(Received for publication, November 16, 1994; and in revised form, January 13, 1995)
From the
The deduced primary sequence of the cytoplasmic protein-tyrosine
kinase domain of the insulin receptor contains a conserved kinase
homology region (receptor residues 1002-1257) flanked by a
juxtamembrane region and a C-terminal tail. A soluble 48-kDa derivative
(residues 959-1355) containing these regions but lacking the first six
residues of the juxtamembrane region had earlier been synthesized in
Sf9 cells using a baculovirus expression system. The catalytic core of
the kinase domain was studied first by proteolytic analysis of the
48-kDa kinase and then by expressing a series of truncated kinase
domains in transiently transfected COS cells. Based on these studies,
two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283)
kDa, respectively, were overexpressed in Sf9 cells. Biochemical
characterization of the 35-kDa kinase revealed that the core kinase
conserved the major functional properties of the native receptor kinase
domain. Activity of the 35-kDa kinase toward a synthetic peptide
increased more than 200-fold upon autophosphorylation, which occurred
exclusively at Tyr-1158, Tyr-1162, and Tyr-1163; the largest increase
was observed between bis- and trisphosphorylation of the kinase. The
activated 35- and 48-kDa kinases were similar with respect to specific
activity and ATP and Mg requirements for peptide
phosphorylation. Moreover, autophosphorylation appeared to initiate
predominantly at Tyr-1162, immediately followed by phosphorylation at
Tyr-1158 and then at Tyr-1163. The rate of autophosphorylation was
dependent on enzyme concentration, consistent with a trans-phosphorylation mechanism. Finally, the 35-kDa kinase
was crystallized, making possible elucidation of its three-dimensional
structure by x-ray crystallography.
The insulin receptor is a disulfide-linked heterotetrameric
transmembrane glycoprotein composed of two extracellular -subunits
and two
-subunits that contain a single trans-membrane domain and
a cytoplasmic protein-tyrosine kinase domain(1, 2) .
The binding of insulin to the
-subunit of the receptor on intact
cells immediately activates the proteintyrosine kinase activity in the
-subunit, resulting in rapid autophosphorylation of multiple
tyrosine residues: Tyr-1158, Tyr-1162, and Tyr-1163 in the kinase
homology region; Tyr-1328 and Tyr-1334 in the C-terminal tail; and one
or more of Tyr-965, Tyr-972, and Tyr-984 in the juxtamembrane region (3, 4, 5) . Autophosphorylation of the three
core tyrosines (1158, 1162, and 1163) results in activation of the
kinase toward exogenous substrates(5, 6, 7) .
Substantial evidence has accumulated suggesting that the
insulin-stimulated protein-tyrosine kinase activity of the insulin
receptor is essential for mediation of insulin
action(8, 9, 10, 11) . Insight into
the structure and function of the kinase domain is thus critical for
understanding insulin action.
The cytoplasmic protein-tyrosine kinase domain of the insulin receptor has been expressed as an active soluble kinase in both stably transfected mammalian Chinese hamster ovary cells (12) and insect Sf9 cells(13, 14) , demonstrating that the cytoplasmic kinase domain is capable of autonomous function. Furthermore, a soluble 48-kDa derivative, which contains the cytoplasmic domain lacking the first six residues after the transmembrane domain (residues 959-1355), has been shown to exhibit the major functional properties of the native receptor kinase(13, 15, 16) . Overproduction of this soluble enzyme has made possible structure and function studies of the kinase domain by biochemical as well as biophysical approaches including NMR spectroscopy(17, 18, 19) .
However, despite the increasing body of data derived from in
vitro studies of the soluble kinase, the kinase domain is poorly
characterized in structural terms. The 48-kDa kinase has been found to
be resistant to crystallization, most likely due to the presence of
unstructured segments in the molecule. ()Furthermore, no
crystallographic structure of a protein-tyrosine kinase has been
determined that could be used to construct, by homology, a model of the
insulin receptor kinase. In fact, the four protein kinases for which
the three-dimensional structures have been reported all belong to the
family of protein serine/threonine kinases (cAMP-dependent kinase,
cyclin-dependent kinase 2, the mitogen-activated protein kinase Erk2,
and twitchin kinase) ((20) -23, reviewed in (24) ). Engineering new derivatives of the insulin receptor
kinase domain suitable for x-ray analysis is thus required to determine
the three-dimensional structure of this kinase domain, which is of
fundamental importance for understanding its mechanism of catalysis.
