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* Division of Immunochemistry and
Division of Molecular Biophysics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
1Correspondence: Division of Immunochemistry Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69120 Heidelberg, Germany. E-mail: W.Droege{at}dkfz-heidelberg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ATP indicated that the
nonphosphorylated IRK (IRK-0P) is relatively inactive, and
crystallographic data indicated that the ATP binding site of IRK-0P is
blocked by its activation loop. We now show that phosphocreatine (PCr)
in combination with hydrogen peroxide serves as an alternative
phosphate donor and that ATP and PCr use distinct binding sites.
Whereas phosphorylation of the IRK by ATP is inhibited by the
nonhydrolyzable competitor adenylyl-imidodiphosphate, phosphorylation
by PCr is enhanced. The IRK mutant Tyr1158Phe showed no
phosphorylation with PCr but almost normal phosphorylation with ATP,
whereas Tyr1162Phe was phosphorylated well with PCr but less then
normal with ATP. 3-Dimensional models of IRK-0P revealed that the
conversion of any of the four cysteine residues 1056, 1138, 1234, and
1245 into sulfenic acid produces structural changes that bring Tyr1158
into close contact with Asp1083 and render the well-known catalytic
site at Asp1132 and Tyr1162 accessible from a direction that differs
from the known ATP binding site. The mutant Cys1138Ala, in contrast,
showed relatively inaccessible catalytic sites and weak catalytic
activity in functional experiments. Taken together, these findings
indicate that `redox priming' of the IRK facilitates its
autophosphorylation by PCr in the activation loop.—Schmid, E.,
Hotz-Wagenblatt, A., Hack, V., Dröge, W. Phosphorylation of the
insulin receptor kinase by phosphocreatine in combination with hydrogen
peroxide: the structural basis of redox priming.
Key Words: insulin responsiveness • redox regulation • signal transduction • tyrosine kinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6)
. Early in NIDDM, insulin resistance is most
pronounced in skeletal muscle, i.e., the tissue responsible for
70–90% of the glucose disposal after carbohydrate ingestion
(7
8
9
10)
. A muscle-specific insulin receptor (IR) knockout
was shown to exhibit features of NIDDM (11)
. The cause of
the insulin resistance in NIDDM and old age is not known but may be
related to the mechanism of IRK activation, which is also poorly
understood. A large body of evidence indicated that the
nonphosphorylated IRK has only weak kinase activity, but,
paradoxically, autophosphorylation at Tyr1158, Tyr1162, and Tyr1163 is
required for kinase activation (12
13
14
15
16
17
18
19)
. It must be
emphasized, however, that all studies of the catalytic activity and of
the autophosphorylation of the IRK were based on experiments with
32P--ATP (see refs 12
13
14
15
16
17
18
19
). This seemed
justified in view of earlier studies showing that ATP is the preferred
phosphate donor for most protein kinase (PK) species and that GTP, UTP,
or ITP donate phosphate to a minor extent (20)
. The
crystal structures of several PKs, including the triple-phosphorylated
insulin receptor kinase (IRK-3P) (19)
, cAPK
(21)
, Lck (22)
, c-Src (23
, 24)
,
Hck (25)
, and FGFRK (26)
, revealed
structurally similar PK domains with similar ATP binding sites. The
nonphosphorylated IRK (IRK-0P) showed a similar crystal structure but
differed with respect to the atypical position of its `activation
loop', which prevents productive ATP binding (27)
. To
reconcile this finding with the requirement for autophosphorylation, it
was hypothesized that the induction of the catalytic activity may
involve transient dislocation of the activation loop including Tyr1162
away from the catalytic site in such a way that Mg-ATP can bind to the
common ATP binding site and have contact to the catalytic site
(27)
. This catalytic site could then engage in
trans-autophosphorylation.
