(Received for publication, August 31, 1995; and in revised form, October 31, 1995)
From the
The nucleotide binding properties of the epidermal growth factor
(EGF) receptor protein-tyrosine kinase were investigated with the
fluorescent nucleotide analog
2`(3`)-O-(2,4,6-trinitrophenyl)adenosine 5`-triphosphate
(TNP-ATP). TNP-ATP was found to be an active substrate for the
autophosphorylation reaction of the recombinant EGF receptor
protein-tyrosine kinase domain (TKD). Whereas the V for the TNP-ATP-dependent autophosphorylation reaction was
approximately 200-fold lower than that of ATP, the K
for this reaction was similar to that observed with ATP. The
nucleotide analog was also shown to be an inhibitor of the
ATP-dependent autophosphorylation and substrate phosphorylation
reactions of the TKD. Spectroscopic studies demonstrated both a high
affinity binding of TNP-ATP to the recombinant TKD and a markedly
enhanced fluorescence of the bound nucleotide analog. The fluorescence
of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which
enabled determination of the dissociation constants for both ATP and
the Mn
complex of ATP. A truncated form of the EGF
receptor TKD lacking the C-terminal autophosphorylation domain
exhibited an enhanced affinity for TNP-ATP, which indicated that the
autophosphorylation domain occupied the peptide substrate binding site
of the TKD and modulated the binding of the nucleotide substrates.
A large variety of polypeptide growth factor receptors (1, 2) and oncogene products (3) possess an intrinsic protein-tyrosine kinase activity that is known to be critical to their diverse cellular functions. Each of these enzymes catalyzes the ATP-dependent phosphorylation of tyrosine residues in peptide substrates and most undergo an autophosphorylation reaction, by which specific tyrosine residues within their primary structures become phosphorylated. In some cases, autophosphorylation has been shown to regulate the enzymic activity of protein-tyrosine kinases. In other cases, e.g. for the variety of polypeptide growth factor receptors with protein-tyrosine kinase activity, autophosphorylation is known to trigger the formation of signal transduction complexes(1, 2, 4) . Here the phosphorylated protein-tyrosine kinases interact with cellular target proteins that specifically recognize and bind to phosphorylated tyrosine residues(5) .
Whereas protein-tyrosine kinases constitute an
important family of enzymes with well studied involvement in numerous
cell growth control processes, including significantly the aberrant
growth of cancerous cells, much remains to be determined about their
enzymic mechanisms. Studies of the steady state kinetics of these
enzymes have been
reported(6, 7, 8, 9, 10) .
However, the mechanisms of the substrate phosphorylation and
autophosphorylation reactions of protein-tyrosine kinases are not
understood at the molecular level. Also, the structural changes in the
active site that accompany the activation of protein kinase activity by
various agents have not been characterized. In the present work, we
have investigated the potential of the fluorescent nucleotide analog
2`(3`)-O-(2,4,6-trinitrophenyl)adenosine 5`-triphosphate
(TNP-ATP) ()as a molecular probe for enzymological studies
of protein-tyrosine kinases. We have determined that TNP-ATP binds to
the active site of the epidermal growth factor (EGF) receptor
protein-tyrosine kinase, shows a markedly enhanced fluorescence when
bound to the active site, and is also a substrate for the
autophosphorylation reaction of the protein kinase. Comparison of the
affinities of TNP-ATP for full-length and C-terminally truncated
recombinant EGF receptor protein-tyrosine kinase domains indicated that
the C-terminal autophosphorylation domain may occupy the active site at
equilibrium. The fluorescent nucleotide analog was also exploited in
the determination of the affinities of binding of both ATP and the
Mn
complex of ATP to the kinase active site. The
TNP-ATP nucleotide analog therefore appears to have much potential as a
molecular probe for investigating the structure and function of
protein-tyrosine kinases.