In the present study, we determined the catalytic core of the kinase domain first by proteolytic analysis of the 48-kDa kinase and then by expressing truncated kinase domains in tran-siently transfected COS cells. The results derived from these experiments provide critical information concerning the subdomain organization of the kinase domain. To produce large quantities of the core kinases, we have expressed two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, using the baculovirus expression system. In addition, biochemical characterization of the 35-kDa kinase reveals that the core kinase exhibits functional properties characteristic of those of the native receptor kinase. Finally, the 35-kDa kinase was found to be appropriate for crystallization and x-ray analysis.
Three other double-stranded synthetic oligonucleotides extend the 5`
end of the cDNA insert to the codons coding for Val-983, Val-985, and
Val-991, respectively. The resulting plasmids, designated peNT24,
pe
NT30, pe
NT32, and pe
NT38, encode the four N-terminally
truncated kinase domains lacking 24, 30, 32, and 38 N-terminal residues
of the cytoplasmic domain, respectively (see Table 1). The
plasmid pe
NT24 contains an additional substitution of Tyr-984 with
Phe. The cDNA 5` end regions of these plasmids containing the sequences
shown above were confirmed by DNA sequencing.
position corresponding to Ala-1284 or Asp-1265 using an
oligonucleotide-directed mutagenesis system (Bio-Rad). Mutant cDNAs
were screened by sequencing of the mutated regions. The BstXI/XbaI (bp 3439-4443) fragment containing
either the Ala-1284 or Asp-1265 mutation in M13 was sequenced to ensure
that no other mutation had occurred and then isolated from M13 to
replace the corresponding fragment in peiBgl. The resulting plasmids,
designated peCT72 and pe
CT91, encode receptor residues
Gly-959 to Lys-1283 and Gly-959 to Lys-1264, respectively. These
expression plasmids were characterized by restriction mapping and
sequencing of the mutated region to ensure that the mutation had been
introduced correctly.
The resulting plasmid, designated pVLNT25CT72, contains a
truncated receptor cDNA, which encodes receptor residues Val-978 to
Lys-1283 with the additional substitutions of Cys-981 with Ser and
Tyr-984 with Phe, and is placed under the transcriptional control of
the polyhedrin promoter.
The plasmid pVLNT32CT72, which encodes
receptor residues Val-985 to Lys-1283, was constructed by replacing the NcoI/XbaI cDNA insert in the baculovirus transfer
plasmid pVL
NT25CT72 with the corresponding fragment isolated from
the expression plasmid pe
NT32CT72 described above.
Transfer of
the receptor cDNA from the recombinant baculoviral transfer plasmid to
the baculovirus Autographa californica nuclear polyhedrosis
virus DNA was performed by homologous recombination into the native
polyhedrin gene after calcium phosphate cotransfection into insect Sf9
cells with a modified baculovirus DNA, which allows one to achieve
almost 100% recombination efficiencies (BaculoGold transfection kit,
Pharmingen). Sf9 cells were generously provided by Dr. Max Summers
(Texas A & M University) and were maintained as
described(27) . After transfection, recombinant viruses were
isolated by plaque assay(27) . Six independent plaques were
screened for recombinant protein expression by immunoblot and the
kinase activity assay described above. They all exhibited similar
recombinant protein expression levels. Two recombinant viruses thus
obtained, AcNT25CT72 and Ac
NT32CT72, were used for the
studies described herein.
Autophosphorylation reactions
included 50 mM Hepes, pH 7.5, 2 mM dithiothreitol,
MgCl or MnCl
, and ATP at indicated
concentrations. Reactions were terminated by adding Laemmli sample
buffer or by adding EDTA to 20 mM, and phosphoproteins were
separated by SDS-PAGE or by native PAGE (Bio-Rad minigels; all the
procedures are identical to those of SDS-PAGE, except that SDS is not
included in buffers and the gels are run at 4 °C).