However, because Tyr1162 is located in the original IRK-0P structure in
a position next to the catalytic amino acid Asp1132 (27)
,
this configuration seems ideally suited for
cis-autophosphorylation. In this study, therefore, we
considered the alternative possibility that an unidentified phosphate
donor may bind to a site different from the common ATP binding site and
phosphorylate Tyr1162 without requiring the dislocation of the
activation loop. To test this hypothesis, we studied alternative
phosphate donors and focused on the predominant skeletal muscle
phosphagen phosphocreatine (PCr). Because autophosphorylation of the IR
was previously shown to be facilitated by hydrogen peroxide (HP)
(28
29
30
31
32)
and strongly enhanced by procedures that decrease
intracellular levels of glutathione (GSH) (33)
, we
pretreated the cells with buthionine sulfoximine (BSO), a specific
inhibitor of GSH biosynthesis (34)
, and treated the
immunoprecipitated IR routinely with HP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or with the
IR mutants Tyr1158Phe or Tyr1162Phe (36)
(kindly provided
by Dr. T. Pillay, University of Nottingham Medical School, Nottingham,
U.K., and Dr. P. Wilden, University of Iowa) were cultured in F12
medium with 10% fetal calf serum, glutamine, and antibiotics to total
confluence and moderately starved in order to decrease intracellular
PCr level. For the last 17 h before lysis, the cells were further
cultured in serum-free modified NCTC 135 medium (37)
in
the presence of 30 µM BSO (Sigma, St. Louis, Mo.).
Transient transfection of CHO cells
CHO cells were transiently transfected with pECE expression
vectors containing either wild-type human IR or IR cDNA
containing the single ß chain mutations Cys981Ala, Cys1056Ala,
Cys1138Ala, Cys1234Ala, Cys1245Ala, and Cys1308Ala
(38)
(kindly provided by Dr. C. W. Ward, CPIRO,
Parkville, Australia). We confirmed the catalytically inactive
Cys1138Ala mutation by DNA sequencing. In addition,
Xbal-BamHI fragments were prepared to confirm the wild-type
and mutated hIR DNA. Transfection was done with 2 µg wild-type or
mutant DNA by using the Lipofectamine technique (Gibco BRL, Grand
Island, N.Y.) according to the manufacturer's protocol. After
incubation for 48 h in F12 medium, the cells were incubated for an
additional 17 h in serum-free modified NCTC135 medium containing
30 µM BSO.
Insulin stimulation, cell lysis, IR precipitation, in
vitro kinase assay, and detection of tyrosine phosphorylation
The cells were finally washed with cold phosphate-buffered
saline/0.4 mM EDTA and lysed with 0.1% sodium dodecyl sulfate (SDS)
and 0.1% DOC, as described previously (33)
The IR was
immunoprecipitated with the monoclonal hIR ß chain-specific antibody
Ab-1 (Oncogene Science, Uniondale, N.Y.) and washed as described
(33)
. If indicated, the immunoprecipitates were then
incubated with 3 or 100 nM human recombinant insulin (Sigma) for 30 min
on ice. After pelleting, the immune complexes were further incubated
for 20 min at 30°C in kinase buffer (33)
without
manganese chloride either with graded concentrations of PCr (Sigma), 2
mM phosphoenol pyruvate (Sigma), 100 µM ATP (Sigma), or other
nucleotides in the presence of 25 µM HP (Merck; Darmstadt, Germany)
unless indicated otherwise. The immune complexes were finally washed
with ice-cold kinase buffer and subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) and Western blotting. After probing for tyrosine
phosphorylation with the monoclonal antiphosphotyrosine antibody 4G10
(Upstate Biotechnology, Lake Placid, N.Y.; BioMol, Hamburg, Germany),
IR expression was determined with a polyclonal anti-ßIR antibody
(Santa Cruz Biotechnology, Heidelberg) as described (33)
.
In some cases the immunoprecipitates were subjected to the in
vitro kinase assay (incorporation of
32P--ATP), as described elsewhere
(33)
, and again probed for IR expression.
3-Dimensional modeling of the nonphosphorylated IR ß chain and
of derivatized proteins
The 3-dimensional structures of the IRK domain and its
derivatives were modeled on an Indigo II workstation (Silicon
Graphics). The starting coordinates of nonhydrogen atoms were taken
from the Protein Data Bank entry 1irk (27)
. Atoms not
reported in Protein Data Bank 1irk were inserted with the help of the
INSIGHT II library and hydrogen atoms were added. This structure was
the basis for all subsequent modifications. Energy minimization was
performed on an IBM-SP2 workstation using the DISCOVER module of the
INSIGHT II program (Molecular Simulation BIOSYM Technologies Inc.) and
the conjugate gradient method for energy minimization. The generation
of hydrogen atoms and the automatic assignment of atom potential types
and partial charges were achieved using the consistent valence force
field (39)
. The polar amino acids of the molecule were in
the uncharged form and a dielectric constant of 1 was used, because the
water in the electrostatic environment was expected to inhibit
interactions at the surface. During the first 100 steps of
minimization, only the hydrogen atoms were allowed to move in order to
avoid large forces at the backbone just because of the newly added
hydrogens. For the next 100 steps, all C
atoms were tethered using a
harmonic potential with the force constant of 100 kJ. Full minimization
of all atoms was carried out until the maximum force value reached a
value below 0.001. The figures of the minimized structures were
obtained by RASMOL (Roger Sayle).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was prepared and incubated with insulin
and either 0.1 mM ATP, GTP, UTP, CTP, 2 mM phosphoenolpyruvate or 1–40
mM PCr in combination with 25 µM HP as described in Materials and
Methods. The resulting tyrosine phosphorylation was then determined
with the anti-phosphotyrosine antibody 4G10. Experiments with the most
abundant mammalian phosphagen (i.e., PCr) revealed that
tyrosine phosphorylation of the IRß chain does indeed occur at
physiologically relevant concentrations of 5–40 mM PCr in the
presence, but not in the absence, of HP (Fig. 1
A, B). When CHO cells were transiently transfected in
parallel with hIR DNA or DNA from the structurally related src family
kinase fynB, PCr-dependent tyrosine phosphorylation was seen in the IR
but not in the fyn protein (Fig. 1C
).
