Oligomerization-induced Modulation of TPR-MET Tyrosine Kinase Activity*
John L. Hays
and
Stanley J. Watowich
From the
Department of Human Biological Chemistry and Genetics and the Sealy
Center for Structural Biology, University of Texas Medical Branch, Galveston,
Texas 77555-0645
Received for publication, October 17, 2002
, and in revised form, March 31, 2003.
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ABSTRACT
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Phosphorylation, although necessary, may not be sufficient to fully
activate many receptor tyrosine kinases (RTKs). Oligomerization-induced
conformational changes may be necessary to modulate the kinetic properties of
RTKs and render them fully functional. To investigate this regulatory
mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative
of the RTK c-MET, has been expressed and purified for quantitative enzymatic
analysis. This naturally occurring oncoprotein contains the cytoplasmic domain
of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR).
cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified
for comparative enzymatic analysis. ATP and peptide substrates have been
kinetically characterized for both TPR-MET and cytoMET. Significantly,
phosphorylated TPR-MET has smaller Km values for
ATP (Km,ATP) and peptide substrates
(Km,peptide) and a larger
kcat relative to phosphorylated cytoMET. This provides the
first direct evidence that receptor oligomerization and not simply activation
loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation
constants (Kd,ATP) for the two enzymes
also displayed significant differences. In contrast, the
KI values for the ATP competitive inhibitor
staurosporin are similar for the two phosphorylated enzymes. These results
suggest that much of the oligomerization-induced kinetic changes occur with
respect to peptide substrate binding or catalytic efficiency. The possibility
that oligomerization-induced conformational changes occur within the
cytoplasmic domain of receptor tyrosine kinases has significant implications
for structure-based design of RTK inhibitors and the development of a detailed
mechanistic model of RTK activation.
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INTRODUCTION
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Stimulation of receptor tyrosine kinases
(RTKs)1 by ligand
binding initiates an intracellular signaling cascade that can induce cellular
differentiation, proliferation, and/or migration, among other cellular
responses (1). Binding of a
cognate extracellular ligand to a monomeric RTK induces receptor
oligomerization and, in turn, autophosphorylation of tyrosine residues within
the cytoplasmic tail of the receptor
(13).
The phosphorylated form of the receptor recruits signaling molecules through
Src homology 2 or phosphotyrosine binding domains, and subsequent activation
of these molecules initiates intracellular signaling cascades
(1,
4).
The molecular details of how RTK oligomerization regulates its function are
incompletely understood. It is known that receptor oligomerization enhances
autophosphorylation of tyrosine residues within the kinase regulatory domain
and that this phosphorylation results in localized conformational changes that
increase the kinase activity of the receptor
(57).
However, regulatory domain phosphorylation, although necessary, may not be
sufficient to fully activate the kinase
(8,
9). Additional
oligomerization-induced conformational changes may be necessary to increase
the kinase catalytic activity and render the RTK fully functional. Evidence
for this level of regulation is provided by studies with phosphatase
inhibitors such as peroxovanadate compounds. Posner et al.
(7) demonstrated that
unstimulated insulin receptors possess basal kinase activity but are unable to
fully mimic ligand-stimulated insulin receptor autophosphorylation and
signaling in cultured hepatoma cells or hepatic microsomes following
peroxovanadate treatment (10).
Similarly, pervanadate treatment of the platelet-derived growth factor (PDGF)
receptor is able to induce autophosphorylation in the absence of PDGF but is
unable to increase the activity of the receptor toward exogenous substrate
without the presence of PDGF
(9). Studies with polyclonal
antibodies have qualitatively shown that a phosphorylated monomeric receptor
does not possess the ability to phosphorylate exogenous substrate in the same
manner as the oligomeric receptor
(8). All of these studies would
indicate that there is some unique functional characteristic contained within
the oligomeric RTK that is not present in the monomeric receptor.
Biochemical characterization of RTKs has been performed using both
recombinant truncated kinase domains and cytoplasmic domains
(5,
1114).
Cheng and Koland (14) showed
that the cytoplasmic domain of the EGF receptor has an almost 10-fold greater
Kd for an ATP analog than a carboxyl terminus
deletion mutant of the cytoplasmic tail
(14), demonstrating that the
isolated kinase domain may not be a sufficient model for receptor function.
Murray et al. (5) were
able to determine kinetic parameters for phosphorylated and unphosphorylated
Tie2 cytoplasmic kinase domain and showed that phosphorylation resulted in a
25-fold decrease in substrate Km. Parast
et al. (12) also
showed an order of magnitude increase in the catalytic activity of the
phosphorylated VEGFR2 tyrosine kinase domain versus the
non-phosphorylated receptor. These studies demonstrated the importance of the
phosphorylation state of RTKs in modulating their kinase activity, but they
did not address the issue of oligomerization. The question remains, does the
activity of phosphorylated RTK monomer accurately represent the
ligand-activated state of the RTK? If not, what are the differences between
the oligomeric and monomeric forms of the receptor?
