(Received for publication, June 23, 1995; and in revised form, August 9, 1995)
From the
The phosphorylating and transforming activities of c-Src are
negatively regulated by phosphorylation at Tyr-527 near its carboxyl
terminus. Previous studies have indicated that c-Src preferentially
autophosphorylates Tyr-416, a residue in the middle of the catalytic
domain, in vitro, and that Tyr-527 is phosphorylated by the
carboxyl-terminal Src kinase, Csk. However, indirect evidence suggests
that c-Src may also autophosphorylate Tyr-527 as part of a negative
feedback loop. While some in vivo evidence suggests that
Tyr-527 can be autophosphorylated in an intermolecular interaction, it
has not previously been possible to directly demonstrate significant
autophosphorylation in vitro. Here we show that c-Src purified
from recombinant bacteria can autophosphorylate Tyr-527 to high levels in vitro when incubated with sufficiently high concentrations
of ATP (K(Mg
/ATP)
20
µM) that are well above those that have been used
previously. In vitro Tyr-527 autophosphorylation can occur
both as an intra- and intermolecular interaction; higher enzyme
concentrations are required for intermolecular Tyr-527 phosphorylation
than for Tyr-416 autophosphorylation. These results support the
possibility that, like G-proteins, c-Src can switch itself off in
vivo by its own enzymatic activity.
c-Src activity is negatively regulated in vivo primarily by phosphorylation of Tyr-527 (located near the carboxyl terminus) to high levels (>90%)(1) . Tyr-527 can be phosphorylated by Csk, and its phosphorylation is reduced 2-5-fold in transgenic Csk knock-out mice(2, 3) . However, some evidence points to a role for Tyr-527 autophosphorylation. Autophosphorylation could account for the 20-50% of the Tyr-527 phosphorylating activity that remains in mouse cells lacking Csk (2, 3) and for the fact that the ability of Csk to phosphorylate Tyr-527 in carboxyl-terminal mutants of c-Src does not correlate with the level of Tyr-527 phosphorylation in fibroblasts(4) . Furthermore, phosphorylation of Tyr-527 is decreased in kinase-defective c-Src mutants, particularly during mitosis(5, 6) . These observations suggest the existence of a negative feedback loop that might be mediated by Tyr-527 autophosphorylation(7) .
Some indirect evidence has been presented to suggest that c-Src might autophosphorylate Tyr-527(8) , but direct evidence for autophosphorylation of Tyr-527 in vitro is lacking. When incubated with ATP in vitro, c-Src preferentially autophosphorylates Tyr-416, a residue in the center of the catalytic domain, and not Tyr-527(9, 10) . However, all in vitro Src-autophosphorylation studies of which we are aware have used ATP concentrations in the low micromolar range, presumably because of technical limitations imposed by the use of radioactive isotopes. Physiological ATP concentrations are significantly higher (>1 mM), and it is possible that Tyr-527 autophosphorylation might proceed efficiently at these concentrations. To unambiguously resolve this issue, we have investigated the ability of c-Src to autophosphorylate Tyr-527 in vitro using both immunoprecipitated proteins and proteins purified from recombinant bacteria (which are believed to contain no other protein-tyrosine kinases). We demonstrate that c-Src can extensively phosphorylate Tyr-527 by intra- as well as intermolecular mechanisms, implying that isolated c-Src molecules can down-regulate their own activity.
All purification steps were carried out at 4 °C.
Proteins were precipitated from the supernatant by adding solid
(NH)
SO
to 70% saturation, stirring
for 2 h, and then centrifuging for 30 min at 15,000
g.
The pellet was resuspended in 20 ml of 20 mM Tris-HCl, pH 7.4,
10 mM thioglycerol, 1 mM phenylmethylsulfonyl
fluoride, 10% glycerol and dialyzed overnight against the same buffer.
c-Src(Y416F) was purified from the dialyzed sample by affinity
chromatography on a polyE4Y1-Sepharose affinity column as described
previously(15) . Most of the c-Src(Y416F) kinase activity
emerged in the 0.3 M NaCl eluate. This was used for subsequent
immunoaffinity chromatography (16) on a column prepared by
coupling anti-Src MAb327 to CNBr-activated Sepharose 4B (Sigma).
