
localization and cell transformation (19,20). ras proteins
bind GDP and GTP with high affinities (21-23) and
display a low level of GTPase activity (24-27). In addition, the viral rasH and rasK p21s possess a GTPdependent autokinase activity that phosphorylates threonine-59 both in vitro and in vivo (28). This autophosphorylation likely results from threonine-59 serving as
an alternative to water as a phosphate acceptor during
GTP hydrolysis. The physiological significance of this
reaction, however, has not been evaluated.
The membrane localization, guanine-nucleotide binding, and GTPase activities of p21 are reminiscent of the
a-subunits of the G proteins, which are involved in
transduction of signals from a variety of membrane
receptors to enzymes which function in the metabolism
of second messengers, including hormone-sensitive adenylate cyclase (29), cyclic GMP phosphodiesterase
(30), and phospholipase C (31, 32). It is thus an attractive hypothesis that ras proteins function analogously
to the G proteins as signal transducing molecules,
although neither the putative receptors nor effectors
INTRODUCTION
The ras genes were first identified as the transforming
genes of Harvey and Kirsten sarcoma viruses (1). Cellular homologs of ras were subsequently detected as
activated transforming genes in human neoplasms by
the ability of some tumor DNAs to induce transformation of NIH 3T3 cells (2-4). To date, three cellular ras
genes {rasH, rasK, and ras") have been identified as
activated transforming genes in approximately 10-20%
of a wide variety of human neoplasms including carcinomas, sarcomas, neuroblastomas, leukemias, and
lymphomas. Although the role of these genes in the
pathogenesis of neoplasms is unclear, their frequent
activation suggests that they can contribute to some
stage of the development of tumors originating from
many different cell types.
0888-8809/87/0127-0136$02.00/0
Molecular Endocrinology
Copyright © 1987 by The Endocrine Society
127
MOL ENDO-1987
128
with which ras might interact have been identified in
mammalian cells.
In vitro mutagenesis has been widely used to identify
functional domains of proteins and to correlate biochemical and biological activities. In the case of ras
(and of the G proteins) it is of interest to define protein
domains involved in interactions with guanine nucleotides, which presumably regulate the activity of these
transducing molecules, and to identify domains which
may be involved in interactions with other cellular proteins which could constitute regulators or effectors in
signal transduction pathways.
In this manuscript, we review recent published (33)
and unpublished studies in which we have developed
assays to screen directly for mutant ras genes encoding
proteins which are defective either in one of the known
biochemical activities of p21 or in its known biological
activity, cell transformation. The GTP binding (33) and
GTP-dependent autokinase activities of ras proteins are
assayed on lysed bacterial colonies expressing p21.
Mutants deficient in these activities can then be identified after random mutagenesis of a ras bacterial expression vector. In a complementary approach, a retroviral
shuttle vector has been used to isolate mutants of ras
defective in transformation of NIH 3T3 cells. These
strategies have the advantage of permitting efficient
screening of a randomly mutagenized population to
directly isolate mutants with the desired phenotype.
Mutations affecting a given activity can thus be identified without prior prediction of the relevant sites, as is
required for site-directed mutagenesis. Furthermore,
since random mutagenesis introduces point mutations
throughout the molecule of interest, multiple active
domains can be identified without introducing severe
alterations in protein structure, as is commonly the case
with deletion or linker-scanning mutagenesis. The biochemical and biological mutagenesis screening procedures employed here may be applicable to studies of a
variey of other proteins, including the G proteins, which
display related biochemical activities or for which biological assays can be developed.
Vol. 1 No. 2
from pTR 1340 containing a tac promoter and origin of
replication and ampicillin resistance gene from pBR322.
A similar construct was used to express cellular rasH
p21. The pXVR constructs were transformed into the
Escherichia coli strain PR13-Q, which overproduces lac
repressor to partially repress the tac promoter and
hence maintain cell viability. Upon induction with isopropylthio-/3-D-galactoside (IPTG), large amounts of p21
were synthesized, accounting for approximately 30%
of total bacterial protein (Fig. 1A). A simple extraction
procedure, adapted from previous studies (27), was
used to enrich p21 to greater than 80% purity (Fig. 1B).