To determine
the autophosphorylation sites of the soluble kinases, the P-labeled kinases were excised from the untreated wet gel,
electroeluted from the gel, digested with tosylphenylalanyl
chloromethyl ketone-trypsin, and subjected to either phosphoamino acid
analysis or two-dimensional phosphopeptide mapping as
described(28) .
Exogenous kinase activity assays were
performed with 50 mM Hepes, pH 7.5, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, MgCl and ATP at indicated concentrations, and 1 mM FYF.
Initial velocities were measured during the first 10% of substrate
phosphorylation.
A
number of other proteolytic enzymes including papain, elastase, and
subtilisin were tested for their ability to generate a stable, active
kinase fragment. Elastase treatment generated the smallest active
fragment with a molecular mass of 35 kDa. The N-terminal residue
was identified by gas phase protein microsequencing as Ser-982.
Furthermore, the sequence of peptides generated by treatment of the
35-kDa fragment with cyanogen bromide and trypsin revealed that the C
terminus of this elastase-generated kinase derivative was between
Glu-1286 and Glu-1291.
These proteolytic studies identified a catalytic core within the kinase domain of the insulin receptor that is flanked on both sides by protease-sensitive regions. With the aim of generating a core kinase, we first determined the N- and C-terminal boundaries of the catalytic core of the kinase domain by expressing the N- or C-terminally truncated kinase domains in COS cells. The N- and C-terminal deletions that did not result in the loss of protein stability and kinase activity were then selected to design final core kinases. Table 1summarizes the procedure and results.
To assess the expression of truncated kinase domains in transiently transfected COS cells, total cell lysates were examined by SDS-PAGE followed by Western blot analysis using antibody 766 directed against the 48-kDa soluble kinase. As illustrated in Fig. 1A, a specific band was observed in cells transfected with each of the expression plasmids encoding the N-terminally truncated kinase domains as well as for the 48-kDa kinase but not in non-transfected cells. This result indicates that these four truncated kinase domains are all expressed as a soluble protein in the cytoplasm of transfected cells (Table 1).
Figure 1:
Expression analysis of
the N- or C-terminally truncated kinase domains in transfected COS
cells. Non-transfected COS cells (COS), transfected COS cells
that transiently express the 48-kDa kinase (48k), and
N-terminally (NT) or C-terminally (
CT)
truncated kinase domains were analyzed as follows (see ``Materials
and Methods'' for detailed description). The truncated kinase
domains lack 24 (
24), 30 (
30), 32 (
32), or 38 (
38) residues from the N
terminus or 72 (
72) or 91 (
91) residues
from the C terminus of the cytoplasmic domain. A, immunoblot
analysis. Cell extracts containing
50 µg of proteins were
loaded on 12% SDS gel followed by immunoblotting with antibody 766
directed against the 48-kDa soluble kinase. The positions of the
molecular mass markers (in kDa) in this and the following figures are
indicated on the left. B, autophosphorylation assay.
Recombinant proteins were immunoprecipitated from cell extracts and
were incubated with [
-
P]ATP followed by
SDS-PAGE and autoradiography.
To
determine whether these expressed truncated kinase domains are active,
cell lysates were immunoprecipitated with antibody 766 and then
incubated with [-
P]ATP (see
``Materials and Methods''). As shown in Fig. 1B, in the case of the three truncated kinase
domains,
NT24,
NT30, and
NT32 as well as the 48-kDa
kinase, a single phosphoprotein band was observed that was not visible
for the largest N-terminally deleted kinase domain,
NT38, as for
the control immune complex with non-transfected COS cells. Furthermore,
inclusion of the synthetic peptide FYF in phosphorylation reactions
resulted in the incorporation of
P into this peptide for
the three autophosphorylated truncated kinase domains as well as for
the 48-kDa kinase (data not shown). Again, the
NT38 kinase had no
detectable exogenous kinase activity. These results indicate that the
deletion of 24, 30, or 32 residues from the N terminus of the
cytoplasmic domain results in an active soluble kinase, whereas the
deletion of 38 residues completely inactivates the kinase domain.
We
cannot rule out the possibility that the polyclonal antibody 766
inhibits the kinase activity of the NT38 kinase in the immune
complex. However, this antibody does not inhibit the kinase activity of
all other truncated kinase domains, particularly the
NT32 kinase,
which contains only six additional N-terminal residues, suggesting that
the inactivation is an intrinsic property of the
NT38 kinase and
that deletion of these six residues disrupts a critical structural
feature of the kinase domain that is outside of the kinase homology
region.