Fractions containing protein-tyrosine kinase activity were pooled,
aliquoted, and stored at -80 °C.
Wild-type c-Src for in vitro autophosphorylation experiments was purified from
baculovirus-infected Sf9 insect cells (generously provided by R. Clark
and F. McCormick, Cetus) as described by Zhang et al.(15) followed by immunoaffinity chromatography as
described above. In vivoP-labeled wild-type
c-Src for tryptic phosphopeptide standards was prepared from
NIH(pMcsrc) c-Src overexpresser cells as described(11) .
Figure 1:
Autophosphorylation of
immunoprecipitated Src at Tyr-527. Src was immunoprecipitated from
overexpresser cell lysates with or without prior in situ dephosphorylation and in the presence or absence of FSBA (which
irreversibly inactivates protein kinases). Where indicated, lysates
from different overexpressers were mixed after incubation with anti-Src
antibody and before precipitation with rabbit anti-mouse-protein A
Sepharose beads. Immunoprecipitated Src autophosphorylation was assayed
with 200 µM ATP in the absence (odd lanes) or
presence (even lanes) of 5 mM MgCl.
Antiphosphotyrosine immunoblots are shown. Lanes 1 and 2, untreated c-Src(Y416F); lanes 3 and 4,
dephosphorylated c-Src(Y416F); lanes 5 and 6,
FSBA-treated, dephosphorylated c-Src(Y416F); lanes 7 and 8, FSBA-treated, dephosphorylated c-Src(Y416F)
co-immunoprecipitated with c-Src(Y416F/Y527F); lanes 9 and 10, untreated c-Src(Y416F/Y527F). Reactions were normalized to
contain equal amounts of each Src protein (i.e. lanes 7 and 8 contained twice as much total Src protein as the other
lanes); this was verified by probing duplicate blots with anti-Src
antibody (not shown).
In the presence of 200
µM Mg/ATP, immunoprecipitated,
dephosphorylated c-Src(Y416F) autophosphorylated at Tyr-527 to levels
similar to the steady-state levels observed in vivo (Fig. 1, lanes 1-4). This level has been
shown by different methods to be
>90%(4, 6, 20) . Autophosphorylation at
Tyr-527 by c-Src(Y416F) was completely abolished by pretreatment with
FSBA (lanes 5 and 6). However, stoichiometric
phosphorylation of FSBA-treated, kinase-inactive c-Src(Y416F) was
observed in the presence of co-immunoprecipitated kinase-active
c-Src(Y416F/Y527F) (lanes 7 and 8). This indicated
that c-Src(Y416F/Y527F) was able to phosphorylate Tyr-527 of
c-Src(Y416F) in an intermolecular interaction (in trans). The
level of intermolecular Tyr-527 phosphorylation was similar to the
level of Tyr-527 autophosphorylation observed with kinase-active
c-Src(Y416F) alone (cf. lanes 4 and 8). We
conclude that the in vitro reaction can phosphorylate most of
the available Tyr-527 residues.
Tyr-527 autophosphorylation of the
c-Src(Y416F) mutant exhibited a K for
Mg
/ATP of approximately 20 µM (data not
shown). In agreement with previous results, little autophosphorylation
was detected at ATP concentrations below 10 µM.
Autophosphorylation of Tyr-527 (in c-Src (Y416F)) was at least 2-fold
slower than autophosphorylation of Tyr-416 (in c-Src (Y527F)) at 200
µM ATP (data not shown). These results suggest that c-Src
can autophosphorylate Tyr-527 to high levels at physiological ATP
concentrations, and that this can occur by an intermolecular
interaction. However, in spite of stringent immune complex washing
conditions, as with prior studies, the possibility that the
immunoprecipitates contained Csk or another contaminating exogenous
Tyr-527 kinase could not be formally excluded.