Isolation of rasH Mutants Deficient in Guanine
Nucleotide Binding
The isolation of ras GTP-binding mutants has been
previously reported (33). Bacteria were transformed
with pXVR and replica plated on nitrocellulose filters.
Expression of p21 was induced by incubating one set
of filters with IPTG and bacterial colonies were lysed
such that bacterial protein was bound to the filters.
Filters were then incubated with 10~8 M 32P-GTP,
washed with Tris-buffered saline (TBS), and autoradi-
B
RESULTS
Bacterial Expression of rasH
The screening strategies used to isolate ras mutants
which are deficient in interactions with guanine nucleotides rely on the development of assays to detect these
biochemical activities of p21 directly on lysed bacterial
colonies expressing ras protein. As described (33), a
rasH expression vector designated pXVR, which directs
the synthesis of authentic viral rasH p21, was constructed by ligating three elements: 1) a 720 base-pair
restriction fragment encoding all but the first five amino
acids of the viral rasH gene, 2) a 32 base-pair synthetic
oligomer containing a bacterial Shine-Delgarno sequence followed by a six base-pair spacer plus the first
five codons of viral rasH, and 3) a plasmid backbone
P21
Fig. 1. Expression of ras Proteins in Bacteria
Bacteria were induced to synthesize p21 by incubation with 5
mM IPTG at 37 C. After 1 h, cells were collected by centrifugation and either dissolved directly in SDS-polyacrylamide gel
electrophoresis sample buffer (A) or used for extraction of p21
as outlined in Materials and Methods (B). The samples were
then electrophoresed in 7.5-15% SDS-polyacrylamide gels.
Lane a, PR13-Q bacteria; lane b, PR13-Q transformed by
pXVR; lane c, by pXVR mutant asparagine-119; lane d, by
pXVR mutant threonine-83; lane e, by pXVR mutant isoleucine144.
Functional Assays for ras Protein
129
ographed to visualize GTP binding to lysed bacterial
colonies. Results of a representative experiment are
presented in Fig. 2. Each bacterial colony transformed
with pXVR bound GTP whereas no GTP binding to
control colonies lacking the ras expression vector was
detected.
To generate binding mutants, the ras expression
vector was randomly mutagenized with hydroxylamine,
which converts C to T (34), before transforming PR13Q bacteria. The extent of mutagenesis was monitored
as a function of time by quantifying the efficiency of
transformation of E. coli to ampicillin resistance. After
transforming bacteria with a pool of DNA which was
mutagenized to a 40% colony survival rate, bacterial
colonies were screened for a loss in GTP-binding activity by the colony binding assay.
Six mutants were identified after screening 1500
colonies. Three contained nonsense mutations resulting
in the expression of truncated forms of p21. As shown
in Fig. 1, the remaining three mutants encoded full
length p21 molecules and were thus chosen for further
characterization. A summary of their properties, which
has been reported in detail elsewhere (33), is presented
in Table 1.
In all three cases, nucleotide sequencing of the entire
coding region of the mutant ras genes revealed only
single point mutations which resulted in amino acid
substitutions at position 83, 119, or 144. The mutant
p21s displayed equilibrium binding affinities for both
GTP and GDP which were 25- to 100-fold lower than
the GTP and GDP binding affinities of wild type p21. In
each case, the reduction in equilibrium-binding affinity
could be accounted for by increased rates of dissociation of the p21-GTP complex. Interestingly, each of
these GTP-binding deficient mutants induced transformation of NIH 3T3 cells with efficiencies indistinguishable from that of the wild type viral rasH gene.
Isolation of rasH Mutants Deficient in GTPDependent Autokinase
In addition to GTP binding, all ras proteins catalyze the
hydrolysis of bound GTP at a low rate (1-10 mmol/
min-mol GTP bound). In the case of the viral rasH and
rasK proteins, autophosphorylation of a threonine at
position 59 may also result from this activity. Because
of the low level of GTP hydrolysis, we have not been
able to assay ras GTPase directly on lysed bacterial
colonies. However, the bacterial colony GTP-binding
assay has been extended to simultaneously assay the
GTP-dependent autokinase activity of viral rasH p21.