Alternatively,
the autophosphorylation activity of TKD61 with ATP or TNP-ATP as
substrate was assayed by an immunological method, employing the
recombinant antiphosphotyrosine/horseradish peroxidase conjugate RC20.
TKD61 protein (1.0 µg) was preincubated as described above, and the
reaction was initiated by addition of either ATP or TNP-ATP to the
indicated concentration (final volume 36 µl). After a 10-s
incubation with ATP as substrate, or a 15-min incubation with TNP-ATP
as substrate, the reactions were quenched by addition of SDS-PAGE
sample buffer, and samples were subjected to SDS-PAGE (12% gel).
Resolved proteins were electrophoretically transferred to an
Immobilon-P membrane (Millipore) and blotted with the RC20 reagent at a
1:2,500 dilution. Bound RC20 was detected with the enhanced
chemiluminescence (ECL) method (Amersham), and autophosphorylation was
quantified by densitometry of the resulting ECL lumigrams. Parallel
assays of TKD61 samples incorporating known quantities of
[P]phosphotyrosine were used to generate a
standard curve, which allowed conversion of ECL intensities to absolute
values of phosphotyrosine incorporation.
The affinity of the interaction of TNP-ATP with each of the TKD proteins was determined by titrating a fixed quantity of the TKD protein with increasing concentrations of the fluorescent nucleotide, while recording the fluorescence intensity at the wavelength maximum of the emission spectrum of enzyme-bound TNP-ATP (540 nm) with excitation at 418 nm. A control TNP-ATP titration was performed without addition of the TKD protein, and the obtained fluorescence intensity data were subtracted from that recorded in the presence of TKD protein. The enhancement of fluorescence occurring in the presence of the TKD protein was assumed to be proportional to the quantity of enzyme-bound TNP-ATP and was plotted as a function of total concentration added TNP-ATP (see Fig. 5). The titration curve would then obey the equation
Figure 5:
Affinity of TNP-ATP/EGF receptor TKD
interactions. A, titration of the EGF receptor TKD proteins
with TNP-ATP. Aliquots of TNP-ATP stock solutions were diluted into
solutions of either TKD61 or TKD38, each at 0.5 µM, and
the fluorescence intensity (excitation 418 8 nm; emission 540
8 nm) was recorded after each addition. Blank TNP-ATP
titrations were performed in an identical manner but without added TKD
protein. The fluorescence intensities shown are the differences between
TKD61 (
) or TKD38 (
) and blank titration data. Results of
three independent titrations of TKD61 and two titrations of TKD38 are
included. The titration data were analyzed by curve-fitting to
determine the dissociation constant characterizing the interaction and
to generate the theoretical curves shown (see ``Experimental
Procedures''). B, titration of the TKD61 (
) or
TKD38 (
) proteins with TNP-ATP in the presence of
Mn
. Experiments were performed and analyzed as in A, except that 100 µM MnCl
was
included in the the assay medium to support the formation of the
Mn
TNP-ATP complex.
where E and T
are the
total concentrations of added TKD protein and TNP-ATP nucleotide,
respectively, K
is the dissociation constant
characterizing the interaction, and
F
is the
difference in the intrinsic fluorescence yields of enzyme-bound and
free TNP-ATP. This equation was fit to the titration data with a
nonlinear least squares algorithm and with K
, E
, and
F
as adjustable parameters. The best-fit
parameters were used to generate the theoretical curves shown in Fig. 5. Dissociation constants for binding of the Mn
complex of TNP-ATP (Mn
TNP-ATP) were determined by TNP-ATP
titrations performed in the presence of a fixed concentration of free
Mn
ion.
Given the known values for K, the dissociation constants for binding of
ATP to the TKD proteins (K
) were determined by
titrating mixtures of TNP-ATP and TKD with increasing concentrations of
ATP, while monitoring the fluorescence of bound TNP-ATP (see Fig. 6). was fit to the titration data with K`
= K
(1 +
[ATP]/K
) substituted for K
, and with K
and
F
treated as adjustable parameters.
