(Received for publication, February 7, 1995; and in revised form, May 30, 1995)
From the
Two activated transforming mutants of human
pp60 were found to possess single
point mutations within the regulatory carboxyl terminus (E527K in CY
CST201) and the kinase domain (E381G in WO CST1), respectively, that do
not directly interfere with either the regulatory c-Src kinase (CSK)
phosphorylation site (Tyr
) or the SH2/3 domains. In
vivo, both mutant proteins are hypophosphorylated on their
carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src
proteins exhibited resistance to inactivation by CSK relative to
wild-type Src. Under these in vitro conditions, E381G c-Src
was found to be phosphorylated by CSK to wild-type levels, while E527K
c-Src was not detectably phosphorylated. The ability of CSK to
phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src
was also impaired, suggesting that CSK is unable to recognize E527K
c-Src as an efficient substrate. In the case of E381G c-Src,
examination of whether its SH2/3 domains were accessible to the
carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced
ability of autophosphorylated E381G c-Src to bind to a synthetic
phosphopeptide modelled from the SH2-binding region of polyoma middle-T
antigen which binds to Src SH2 with high affinity. This suggests that
the E381G c-Src mutation results in an altered or reduced accessibility
of the SH2 domain of the autophosphorylated form of E381G c-Src and may
represent a previously undescribed mode of Src activation. Further
study of these and other Src mutants may offer additional new insights
into the regulation of ``Src family'' kinases.
pp60, or Src, is a
membrane-associated tyrosine kinase and the cellular homologue of the
highly oncogenic form, pp60
, or
v-Src, that is encoded by Rous sarcoma virus.
pp60
, and the other related members
of the ``Src family'' which includes Src, Lck, Fyn, Lyn, Hck,
Fgr, Blk, Yrk, and Yes, are thought to be key components in signal
transduction pathways that relay signals received at the cell membrane
to the cytoplasm and nucleus(1) . Current investigations have
identified the particular importance of Lck in T-cell receptor (2) and Src in platelet-derived growth factor receptor (3) pathways. Src family members have the potential to become
transforming proteins as a result of regulatory defects, and
pp60
, in particular, has been
implicated in the development of human breast and colon
cancer(4, 5) .
pp60 kinase activity is tightly controlled in vivo, and
various studies have elucidated the importance of a highly conserved
carboxyl-terminal tyrosine regulatory site (Tyr
in
chicken pp60
, Tyr
in
human pp60
). Src proteins which
lack this tyrosine due to mutation exhibit elevated kinase activity in vivo and the ability to cause cellular transformation and
tumor formation(6, 7) . Activation has also been
observed in other Src family members with similar
mutations(8, 9, 10) , emphasizing the
importance of this region and suggesting the presence of a common
regulatory mechanism.
The mechanism by which phosphorylation at
Tyr regulates Src activity has been addressed in several
laboratories(11, 12, 13, 14, 15, 16) .
The currently accepted theory proposes an intramolecular interaction of
the phosphorylated carboxyl-terminal tyrosine with the SH2/SH3 domains
of Src such that the enzyme activity is inhibited. Recently, a protein
tyrosine kinase, CSK, (
)that can phosphorylate Tyr
and inactivate Src activity has been
identified(17, 18, 19, 20, 21) .
CSK can also regulate other Src family members, including Lck, Fyn, and
Lyn, by phosphorylation of the corresponding carboxyl-terminal tyrosine
residue(17, 22) . Src, Fyn, and Lyn activity is
elevated in CSK
mice(23, 24) , supporting the in vivo importance of CSK and implying a common mode of regulation for
several Src family members.
Interestingly, activating mutations in
the Src family of tyrosine kinases are not limited to mutations that
remove the CSK tyrosine phosphorylation site. Activating mutations have
been found throughout Src, with the exception of the unique
region(25) . In our laboratory, we have used a
retroviral-mediated selection procedure to isolate and characterize a
number of activated transforming mutants of human Src that do not
directly alter Tyr(7) and have begun biochemical
studies using highly purified forms of Src to examine how these
mutations alter Src tyrosine kinase activity and make Src refractory to
normal regulation by CSK.