CONTROL
pXVR
Fig. 2. GTP Binding to Bacterial Colonies Expressing p21
PR13-Q bacteria transformed with pXVR or nontransformed control bacteria were plated on nitrocellulose filters, lysed, used in a
GTP binding assay, and autoradiographed.
Table 1. Summary of Properties of GTP-Binding Mutants of rasH
Amino Acid
Substitution
none
ala-83+thr
asp-119-»asn
thr-144-*ile
Binding Affinity
1 X 10" 8
3 x 10~7
1 x 10"6
2.5 x 10"6
Dissociation Rate
(K_,) (min-1)
Transforming Activity
(foci/ng DNA)
0.02
0.3
2.0
0.7
5
5
5
5
Data on the binding affinities for GTP and GDP, the dissociation rates (k_i) of bound GTP from p21, and the transforming activities
of mutant ras genes assayed by transfection of NIH 3T3 cells are summarized from Ref. 33.
MOL ENDO-1987
130
These experiments were performed as described
above except that 7-32P-GTP was used and the incubation was performed at 37 C. Thus, after removing
unbound nucleotide, the signal observed represented
bound GTP plus 32P covalently attached to p21 (Fig. 3,
binding and autophosphorylation). At this point, GTPbinding mutants could be isolated as described above,
since mutants that failed to bind GTP would also lose
autophosphorylation activity. The same filters were
then washed with 10% trichloroacetic acid (TCA) so
that only covalently bound 32P remained on the filters
(Fig. 3, autophosphorylation). This signal did in fact
result from autophosphorylation since it was not detected when the incubation with GTP was performed
at 4 C (Fig. 3).
Autophosphorylation mutants were then isolated by
screening for bacterial colonies that bound GTP but
failed to yield an autophosphorylation signal. Figure 3
illustrates the isolation of one of two such mutants
characterized to date.
As was the case for GTP-binding mutants, nucleotide
sequencing revealed that each of the autokinase mutants was the result of a single nucleotide change. In
one case, the lack of autokinase was a consequence
of mutating codon 59 from ACA to ATA, so that the
mutant gene encoded isoleucine rather than threonine
BINDING
•
AUTOPHOSPHORYLATI ON
Vol. 1 No. 2
at this position. Since threonine-59 is the acceptor for
p21 autophosphorylation, this mutation would be expected to abolish the activity. The second mutant resulted from changing codon 7 from GTG to ATG, so
that the mutant gene encoded methionine instead of
valine.
The methionine-7 p21 was unaltered in either GTPbinding affinity or rate of dissociation of the p21-GTP
complex. To determine the autokinase activity of methionine-7 p21, partially purified protein was incubated
at 37 C for 15-120 min with Y- 3 2 P-GTP, electrophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide
gels and autoradiographed to detect 32P-labeling of p21
(Fig. 4). These experiments indicated that the autophosphorylation activity of methionine-7 p21 was 5- to
10-fold lower than that of wild type viral ras" p21. The
methionine-7 mutant induced transformation of NIH 3T3
cells with an efficiency indistinguishable from that of
wild type viral rasH, indicating that this reduction in
autokinase did not affect transforming activity.
Mutants of rasH Deficient in Cell Transformation
In addition to isolation and analysis of mutants defective
in interactions with guanine nucleotides, an independent
approach to study ras function is the identification of
A U T 0 P N OtPMOft Y L A TI O N
37°C
4°C
%«•%%
Fig. 3. Assay of GTP-Dependent Autokinase Activity on Bacterial Colonies Expressing p21
PR13-Q bacteria transformed by mutagenized pXVR were plated on nitrocellulose filters, lysed, and incubated with 7-32P-GTP at
either 37 C or 4 C. Filters were then washed with TBS and autoradiographed for 4 h (designated binding + autophosphorylation).
The same filters were then washed with 10% TCA and autoradiographed for 16 h (designated autophosphorylation). A mutant
colony defective in autophosphorylation is circled.
Functional Assays for ras Protein
131
p21
B
pp21
Fig. 4. Autophosphorylation Activity of Methionine-7 p21
P21 s were partially purified from PR13-Q bacteria transformed
with vectors which expressed cellular rasH (CRH), viral rasH
(VRH), or methionine-7 mutant viral rasH (MET-7) protein. The
partially purified p21s were incubated with 2 x 10~6 M 7-32PGTP at 37 C for 15 (lane a), 30 (lane b), 60 (lane c), and 120
(lane d) min. Aliquots were electrophoresed in 7.5-15% SDSpolyacrylamide gradient gels which were stained with Coomasie blue (A) and autoradiographed (B).