Dissociation constants for binding of the Mn
complex
of ATP (Mn
ATP) were determined similarly by ATP titrations in
which the concentration of free Mn
ion was held
constant (cf. (13) ).
Figure 6:
Effects of ATP upon the fluorescence of
the TNP-ATP/TKD61 complex. A, as described in Fig. 5, a
solution of TNP-ATP (0.1 µM) and TKD61 (1.0
µM) was prepared, and the fluorescence of TNP-ATP was
recorded as ATP was added to increasing concentrations. The
fluorescence intensity data were corrected by subtracting the
contributions of the TKD61 protein and free TNP-ATP and hence represent
the enhancement of fluorescence occurring with the TNP-ATP/TKD61
interaction. Results of three independent experiments are included. The
data were analyzed as described under ``Experimental
Procedures'' to yield estimates for the dissociation constant for
ATP binding (see Table 1) and the best-fit theoretical curve
shown. B, effect of MnATP on the fluorescence of the
Mn
TNP-ATP/TKD61 complex. Titrations were performed and analyzed
as in A, except that a TNP-ATP concentration of 1.0 µM was employed. Increasing concentrations of MnCl
and
ATP were added, so that the concentration of free Mn
was held constant at 50 µM as the Mn
ATP
concentration was increased. Concentrations of Mn
ATP complex and
free Mn
were calculated as described(13) .
Results of three independent experiments are
included.
The effect of the peptide
substrate tyrsub (S) on the affinity of nucleotide for the TKD38
protein was analyzed by determining the apparent dissociation constant
for TNP-ATP (K`) in the presence of varying
concentrations of the peptide (see Fig. 7). The peptide appeared
not to compete directly with TNP-ATP for binding to the TKD38 protein,
but instead reduced the affinity of binding of TNP-ATP. Assuming that S
and TNP-ATP bound reversibly to the TKD protein, the dependence of K`
on the concentration of S would be given
by
Figure 7:
Effect of a peptide substrate on the
affinity of the TNP-ATP/TKD38 interaction. The TKD38 protein (0.5
µM) was titrated with TNP-ATP in the presence of various
fixed concentrations of the peptide tyrsub, and the fluorescence
intensity was recorded after each TNP-ATP addition as described in Fig. 5. Shown are titrations performed in the absence () or
presence (
) of 20 µM tyrsub. For each tyrsub
concentration, the apparent dissociation constant for TNP-ATP binding (K`
) was determined by fitting of , and the theoretical curves shown were generated. The inset shows the dependence of K`
upon the concentration of tyrsub and the theoretical curve
yielded by fitting of (see ``Experimental
Procedures'').
where K and K
are the intrinsic dissociation constants for TNP-ATP and S,
respectively, and
is the factor by which K`
is enhanced in the presence of a saturating concentration of S. K`
was determined at varying concentrations
of S, and was fit to these data with K
, K
, and
as
adjustable parameters (see Fig. 7).
The dependence of the rate of
autophosphorylation of the TKD61 protein upon the concentration of ATP
is shown in Fig. 1, A and B. Similar
autophosphorylation kinetics were observed when the reaction was
monitored by assaying the incorporation of P
into the protein with the substrate
[
-
P]ATP (Fig. 1A, K
= 6.0 ± 0.8 µM, n = 3) or by quantifying enzyme-associated phosphotyrosine
with an immunoblotting method, employing a recombinant
antiphosphotyrosine reagent (Fig. 1B, K
= 5.5 ± 1.4 µM, n =
4). The latter method was subsequently used to detect the
autophosphorylation of the TKD61 protein with the substrate TNP-ATP (Fig. 1C). Although significantly greater incubation
times were necessary to generate detectable TKD61-associated
phosphotyrosine with the substrate analog, the protein was shown to be
autophosphorylated with a similar nucleotide concentration dependence (K
= 4.4 ± 0.5 µM, n = 6). The maximal velocity for the
autophosphorylation of the TKD61 protein with TNP-ATP as the substrate
was subsequently directly assayed (Fig. 2). Whereas the
ATP-dependent reaction exhibited a V
of 2.3
min
(see Fig. 1A), the
TNP-ATP-dependent reaction was approximately 200-fold slower with a V
of 0.010 min
. The results
of these experiments indicated that TNP-ATP can substitute for ATP as a
substrate for the autophosphorylation reaction of the EGF receptor
protein-tyrosine kinase. Although the K
for the
nucleotide analog was similar to that of the authentic substrate, the
velocity of the autophosphorylation reaction was dramatically reduced
with the TNP-ATP substrate.