|
A substantial degree of IRK phosphorylation was also seen with other
nucleotides and phosphoenolpyruvate. However, in all these cases (not
shown) as well as in the case of ATP (Fig. 2
), phosphorylation was inhibited by the nucleotide analog
adenylyl-imidodiphosphate (AMP-PNP). Phosphorylation by PCr, in
contrast, was enhanced by AMP-PNP. With the relatively low
concentration of 3 nM insulin (Fig. 2)
or in the absence of insulin
(Fig. 3
), the PCr-dependent IRK phosphorylation was barely detectable without
AMP-PNP but quite substantial in the presence of AMP-PNP. In the
phosphorylated IRK, AMP-PNP binds to the adenosine binding site
(19)
and therefore competitively inhibits the
phosphorylation by ATP or other phosphate donors that bind to this
site. In the nonphosphorylated IRK, in contrast, the activation loop
separates the adenosine binding groove from the catalytic site
(27)
. In this case, binding of AMP-PNP to the adenosine
binding groove would be expected not to interfere with the binding of
another phosphate donor to the catalytic site. The enhancement of the
PCr-dependent phosphorylation by AMP-PNP therefore suggested that
1) PCr is not acting via the ATP binding site, 2)
PCr is most probably involved in the initial phosphorylation of the
nonphosphorylated IRK, and 3) the PCr-dependent
phosphorylation may be synergistically enhanced by an adenosine
nucleotide. This possibility remains to be investigated.
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Failure of PCr to phosphorylate the IRK mutant Tyr1158Phe
To determine whether PCr may phosphorylate preferentially or
exclusively Tyr1162 (see opening paragraphs), we studied CHO cells
stably transfected with DNA from the hIR mutant Tyr1162Phe. CHO cells
transfected with the mutant Tyr1158Phe and wild-type IRK were included
as controls. These experiments confirmed and extended previous studies
(36)
showing that the ATP-dependent phosphorylation was
strongly decreased in the case of the mutant Tyr1162Phe but, on
average in our experiments, only moderately decreased in the case
of Tyr1158Phe (Fig. 3A
). The PCr-dependent phosphorylation,
in contrast, was clearly detectable in the case of the Tyr1162Phe
mutant but undetectable in the case of the Tyr1158Phe mutant. This
finding led to the unexpected conclusion that Tyr1158 rather than
Tyr1162 may serve as the (primary) PCr-dependent phosphorylation sites.
AMP-PNP enhanced PCr-dependent phosphorylation of the wild-type IRK but
not of the two mutant proteins (Fig. 3B
).
The structural basis of `redox priming'
The conclusion that Tyr1158 serves as the primary target of the
PCr-dependent phosphorylation was not easily reconciled with the
calculated distance between Tyr1158 and the nearest aspartate group. In
the activated IRK-3P protein (19)
, the hydroxyl group of
the substrate tyrosine is in close contact with the carboxylate group
of the catalytic amino acid Asp1132 (O
-O
2:2.7 Å). A similar
distance of 2.6 Å was found between O of Tyr1162 and O2 of
Asp1132 in the nonphosphorylated IRK structure (27)
(see
also Fig. 4
A). In contrast, the computed distance between O of Tyr1158 (gray ring
in the center of Fig. 5
A) and the carboxylate group (light blue) of the nearest
aspartate (Asp1083) in the 3-dimensional model of the nonphosphorylated
IRK structure (1irk without mercury; see Materials and Methods) is
greater than 4 Å (Table 1
). Therefore, we used 3-dimensional modeling of derivatized IRK protein
structures to determine whether the interaction of the IRK with HP may
cause a structural alteration that moves the hydroxyl group of Tyr1158
into the proper distance of the carboxylate group of Asp1083.