Immunoblot analysis detected a specific band for
the CT72 kinase with an apparent molecular mass of 38 kDa (Fig. 1A), and this expressed protein was found to
display both autophosphorylation (Fig. 1B) and
exogenous kinase activities (data not shown), indicating that the
CT72 kinase is expressed as a soluble active kinase in COS cells.
In contrast, expression of the
CT91 kinase in COS cells was hardly
detectable by immunoblot (Fig. 1A), and the
autophosphorylation assay following immunoprecipitation failed to
detect any phosphoprotein in this case (Fig. 1B),
suggesting that this truncated kinase domain expressed in COS cells is
unstable. Thus, the limited tryptic proteolysis of the 48-kDa soluble
kinase most probably occurs after Lys-1283, and this deletion (72
residues) is larger than the largest C-terminal deletion (69 residues)
so far reported (32) that does not result in the loss of
protein stability and kinase activity of the protein-tyrosine kinase
domain.
Figure 2:
Expression analysis of the soluble core
kinases in transfected COS cells. The soluble cell extracts of
transfected COS cells that transiently express the 48-kDa kinase
() or one of the two core kinases
NT24CT72 (
) and
NT32CT72 (
) were separated by MonoQ FPLC followed by
exogenous kinase activity assay performed on each fraction as described
under ``Materials and Methods.'' The activity is expressed as
radioactivity incorporated into the exogenous substrate
FYF.
Figure 3:
Expression analysis of the soluble core
kinases in Sf9 cells infected with recombinant baculovirus. A,
immunoblot analysis of the soluble cell extracts. Cell extracts
(100 µg of proteins) of recombinant virus-infected Sf9 cells
expressing one of the two core kinases,
NT25CT72 (lane 1)
and
NT32CT72 (lane 2), or of wild-type virus-infected Sf9
cells (lane 3) were loaded on 12% SDS gel followed by
immunoblotting with antibody 766. Lanes 4 and 5 show
the MonoQ FPLC-purified core kinases
NT24CT72 and
NT32CT72
(
200 ng of protein) expressed in transfected COS cells. The
positions of the core kinases (in kDa) are indicated on the right. B, Coomassie Blue-stained SDS-PAGE of the
purified soluble kinases. Purification of the 48-kDa kinase (lane
1) and the two core kinases from infected Sf9 cells,
NT25CT72 (lane 2) and
NT32CT72 (lane 3), was carried out
as described under ``Materials and Methods.'' A protein
sample of 2 µg for each kinase was subjected to SDS-PAGE in a 12%
gel. The positions of the soluble kinases (in kDa) are indicated on the right.
These two transfer plasmids were then used to obtain two recombinant
viruses, AcNT25CT72 and Ac
NT32CT72 (see ``Materials and
Methods''). A recombinant protein of 35 or 34 kDa was specifically
recognized by antibody 766 in cells infected with Ac
NT25CT72 or
Ac
NT32CT72, respectively, but not in cells infected with wild-type
virus (Fig. 3A). Exogenous substrate phosphorylation
was also detectable in crude cell extracts of infected cells (data not
shown), indicating that the core kinases are both stable and active
when expressed in both insect and COS cells. The maximal expression
level of the core kinases in Sf9 cells was found at 64 and 72 h
postinfection, instead of at 42 h as for the 48-kDa kinase.
Purification of the core kinases from infected cells was carried out
on a MonoQ anion exchange FPLC column followed by gel filtration
chromatography (see ``Materials and Methods''). The final
purified sample migrated as a single band of apparent molecular mass of
35 (NT25CT72) or 34 (
NT32CT72) kDa on SDS-PAGE (Fig. 3B). The purity of the sample was estimated to be
95% based upon densitometry scanning of Coomassie Blue-stained
gels. On gel chromatography, the core kinases elute at positions of 38
(
NT25CT72) and 36 (
NT32CT72) kDa between ovalbumin (43 kDa)
and chymotrypsinogen A (25 kDa) (data not shown), indicating that the
core kinases are monomeric enzymes. The purified core kinases can be
stored at 4 °C for several months without loss of activity, whereas
the 48-kDa kinase slowly undergoes degradation. The recombinant
proteins account for 2-5% of the total cell proteins in infected
cells. This high level of expression combined with the two-step
purification procedure resulted in an overall yield of about 1 mg of
purified enzyme for a typical preparation of 200 ml of culture (2
10
cells/ml).