Fig. 5. The viral rasH gene was inserted into the Ba/nH1
site of pZIPneoSV(X)1 to generate the plasmid designated pZIPneoras. Plasmid DNA was treated with hydroxylamine to a 10% bacterial colony survival rate and
the mutagenized plasmid was used to transform the \j/2 helper cell line to generate a retrovirus stock. NIH
3T3 cells were then infected with the mutagenized
ZIPneoras virus and cloned in medium containing G418
to select for cells converted to G418 resistance. More
than 95% of G418-resistant colonies infected with nonmutagenized ZIPneoras virus were morphologically
transformed. However, after infection with mutagenized
virus stocks, morphologically normal G418-resistant
colonies were observed with a frequency of about 10%.
One trivial explanation for the appearance of nontransformed colonies after infection with mutagenized
ZIPneoras virus was that the cells did not produce p21.
This could occur either as a consequence of non-sense
mutations in the p21 coding region or regulatory mutations which block p21 synthesis. Since the ras gene
is transcribed using the same promoter in the retroviral
long terminal repeat as is used for viral replication,
transcriptional regulatory mutations should not be maintained in mutagenized viral stocks. To evaluate p21
expression, 10 colonies of morphologically nontransformed cells were labeled with 35S-methionine and p21
was assayed by immunoprecipitation. Six colonies produced p21 at levels similar to cells which were morphologically transformed by infection with ZIPneoras virus
(representative results are shown in Fig. 6). Thus, in
these cases, the transformation defectiveness of the
mutant viruses was not a consequence of failure to
synthesize p21 in normal amounts.
Mutagenize pZIPneoras
4Transfect $2 cells
4-
mutants defective in cell transformation. Although overlapping mutants might be identified by these two approaches, transformation-defective mutants might also
identify additional functional domains, including those
involved in interaction with effector molecules.
We have used a retroviral shuttle vector,
pZIPneoSV(X)1 (35), to facilitate generation and analysis of transformation-defective mutants. The advantageous features of this vector are: 1) Moloney murine
leukemia virus long terminal repeats and packaging
sequences, allowing propagation as a replication-defective retrovirus; 2) the neor gene, allowing selection for
resistance to the antibiotic G418 in mammalian cells
and to kanamycin in bacteria; 3) a pBR322 origin of
replication, allowing propagation as a bacterial plasmid;
and 4) a SV40 origin of replication, allowing amplification
and efficient recloning of DNA from transformed mammalian cells.
The general scheme for isolation and analysis of
transformation-defective ras mutants is illustrated in
Collect mutagenized ZIPneoras virus stock
4Infect NIH 3T3 cells
4Select G418-resistant cells
Screen for morphologically non-transformed colonies
4Fuse with Cos-7 cells
4Transform E. coli
4Isolate mutant ras plasmid
Fig. 5. Scheme for Isolation of Transformation-Defective Mutants
MOL ENDO-1987
132
a bed
Vol. 1 No. 2
notype, the recovered ras genes were reintroduced into
the ZIP vector and assayed by transfection of NIH 3T3
cells. The transforming activities of threonine-146 and
serine-34 mutant rasH genes were 100 and 1 foci/jig
DNA, respectively, in contrast to a transforming efficiency of 2 x 103 foci/Mg DNA for wild type viral rasH.
The transforming efficiency of the serine-34 mutant was
lower than that of normal cell rasH, which induced
transformation with an efficiency of approximately 10
foci/jig DNA in the ZIP vector.
Serine-34 p21 was unaltered in GTP-binding affinity,
GTP dissociation rate, GTPase activity, stability, or
membrane localization in infected cells as compared to
wild type viral rasH p21. In contrast, threonine-146 p21
was defective in guanine nucleotide binding, displaying
a dissociation consant (Kd) of approximately 10" 6 M for
both GTP and GDP. The dissociation rate of GTP from
threonine-146 p21 was similarly increased 100-fold
compared to wild type viral rasH p21. Neither GTPase
activity, stability, nor membrane localization of p21 were
affected by the threonine-146 mutation.