To
measure the effects of autophosphorylation or CSK phosphorylation on
Src's ability to bind to the middle-T phosphopeptide resin, 100
ng of Src was incubated in the absence or presence of 25 µM ATP and 300 ng of CSK in 50 µl of kinase reaction buffer
containing 50 mM Hepes, pH 7.4, 5 mM MgCl, 0.15 M NaCl, 1 mM DTT, and
0.02% Nonidet P-40 for 30 min at 30 °C. EDTA was then added to a
final concentration of 20 mM, and the ability of the Src to
bind middle-T phosphopeptide was measured by adding one-fifth of each
kinase reaction (10 µl) to 10 µl (packed volume) of middle-T
phosphopeptide beads that had been washed and resuspended in 30 µl
of binding buffer containing 50 mM Hepes, pH 7.4, 1 mM DTT, 0.1% Nonidet P-40, 10% glycerol, 0.10 M NaCl, and
0.1% (w/v) bovine serum albumin. Following a 1-h incubation at 4 °C
on a rotator, the supernatant was removed and the beads were washed
three times with binding buffer. The resin was then resuspended in
SDS-PAGE sample buffer, and an aliquot was subjected to SDS-PAGE,
transferred to nitrocellulose, and blotted with 2-17 anti-Src antibody.
Cloning and sequencing the Src
gene of two of the viruses, WO CST1 and CY CST201, that were isolated
either in the absence or presence of 5-azacytidine, respectively,
revealed that CY CST201 (which will subsequently be referred to as
E527K c-Src) possessed only a single nucleotide mutation (G
A) that resulted in a single amino acid change
(Glu
Lys) near the carboxyl terminus and 3 amino
acids amino-terminal from the CSK phosphorylation site (Fig. 1).
WO CST1 (which will subsequently be referred to as E381G c-Src) was
also found to contain only a single nucleotide mutation (A
G) that caused a single amino acid change (Glu
Gly), but it was located within the kinase domain.
Figure 1:
The domain structure
of human pp60 and the location of
two activating mutations. Src and Src family members can be subdivided
into several structural domains based on amino acid homology between
members. From left to right,
pp60
consists of a myristoylated
amino terminus, a unique region which is poorly conserved among family
members, a SH3 domain, a SH2 domain, a tyrosine kinase domain
containing an autophosphorylation site, and a regulatory carboxyl
terminus containing a regulatory tyrosine phosphorylation site. The
activating mutations present in WO CST1 (CST1) and CY CST201 (201a2) Src are shown.
Cyanogen bromide cleavage of in vivo [P]orthophosphate-labeled Src
immunoprecipitated from virally infected CEF cells was used to generate
Src fragments that contain the regulatory phosphorylation sites of
Src(31) . Fig. 2A shows that both E381G c-Src
and E527K c-Src were hypophosphorylated on the 5.2-kDa peptide
containing Tyr
and hyperphosphorylated on the 7.8-kDa
peptide containing Tyr
relative to wild-type Src. In
addition, the two mutant proteins had 10- and 29-fold elevations (E527K
c-Src and E381G c-Src, respectively) in tyrosine kinase activity
relative to wild-type Src when immunoprecipitated from CEF cells under
conditions which preserve the regulatory phosphorylation sites (Fig. 2B).
Figure 2:
Characterization of the in vivo phosphorylation status and activity of normal and activated Src.
CEF cells (75% confluent) infected with wild-type, WO CST1 (E381G), or
CY CST201 (E527K) virus were lysed directly in RIPA buffer (for
activity measurement) or were incubated for 7 h in the presence of
[P]orthophosphate prior to lysis in RIPA buffer
(to examine phosphorylation sites). Aliquots of cell lysate containing
equal amounts of Src (as deduced by immunoblotting) were
immunoprecipitated with 2-17 anti-Src antibody. A, the
immunoprecipitated Src from the metabolically labeled plates was
subjected to SDS-PAGE, the Src band was localized by autoradiography,
excised, and subjected to CNBr cleavage, and the cleaved products were
separated by SDS-PAGE (22% gel). An autoradiogram is shown. B,
the immunoprecipitated Src from the unlabeled plates was assayed for
tyrosine kinase activity using the cdc2 substrate peptide as described
in the CSK inactivation assay.