Figure 1:
Comparison of ATP and
TNP-ATP as substrates for the EGF receptor autophosphorylation
reaction. A, autophosphorylation kinetics of the recombinant
TKD61 protein with [-
P]ATP as substrate.
The TKD61 protein was incubated in the presence of 10 mM
MnCl
and varying concentrations of
[
-
P]ATP for 10 s at room temperature, and
incorporation of
P
into the TKD61 protein was
assayed. Nonlinear least squares fitting of the phosphorylation data
yielded values for the K
and V
of the reaction and the theoretical curve
shown. B, autophosphorylation kinetics of the TKD61 protein as
assayed by antiphosphotyrosine immunoblotting. The TKD61 protein was
incubated as in A, but with unlabeled ATP as substrate. The
reaction products were subjected to SDS-PAGE, immunoblotted with the
recombinant antiphosphotyrosine reagent RC20, and visualized with
enhanced chemiluminescence (ECL) detection (see inset).
Relative intensities of the antiphosphotyrosine signals were determined
by densitometric analysis of the resulting ECL lumigrams, and the
apparent K
value was obtained as above. C, autophosphorylation of TKD61 with TNP-ATP as substrate.
Experiment was performed as in B, except that TNP-ATP was
substituted for ATP and samples were incubated for 15 min at room
temperature. Antiphosphotyrosine was detected by immunoblotting (see inset), and the K
value for
TNP-ATP was determined.
Figure 2:
Velocity of the TNP-ATP-dependent
autophosphorylation reaction. A, calibration of the
antiphosphotyrosine signal with known quantities of P
-labeled TKD61 protein. The TKD61 protein was
incubated with [
-
P]ATP, and the
incorporation of
P
was assayed as described in Fig. 1A. The indicated quantities of labeled TKD61
protein were then subjected to SDS-PAGE, antiphosphotyrosine
immunoblotting, and densitometric analysis. The slope of the fitted
line indicates the relationship between antiphosphotyrosine signal
intensity and phosphotyrosine content. B, the TKD61 protein
was incubated at room temperature in the presence of 10 mM MnCl
and 50 µM TNP-ATP for the indicated
time intervals. Absolute phosphotyrosine content of the TKD61 protein
was quantified by immunoblotting and use of the standardization in A. The slope of the fitted line is the velocity of the
TNP-ATP-dependent autophosphorylation reaction under the conditions of
the assay (0.010 min
).
Figure 3:
TNP-ATP as an inhibitor of the
ATP-dependent autophosphorylation and substrate phosphorylation
reactions of the EGF receptor protein-tyrosine kinase. A, the
TKD61 protein was assayed for autophosphorylation activity as described
in Fig. 1A in the presence of 10 mM MnCl and varying concentrations of ATP (5-100 µM)
and fixed concentrations of the inhibitor TNP-ATP: 0 µM
(
), 10 µM (
), 20 µM (
), 50
µM (
), and 100 µM (
).
Double-reciprocal plots of the velocity data are shown. The velocity
data obtained at each TNP-ATP concentration were fit by nonlinear least
squares to determine the apparent K
and V
for ATP, and the lines shown represent the
linear transformation of the best-fit hyperbolic curves (see
``Experimental Procedures''). B, the TKD61 protein
was assayed for substrate phosphorylation activity as in A with the substrate GAT (0.4 g/liter) added and incubations of 10
min at room temperature. Assays were performed with varying ATP
concentrations and fixed concentrations of TNP-ATP: 0 µM (
), 20 µM (
), 50 µM (
),
80 µM (
), and 100 µM (
). The
velocity data were analyzed as described in A.