The TPR-MET oncoprotein provides a unique opportunity to address this
question. TPR-MET is a naturally occurring oncoprotein resulting from a fusion
between TPR (a coiled-coil domain derived from the nuclear pore complex) and
the cytoplasmic domain of c-MET
(6,
15,
16)
(Fig. 1). c-MET, the RTK for
hepatocyte growth factor/scatter factor, has been shown to be involved in
angiogenesis, placental and liver development, B-cell differentiation, and
embryogenesis, and altered c-MET function has been implicated in multiple
neoplastic disorders (17).
Targeted overexpression of c-MET has also been shown to cause hepatocellular
carcinoma in transgenic mice
(18). The TPR-MET oncoprotein
is oligomerized through the TPR domain, which results in a constitutively
active tyrosine kinase (6) that
has the ability to produce mammary hyperplasia and carcinoma, as well as
multiple other neoplasms in transgenic mice
(18). The isolated cytoplasmic
domain of c-MET (cytoMET) has kinase activity; however, this protein cannot
transform NIH3T3 cells and is, therefore, functionally inactive
(6). These studies suggest
there may be kinetic differences between functionally active TPR-MET and
functionally inactive cytoMET. Here we present a detailed analysis that
conclusively shows phosphorylated TPR-MET and phosphorylated cytoMET have
significantly different kinetic properties. Because these two proteins differ
in their oligomerization state, this implies that oligomerization-induced
conformational changes modulate the activity of RTKs, and these changes are
necessary to fully activate the receptor.

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FIG. 1. Schematic of c-MET, TPR-MET, and cytoMET. TPR-MET is derived by the
fusion of a large portion of the cytoplasmic domain of the c-MET receptor with
the coiled coil motif of TPR. The cytoMET protein is the c-MET-derived portion
of TPR-MET. LBD, extracellular ligand binding domain; TM,
transmembrane region; L, leucine zipper; KD, kinase domain.
Y1232, Y1233, Y467, and Y468 refer to tyrosine residues
within the activation loop of the kinase domain as numbered in either c-MET
(Y1232, Y1233) or TPR-MET and (Y467, Y468). Y1349, Y1356, Y482, and
Y489 similarly refer to tyrosine residues within the
carboxyl-terminal tail of the cytoplasmic domain of c-MET or TPR-MET.
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EXPERIMENTAL PROCEDURES
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Materials and ReagentsAll restriction endonucleases were
from New England BioLabs, Taq pfu was from Promega, the Bac-to-Bac
baculovirus expression system, SF-900II serum-free medium, fetal bovine serum,
and 100x antibiotic/antimycotic were purchased from Invitrogen. ATP,
NaVO4, DTT, HEPES, and staurosporin were purchased from Sigma.
[
-32P]ATP was purchased from ICN. Sol Grade CHAPS was
purchased from Anatrace. Guanidine hydrochloride was purchased from Roche
Applied Science. SAM2 biotin capture membrane and SignaTECT 1
(SignaTECT protein tyrosine kinase assay system, substrate 1,
biotin-EEEEAYGWLD) were purchased from Promega. Complete protease inhibitor
mixture was purchased from Roche Applied Science. PepTyr-489
(biotin-DSDVHVNATYVNVKCVAP) was synthesized and purified in the
University of Texas Medical Branch (UTMB) Protein Chemistry Core Facility, and
its identity was verified by mass spectroscopy. MANT-AMPPnP (a fluorescent
non-hydrolyzable derivative of ATP) was synthesized and purified by the UTMB
Organic Chemistry Core Laboratory, and its identity was verified by mass
spectroscopy. Anti-human Met antibody was purchased from Santa Cruz
Biotechnology, Inc. (sc-161), and anti-phosphotyrosine (clone 4G10) and the
anti-phosphorylated MET activation loop antibodies were from Upstate
Biotechnology, Inc.
Construction of Recombinant Baculovirus VectorsThe
tpr-met oncogene was a gracious gift from Dr. Morag Park (McGill
University). Both tpr-met and cytomet were amplified using
PCR of the tpr-met containing pXM vector. A carboxyl-terminal
hexahistidine tag and BamHI and EcoRI restriction sites were
added during PCR. The PCR product was gel-purified, digested, and ligated into
the pFastBac shuttle plasmid. The sequence was verified in the shuttle plasmid
using automated sequencing (UTMB Protein Chemistry Core Laboratory). Clones
with verified sequences were transformed into DH10Bac cells; transposition
into baculovirus DNA was monitored by loss of
-galactosidase activity.
Recombinant baculovirus DNA was purified by isopropanol precipitation and
successive ethanol washes and used for transfection of confluent Sf9 cells.
The recombinant baculovirus obtained from the Bac-to-Bac cloning protocol was
amplified to a titer of >1 x 108 pfu/ml before infections
for protein expression. Amplifications were carried out using 5 x
105cells/ml and a multiplicity of infection of 0.05 for 96 h at 27
°C. Titer was verified using serial dilution plaque assays in
duplicate.