Figure 3: Purification of Src. Src was purified from either baculovirus-infected Sf9 cells containing wild-type (CS) or E381G c-Src (CST1) or retrovirus-infected CEF cells containing E527K c-Src (201a2) by immunoaffinity chromatography. A, the purified protein was resolved on 9% SDS-PAGE and stained with Coomassie Blue. Lanes 1, 3, and 5 contain 84 ng, and lanes 2, 4, and 6 contain 120 ng of Src protein, respectively. B, 5.0 ng of each purified Src protein or an equivalent amount of Src immunoprecipitated with 327 anti-Src antibodies from lysates made from human foreskin fibroblasts (33) was resolved on 8% SDS-PAGE, transferred to nitrocellulose, and blotted with either antiphosphotyrosine or 327 anti-Src antibodies. The dark band beneath the Src band in the human foreskin fibroblast lane is immunoglobulin heavy chain.
The purified Src protein was next examined for tyrosine
kinase activity by its ability to phosphorylate a highly Src
family-specific synthetic peptide substrate modelled against residues
6-20 of cdc2 ([K]cdc2(30) ).
Wild-type and E527K c-Src demonstrated equivalent activities against
the cdc2 peptide at all concentrations tested up to 1100 µM (Fig. 4). Interestingly, E381G c-Src was approximately
2-4-fold more active at phosphorylating the peptide relative to
the other two purified proteins. This difference was observed in
several Src preparations and was observed also when using Src
immunoprecipitated directly from crude Sf9 cell lysates (results not
shown).
Figure 4:
Phosphorylation of the cdc2 peptide by
purified normal and activated Src. The activity of wild-type (),
E381G c-Src (
), and E527K (
) c-Src was measured by the
incubation of 1.5 ng of Src with cdc2 peptide in kinase buffer (see Src Inactivation Assay under ``Materials and
Methods'') for 15 min. The reactions were stopped, an aliquot was
spotted onto p81 phosphocellulose, and the incorporated radioactivity
was quantitated with a scintillation counter. Each point represents the
mean of duplicate assays ± S.D.
Figure 5:
Inactivation of normal and activated Src
by CSK. 10 ng of purified wild-type (), E381G c-Src (
), or
E527K (
) c-Src was incubated together with CSK in the presence of
ATP for 30 min at 30 °C. cdc2 substrate peptide was then added and
incubated for an additional 15 min to measure Src kinase activity. Each
point represents the mean of duplicate assays ± S.D. Error
bars are not shown when less than the width of the data
symbol.
Figure 6: Phosphorylation of wild-type and activated Src by CSK. 10 ng of wild-type (CS), E381G c-Src (CST1), or E527K c-Src (201a2) was incubated in the absence or presence of 300 ng of CSK under identical incubation conditions as described in Fig. 5except for the use of higher specific activity ATP (approximately 17 µCi/2500 pmol). The reaction contents were subjected to SDS-PAGE, the Src band was excised and subjected to CNBr cleavage, and the cleaved protein was separated by SDS-PAGE on a 22% gel.
Phosphorylation assays using CSK indicated that there was a marked inability of CSK to phosphorylate the mutant peptide (mfcp) compared to the normal peptide (fcp) (Fig. 7). At the highest concentration of peptide employed in the assay (1200 µM), there was a 10-fold difference in the velocity of the reaction. This result is consistent with our observation showing the inability of CSK to phosphorylate E527K c-Src protein.
Figure 7:
Phosphorylation of Fyn carboxyl-terminal
peptides by CSK. 75 ng of CSK was incubated with either a human Fyn
carboxyl-terminal peptide (fcp) () or a mutant peptide (mfcp)
(
) which contained the corresponding E527K c-Src mutation. The
reactions were stopped following a 15-min incubation, and the
incorporated radioactivity was quantitated by a scintillation counter.
Each point represents the mean of duplicate assays ±
S.D.