Plasmid pLS4, which contains a lac UV5 promoter linked to the v-src gene, has previously been used for expression of v-Src in bacteria(21, 22) . pLSc416F was constructed by replacing the v-src gene with the c-src(Y416F) gene (from plasmid pRLc416F, generously provided by B. C. Cobb, University of Virginia). The NaeI-MluI and MluI-Tth111I fragments containing the c-src(Y416F) coding region were isolated from pRLc416F and ligated into the homologous NaeI and Tth111I sites in pLS4.
c-Src(Y416F) was purified from lysates of pLSc416F-transformed
DH10B cells as described under ``Experimental Procedures.''
Western blots with anti-phosphotyrosine antibody showed that the
purified protein contained negligible phosphotyrosine, possibly because
of dephosphorylation during purification in the absence of phosphatase
inhibitors (data not shown). The specific activity of the purified
protein, using polyE4Y1 random oligomers of glutamate and tyrosine as
substrate, was approximately 230 nmol P
incorporated/min/nmol of c-Src(Y416F).
Autophosphorylation assays
were conducted using radioactively labeled
[-
P]ATP to permit the position(s) of the
autophosphorylated site(s) to be precisely mapped. Wild-type c-Src
(purified from baculovirus) was autophosphorylated and analyzed in
parallel as a control. Examination by SDS-PAGE and autoradiography
showed, as expected, that Src was the only labeled protein present
(data not shown). Autophosphorylated c-Src(Y416F) was subjected to
multiple analyses to identify the autophosphorylated residue(s) (Fig. 2). Phosphoamino acid analysis showed that only
phosphotyrosine was present (Fig. 2A). Cleveland
partial digests with S. aureus V8 protease showed that the
phosphorylated residue(s) were located within the V2 fragment
consisting of the carboxyl-proximal 26-kDa fragment of the protein (Fig. 2B). No autophosphorylation in the amino-proximal
34-kDa region was detected under the conditions used. Two-dimensional
tryptic phosphopeptide analyses of autophosphorylated c-Src(Y416F)
revealed a single spot corresponding to the peptide containing
phosphotyrosine 527 (Fig. 2C). Tryptic phosphopeptide
maps of in vivo phosphorylated (Fig. 2D) and in vitro autophosphorylated (Fig. 2E)
wild-type c-Src were used to provide standards for the locations of the
phosphotyrosine 527- and phosphotyrosine 416-containing peptides,
respectively. Spots corresponding to phosphotyrosine 527 as well as to
phosphotyrosine 416 were observed with in vitro autophosphorylated wild-type c-Src (Fig. 2E).
(Because of unavoidable inefficiencies in digestion and recovery of the
tryptic peptides, the relative intensities of the spots do not
necessarily correspond to the relative phosphorylation levels.) We
conclude that c-Src(Y416F), purified from a source containing no other
protein-tyrosine kinase activity, autophosphorylates itself primarily
at Tyr-527 in the presence of sufficient (
20 µM) ATP
concentrations.
Figure 2:
Analysis of autophosphorylated
c-Src(Y416F) purified from recombinant bacteria. Purified,
dephosphorylated c-Src(Y416F) (5 ng) was autophosphorylated by
incubation in 50 µl of a buffer containing 20 µM
[-
P]ATP (8,600 cpm/pmol) and 10 mM MgCl
(30 min at room temperature). Wild-type c-Src was
used in parallel for comparison. Autophosphorylated
P-labeled Src proteins were subjected to subsequent
analyses as described under ``Experimental Procedures.'' A, phosphoamino acid analysis. Lane 1, c-Src(Y416F); lane 2, wild-type c-Src. The positions of the nonradioactive
phosphoserine (P-S), phosphothreonine (P-T), and
phosphotyrosine (P-Y) standards visualized by ninhydrin
staining are indicated by the dotted circles. B,
autophosphorylated bands were excised following SDS-PAGE and analyzed
by partial digestion with 130 ng of S. aureus V8 protease
during 12.5% SDS-PAGE as described previously(19) . V1, V2, V3, and V4 indicate the
positions of the 34-kDa amino-terminal and 26-kDa carboxyl-terminal
fragments and the 18- and 16-kDa amino-terminal V1 subfragments, respectively. Lane 1, c-Src(Y416F); lane 2, c-Src. C-E, bands containing
autophosphorylated c-Src(Y416F) (panel C), autophosphorylated
c-Src (panel D), and in vivo phosphorylated c-Src
from mouse fibroblasts were eluted from gels and digested with trypsin.