Protein Expression and PurificationBoth TPR-MET and cytoMET
proteins were expressed and purified using similar protocols. Sf9 insect cell
lines were propagated at 27 °C in spinner flasks using SF-900II serum-free
medium supplemented with 5% fetal bovine serum and 0.5x
antibiotic/antimycotic; cell density was maintained between 5 x
105 and 3 x 106 cells/ml for propagation. Protein
expression was initiated by infecting Sf9 cells (2 x 106
cells/ml) with recombinant baculovirus at a multiplicity of infection of 5.
Cells were pelleted 72 h post-infection and stored at 78 °C. Frozen
cell pellets were thawed on ice, resuspended with chilled TBSC (50
mM Tris, pH 7.5, 150 mM NaCl, 0.5% CHAPS) supplemented
with 1 mM DTT and 1x complete protease inhibitor mixture, and
lysed in a Dounce homogenizer. The lysate was cleared by centrifugation 30,000
x g for 30 min at 4 °C. The cleared lysate was incubated
overnight at 4 °C with pre-equilibrated Ni-NTA beads (Qiagen). After
binding, the beads were washed successively with TBSC + 250 mM NaCl
and TBSC + 500 mM NaCl + 5 mM imidazole. Protein was
eluted with TBSC + 500 mM imidazole. Purification was monitored by
SDS-PAGE with the resulting bands visualized with Coomassie Blue staining.
Protein concentrations were determined by absorbance at
A280, with molar extinction coefficients calculated from
the aromatic residue content of the proteins
(19).
To ensure complete autophosphorylation of the recombinant kinases, the
eluent was dialyzed at 4 °C against PBSC (50 mM sodium
phosphate, pH 6.5, 150 mM NaCl, 0.5% CHAPS, 1 mM DTT)
supplemented with 50 µM ATP, 25 mM MgCl2,
and 5 mM MnCl2. A second dialysis was performed against
PBSC and 5 mM EDTA. After dialysis, the proteins were further
purified over a gel filtration column (Phenomenex BIO-SEP SEC-3000 column) in
PBSC + 5 mM EDTA or an anion exchange (POROS 10HQ) column with salt
as the eluent. Both proteins were characterized with gel filtration
chromatography (Phenomenex BIO-SEP SEC-3000 column) to determine their
oligomerization state.
Fluorescence Studies of TPR-MET and cytoMETBinding of
MANT-AMPPnP (fluorescent-tagged non-hydrolyzable ATP) to purified TPR-MET and
cytoMET was measured using a Fluorolog-3 spectrofluorometer, model
FL322. Quenching of the fluorescence from the five common tryptophan
residues found in both TPR-MET and cytoMET was measured for increasing
concentrations of MANT-AMPPnP. All data were measured at 10 °C with an
excitation wavelength (
exc)of290 nm, and emission data were
collected from 300 to 387 nm for both the sample and a reference cuvette with
buffer and ligand. Fluorescence was corrected for inner filter effects by
measuring the absorbance of the protein/ligand solutions at the excitation and
emission wavelengths. The quenching of tryptophan fluorescence occurring upon
the addition of MANT-AMPPnP was assumed to be proportional to the amount of
protein-bound MANT-AMPPnP present. The data were fitted using the following
equation to obtain dissociation constants:
F/F
o = 1
{(Q[MANT-AMPPnP])/(Kd + [MANT-AMPPnP])}.
Where
F represents the fluorescence of the sample minus the
background cuvette, F o is the initial protein
fluorescence in the absence of ligand, Q is the maximum quenching of
the protein during the experiment, and Kd is the
dissociation constant. The data were plotted as
F/Fo versus [MANT-AMPPnP] and fit using
non-linear least squares regression analysis to the above equation with the
GraphPad Prism program and Kd and Q as
fitting parameters. To determine the dissociation constant for ATP
(Kd,ATP), titrations of enzyme with
MANT-AMPPnP were performed in the presence of a fixed amount of ATP. The
Kd,ATP can then be determined by
solving the equation: Kd, apparent =
Kd,MANT{1
([ATP]/Kd,ATP)}. To determine total
active protein, the quenching of total fluorescence was assumed to be
proportional to the amount of MANT-AMPPnP bound to the protein, and the
dissociation constant was determined in independent experiments. The total
protein ([Et]) can then be found by solving the
quadratic equation: (1
F)/(1
Fmax) = {([Et] +
[MANT-AMPPnPt] + Kd)
(([Et] + [MANT-AMPPnPt] +
Kd)2
4[Et][MANT-AMPPnPt])1/2}/(2[Et]).