Figure 8: Binding of Src to middle-T phosphopeptide resin. Wild-type (CS) and E381G c-Src (CST1) were preincubated under the conditions described under ``Materials and Methods.'' The resulting unphosphorylated (-ATP, -CSK), autophosphorylated (+ATP, -CSK), or CSK-phosphorylated (+ATP, +CSK) wild-type (CS) or E381G c-Src (CST1) was then incubated with immobilized phosphopeptide derived from polyoma middle-T antigen. The middle-T phosphopeptide resin was washed, resuspended in SDS-PAGE sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and blotted with 2-17 anti-Src antibody. The two NSB lanes contain wild-type (-ATP, -CSK, left lane) and E381G c-Src (-ATP, -CSK, right lane) that was bound to resin lacking peptide.
We have isolated a number of mutant viruses harboring the Src oncogene that, as a result of mutations within Src which activate the tyrosine kinase activity of the enzyme, are now capable of morphological transformation of CEF cells. These activating mutations were found throughout the Src molecule, with the exception of the unique domain (results not show), and we are currently examining how these mutations activate Src in hopes of furthering our understanding of Src regulation.
In this report, two mutant forms of Src were examined and were found to be resistant to inactivation by CSK. Cloning and dideoxy-DNA sequencing revealed that one mutant, WO CST1, possessed only a single point mutation within the kinase domain resulting in a single amino acid substitution (E381G). The other, CY CST201, possessed only a single point mutation resulting in a single-amino acid substitution (E527K) that was localized near the carboxyl terminus and only 3 amino acids amino-terminal to the CSK phosphorylation site. Both mutations resulted in vivo hypophosphorylation of the regulatory phosphorylation site of Src accompanied by an elevation in Src tyrosine kinase activity. Previously, a mutation corresponding to the mutation in WO CST1 and mutations corresponding to the mutation in CY CST201 have been observed in the context of chicken Src(36, 37, 38) . In both cases, the mutations were also thought to be responsible for the observed Src activation.
To further characterize the defects in Src regulation, we have purified both normal and mutant Src to homogeneity and have examined their susceptibility to both inactivation and phosphorylation by the important regulatory enzyme CSK. In activation assays, we found that both mutants were resistant to inactivation by CSK relative to wild-type Src. Interestingly, E381G c-Src became phosphorylated to wild-type levels in the absence of significant changes in tyrosine kinase activity. E527K c-Src could not be detectably phosphorylated by CSK under our soluble in vitro conditions.
The mutation in
E527K c-Src results in a rather drastic charge change of +2 (Glu
Lys) only 3 amino acids amino-terminal to the CSK
phosphorylation site and involves a residue that is highly conserved
among all Src family members. We hypothesized that the change in charge
interfered directly with the ability of CSK to phosphorylate the
neighboring Tyr
residue. In order to test this
possibility directly, we synthesized a peptide modelled against the
carboxyl terminus of Src family members. The carboxyl-terminal region
of Src family members is highly conserved, and we have used a specific
substrate peptide (fcp) derived from the carboxyl terminus of wild-type
human Fyn (residues 503-537) to assay CSK during its purification
from bovine thymus (results not shown). This region of Fyn is identical
with the corresponding Src region with the exception of four conserved
amino acid changes. We found that when the E527K c-Src mutation was
incorporated into the peptide, this led to a dramatic decrease in its
ability to be phosphorylated by CSK that was consistent with our
results using the intact Src protein and suggests that the mutation
causes an alteration in substrate recognition that can be localized to
this 30-amino acid region of Src. The resistance of Tyr
to phosphorylation by CSK in E527K c-Src also explains the in
vivo hypophosphorylation at this site that we have observed
(results not shown).
Supporting evidence for the role of Glu in efficient substrate recognition has been obtained through
examination of mutations corresponding to the E527K c-Src mutation in
the context of chicken Src, but in the presence of additional
carboxyl-terminal mutations which may complicate
interpretations(34, 37) . Reduced in vivo phosphorylation was observed on the carboxyl-terminal regulatory
tyrosine as well as resistance to CSK phosphorylation in vitro whenever the acidic residue amino-terminal to Tyr
was mutated, suggesting that the presence of Glu
(in chicken) appeared to be essential for both efficient
autophosphorylation and CSK phosphorylation of
Tyr
(34, 37) .