The nucleotide analog was also tested as an inhibitor of
the ATP-dependent substrate phosphorylation activity of the EGF
receptor protein-tyrosine kinase. In this case, TNP-ATP was seen to be
a mixed inhibitor with respect to ATP (see Fig. 3B)
and, hence, exerted effects on both the apparent K for ATP and the apparent V
. Secondary
plots of the inhibition were again nonlinear (data not shown), so that
a K
characterizing the inhibition could not be
determined. The K
for ATP in the substrate
phosphorylation reaction under the conditions of the inhibition
experiment was found to be
30 µM, and significant
inhibition of the reaction occurred with TNP-ATP concentrations
exceeding this value. It is possible that the mixed inhibition kinetics
observed resulted from a simple competition between ATP and TNP-ATP at
the nucleotide binding site combined with a second inhibitory
interaction occurring at higher concentrations of the nucleotide
analog.
The fluorescence emission spectrum of TNP-ATP was recorded in the presence and absence of each of the TKD proteins. In the presence of either the TKD61 protein (Fig. 4A) or the C-terminally truncated TKD38 protein (Fig. 4B), the fluorescence of the nucleotide analog was significantly enhanced, and the wavelength maximum was blue-shifted from 555 to 540 nm. Under the conditions of these experiments, the TNP-ATP nucleotide concentration exceeded that of the added TKD38 or TKD61 protein, so that the enhancement of TNP-ATP fluorescence that occurred upon interaction with the TKD proteins was underestimated. Related experiments indicated that the fluorescence emission of TNP-ATP at 540 nm was enhanced approximately 9-fold and 17-fold when the nucleotide was associated with the TKD61 and TKD38 proteins, respectively (data not shown).
Figure 4: Fluorescence analysis of TNP-ATP/EGF receptor TKD interactions. A, the fluorescence emission spectrum of TNP-ATP (1.0 µM) in 20 mM sodium Hepes, 10% (v/v) glycerol, pH 7.4 with (-) or without (- - -) added TKD61 protein (0.5 µM). Samples were held at 22 °C, and fluorescence was recorded with excitation at 418 nm and with 8 nm excitation and emission band passes. Each TNP-ATP spectrum shown was corrected by subtraction of an appropriate blank spectrum, that of either a buffer blank or buffer with added TKD61 protein. B, the fluorescence emission spectrum of TNP-ATP (1.0 µM) in the presence (-) or absence(- - -) of the TKD38 protein (0.5 µM) recorded as described in A.
Fig. 5A shows the results of titrations in which fixed
concentrations of the TKD proteins were treated with increasing
concentrations of TNP-ATP and nucleotide fluorescence was monitored.
From these spectroscopic data, it was possible to estimate the
dissociation constants for the interactions between TNP-ATP and each of
the recombinant TKD proteins (see ``Experimental
Procedures''). Under the conditions of these experiments, TNP-ATP
bound with high affinity to both the TKD61 (K =
0.43 ± 0.22 µM, n = 3) and TKD38 (K
= 54 ± 15 nM, n = 2) proteins. Hence, the truncated TKD38 protein bound
TNP-ATP with a significantly higher affinity than did the TKD61
protein.