Kinetic AssaysAll kinetic assays were performed at room
temperature unless otherwise noted. Ni-NTA-purified TPR-MET (or cytoMET) was
pre-incubated for 10 min at 4 °C in kinase reaction buffer (50
mM HEPES, pH 7.3, 100 mM NaCl, 25 mM
MgCl2, 5 mM MnCl2, 5 mM
-glycerophosphate) with 1 µM ATP and 10 mM
freshly prepared DTT added. Kinase reactions were carried out in kinase
reaction buffer supplemented with 0.1 mM NaVO4, 1 pmol
[
-32P]ATP and 1 mM DTT, with ATP and
tyrosine-containing substrate added to desired concentrations. Reactions were
initiated by addition of enzyme (final concentration 110 nM)
to a complete reaction mixture. At time points during the steady state linear
range of the reaction, aliquots were removed from the reaction mix, quenched
with 5 M guanidine hydrochloride, and applied to
streptavidin-coated polyvinylidene difluoride membranes (SAM2
biotin capture membrane; Promega). The membranes were sequentially washed with
2 M NaCl, 2 M NaCl + 1% H3PO4,
distilled water, and 70% EtOH. Membranes were dried, and incorporated
radioactivity was measured using Cerenkov counting on a Hewlett Packard LS300
liquid scintillation counter. Non-weighted least squares linear regression
lines were calculated for the linear range of each of individual reaction, and
the program GraphPad Prism was used to calculate steady state rates. Each time
point was measured in duplicate, and each linear range contained from four to
eight points. Kinetic parameters were calculated from nonlinear least squares
fitting to a Michaelis-Menten equation using the program GraphPad Prism.
Inhibitor StudiesTo determine the
KI of the known ATP competitive inhibitor
staurosporin, linear ranges were calculated (as described above) in the
presence of a known amount of inhibitor. Staurosporin was initially dissolved
to a final concentration of 1 mM in Me2SO and then
subsequently diluted into kinase reaction buffer at the desired final
concentration. Steady state rates were compiled to generate rate curves, and
unweighted non-linear least squares regression analysis was used to determine
. Determining this value at
multiple inhibitor concentrations allowed calculation of
KI by comparing to the previously calculated
Km for ATP using the formula:
(22).
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RESULTS
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Purification of TPR-MET and cytoMETThe expression of
carboxyl-terminal hexahistidine-tagged TPR-MET and cytoMET in Sf9 cells was
accomplished by infection with recombinant baculovirus as described under
"Experimental Procedures." Both proteins were isolated from the
soluble fraction of the cell lysate and were purified to
95% homogeneity
as evaluated by Coomassie Blue staining
(Fig. 2).
Fig. 2B shows a
Western blot analysis of both TPR-MET and cytoMET after purification. Both
proteins positively react against a commercially available anti-human c-MET
antibody that was derived from a peptide corresponding to the
carboxyl-terminal tail of the c-MET receptor. Both TPR-MET and cytoMET are
phosphorylated as demonstrated from their recognition by anti-phosphotyrosine
antibody 4G10 and an antibody specific for the phosphorylated activation loop
of cMET. Furthermore, incubation of this phosphorylated enzyme with
[
-32P]ATP revealed no further incorporation of phosphate
into either TPR-MET or cytoMET. This result is expected, because both proteins
were incubated from 4 h to overnight at 4 °C with ATP and MgCl2
during the purification process (see "Experimental
Procedures").

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FIG. 2. Composite SDS-PAGE analysis of the purification of TPR-MET and
cytoMET. TPRMET was expressed in Sf9 cells using a baculovirus vector as
described under "Experimental Procedures." Panel A, lane
1, TPR-MET whole cell lysate after recombinant baculovirus infection for
96 h. Lane 2, elution from Ni-NTA column. Lane 3, fraction
from BIOSEP SEC-S300 gel filtration column. lane 4, whole cell lysate
after recombinant baculovirus infection for 96 h. Lane 5, elution
from Ni-NTA column. Lane 6, fraction from BIOSEP SEC-S300 gel
filtration column. Panel B, Western blot analysis of TPR-MET and
cytoMET. Lanes 13, purified TPR-MET. Lanes 46,
purified cytoMET. Lanes 1 and 4 were probed with anti-MET,
lanes 2 and 5 were probed with anti-phosphotyrosine, and
lanes 3 and 6 were probed with anti-phosphorylated MET
activation loop.
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Enzymatic characterization showed that kinetic variables for each enzyme
recovered after elution from either the Ni-NTA or gel filtration columns were
similar (data not shown). Both proteins were subjected to analysis on gel
filtration columns to determine their oligomerization state. TPR-MET
(molecular mass,
62 kDa) eluted at a volume corresponding to an observed
molecular mass between 100 and 250 kDa; no protein was apparent in the void
volume or in fractions corresponding to monomer molecular mass. This size
range would correspond to an oligomer of a dimer, trimer, or tetramer for the
TPR-MET protein. Different concentrations of TPR-MET were used in the kinase
assays, and each time identical Km,ATP
and Km,peptide were obtained. Thus, even if
multiple oligomerization states were present their kinetic properties were
identical. cytoMET (molecular mass,
43 kDa) eluted at a volume
corresponding to a molecular mass of
40 kDa, consistent with a monomeric
cytoMET species.