DISCUSSION
Fig. 6. Expression of p21 in NIH Cells Infected with Transformation-Defective Viral rasH Mutants
Cells were labeled with 35S-methionine. Cell extracts were
immunoprecipitated with anti-p21 monoclonal antibody YA6259 and electrophoresed in 7.5-15% linear gradient SDSpolyacrylamide gels. Lane a, NIH cells infected with wild type
ZlPneoras virus; lane b, NIH cells infected with ZlPneoras virus
mutant threonine-146; lane c, NIH cells infected with ZIPneoras
virus mutant serine-34; lane d, uninfected NIH 3T3 cells.
The SV40 and pBR322 origins of replication in
pZIPneoSV(X)1 permit efficient recloning of the mutant
retroviruses from mammalian cells. Therefore, to identify the mutations in the transformation-defective rasH
mutants, the morphologically nontransformed cells
were fused to Cos-7 cells. The Cos-7 cell line produces
SV40 T antigen which acts to drive replication from the
SV40 origin, resulting in the production and excision of
multiple unintegrated copies of the ZIPneoras DNA.
Unintegrated DNA was then extracted from the heterokaryons and used to transform E. coli, thereby recovering the mutant ras genes as bacterial plasmids.
Two independent mutations were identified in the six
ras genes recovered in these experiments. One mutant
encoded threonine in place of alanine at position 146.
The other five recovered genes contained identical double mutations which changed codon 34 from CCC to
TCT, encoding serine in place of praline. The identification of five isolates of the same double mutant suggests that there was a selective enrichment for this
mutant virus during replication of the mutagenized viral
stock.
To verify that these single amino acid substitutions
were responsible for the transformation-defective phe-
We have reviewed strategies for the isolation of mutant
ras genes encoding proteins which are deficient either
in interactions with guanine nucleotides or in induction
of cell transformation. In both cases, the approach has
been to use random mutagenesis coupled with screening procedures designed for efficient isolation and analysis of mutants with the desired phenotype. Interactions
of ras with guanine nucleotides, including GTP binding
and GTP-dependent autokinase, have been detected in
lysed bacterial colonies expressing ras protein. The
transforming activity of ras in NIH 3T3 cells has been
assayed using a retroviral shuttle vector that permits
efficient recloning from infected cells for analysis of
mutant ras genes.
Each of the assays employed has a low background
of false negatives (less than 5%) so that it is possible
to rapidly screen a large number of mutagenized plasmids. Consequently, a low rate of mutagenesis can be
used to ensure that most mutants result from only
single point mutations. This was demonstrated to be
the case for all but one of the mutants isolated in the
present experiments, including mutants defective in
GTP binding, autokinase, and cell transformation.
A major advantage of the ability to efficiently isolate
mutants after random mutagenesis is that this approach
does not require prior prediction of target sequences,
as is the case for site-directed mutagenesis. Thus it is
possible to directly identify multiple molecular domains
involved in a given biochemical or biological activity. For
example, we have identified mutations in three regions
of ras (amino acids 83,119, and 144) which affect GTP
binding. Two of these amino acids, 83 and 119, occur
in areas of strong homology between ras and other
guanine-nucleotide binding proteins including the G proteins, transducin and bacterial elongation and initiation
factors (36-38). Based on these homologies, one might
Functional Assays for ras Protein
have predicted these two domains as targets for sitedirected mutagenesis and this has in fact been done
for ras amino acids 116-119 (39-42). In contrast, the
GTP-binding mutations at amino acids 144 and 146
occur in a region which is not significantly homologous
to other GTP-binding proteins and would not represent
a readily predicted target for site-directed mutagenesis.
Likewise, there is no clear basis for prior prediction of
the effect of mutation at amino acid 7 on GTP-dependent autokinase activity or for the isolation of a transformation-defective mutant altering amino acid 34.