For studies of the core kinases to be relevant to the kinase in the native receptor, the core kinases must exhibit major functional properties of the native receptor kinase. The most important characteristic of the insulin receptor kinase is the activation of its catalytic activity toward exogenous substrates by autophosphorylation of the three core tyrosines of the kinase(3, 4, 5, 6, 7) . To examine whether the core kinases preserve these functional properties, we have investigated the correlation between the autophosphorylation state of the enzyme and its activation toward peptide substrate FYF and have determined the autophosphorylation sites. Furthermore, we have also examined the order and the mechanism of autophosphorylation at the three core tyrosines. Most of the enzymatic characterizations were performed on the 35-kDa kinase.
Figure 4:
Activation by autophosphorylation of the
35-kDa kinase. Enzyme (2 µM) was incubated in the presence
of 4 mM [-P]ATP and 8 mM MgCl
. At the indicated times, aliquots of reactions
were terminated by addition of 20 mM EDTA. The
P
incorporated into the kinase (
) was determined after SDS-PAGE in
a 12% gel followed by scintillation counting of the Coomassie
Blue-stained bands. The exogenous kinase activity (
) was measured
with diluted aliquots in a 10-min assay with 1 mM FYF, 0.25
mM [
-P]ATP, and 8 mM MgCl
. Stoichiometry of phosphorylation of the kinase
was calculated from the specific activity of the
[
-P]ATP and the amount of the kinase
present in each sample. The exogenous kinase activity is expressed as
the percentage of the measured maximal activity, which was achieved
after 25 min of autophosphorylation.
Because several phosphate molecules were incorporated into the core kinase during the course of activation, we attempted to separate the multiple forms of phosphorylated enzyme by native PAGE, which separates the proteins by their charge as well as by their size. As illustrated in Fig. 5A, the unphosphorylated kinase migrated as a single major band, and three additional bands, apparently containing one, two, or three phosphate groups, appeared progressively during the course of autophosphorylation. The unphosphorylated kinase also displayed a minor band under the major band. This minor band, representing less than 10% of the unphosphorylated kinase, disappeared upon autophosphorylation and was not detectable on SDS-PAGE, suggesting that the minor band results from a conformational change or from covalent modifications that modify the charge of the molecule. The three phosphorylated forms, designated 35-kDa pY1, 35-kDa pY2, and 35-kDa pY3, correspond in fact to the mono-, bis-, and trisphosphorylated kinases on the three core tyrosines (see below).
Figure 5:
Native PAGE analysis of the
autophosphorylation cascade of the 35-kDa kinase and correlation with
enzyme activity. A, autophosphorylation of the 35-kDa kinase
was carried out as described in the legend of Fig. 4, and, at
indicated times, aliquots of each reaction were analyzed by 7.5% native
PAGE followed by Coomassie Blue staining. B, correlation
between the exogenous kinase activity and mono-, bis-, and
trisphosphorylations of the 35-kDa kinase. Exogenous kinase activity
() was measured as described in Fig. 4and is expressed as
the percentage of the measured maximal activity. The quantities of
monophosphorylated (
), bisphosphorylated (
), and
trisphosphorylated (
) forms were determined by densitometry
scanning or by Cerenkov counting of the bands (35-kDa pY1, 35-kDa pY2,
and 35-kDa pY3; both methods yielded similar results) and are expressed
as the percentage of the amount of the kinase present in each sample
(sum of all forms including the unphosphorylated
form).