Binding StudiesBinding studies of TPR-MET and cytoMET for
MANT-AMPPnP allowed determination of substrate affinities and subsequent
determination of the binding active enzyme fraction in the purified protein
samples. The latter measurement is necessary to accurately determine the
kcat value for TPR-MET and cytoMET. The quenching of
intrinsic tryptophan fluorescence by MANT-AMPPnP was measured for both
phosphorylated TPR-MET and cytoMET. Four of the five tryptophan residues are
within the carboxyl-terminal kinase domain of the both TPR-MET and cytoMET, as
determined by sequence analysis and comparison with the known structure of the
insulin receptor (20). These
residues were accessible to quenching by the MANT nucleotide whose absorption
maximum at 355 nm overlaps with the tryptophan emission maximum in both
TPR-MET and cytoMET at 335 nm. A fixed amount of protein was titrated with
increasing amounts of MANT-AMPPnP, and the quenching was followed from 300 to
387 nm, to avoid interference with MANT emission
(Fig. 3A). The area
under the curve was integrated, and, after inner filter effects were accounted
for, normalized fluorescence was plotted against [MANT-AMPPnP], and the data
were fit with non-linear least squares regression analysis
(Fig. 3, B and
C). Results from the binding studies are listed in
Table I. The binding constants
for MANT-AMPPnP (Kd,MANT) are 9.1
µM for TPR-MET and 8.7 µM for cytoMET, which are
statistically identical for the two enzymes. To determine ATP binding
constants (Kd,ATP), the titrations
were repeated with 10 µM ATP added. The apparent
Kd with ATP present, when coupled to the known
Kd,MANT can yield the
Kd,ATP as described under
"Experimental Procedures." This analysis reveals a slight decrease
in Kd,ATP when comparing the
oligomeric TPR-MET to the monomeric cytoMET indicating an
oligomerization-dependent change in the conformation of the ATP binding site,
independent of phosphorylation of the activation loop.
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TABLE I Binding of TPR-MET and cytoMet
TPR-MET and CytoMet were analyzed by the protocols described under
"Experimental Procedures." The Kd values
reported represent the Kd ± S.E. for unweighted
nonlinear least squares regression analysis of data from
Fig. 3. Two-tailed p
values were calculated within the Graphpad Prism program using paired
t tests.
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Kinetic ActivityTo ensure complete autophosphorylation of
the enzymes and to separate the autophosphorylation reaction from the
exogenous substrate kinase reactions, both TPR-MET and cytoMET were separately
incubated with ATP, MgCl2, and MnCl2 prior to use in
kinetic studies. The steady state kinetic properties of the fully
phosphorylated form of TPR-MET and cytoMET with respect to ATP and exogenous
tyrosine substrates were examined. TPR-MET and cytoMET were both recognized by
phosphotyrosine-specific antibodies before the kinase reaction. However,
neither enzyme underwent additional autophosphorylation during the course of
these reactions, as demonstrated by the inability of either enzyme to
incorporate phosphate from radiolabeled [
-32P]ATP (data not
shown). It was also necessary to maintain adequate reducing conditions (DTT
concentration > 1mM) to ensure full activity of the kinases.
Thus, both enzymes were reduced and fully phosphorylated before determining
their kinetic properties with exogenous substrates.
The steady state linear range for formation of phosphorylated exogenous
substrate occurred between 2 and 50 min for both phosphorylated TPR-MET and
cytoMET. This range is similar to the linear range reported for
ligand-stimulated immunoprecipitated c-MET
(11). Linear ranges were
analyzed using an unweighted linear least squares regression analysis within
the GraphPad Prism program (see Fig.
4A and
5A). For each
reaction, between four and eight separate time points were examined in
duplicate to determine the reaction rate; all regression lines fit the data
with r2 values greater than 0.9.
Fig. 4A and
Fig. 5A show
representative kinetic data with fixed enzyme and ATP concentrations and
increasing SignaTECT 1 peptide concentrations. The slopes of the calculated
regression lines provided steady state velocities from which to construct
Michaelis-Menten rate curves (see Fig.
4B and Fig.
5B) for SignaTECT 1. An identical method was used to
construct rate curves for the tyrosine-containing peptide substrate,
PepTyr-489, and ATP (see Fig. 4, C
and D and Fig. 5,
C and D). Each data point in the
Michaelis-Menten rate curves represents the mean of two to five independent
experiments. Michaelis-Menten equations were fit to the experimental data
using unweighted non-linear least squares regression, and kinetic parameters
were derived from the curve for each kinase reaction.

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FIG. 4. Kinetic data for TPR-MET. The linear range of the kinase reaction is
shown in panel A. SignaTECT 1 was used at the indicated
concentrations with ATP being held constant at 400 µM. Each time
point was measured in duplicate for a given experiment with the data
representing the mean ± S.D. Panels BD show
rate curves for indicated substrates derived from linear ranges as described
under "Experimental Procedures." Each point on a given rate curve
represents one to five independent experiments at that given
concentration.
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FIG. 5. Kinetic data for cytoMET. The linear range of the kinase reaction is
shown in panel A. SignaTECT 1 was used at the indicated
concentrations with ATP being held constant at 400 µM. Each time
point was measured in duplicate for a given experiment with the data
representing the mean ± S.D. Panels BD show
rate curves for indicated substrates derived from linear ranges as described
under "Experimental Procedures." Each point on a given rate curve
represents one to five independent experiments at that given
concentration.