Another feature of the random mutagenesis strategies is that the number of amino acids involved in a
given activity can be estimated from the frequency of
isolation of mutants. A good example of this is the
isolation of GTP-binding mutants. The frequency of
missense mutants defective in GTP binding was approximately equal to the frequency of colonies which
did not produce full-length ras protein. The latter class
presumably was a consequence of non-sense mutations, which can be induced by hydroxylamine at 10
rasH codons. Thus, one would predict there are approximately 10 missense mutations which can be induced by hydroxylamine to result in a ras gene encoding
a protein defective in nucleotide binding.
The relationship between the biological and biochemical activities of p21 are complex and remain to be fully
elucidated. Mutations at positions 12 and 61, which
activate the transforming potential of p21, do not alter
the affinity or specificity of p21 for binding guanine
nucleotides (23, 43) but do reduce the GTPase activity
of p21 by a factor of 5-10 (24-27, 43, 44). However,
analysis of ras genes differing by seventeen different
amino acid substitutions at amino acid 61 indicated that
reduced GTPase did not correlate quantitatively with
the transforming potency of the mutated genes (43).
Furthermore, two mutated ras genes, proline-61 and
glutamic acid-61, displayed reduced GTPase but no
activation of transforming potential, indicating that reduction in GTPase was not sufficient to activate the
transforming potential of p21 (43). Conversely, it has
been reported that a rasH gene activated by the substitution of threonine for alanine at position 59 displays
normal GTPase activity (45). Taken together, these
observations indicate that mutations at ras codons 12
and 61 can lead to reduced GTPase and activation of
transforming potential, but that a reduction in GTPase
activity is neither necessary nor sufficient for ras activation.
We have identified a mutation at position 7 which
reduces the GTP-dependent autokinase of viral rasH
p21 5- to 10-fold without altering its GTP-binding properties or transforming activity. If autophosphorylation of
viral rasH threonine-59 results from this threonine serving as an alternative to water as a phosphate acceptor
for GTP hydrolysis, then one would predict that amino
acid 7 is also involved in the GTPase reaction. It will
thus be of interest to introduce the methionine-7 mutation into normal cellular rasH to investigate its effect
on GTPase and to determine whether it activates transforming potential.
133
Mutations which reduce the affinity of p21 for GTP
by up to 5000-fold do not necessarily reduce the transforming efficiency of activated viral or cellular rasH
genes (33, 39, 40, 42). Because of the high concentrations of intracellular GTP [in the millimolar range (46)],
at least some of these GTP-binding mutants still appear
to bind GTP in vivo (33). However, it is clear that high
affinity GTP binding is not required for transformation
by activated ras genes. This may reflect permanent
mutational activation rendering the protein insensitive
to normal regulation by guanine nucleotide binding.
In contrast, two previously reported mutants (47) and
the threonine-146 mutant described here reduce both
guanine nucleotide binding and transforming activity of
viral rasH p21. Since the threonine-146 p21 is reduced
only 100-fold in affinity for GTP, the reduction of transforming activity does not appear to be a direct consequence of reduced GTP binding. Rather, these results
suggest that some mutations which affect GTP binding
may also exert independent effects on conformation
and biological activity of p21.
If p21 function is analogous to that of the G proteins,
one would predict that the activity of p21 is normally
regulated by the kinetics of guanine nucleotide exchange. In particular, physiological activation of p21 is
expected to be mediated by interaction with a cell
surface receptor that promotes the exchange of GTP
for bound GDP. The high equilibrium binding affinity of
GDP and GTP by p21 reflects a low dissociation rate
of bound nucleotide, the physiological function of which
is thought to be stabilization of the p21-GDP complex
until receptor-stimulated guanine nucleotide exchange
occurs. Mutations which alter the equilibrium affinity of
p21 for GTP do so primarily by increasing the rate of
dissociation of bound guanine nucleotide (33). Thus,
although these mutations did not necessarily reduce
the transforming activity of p21s which were already
activated by other mutations, they might be expected
to impair the regulation of normal p21 functon. Indeed,
some mutations which reduce guanine nucleotide binding have been found to increase the transforming activity of normal cell p21, suggesting that a decreased
stability of the p21-guanine nucleotide complex can
result in constitutive p21 activation (40, 42). However,
the alternative possibility that activation of transforming
potential by these mutations is a result of effects on
p21 conformation has not been excluded. Clearly, further analysis of the relationship between biological activity and interactions with guanine nucleotides is
needed to understand the role of guanine nucleotides
in regulation of p21 function.