The affinity of the TNP-ATP/TKD61 interaction was
significantly greater than that indicated by the K for TNP-ATP in the autophosphorylation reaction of TKD61 (see
above), which might have been due to the absence of an activating
divalent metal ion in the fluorescence assay medium (see
``Discussion''). Subsequent titrations were performed in the
presence of a minimal concentration (100 µM) of
Mn
, sufficient to promote the formation of a
Mn
TNP-ATP complex (see Fig. 5B). Higher
concentrations of Mn
were found to cause protein
aggregation under the conditions of the fluorescence assays (data not
shown). These titrations allowed the determination of the affinities of
Mn
TNP-ATP for both the TKD61 (K
= 1.9
± 1.6 µM, n = 2) and TKD38 (K
= 0.36 ± 0.28 µM, n = 2) proteins. The K
characterizing the Mn
TNP-ATP interaction with TKD61 was
similar to the K
for the nucleotide in the
TNP-ATP-dependent autophosphorylation reaction of TKD61 (see Table 1), which was consistent with the assumption that the
Mn
TNP-ATP complex was the active substrate in the
autophosphorylation reaction. As was the case with the free TNP-ATP
molecule, the Mn
TNP-ATP complex was apparently bound with
significantly higher affinity by TKD38 than by TKD61.
The data of Fig. 7were analyzed in terms of a model
in which TNP-ATP and the peptide substrate bound to distinct sites on
the TKD38 protein, but exerted a mutual negative binding interaction
characterized by the parameter of (see Experimental
Procedures). Fitting of to the K`
versus tyrsub concentration data yielded estimates for
(5.6) as well as K
, the dissociation
constant for tyrsub binding to the TKD38 protein in the absence of
nucleotide (3.8 µM). The dissociation constant for TNP-ATP
binding in the presence of a saturating concentration of tyrsub
(
K
) determined by the fitting was 0.86
µM. This value was similar to the dissociation constant
characterizing the binding of TNP-ATP to the C-terminally complete
TKD61 protein (0.43 µM, see Table 1). Apparently,
the peptide substrate, when bound to the active site of the truncated
TKD38 protein, mimicked the C-terminal autophosphorylation domain of
the TKD61 protein. Occupancy of the peptide binding site of the protein
kinase with either the C-terminal autophosphorylation domain in the
context of TKD61 or an exogenous peptide substrate in the case of TKD38
resulted in a similar reduction of the affinity of TNP-ATP binding. In
related experiments (data not shown), both the peptide substrate GAT
and MBP-B3-11, a recombinant fusion protein incorporating
sequences from the C-terminal autophosphorylation domain of the ErbB3
protein and previously characterized as a high-affinity substrate for
the EGF receptor protein-tyrosine kinase(16) , were shown to
also attenuate the binding of TNP-ATP to the TKD38 protein.
The trinitrophenyl analogs of both ATP and GTP have been
successfully employed in mechanistic investigations of a variety of
nucleotide-dependent enzymes. In the present work, we have explored the
potential of TNP-ATP as a substrate analog and spectroscopic probe for
protein-tyrosine kinases. The relatively well characterized EGF
receptor protein-tyrosine kinase, generated and purified as a
recombinant protein, was chosen for these studies. Two forms of the
protein-tyrosine kinase were employed here: TKD61, the recombinant
cytoplasmic domain of the receptor protein, which contained a
functional protein kinase domain and an intact C-terminal
autophosphorylation domain; and TKD38, a truncated cytoplasmic domain
protein, lacking the C-terminal autophosphorylation domain of the
native receptor protein. Although these TKD proteins both lacked the
extracellular growth factor binding domain of the EGF receptor, in the
presence of the millimolar concentrations of Mn, they
exhibited levels of protein-tyrosine kinase activity similar to that of
the purified holoreceptor. These recombinant proteins were also
available in quantities sufficient to facilitate spectroscopic studies
of their interactions with the fluorescent nucleotide analog.
Initial experiments demonstrated that TNP-ATP could serve as a
functional substrate analog of ATP in the autophosphorylation reaction
of the EGF receptor protein-tyrosine kinase. The nucleotide analog
supported the autophosphorylation of the TKD61 protein with a
concentration dependence similar to that of the authentic nucleotide.