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Table II summarizes the
Km values for ATP
(Km,ATP). When measuring the influence
ATP has on the reaction rate for phosphorylation of exogenous substrates, the
ATP concentration was varied from 2 to 800 µM whereas the
peptide substrate (SignaTECT 1 or PepTyr-489) concentration was held constant
at 400 µM. Both TPR-MET and cytoMET followed Michaelis-Menten
kinetics with respect to ATP at constant peptide substrate concentrations. The
Km,ATP value for TPR-MET was
determined to be 35.7 µM, which is in agreement with the
reported value for immunoprecipitated c-MET
(11). The
Km,ATP value for cytoMET was 70.4
µM, a factor of two increase compared with TPR-MET and a
difference that was statistically significant. The value of
Km,ATP was independent of peptide
substrate used in the kinase reaction. The TPR-MET and cytoMET
Km,ATP values are well below reported
intracellular concentrations of ATP; thus, ATP concentration would likely not
be a significant regulatory mechanism for activation of the enzyme.
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TABLE II Kinetic parameters of TPR-MET and cytoMet
TPR-MET and CytoMet were analyzed by the protocols described under
"Experimental Procedures." The Km values
reported represent the Km ± S.E. for unweighted
nonlinear least squares regression analysis of data from Figs.
4 and
5. kcat
values were calculated by taking Vmax derived from kinetic
assays on protein preparations immediately after binding studies to determine
the active fraction. Two-tailed p values were calculated within the
Graphpad Prism program using paired t tests.
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The Km values for peptide substrates
(Km,peptide) are summarized in
Table II. Several peptide
substrates were tested for their activity with TPR-MET and cytoMET.
PepTyr-489, a biotinylated peptide (residues Val-483Pro-497 of TPR-MET)
that includes the in vivo phosphorylation site Tyr-489 in the
carboxyl-terminal domain of TPR-MET, and SignaTECT 1, a biotinylated peptide
containing a single tyrosine residue, were found to be the best substrates out
of a number of different tested peptides for the in vitro kinase
reactions. Both TPR-MET and cytoMET followed Michaelis-Menten kinetics when
varying peptide substrate concentrations from 1 to 1000 µM and
holding ATP concentration constant at 250 µM. The
Km,peptide values of PepTyr-489 and SignaTECT 1
with TPR-MET are 11.8 and 40.7 µM, respectively. In contrast,
the Km,peptide values of PepTyr-489 and SignaTECT
1 with cytoMET are of 67.3 and 231 µM, respectively. The
Km,peptide values, whether the peptide was
derived from the cytoplasmic tail of the receptor (PepTyr-489) or a
nonspecific peptide containing one tyrosine residue, both increased by almost
6-fold for cytoMET compared with TPR-MET
(Table I).
The Vmax, kcat, and
kcat/Km values are reported
for both enzymes in Table II.
The active fraction of protein was defined as that portion able to bind ATP as
determined by fluorescence measurements (see
Table I and
Fig. 3). Typically,
1030% of the purified protein was able to bind nucleotide; the exact
fraction that bound nucleotide was considered active and was determined just
prior to kinetic analysis. Taking the active fraction determined for a
particular sample and the calculated Vmax for a defined
amount of enzyme allowed accurate calculation the kinase
kcat. The measured kcat for TPR-MET
was 1.66 min1 and for cytoMET was 0.569
min1, showing an oligomerization dependant 3-fold
increase in the rate of the reaction. The specificity constant
(kcat/Km) for the oligomeric
TPR-MET increased by greater than 5-fold for ATP and greater than 15-fold for
peptide substrates when compared with cytoMET
(Table II). These data would
suggest there is a significant difference between the functional activity of
oligomeric TPR-MET and monomeric cytoMET proteins, implying an
oligomerization-induced conformational change affects the inherent kinetic
activity of receptor tyrosine kinases.
Inhibitor StudiesTo further understand active site
differences between TPR-MET and cytoMET, the kinetics of a kinase reaction
were performed in the presence of staurosporin. Staurosporin has been
characterized as an ATP competitive inhibitor for multiple kinases
(21), and the crystal
structure for staurosporin within the ATP binding site of tyrosine kinases Src
(Protein Data Bank number 1BYG
[PDB]
) and human lymphocyte-specific kinase (Protein
Data Bank number 1QPJ
[PDB]
) has been determined. Kinase reactions were performed as
above, except that staurosporin was added to the reaction mixture to obtain
final staurosporin concentrations within an order of magnitude of the expected
KI. Reactions were performed with 0, 0.5, and 1
nM staurosporin, 400 µM SignaTECT 1, and ATP
concentrations varying from 10 to 800 µM.
values ranged from
100 to 250 µM for TPR-MET in the presence of inhibitor (see
Fig. 6A and
Table III).
values ranged from
200 to 500 µM for cytoMET in the presence of inhibitor (see
Fig. 6B and
Table III).
KI values were then calculated according to Segel
(22), with
KI values of 0.19 and 0.15 nM obtained
for TPR-MET and cytoMET, respectively
(Table III). This small
difference in KI was not statistically
significant, as judged by detailed error analysis of the data
(Table III).