One transformation-defective mutant, serine-34, was
identified which reduced transforming activity by
greater than 1000-fold. This mutation did not affect the
stability or subcellular localization of p21 in infected
cells nor did it alter interaction of p21 with guanine
nucleotides. Mutants with alterations of p21 amino
acids 35-44 which display similar properties have also
been recently reported by others (48, 49). This region
may thus define a new domain, possibly involved in
interaction of p21 with an effector molecule.
Vol. 1 No. 2
MOL ENDO-1987
134
The assays for interaction of ras with guanine nucleotides described here may be directly applicable to other
cell proteins, such as the G proteins, with similar biochemical properties. More generally, mutants of any
protein expressed in bacteria could be detected as long
as the relevant biochemical activity is retained on nitrocellulose filters. In addition to binding small molecules
and enzymatic activities, such assays might detect
protein-protein (50) and protein-DNA (51) interactions,
or even interactions of proteins with whole cells (52).
The use of a retroviral shuttle vector for efficient mutagenesis and rescue could also be applied to any gene
which induces a biologically detectable phenotype in
cultured cells.
MATERIALS AND METHODS
GTP Binding to Lysed Bacterial Colonies
PR13-Q bacteria, transformed with the pXVR rasH expression
vector, were plated on agar containing ampicillin and kanamycin. PR13-Q was constructed by mating into the strain
PR 13 (53), an episome containing the iQ allele of the i gene
and the kanamycin resistance marker of Tn5. After replica
plating onto nitrocellulose filters, colonies were lysed by exposure to chloroform mist for 10 min, followed by incubation
with gentle shaking overnight (20 C) in 20 nriM Tris-HCI, pH
7.4, 1 mM MgCI2, 150 mM NaCI, 3% BSA, 1 Mg/ml DNase I,
and 40 Mg/ml lysozyme. The filters were then washed by
gentle shaking in 20 mM Tris-HCI, pH 7.4, and 150 mM NaCI
for 1 h at 20 C (50). The filters were then incubated with gentle
shaking at 4 C in TBS (20 mM Tris-HCI, pH 7.4, 500 mM NaCI)
plus 1.0 mM MgCI2) and 10~8 M a-^P-GTP (New England
Nuclear, Boston, MA; 600 Ci/mmol) (10 ml/5 filters). After 1 h,
the filters were washed twice for 10 min each with TBS at 4
C and autoradiographed.
Autophosphorylation Activity on Lysed Bacterial Colonies
To measure autophosphorylation activity and GTP-binding
activity on the same lysed bacterial colonies, the method
described above was altered in the following manner. The
filters were incubated at 37 C in TBS plus 1.0 mM MgCI2 and
10~7 M 7-32P-GTP (New England Nuclear, 10-50 Ci/mmol).
After 1 h, the filters were washed in TBS and autoradiographed. This signal represented bound GTP plus ^ P covalently bound to p21. To measure autophosphorylation activity
independently from binding activity, the same filters were then
washed with 10% TCA at 4 C, which removed noncovalently
bound GTP, and again autoradiographed.
Assays on Soluble p21
To isolate ras proteins for binding and autophosphorylation
assays, the bacterial strain PR13-Q was transformed with
pXVR or mutant v~rasH expression plasmids, grown to an A
590 = 0.25 and then induced to make p21 by the addition of
5 mM IPTG. After 1 h, the cells were collected by centrifugation.
The cell pellet from a 2-ml culture was resuspended in 100 /J
lysis buffer (25 mM Tris-HCI, 0.7 mM Na2HPO4, 5 mM KCI, 0.14
M NaCI, 5 mM EDTA, 10 mM MgCI2, 1 % Triton X-100, 25%
sucrose, and 1 mg/ml lysozyme, pH 7.4) and vortexed vigorously. After freezing and thawing twice, the extract was incubated with DNase I (1 /ig/m' for 10 min at 20 C) and then
centrifuged at 10,000 x g for 5 min (27). The pellet was
washed in 1 % Triton X-100, resuspended in 50 n\ 3.5 M
guanidine hydrocholoride in 20 mM MES [2(A/-morpholine)
ethane sulfonic acid], pH 7.0, and paniculate matter was
removed by centrifugation at 10,000 x g for 5 min (27).