However, the maximal velocity of the reaction with TNP-ATP as substrate
was approximately 200-fold lower than with ATP. Although these results
indicated that TNP-ATP was a rather inefficient substrate for the EGF
receptor protein-tyrosine kinase, they did suggest that the nucleotide
analog bound with high affinity to the enzyme and in the site normally
occupied by ATP. In previous studies of the F-ATPase (17, 18) and aspartokinase I(15) , the TNP-ATP
nucleotide analog was also shown to function as a substrate, but again
with a greatly reduced turnover rate relative to that of ATP.
The observed properties of TNP-ATP as a substrate in the autophosphorylation reaction of the EGF receptor protein-tyrosine kinase suggested that TNP-ATP might be an effective inhibitor of the ATP-dependent reactions of this enzyme. Indeed, TNP-ATP was seen to function as a competitive inhibitor with respect to ATP in the autophosphorylation reaction of the TKD61 protein (see Fig. 3A). TNP-ATP also inhibited the exogenous substrate phosphorylation activity of TKD61, showing mixed-type inhibition kinetics with respect to the substrate ATP (see Fig. 3B). Although no simple mechanism can be forwarded to explain quantitatively the details of the observed TNP-ATP inhibition kinetics, the kinetics were consistent with a competitive interaction between TNP-ATP and ATP at the catalytic site, in conjunction with a second, lower-affinity inhibitory interaction between the TNP-ATP and the TKD61 protein.
Our investigations of the
substrate activities and inhibitor properties of TNP-ATP suggested that
the nucleotide analog bound with high affinity to the catalytic site of
the EGF receptor protein-tyrosine kinase. Given that the TNP-ATP
molecule exhibits an enhanced fluorescence in hydrophobic
environments(19) , fluorescence spectroscopy was used to
monitor the binding of the nucleotide analog to the recombinant EGF
receptor TKD. Initial experiments indicated that the fluorescence of
TNP-ATP was significantly enhanced in the presence of micromolar
concentrations of the TKD61 protein. A subsequent titration revealed an
interaction with a dissociation constant of 0.43 µM. The
truncated TKD38 protein, which lacked the C-terminal
autophosphorylation domain of the EGF receptor, bound the TNP-ATP
molecule with an even greater affinity (K =
54 nM) than did the full-length TKD61 protein.
The K values for TNP-ATP binding to the TKD proteins
were significantly lower than the K
for TNP-ATP
observed in the autophosphorylation studies. As the autophosphorylation
assay medium included 10 mM MnCl
, it was
considered that the true substrate of the TNP-ATP-dependent
autophosphorylation reaction was the Mn
complex of
TNP-ATP (Mn
TNP-ATP). Subsequent TNP-ATP titrations were therefore
performed with Mn
present in the assay medium, at
concentrations sufficient to promote the formation of the
Mn
TNP-ATP complex. From these experiments it was possible to
estimate the dissociation constants for Mn
TNP-ATP binding to
TKD38 (K
= 0.36 µM) and TKD61 (K
= 1.9 µM). The latter
dissociation constant was similar to the K
value
for TNP-ATP in the autophosphorylation reaction (see Table 1). As
was the case for the free TNP-ATP molecule, the Mn
TNP-ATP complex
bound with significantly higher affinity to the TKD38 protein than to
the TKD61 protein.
Given these observations, it hypothesized that
the C-terminal autophosphorylation domain of the TKD61 protein, which
must at least transiently occupy the peptide substrate binding site of
the enzyme, could modulate the binding of nucleotide substrates at the
active site. This hypothesis predicted that peptide substrates of the
EGF receptor protein-tyrosine kinase would in the context of the TKD38
protein also modulate the binding of the TNP-ATP nucleotide. The
peptide substrate, tyrsub, was found to significantly reduce the
affinity of binding of TNP-ATP to the TKD38 protein (see Fig. 7). In the presence of a saturating concentration of
tyrsub, the K for TNP-ATP binding to TKD38 was
increased by a factor of 5.6 and assumed a value of 0.86
µM, similar to the K
for TNP-ATP
binding to the C-terminally complete TKD61 protein (0.43
µM). It was therefore considered that the peptide
substrate, when bound to the TKD38 protein, mimicked the C-terminal
autophosphorylation domain of the TKD61 protein and exerted a similar
negative effect on nucleotide binding affinity. Implicit in this
interpretation of the data is the assumption that the C-terminal domain
of the TKD61 protein occupied the peptide binding site at equilibrium.