The resulting phosphopeptides were separated by electrophoresis
(horizontal direction, anode on the left) and chromatography (vertical
direction). The samples were spotted at the positions marked with arrowheads. Spots 1 and 2 represent the
peptides containing Tyr-416 and Tyr-527,
respectively.
Experiments with purified proteins showed that the K for autophosphorylation of wild-type c-Src at
Tyr-416 was -6 µM Mg
/ATP, while
that for autophosphorylation of c-Src(Y416F) at Tyr-527 was -20
µM.
To
investigate the nature of the autophosphorylation reactions in more
detail, we performed autophosphorylation dilution experiments with
purified wild-type c-Src and c-Src(Y416F). Autophosphorylation of fixed
amounts of these proteins was assayed in different volumes of a buffer
containing 50 µM [-
P]ATP/10
mM Mg
as well as 0.3% sodium deoxycholate
(to minimize the possibility of aggregation). (Both protein
preparations contained no detectable phosphotyrosine and had similar
specific activities for phosphorylation of polyE4Y1 synthetic random
oligomers (3,000-4,000 nmol/min mg).) If autophosphorylation were
intramolecular, we would expect the level to be independent of
concentration; if it were intermolecular, we would expect it to
increase linearly with concentration.
Quantitative analysis of the
autophosphorylation levels by SDS-PAGE and autoradiography yielded the
curves shown in Fig. 3. Incorporation of radioactive P into wild-type c-Src increased roughly linearly with
concentrations in the range of 5-143 ng/ml. This indicates that
much of the Tyr-416 autophosphorylation was intermolecular. The
residual autophosphorylation in the (extrapolated) limit of zero c-Src
concentration is larger than the amount that can be explained by
Tyr-527 autophosphorylation, which suggests, in agreement with previous
results(16) , that there is also an intramolecular Tyr-416
autophosphorylating activity. In contrast, autophosphorylation of
c-Src(Y416F) was essentially independent of concentration. To exclude
the possibility that this result was an artifact induced by
c-Src(Y416F) aggregation, we analyzed the most diluted samples using
Sephadex G-100 Superfine column chromatography in the presence of
protein standards of known molecular weight (glucose oxidase,
-160 kDa, and bovine serum albumin, -66 kDa). Column
fractions were analyzed by SDS-PAGE and either Western blotting with
anti-Src antibody MAb327 or silver staining (to locate the marker
proteins). The Src proteins were detected in fractions containing
proteins with molecular weights
66 kDa, indicating that they were
present as monomers in the reaction. The concentration independence of
Tyr-527 autophosphorylation indicates that most of this activity was
intramolecular under these conditions.
Figure 3:
Effect of protein concentration on c-Src
autophosphorylation at Tyr-416 and Tyr-527. 1 ng of wild-type c-Src (open circles) or mutated c-Src(Y416F) (solid
circles) was incubated at room temperature in buffer containing 50
µM [-
P]ATP (45,000 cpm/pmol)
with 10 mM MgCl
in a total volume of 7, 20, 70, or
200 µl to obtain the indicated enzyme concentrations. After 5
(c-Src) or 10 min (c-Src(Y416F)), the autophosphorylated proteins were
trichloroacetic acid-precipitated and separated by SDS-PAGE, and the
amounts of
P incorporated into the labeled Src bands were
measured using a Betascope (Betagen). Points are averages from two
experiments; errors are approximately
10%.