Fig. 5B shows the kinetic
correlation between the extent of exogenous kinase activity and the
extent of each of the three phosphorylated forms. Within the first 10
min of autophosphorylation, 35-kDa pY1 and 35-kDa pY2 accumulate, which
is associated with a slight increase in the kinase activity (20%
of maximal activation). The trisphosphorylated form, 35-kDa pY3,
appears at 10 min and becomes a major form after 20 min. About 80% of
maximal activation was observed after 10-25 min of
autophosphorylation, indicating that the largest increase in the kinase
activity occurs between bis- and trisphosphorylation of the kinase. The
activities of 35-kDa pY1 and 35-kDa pY2 are estimated to be 2 and 10%
of that of 35-kDa pY3, respectively. This result is consistent with
previous studies showing that trisphosphorylation of the kinase domain
is necessary for complete activation of the insulin
receptor(5, 6, 34) .
We then compared
catalytic properties of the core kinase to those of the 48-kDa kinase.
Previous studies of the 48-kDa kinase indicate that the extent of
activation following autophosphorylation depends on the activation
conditions as well as on the substrate and the conditions for substrate
phosphorylation(15, 19) . Under the activation
conditions described above, about 5 mol of phosphate were incorporated
per mol of the 48-kDa kinase at the maximal activation state, which was
reached after 25-30 min of incubation. The extent of activation
(200-fold) as well as the specific activity at maximal activation
state (
340 nmol/min/nmol) were found to be similar to those of the
core kinase. Requirements for ATP and Mg
of the 35-
and 48-kDa kinases were determined with the activated enzyme
(pre-autophosphorylated for 30 min under the conditions described
above) and 1 mM FYF. The K
for ATP of the
core kinase, determined with 8 mM Mg
, is 189
µM, which is similar to that of the 48-kDa kinase (183
µM). The Mg
activation profiles,
determined with 0.25 mM ATP, are also similar for the 35- and
48-kDa kinases with half-maximal activation of the kinase activity
around 1.5 mM and maximal activation achieved at 8
mM. These observations indicate that the 35-kDa kinase
displays similar catalytic properties to the 48-kDa kinase.
Figure 6:
Two-dimensional tryptic phosphopeptide
analysis of the autophosphorylation cascade of the 35-kDa kinase.
Autophosphorylation was carried out as described in the legend of Fig. 4. P-Labeled kinases at 2 (2`), 5 (5`), 10 (10`), and 90 (90`) min were
electroeluted from SDS-PAGE, digested with trypsin, and then subjected
to two-dimensional thin-layer chromatography as described under
``Materials and Methods.'' The small circles indicate the sample origin and the large circles represent the dinitrophenyl lysine standard. The drawing in the top panel is a key identifying the observed
phosphopeptides. Peptides C0, C1, B2, B3, A1, and A2 are related
peptides containing Tyr-1158, Tyr-1162, and Tyr-1163 phosphorylated on
one (C0 and C1), two (B2 and B3), or three (A1 and A2) tyrosine
residues. The sequence is DIYETDYYR for C1, B3, and A2 or DIYETDYYRK
for C0, B2, and A1(35) .
It should be noted that the bisphosphorylated form is not completely
converted into the trisphosphorylated form, as 35-kDa pY2 represents
20% of the kinase for the most highly phosphorylated state (Fig. 5B). Prolonged incubation resulted in decreased
trisphosphorylation and accumulation of bis-, mono-, and
unphosphorylated forms; the addition of EDTA, which terminates kinase
activity via chelation of Mg, stopped
dephosphorylation of the kinase (data not shown). These observations
are consistent with dephosphorylation of the kinase catalyzed by the
kinase itself, which was also observed for the 48-kDa
kinase(36) . The present study also indicates that the three
core tyrosines are auto-dephosphorylation sites as well as
autophosphorylation sites.
To examine the initiation of autophosphorylation at the three core tyrosines of the 35-kDa kinase, the monophosphopeptides (C0 and C1) generated at 2 min of autophosphorylation were recovered from the thin-layer plate and digested with V8 protease (Fig. 7). The predominant product of V8 digestion of the major monophosphopeptide C1 (>90%) corresponds to the peptide TDYYR, which is phosphorylated at Tyr-1162 or Tyr-1163 (>80%), whereas the peptide DIYE, which is phosphorylated at Tyr-1158, represents less than 20%. Similar results were obtained for C0, the minor species of monophosphopeptides (<10%); the peptide TDYYRK, which is phosphorylated at Tyr-1162 or Tyr-1163, is the predominant product of V8 digestion (>90%), indicating that autophosphorylation initiates at Tyr-1162 or Tyr-1163.