GTP-binding experiments with purified ras protein were
performed as previously described (33). To measure autophosphorylation activity, 5 ^g protein were incubated in 50 n\
20 mM Tris-HCI, pH 7.4, plus 0.2 mM MgCI2> 5 mM dithiothreitol,
20 Mg/ml BSA, and 2 M M 7-32P-GTP (10-50 Ci/mmol) at 37 C
for various times. The samples were then electrophoresed in
SDS-polyacrylamide gels and the gels were dried and autoradiographed.
In Vitro Mutagenesis
Thirty microliters of DNA to be mutagenized (1 mg/ml) were
mixed with 150 M' ethylene glycol and heated to 70 C for 5
min. After removing a 27-^1 aliquant to be used as a nonmutagenized control, 16 pi hydroxylamine solution (0.5-1 M hydroxylamine, 0.2 M Na pyrophosphate) were added to the
remainder of the DNA and the reaction was allowed to proceed
at 70 C. After various times, 30-MI aliquants were removed
and added to tubes containing 80 M' ice-cold stop solution (0.6
M Tris-HCI, pH 8.0,1.0 M NaCI, 20% acetone) (54). The DNAs
were separated from residual hydroxylamine by passage
through 1.0 ml Sephadex-G50 columns equilibrated in 10 mM
EDTA. The degree of mutagenesis was monitored by quantifying the efficiency of transformation of E. coli to ampicillin
resistance.
Immunoprecipitations
NIH cells infected with either wild type or mutant rasH virus
were labeled for 15 h at a density of 106 cells/60-mm dish with
35
S-methionine (250 /iCi/ml, 500 Ci/mmol; New England Nuclear) in media containing 10% calf serum. p21 was immunoprecipitated from these cells with anti-p21 monoclonal antibody YA6-259 (18, 23).
Isolation of Transformation Defective Mutants
Procedures using the ZIP-^-2 system were as described by
Cepko et al. (35). pZIPneoras was mutagenized with hydroxylamine at a rate that yielded 10% colony survival. One
hundred nanograms of this DNA along with 20 /*g carrier NIH
DNA were used to transfect each of three dishes of \p-2 cells
(5 x 105 cells/60-mm dish). After 3 days, the cells were
trypsinized and split into nine 100-mm dishes in the presence
of 100 Mg/ml G418. Drug-resistant colonies (~300) were
pooled 14 days later and culture media containing a stock of
randomly mutagenized ZIPneoras retroviruses was collected.
The titer of virus particules (generally 104/ml) was determined
by infection of NIH 3T3 cells with an aliquot of this media and
quantification of G418 resistant colonies 14 days later. To
screen for transformation-defective retroviruses, NIH 3T3 cells
(2.5 x 105/60-mm dish) were infected with -300 colonyforming units and cloned by subculture into ten 96-well microtiter dishes containing media plus 400 M9/ml G418. Wells were
examined microscopically to identify morphologically nontransformed G418-resistant colonies.
To rescue proviral DNA, nontransformed NIH cells were
mixed in a 1:1 ratio with Cos-7 cells (5 x 105 cells each/60mm dish). Forty eight hours later, the cells were fused by
exposure to 1.0 ml 50% polyethyleneglycol 1000 in Dulbecco's
modified Eagle's medium for 1 min. Forty eight hours after
fusion, the cells were dissolved in 2% SDS + 10 mM Tris, pH
7.4, + 10 mM EDTA; NaCI was added to a final concentration
of 1.25 M, and the sample was incubated at 4 C for 6 h.
Paniculate matter was removed by centrifugation at 30,000
rpm in a Ti50 rotor (Beckman, Fullerton, CA) for 1 h. DNA was
then precipitated from the supernatant with ethanol and used
to transform HB101 to kanamycin resistance.
Functional Assays for ras Protein
Acknowledgments
Received September 5,1986.
Address requests for reprints to: Dr. Geoffrey M. Cooper,
Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, 44 Binney Street, Boston, MA 02115.
* This research was supported by grants from the National
Cancer Institute, a fellowship (to L.A.F.) from the Leukemia
Society of America, and a faculty research award (to G.M.C.)
from the American Cancer Society.
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