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FIG. 6. Staurosporin inhibition of TPR-MET and cytoMET. Rate curves were
generated as described under "Experimental Procedures" using
varying ATP concentrations and constant peptide concentrations (250
µM SignaTECT 1). The inhibitors were mixed at the given
concentrations into the reaction mixture before addition of enzyme. Panel
A, inhibition of TPR-MET with staurosporin. Panel B, inhibition
of cytoMET.
|
|
View this table:
[in this window]
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|
TABLE III Inhibition kinetics of TPRMET and CytoMET
TPR-MET and cytoMET were analyzed as described under "Experimental
Procedures" to generate Michaelis-Menten curves in the presence of
varying concentrations of staurosporin
(Fig. 5). The
values were calculated using
nonlinear least squares regression analysis of the data from
Fig. 5 in the presence of a
known concentration of inhibitor. The KI values were then
calculated using the following equation.
(22). Two-tailed p
values were calculated using a Student's t distribution.
|
|
 |
DISCUSSION
|
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Numerous groups have demonstrated the important role of RTKs in both normal
and diseased tissues
(13).
The mechanism and pathways RTKs use to transduce their signals has been
exhaustively researched, but there is still little known about the actual
molecular mechanism of how RTKs become activated and what is necessary and
sufficient for receptor activation and signaling. To study the effect of
oligomerization on receptor tyrosine kinase activity, we have used a
functionally active fusion protein derivative of the cytoplasmic domain of the
c-MET receptor, TPR-MET, and compared it to a functionally inactive protein,
cytoMET, containing the cytoplasmic domain of the c-MET receptor
(6).
The catalytic activities (kcat) for oligomeric TPR-MET
and monomeric cytoMET have been accurately determined. Significantly,
functionally active TPR-MET has a 3-fold increase in kcat
relative to functionally inactive cytoMET. In addition there was an
17-fold increase in the specificity constant
(kcat/Km) for peptide and a
greater than
6-fold increase in the specificity constant for ATP when
comparing the relative activities of TPR-MET to cytoMET. The observed increase
in specificity constant for TPR-MET relative to cytoMET was independent of
peptide substrate used. This suggests that receptor oligomerization may modify
the conformation of the catalytic site, thus leading to increased activity of
the oligomeric receptor.
To further explore the relationship between oligomerization-induced changes
in binding affinity and kinetic activity, we determined the
Kd,ATP,
Km,ATP, and
Km,peptide values for oligomeric TPR-MET and
monomeric cytoMET. Interestingly, the binding of MANT-AMPPnP was identical for
both enzymes, and the binding affinity of ATP
(Kd,ATP) was
3-fold lower for
TPR-MET when compared with cytoMET. In addition,
Km,ATP was
2-fold lower for
TPR-MET relative to cytoMET. These data suggest that the ATP binding site was
similar for both TPR-MET and cytoMET, and thus, receptor oligomerization may
only slightly modify the conformation of the receptor ATP binding site. In
contrast, Km,peptide was
6-fold lower for
TPR-MET relative to cytoMET, suggesting that oligomerization may induce
conformational changes in the tyrosine substrate binding region. This would
explain the large differences in Km,peptide
values and catalytic activity observed between TPR-MET and cytoMET.
RTKs oligomerize in response to ligand binding, and this process
facilitates autophosphorylation of the receptor on specific tyrosine residues
(3). The question remains, does
receptor oligomerization modulate the phosphorylation of downstream signaling
molecules? Many groups have studied isolated kinase domains or intracellular
regions of RTKs, and their work, as well as work presented here, demonstrates
that isolated kinase domains are sufficient for enzymatic activity in
vitro (5,
12,
13)
(Fig. 5). Rodrigues and Park
(6) demonstrated that the
TPR-MET fusion oncoprotein was kinase-active in vitro and
functionally active in cell culture. In contrast, the monomeric cytoMET was
kinase-active in vitro, but it was functionally inactive in cultured
cells. Other studies (9,
23) have shown that monomeric
RTKs can undergo autophosphorylation in cell culture in the presence of
phosphatase inhibitors. These studies suggest that the monomeric form of the
RTKs possess some basal kinase activity but are unable to fully mimic the
effects of the oligomeric receptors. One possible explanation for this
observation might be that oligomerization creates a local increase in
concentration of tyrosine substrate for the kinase. In this hypothesis, it is
not necessary for the oligomer to have any different kinetic properties from
the monomer, as the increase in concentration of substrates would presumably
increase the activity of the kinase to a sufficient level to induce downstream
signaling. An alternative hypothesis is that oligomerization causes some
structural alterations in the kinase domain that decreases the
Km and increases the catalytic activity of the
receptor such that downstream signaling is initiated. In this hypothesis, a
defined interaction between the oligomeric subunits causes the conformational
change in the receptor and the subsequent increase in its enzymatic activity.
One possible structural explanation for this has been proffered by Hubbard
et al. (2), where they
suggest formation of a dimer may stabilize the kinase activation loop in a
catalysis-favorable conformation. Our data support this latter hypothesis, as
the oligomeric TPR-MET possesses increased catalytic activity, as measured by
a decrease in Km and an increase in
kcat, compared with monomeric cytoMET. These two
hypotheses are not mutually exclusive, as oligomerization likely does induce a
local increase in tyrosine substrate concentration that may then be acted upon
by a kinase with enhanced catalytic activity. It is also important to note
that these experiments do not address the important point of dephosphorylation
of the active kinase. Oligomerization may confer protection from
dephosphorylation, as has been reported for the PDGF receptor
(24), and this mechanism may
play a role in downstream signaling through oligomeric, as opposed to
monomeric, receptors.