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Because HP was previously shown to convert cysteine residues into
cysteine-sulfenic acid derivatives (CyS-H +
H2O2 
CyS-OH +
H2O; see refs 40
, 41
), we determined the
3-dimensional structure of the various sulfenic acid derivatives of the
IR-ß chain by molecular modeling and energy minimization. The
structure `1irk w/o mercury' (Fig. 4A
) was obtained from
the 3-dimensional structures of the IRK domain (27)
(1irk;
not shown here) by removing the ethyl-mercury groups and by subsequent
energy minimization. The 3-dimensional structures Ser981Cys and 981
CyS-OH (not shown) obtained by replacing serine 981 with the authentic
cysteine and cysteine-sulfenic acid, respectively, followed by energy
minimization, had essentially the same structure as `1irk w/o
mercury'. In contrast, the four derivatives 1056 CyS-OH, 1138 CyS-OH,
1234 CyS-OH, and 1245 Cys-OH showed conspicuous structural changes
(Fig. 4B
and Fig. 5B-E
). These four sulfenic
acid derivatives had almost identical 3-dimensional structures, as
illustrated by the superposition in Fig. 4B
. Each of these
four derivatizations moved the hydroxyl group of Tyr1158 into close
contact with the carboxylate group of Asp1083 (O-O2:2.8–2.9 Å,
see Table 1
and Fig. 5
) and rendered the catalytic center at Asp1132
accessible from a direction opposite to that of the common ATP binding
site (Fig. 4)
. The accessibility of the carboxylate group of the
catalytic amino acid Asp1132 and the adjacent hydroxyl group of Tyr1162
together with the positive and negative charges at the surface in the
vicinity of this catalytic center are shown in Fig. 4C-E
.
The 3-dimensional models of the nitric oxide derivatives 1234 CyS-NO
(Fig. 5F
), 1056 CyS-NO, 1138 CyS-NO, and 1245 CyS-NO (not
shown) were similar to the structures of the sulfenic acid derivatives.
The structures of the mutants Cys1234Ala (Fig. 5G
),
Cys1245Ala, and Cys1056Ala (not shown) were also similar to the
corresponding CyS-OH derivatives, whereas Cys1138Ala (not shown)
and the 1234 CyS-OH derivative of Cys1138Ala (Fig. 4E
, Fig. 5H
) had inaccessible catalytic sites.
Defective PCr-dependent and ATP-dependent phosphorylation of the
IRK mutant Cys1138Ala
Experiments with CHO cells (Fig. 6
) and NIH-3T3 cells (not shown) transiently transfected with DNA from
the hIR mutant Cys1138Ala confirmed the prediction from the
3-dimensional modeling that this mutant is poorly autophosphorylated by
PCr and ATP, although the protein is clearly expressed. Essentially
similar results were obtained if the phosphorylation reaction was
performed in the presence of 5 mM AMP-PNP and with 3 µM insulin (not
shown). Cells transiently transfected with the wild-type or other
CysAla mutants, in contrast, showed strong tyrosine phosphorylation
(Fig. 6)
and kinase activity with 32P--ATP
(not shown). These differences were not detected in a previous study of
these mutants (38)
. It is conceivable that in this
previous study the endogenous IR of the host cell might have
contributed to the functions under test.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 27)
, together with our
3-dimensional models, suggest that the IRK exposes sequentially
different binding sites for different phosphate donors. The enhancing
effect of AMP-PNP indicates, moreover, that a physiological adenosine
nucleotide may serve as a positive allosteric regulator of the
PCr-dependent phosphorylation. Regardless of whether this possibility
will be confirmed or not, AMP-PNP appears to generate a steric effect
similar to that induced by insulin or by the mutation Tyr1162Phe.
AMP-PNP, insulin, or said mutation were all found to enhance the
PCr-dependent IRK phosphorylation independent of each other. In
agreement with these findings, the mutant Tyr1162Phe was previously
shown to express a relatively high baseline kinase activity that was
not significantly increased by insulin (42)
.