The negative effects of both tyrsub and the C-terminal
autophosphorylation domain on the binding of TNP-ATP to the TKD
proteins appeared not to result from a direct competition between the
peptide substrates and the nucleotide for a single binding site, as the
presence of a saturating concentration of tyrsub did not abolish
nucleotide binding, but instead resulted in only a finite change in the
nucleotide dissociation constant (see Fig. 7, inset).
Apparently, nucleotide and peptide substrate could simultaneously bind
to the kinase domain catalytic site, as a steady-state kinetics study
of the EGF receptor protein-tyrosine kinase has previously indicated (10) . This study, in which the concentrations of ATP and
peptide substrate were co-varied, also indicated a negative interaction
between nucleotide and peptide binding sites. In the absence of
EGF-stimulation, occupancy of the nucleotide binding site by ATP led to
a 20-fold increase in the K of the peptide
substrate. Interestingly, the EGF-stimulated receptor showed a much
weaker interaction between the peptide and nucleotide binding sites,
which resulted in an effective lowering of the K
for the peptide substrate in the presence of saturating
concentrations of ATP. As the fluorescence experiments described here
were performed under nonactivating conditions, i.e. in the
absence of millimolar concentrations of an activating divalent metal
ion, our observation of a negative interaction between nucleotide and
peptide binding sites was consistent with these previous steady-state
kinetics studies.
The published three-dimensional structures of the kinase domain of the cAMP-dependent protein kinase obtained in the presence and absence of a high affinity peptide substrate indeed indicate that peptide substrates occlude the ATP binding site and could certainly modulate the nucleotide-binding properties of this enzyme(20) . The recently obtained three-dimensional structure of the insulin receptor protein-tyrosine kinase domain (21) shows a general structural similarity to that of the cAMP-dependent protein kinase, which suggests that most protein kinases share basic structural and functional elements. Our hypothesis that the C-terminal autophosphorylation domain of EGF receptor protein modulates the nucleotide-binding properties of TKD is therefore consistent with the presently available structural data. An important remaining question is whether the observed interactions between the C-terminal autophosphorylation domain of the EGF receptor and the active site are intramolecular or intermolecular in nature. Certainly, as the transphosphorylation of wild-type and kinase-deficient EGF receptor proteins has been documented(22) , such intermolecular interactions between EGF receptor proteins would be reasonable to assume. This question could be addressed by investigating the TNP-ATP binding properties of the TKD38 protein in the presence of a mutant TKD61 protein that is devoid of nucleotide binding activity.
In summary, we have demonstrated that the fluorescent nucleotide analog TNP-ATP was a functional substrate for the EGF receptor protein-tyrosine kinase. The analog was seen to be an inhibitor of the ATP-dependent reactions catalyzed by the protein-tyrosine kinase, apparently competing with ATP for occupancy of the nucleotide binding site. The binding of TNP-ATP to recombinant EGF receptor TKD proteins could be directly characterized by fluorescence spectroscopy. The presence of the C-terminal autophosphorylation domain was shown to reduce the affinity of binding of the nucleotide analog. It was hypothesized that the C-terminal autophosphorylation domain of the EGF receptor occupied the peptide substrate binding site of the TKD at equilibrium and lowered the affinity of the nucleotide binding site for TNP-ATP. TNP-ATP was therefore seen to be a useful spectroscopic probe for studying the structure and function of the EGF receptor protein-tyrosine kinase, and would likely be of great utility in examinations of other protein-tyrosine kinases.