In contrast to the
putative mechanism of activation we have proposed with regard to E527K
c-Src, an alternate mode of activation in an activated transforming
mutant of chicken Src that possessed a Glu Lys mutation that
corresponds in location to the E527K c-Src mutation in human Src has
been proposed(38) . Interestingly, no obvious alteration in the in vivo phosphorylation status of the carboxyl-terminal
regulatory tyrosine (Tyr
in chicken) was observed by
these investigators, suggesting that either the NIH 3T3 cells used to
express Src contain additional tyrosine kinases able to phosphorylate
Tyr
or that CSK in these cells has enhanced activity or
abundance relative to what we have observed with E527K c-Src in chicken
embryo fibroblasts and were able to obtain in our in vitro assay. The investigators attributed the activation to a
conformational change in the carboxyl terminus(38) .
Other recent work may also be relevant to these findings. A synthetic phosphorylated Src carboxyl-terminal peptide that contains the same mutation as E527K c-Src binds much less efficiently to a Src SH2-GST fusion protein than the corresponding wild-type peptide(13) . Thus, impairment of Src regulation by this single activating mutation may involve the combined effects of two distinct blockades. The first involves impairment of CSK's ability to phosphorylate Src as suggested by our results. In the event that in vivo conditions might somehow allow CSK to overcome this blockage, as appears to be the case in the NIH 3T3 cells described above, the mutation would also impair the ability of the phosphorylated carboxyl terminus of Src to interact with its SH2 domain.
In contrast to E527K c-Src, the
mechanism underlying the regulatory defect in E381G c-Src does not
appear to involve resistance of Tyr to phosphorylation by
CSK. We observed that E381G c-Src was phosphorylated by CSK in
vitro to an extent similar to wild-type Src, but in the absence of
any observed reduction in Src tyrosine kinase activity. The E381G c-Src
mutation is in the kinase domain, is distant from the SH2/SH3 and
carboxyl-terminal regulatory domains, and may be causing a
conformational change such that: (a) the SH2/SH3 domains and
carboxyl-terminal tyrosine interact in a nonproductive manner that does
not inhibit the Src kinase domain or (b) the SH2/SH3 domains
and carboxyl-terminal tyrosine can no longer interact.
To examine
these two possibilities, we tested the SH2 domain accessibility of both
wild-type and E381G c-Src by examining whether they could bind to a
synthetic phosphopeptide modelled against a region of polyoma middle-T
antigen which binds unoccupied Src SH2 domain with high
affinity(35) . We found that, similar to binding experiments
carried out by others with a carboxyl-terminal Src phosphopeptide (11) , the middle-T phosphopeptide binds unphosphorylated
wild-type Src. When the carboxyl terminus of wild-type Src was
phosphorylated by CSK, the binding of Src to middle-T phosphopeptide
resin was reduced dramatically. E381G c-Src also bound to the middle-T
phosphopeptide resin to an extent similar to wild-type Src when
unphosphorylated, but, unexpectedly, E381G c-Src exhibited reduced
binding upon autophosphorylation alone, in the absence of high levels
of phosphorylation at Tyr. The reduced binding of
autophosphorylated E381G c-Src to the middle-T phosphopeptide resin,
whether in the presence or absence of the CSK phosphorylation at
Tyr
, was not accompanied by any attenuation in E381G
c-Src kinase activity (see Fig. 5and results not shown).
Although these results make it difficult to answer our original
question of whether the SH2 domain of E381G c-Src is occupied by its
phosphorylated carboxyl-terminal tail, they do suggest that, upon E381G
c-Src autophosphorylation, the SH2 domain becomes effectively altered
or conformationally blocked, resulting in our observed reduction in
binding to both exogenously supplied binding sites (the synthetic
phosphopeptide resin) as well as to the endogenously phosphorylated
Tyr
. We have also observed similar reductions in the
binding of a CSK-phosphorylated Fyn carboxyl-terminal peptide to E381G
c-Src upon autophosphorylation (results not shown), suggesting that
this phenomenon might have a role to play in the resistance of E381G
c-Src to inactivation by CSK.