In addition, our studies provide a structural explanation for the
widely observed stimulating effects of oxidants (28
29
30
31
32)
and the effects of SH-reactive reagents on the catalytic activity of
the IRK (43
44
45)
. It may be important in this context that
HP production can also be induced among others by insulin
(46)
. The effect of HP is best explained by the prediction
of the 3-dimensional models that oxidation of any one of several
critical cysteine residues by HP may increase the competence of the
kinase to autophosphorylate the activation loop at Tyr1158 and Tyr1162.
A comparison of Fig. 4A, B
and Fig. 5A-E
suggests that cysteine residues at four different positions of the IRK
domain share the function of preventing the spontaneous
autophosphorylation at Tyr1158 and Tyr1162 and that derivatization of
anyone of these residues by HP activates both catalytic sites. This
effect of HP is therefore proposed to account for the enhancement of
IRK activity by HP as reported previously (28
29
30
31
32)
.
The failure of PCr to phosphorylate the mutant Tyr1158Phe indicates
that Tyr1158 is the first and limiting target. However, the possibility
that Tyr1162 is also phosphorylated by PCr is not formally excluded. If
so, the PCr-dependent phosphorylation of Tyr1162 would appear to depend
strictly on the prior phosphorylation of Tyr1158. Three-dimensional
modeling of the IRK structure monophosphorylated at Tyr1158 indeed
showed a slightly better accessibility of Tyr1162/Asp1132 than the
sulfenic acid derivatives, as illustrated by the wide open angle
between the -helix C (yellow) and the activation loop (orange) in
Fig. 5I
. The view into the putative binding site of the
phosphate donor (not shown) is essentially similar to that of
1234-Cys-OH (Fig. 4D
). Earlier studies of the
autophosphorylation of the IRK provided no explanation for the
physiological function of Tyr1158 (19)
. It was noted,
however, that the ATP-dependent phosphorylation of the mutant
Tyr1158Phe is only moderately decreased on the average of our
experiments (see also ref 36
) despite the complete abrogation of the
PCr-dependent phosphorylation. Whether the induction of the
ATP-dependent catalytic activity of the wild-type IRK, in contrast to
the mutant Tyr1158Phe, has a strict requirement for an initial
PCr-dependent phosphorylation of Tyr1158 or whether the alternative
mechanism proposed by Hubbard et al. (see opening paragraphs) may
operate to some extent remains to be determined. There is the
interesting possibility that both modes of phosphorylation have
different functional consequences. A similar sequence of
phosphorylation events may also be involved in the activation of the
closely related nerve growth factor receptor tyrosine kinase, which
contains three functionally important tyrosine groups (Tyr670, Tyr674,
and Tyr675) in the activation loop (47
48
49)
.
The most important physiological implication of the PCr-dependent
phosphorylation of the IRK may be that the muscle tissue gains an
advantage over the liver with regard to insulin-induced fuel
deposition. In contrast to the rather strong cytoplasmic and
mitochondrial creatine kinase activity and high PCr levels in the
skeletal muscle tissue, the liver has little or no creatine kinase
activity (50
, 51)
. Mitochondrial creatine kinase has been
found in human liver by some authors (50)
but not by
others (51)
. It is therefore conceivable that the
PCr-dependent IRK phosphorylation may give the insulin-induced muscular
glycogen and protein synthesis priority over the utilization of glucose
and amino acids in the hepatic lipogenesis. If so, a decrease in the
muscular PCr-dependent IRK phosphorylation may favor obesity.
Insulin-stimulated kinase activity was previously shown to be
markedly compromised in NIDDM and obesity (1
, 2)
, and
physical exercise was shown to increase insulin responsiveness in NIDDM
(52
53
54)
. The mechanism, however, remained obscure. Our
studies now suggest that this effect may be mediated by reactive oxygen
intermediates that may be increased at least temporarily by physical
exercise (55
56
57
58
59)
. Skeletal muscle type II fibers with
relatively fast contraction times were found to have substantially
lower intracellular GSH levels than type I fibers (60)
.
Last but not least, the HP scavenging enzymes catalase and glutathione
peroxidase were shown to increase significantly with age in rat
skeletal muscle (61
, 62)
. In view of these facts, the
findings in this report may lead to new strategies to ameliorate the
decrease in insulin responsiveness that often accompanies NIDDM,
obesity, cancer cachexia, and old age and may have implications also
for sports medicine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication February 8, 1999. Accepted for publication without revision March 19, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i2. J. Biol. Chem. 272,10135-10143-1 to mediate neuritogenesis. Neuron 12,1-20[Medline]
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