Km,ATP and
Km,peptide values have been measured for several
kinases. Naldini et al.
(11) reported
Km values of 36 µM for ATP and 1.5
mM for tyrosine-containing peptide substrates for both
autophosphorylated immunoprecipitated c-MET and the immunoprecipitated
cytoplasmic portion of the receptor. These reported
Km,ATP values are comparable with our
Km,ATP values for oligomeric TPR-MET.
Differences in substrate sequences likely account for the observed differences
in Km,peptide between TPR-MET and
immunoprecipitated c-MET. Importantly, we see significant differences in
Km,peptide values between oligomeric TPR-MET and
monomeric cytoMET, whereas the immunoprecipitated receptors have identical
Km,peptide values. It is likely that
immunoprecipitation of the receptor modifies its conformation, either through
artificial receptor oligomerization or some other mechanism, and thus
immunoprecipitated receptors do not provide an accurate model of the monomeric
receptor.
Other groups
(810)
have similarly reported that there is a difference in the ability of monomeric
receptors to fully mimic oligomeric receptor activation in vitro and
in cell culture. Posner et al.
(7) demonstrated that
pre-activation of the EGF receptor with epidermal growth factor led to a
decrease in the apparent dissociation constant for both ATP and peptide
substrates compared with non-activated receptors. Together, these data suggest
that oligomerization, in addition to activation loop phosphorylation and
protection from phosphatases
(24), modulates the catalytic
activity of receptor tyrosine kinases. It is likely that oligomerization
induces conformational changes to the catalytic transition state, the ATP
binding pocket, and/or the tyrosine-containing substrate binding site and
therefore is important in determining the catalytic properties of the
enzyme.
The activities of both enzymes were examined in the presence of the ATP
competitive inhibitor staurosporin. A number of compounds that interact with
the kinase ATP binding site have been discovered and kinetically characterized
(21,
25). There is a wealth of
kinetic (26) and
crystallographic data (27,
28) demonstrating the role of
staurosporin and related compounds as ATP competitive inhibitors in protein
tyrosine kinases. In this work, we have shown that staurosporin acts as an ATP
competitive inhibitor for both TPR-MET and cytoMET with statistically
indistinguishable KI values for both enzymes
(Table III). Thus, staurosporin
binding more closely approximates the binding of MANT-ATP to the two kinases
as opposed to the binding of ATP to these kinases, suggesting that
staurosporin binding may be influenced by factors and contacts outside the
ATP-kinase binding interface.
Our data suggest that the increased Km,peptide
of the monomeric receptor relative to the oligomeric receptor results from a
decrease in its catalytic efficiency. However, this does not strictly exclude
the possibility that the monomeric receptor may also have a decreased affinity
for peptide substrates relative to the oligomeric receptor. Previous work on
EGF receptor (7,
11) demonstrated that the
EGF-stimulated receptor acted with the same kinetic mechanism and efficiency
as the nonstimulated receptor, but the affinities for various substrates had
changed. Our work shows that the affinity of the receptor for ATP does not
change significantly during oligomerization, although whether receptor
oligomerization affects its binding affinity for tyrosine-containing
substrates has not been determined at this time. The observed differences in
functional activity between oligomeric and monomeric receptors may therefore
be attributed to conformationally induced differences in their catalytic
activities and, possibly, affinities for tyrosine-containing substrates. The
possibility that oligomerization-induced conformational changes occur within
the cytoplasmic domain of receptor tyrosine kinase has significant
implications for structure-based design of RTK inhibitors and further
development of a detailed mechanistic model of RTK activation.
 |
FOOTNOTES
|
---|
* This work was supported in part by Grant 4952-052 from the Texas Higher
Education Coordinating Board (to S. W.) and by the Sealy Center for Structural
Biology (University of Texas Medical Branch). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Supported by National Library of Medicine Training Grant 2T15LM07093 from
the Keck Center for Computational and Structural Biology. 
To whom correspondence should be addressed: Dept. of Human Biological
Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX
77555-0645. Tel.: 409-747-4749; Fax: 409-747-4745; E-mail:
watowich{at}bloch.utmb.edu.
1 The abbreviations used are: RTK, receptor tyrosine kinase; PDGF,
platelet-derived growth factor; EGF, epidermal growth factor; DTT,
dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ni-NTA,
nickel-nitrilotriacetic acid; MANT-AMPPnP, 2'(or
3')-O-(N-methylanthraniloyl)-
,
-imidoadenosine
5'-triphosphate. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. W. Bolen and Dr. W. Bujalowski for helpful discussions, Dr. M.
Park for providing the tpr-met clone, Dr. S. Srivastava for
scintillation counter access, and Y. Wang for excellent technical
assistance.
 |
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