We do not feel that the difference in
binding of the peptide to Src can be explained by autophosphorylation
of Tyr because during our incubations as carried out in Fig. 8, the site undergoes <10% of the phosphorylation that
it would undergo in the presence of CSK, but its binding to the
phosphopeptide was reduced by 66%. We are currently uncertain as to the
role of Tyr
phosphorylation. Results obtained from in
vitro experiments ( Fig. 6and results not shown)
demonstrating that the ability of CSK to inhibit wild-type Src activity
appears independent of the extent of autophosphorylation on
Tyr
, suggest that phosphorylation of Tyr
in
wild-type Src does not desensitize wild-type Src to CSK. This does not
exclude the possibility that the E381G c-Src mutation alters Src in
such a way that it now responds to Tyr
phosphorylation.
In addition to the binding alterations in E381G c-Src we have measured in vitro using a phosphorylated peptide, it is possible that similar changes in the ability to bind tyrosine-phosphorylated molecules may also occur in vivo, resulting in reductions in the ability of E381G c-Src to bind proteins that have been reported to be found in association with Src, such as Fak, p130, and p110(39, 40, 41, 42) . Even if there is impairment in in vivo SH2 binding by E381G c-Src, it has not completely impaired the ability of E381G c-Src to transform chicken embryo fibroblasts; a finding not unlike that reported by Seidel-Dugan et al.(43) , in which various mutant forms of wild-type Src lacking large regions of SH2 and SH3 have elevated tyrosine kinase activity and are still fully capable of cellular transformation.
Based on structure-function and x-ray
crystallographic analysis of the cAMP-dependent protein kinase
catalytic domain (44) and Src family kinase domain homology
with cAMP-dependent protein kinase(45) , the E381G c-Src
mutation at Glu appears to be located only 6 amino acids
amino-terminal from the highly conserved kinase catalytic loop.
Interestingly, an amino acid corresponding to Glu
in
human Src is present in all Src family members but is not conserved in
other tyrosine or serine/threonine kinases. The mutation is also close
enough to the Src autophosphorylation site (Tyr
) and to a
regulatory region (residues 424-428 in
pp60
) in the kinase domain of Src (46) that it might interfere with several regulatory features
of the protein.
Resistance of Src mutants to the effects of the
regulatory phosphotyrosine have been reported by other investigators
examining activated Src mutants possessing mutations in either SH2 or
SH3. It has been demonstrated recently that mutations in either the SH2
or SH3 domain leave Src resistant to CSK regulation without altering
the ability of Src to be phosphorylated by
CSK(12, 15, 16) . The results suggest that
mutations in the SH2/SH3 domains have the potential to interrupt
interactions with the carboxyl-terminal phosphotyrosine, presumably
through direct disturbance of the SH2/3 domains, resulting in
constitutive Src activation. The E381G c-Src mutation in Src is similar
in that it also is resistant to inactivation by CSK even though it can
become phosphorylated at Tyr. What makes this mutation
interesting and distinct from the SH2/3 domain mutations is that it is
distant from both SH2/3 and the carboxyl-terminal regulatory tyrosine
and does not directly interfere with these two domains. Its effects are
likely reflected in the overall structure of the Src molecule and
represent an alternate mechanism for Src activation that has not
currently been described or analyzed biochemically.
In this
manuscript, we have demonstrated the resistance of two activated
transforming mutants of pp60 to regulation
by CSK. It is apparent from these studies and others that mutations in
the Src family act to usurp or modulate the ability of CSK to regulate
tyrosine kinase activity. The mechanisms behind these regulatory
defects appear to fall into specific categories depending upon the
location and nature of the mutation. We have identified two new
activated mutants of human Src exhibiting two different defects in Src
regulation and have noted important differences between the two mutant
Srcs and wild-type Src. One of these alters the ability of CSK to
phosphorylate Src, and the other inhibits the CSK phosphorylation from
regulating Src activity through an apparent conformational change
induced by E381G c-Src autophosphorylation. These studies should
provide interesting insights into Src enzyme regulation and into the
mechanisms responsible for mutational activation of Src.