Figure 7:
V8 protease analysis of mono- and
bis-phosphopeptides. Phosphopeptides were isolated from two-dimensional
tryptic phosphopeptide maps (Fig. 6) of the 35-kDa kinase at 2
(C0 and C1) and 5 (B2 and B3) min of autophosphorylation. The samples
were digested with protease V8, which cleaves the tryptic peptides at
Glu-1159. The resulting V8 peptides were separated by thin-layer
electrophoresis as described (see ``Materials and Methods'').
The positions of the origin and the V8 peptides are indicated. The
sequences of the V8 peptides with phosphorylated tyrosine () are
shown.
Monophosphorylation of the kinase is immediately followed by
bisphosphorylation, as the monophosphorylated form never accumulated to
high amounts (maximal level is 30% of the total kinase, Fig. 5B). The bis-phosphopeptides (B2 and B3) generated
at 5 min of autophosphorylation were examined by V8 digestion. At this
time point, the trisphosphorylated form was undetectable (Fig. 5A). As shown in Fig. 7, V8 digestion of
B3 (
85% of bis-phosphopeptides) gave two predominant products, one
corresponding to Tyr-1158 phosphorylation (
42%) and another to
Tyr-1162 or Tyr-1163 phosphorylation (
55%). The peptide
phosphorylated at both Tyr-1162 and Tyr-1163 represents less than 3%.
Similar results were obtained for B2 (
15% of bis-phosphopeptides).
The proportion of the peptides phosphorylated at Tyr-1158, Tyr-1162 or
Tyr-1163, or both Tyr-1162 and Tyr-1163 is 44:46:10. Taken together,
the bis-phosphopeptides were predominantly phosphorylated at Tyr-1158
and either Tyr-1162 or Tyr-1163, whereas only <5% of the
bis-phosphopeptides were phosphorylated at Tyr-1162 and Tyr-1163.
Tyr-1158 is thus the major site for the addition of the second
phosphate group to the kinase.
Trisphosphorylation of the kinase
occurs late in the reaction cascade, as the bisphosphorylated form
accumulated to as high as 75% of the total kinase when the
trisphosphorylated form appeared at 10 min (Fig. 5B).
Tyr-1163 phosphorylation seems to favor cleavage at Lys-1165 versus Arg-1164(35) . To examine the level of Tyr-1163
phosphorylation in the bisphosphorylated kinase that is predominantly
phosphorylated at Tyr-1158 and either Tyr-1162 or Tyr-1163, we have
compared the ratio of bis-phosphopeptides B2/B3 with that of
tris-phosphopeptides A1/A2 (B2 and A1 result from Lys-1165 cleavage,
and B3 and A2 are from Arg-1164 cleavage). The ratio of B2/B3 (0.18
± 0.02) determined at early time points (5 and 10 min) is
considerably lower than that of A1/A2 (0.62 ± 0.05) determined
at 10, 20, and 30 min, suggesting that the level of Tyr-1163
phosphorylation is relatively lower in the bisphosphorylated kinase
compared with the trisphosphorylated kinase. Thus, Tyr-1163 is probably
phosphorylated last, after Tyr-1158 and Tyr-1162. Interestingly, the
bis-phosphopeptides that were generated at late time points (>90
min) while auto-dephosphorylation of the kinase occurred were found
predominantly phosphorylated at Tyr-1162 and Tyr-1163 (70% of
bis-phosphopeptides). This is consistent with the finding that the
observed ratio of B2/B3 was as high as that of A1/A2 (Fig. 6).
Our results thus suggest that the autophosphorylation cascade of the
core tyrosines initiates at Tyr-1162, immediately followed by
phosphorylation at Tyr-1158 and then at Tyr-1163.
Figure 8:
Concentration dependence of
autophosphorylation of the 35-kDa kinase. Enzyme at indicated
concentrations was incubated for 10 min with 1 mM [-
P]ATP and 8 mM MgCl
(
) or 4 mM MnCl
(
). The
incorporation of
P into the kinase was determined as
described in the legend of Fig. 4.
Figure 9:
Crystal of the 35-kDa kinase obtained by
macroseeding. The dimensions are 0.6
0.6
0.07
mm.