Previous studies showing that kinase-active but not
kinase-defective c-Src is phosphorylated at Tyr-527 when expressed in
yeast have suggested that c-Src can autophosphorylate this
site(8) . However, a clear demonstration of Tyr-527
autophosphorylation in the absence of potentially interfering cellular
tyrosine kinases has been lacking. While autophosphorylation at Tyr-416
is readily observed in c-Src immunoprecipitates, significant Tyr-527
phosphorylation in vitro has not previously been observed. We
have now shown that Tyr-527 can be autophosphorylated to high levels by
c-Src in vitro. This was demonstrated with protein purified
from recombinant E. coli, thereby removing the possibility
that the phosphorylation was catalyzed by an associated
protein-tyrosine kinase. The Tyr-527 autophosphorylation reaction has a K for ATP of about 20 µM in the
presence of 5-10 mM MgCl
. This is
significantly higher than the ATP concentrations used in previous
published studies of c-Src autophosphorylation, which probably explains
why this in vitro reaction has not previously been observed to
occur at a significant level. However, the K
is
much lower than physiological ATP concentrations, so Tyr-527
autophosphorylation may well have physiological significance. In
particular, it may account for the residual Tyr-527 phosphorylation
observed in Csk-deficient mouse cells (2, 3) and for
the decreased Tyr-527 phosphorylation level observed with
kinase-defective c-Src during mitosis(5) .
Even at high ATP concentrations, the Tyr-527 autophosphorylation rate is a few-fold slower than the Tyr-416 autophosphorylation rate. However, this still represents a significant activity that is capable of phosphorylating most of the available sites, at least in vitro. The ratio of autophosphorylation rates depends on the Src concentration since the Tyr-416 autophosphorylation rate is concentration-dependent, presumably a result of significant intermolecular autophosphorylation. The detection of intermolecular autophosphorylation in the dilution experiment is gratifying and consistent with earlier observation of intermolecular phosphorylation at this site in mixed immunoprecipitates and in cotransfected yeast (8) . While the data suggest that some intramolecular autophosphorylation also takes place, our results contrast with earlier dilution studies with v-Src (23) and c-Src (16) (covering the same range of concentrations), which concluded that Tyr-416 autophosphorylation is strictly intramolecular. We do not know the reason for this difference.
In contrast with Tyr-416 autophosphorylation, Tyr-527 autophosphorylation was primarily intramolecular within the concentration range tested in the dilution experiments. While our immune complex mixing experiments and previous yeast co-transfection experiments (8) indicate that Tyr-527 can be autophosphorylated by an intermolecular interaction (presumably occurring at higher effective concentrations than those tested here), the dilution experiment results show that significant rates of Tyr-527 autophosphorylation can be achieved in a unimolecular interaction. This implies that negative feedback by Tyr-527 autophosphorylation need not depend on local accumulation of Src molecules.
The rate of
unimolecular Tyr-527 autophosphorylation measured using purified
protein was -0.2 mol PO/mol of Src/min, corresponding
to a t
for Tyr-527 autophosphorylation of
35 min. Thus, c-Src, even in the absence of exogenous regulatory
proteins like Csk, will only remain in an active state for a finite
period of time after activation and Tyr-527 dephosphorylation. Although
the Tyr-527 autophosphorylation rate is low, it may be functionally
important if the competing dephosphorylation rate is also slow. This
seems likely since dephosphorylation of Tyr-527 proceeds much more
slowly in vitro than dephosphorylation of Tyr-416, (
)presumably because of protection by intramolecular
association of phosphotyrosine 527 with the c-Src SH2 domain. In this
regard, c-Src autoregulation may be similar to that seen in G proteins
that have an intrinsic mechanism for time-delayed down-regulation. It
will be important to determine whether there are proteins that act
analogously to GTPase-activating proteins to accelerate Tyr-527
autophosphorylation and c-Src kinase down-regulation. Such proteins
would clearly possess tumor suppressor potential. Finally, our results
place the model of a negative feedback loop involving direct c-Src
auto-regulation on firmer ground.