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From the
We have recently reported that the Arg
Activation of the insulin receptor (IR)
Both cAMP-dependent
protein kinase (PKA) and Ca
We have previously reported a family of patients with a clinically
common form of non-insulin-dependent diabetes mellitus associated with
a point mutation in the IR gene leading to Arg
To address
further the role of the RK motif in IR function, we have now created
two additional mutant IRs: a single mutant, in which the second basic
residue in the RK motif (Lys
NIH-3T3 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
newborn calf serum. Transfections were carried out by the calcium
phosphate method (26) as described previously(27) . G418
(Life Technologies, Inc.) was used at the effective dose of 0.3 mg/ml.
Individual G418-resistant clones were isolated and screened by
Partially purified receptor
preparations were obtained by applying Triton X-100 solubilized cells
on 1-ml WGA-Sepharose columns. Bound glycoproteins were then eluted by
0.3 MN-acetylglucosamine. Insulin binding activity
in the WGA eluate was determined by incubating 40 µl of WGA eluate
with 1 ml of binding buffer (20) containing 45,000 cpm/ml
Cell surface insulin binding assays were performed on confluent
monolayers of cells. Cells were incubated in 3 ml of binding buffer
(15,000 cpm/ml) for 3 h at 15 °C in the presence of 0-1
µg/ml unlabeled insulin. Unbound radioactivity was eliminated
rapidly by repeated washes with 5 ml of ice-cold phosphate-buffered
saline, and cells were solubilized in 1 ml of 0.1% SDS. Radioactivity
in the lysates was quantitated in a
PKA
phosphorylation reactions were performed by incubating 20 fmol of IRs
with PKA in a final volume of 100 µl of buffer containing 150
mM NaCl, 50 mM Hepes, pH 7.6, 0.02% Triton X-100, 2
mM MgCl
We have shown previously that the RK motif is highly
conserved in the IR family of tyrosine kinases, suggesting an important
role in the function of these kinases(20) . A naturally
occurring mutation in the IR gene(18) , leading to replacement
of the Arg
In the present work, to elucidate
the role of the RK motif in IR function, we have generated one
additional single mutant receptor in which the second basic residue of
the RK motif (Lys
We have hypothesized that this role of the RK region could
be exerted by allowing the receptor to interact with receptor
regulatory proteins, by destabilizing a specific conformation that is
critical for receptor phosphotransferase activity or by both
mechanisms. Previous reports by several laboratories (8, 9, 10, 11) have shown that the IR is
Ser- and Thr-phosphorylated by both PKA and PKC. It has been shown that
phosphorylation of the receptor by PKA and PKC occurs in the absence of
insulin(8, 9, 11) , increases upon insulin
stimulation of the cells(8, 13) , and inhibits IR kinase
and signaling(8, 9, 10, 11) . However,
the molecular details of receptor interaction with PKA and PKC,
including the Ser and Thr residues on the receptor whose
phosphorylation negatively affects its signaling, have not been
identified. Consistent with previous reports(8) , in the present
study we show that preincubation of
The mechanism
through which the intact RK motif allows receptor phosphorylation by
PKC and PKA might include direct activation of these kinases through
the RK as well as accomplishment of receptor binding to PKC and PKA in
a sterically favorable position for receptor phosphorylation. Although
direct activation of PKC by Arg-rich peptides has ben
reported(34) , this is unlikely in the case of the receptor RK
motif for the following reasons. (i) At variance from PKC activation by
Arg-rich peptides(34) , receptor phosphorylation by PKC is
strictly Ca
As
reported previously for the naturally occurring single mutant
receptor(19, 20) , we now show that insulin is unable to
stimulate kinase activity and metabolic signaling also in the second
single mutant receptor. At variance, in spite of a basal activity that
is increased similarly to the single mutants, the double mutant is
fully responsive to insulin in terms of autophosphorylation, kinase
activity, and metabolic signaling. Thus, in all of these mutants, lack
of receptor phosphorylation by PKA and PKC may determine their
increased basal kinase activity and signaling. However, a more complex
change appears to occur in the two single mutants accounting for their
additional inability to transduce insulin signal. We would like to
speculate that replacement of a single residue in the RK motif by a
noncharged amino acid may induce structural changes of the activation
loop of the receptor which differ from those occurring upon
simultaneous replacement of both the Arg and the Lys and account for
the observed differences in autophosphorylation of the single and
double mutants. As we report in the present paper, autophosphorylation
of the key tyrosines (Tyr
Besides controlling insulin-independent receptor
activity by regulating IR interaction with PKA and PKC, the RK motif
might also play an important structural role that allows normal insulin
regulation of the kinase.
We are grateful to Dr. S. Gammeltoft (Bispebjerg
Hospital, Copenhagen) for generously donating the WT IR cDNA, to Dr. E.
Appella (National Cancer Institute) for synthesizing receptor peptides,
and to Dr. P. A. Temussi (University of Naples School of Chemistry) for
valuable help in constructing the molecular models. We also thank Drs.
S. M. Aloj, E. Consiglio, and G. Salvatore (University of Naples
Medical School) for continuous support and advice during the course of
this work and Dr. D. Liguoro for technical help.
Volume 270,
Number 26,
Issue of June 30, pp. 15844-15852, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Gln
insulin receptor mutation (QK single mutant) alters a conserved motif
(RK motif) immediately next to the key tyrosine phosphorylation sites
(Tyr
, Tyr
, Tyr
) of the
receptor and constitutively activates its kinase and metabolic
signaling. To investigate further the function of the RK motif, we have
expressed two additional mutant insulin receptors: a single mutant, in
which the second basic residue in the RK motif (Lys
) was
substituted (RA mutant); and a double mutant, in which both the Arg and
the Lys residues were replaced with noncharged amino acids (QA mutant).
As compared with the transfected wild-type receptors (WT), both the
single and the double mutant receptors were normally synthetized and
transported to the plasma membrane and bound insulin normally. Whereas
the double mutant receptor exhibited preserved insulin-dependent
autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of
these functions were grossly impaired in the two single mutant
receptors. Two-dimensional analysis of tryptic phosphopeptides from
receptor
-subunits revealed that decreased autophosphorylation of
the single mutant receptors mainly involved regulatory
Tyr and carboxyl-terminal Tyr
.
At variance with the insulin-stimulated, insulin-independent tyrosine
kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both
the double and the single mutants. All mutant receptors induced a
2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells.
Treatment of WT transfected cells with
12-O-tetradecanoylphorbol-13-acetate or 8-bromo-cAMP increased
insulin receptor phosphorylation by 3-fold. No phosphorylation was
observed in cells expressing the two single or the double mutant
receptor. Consistently, purified preparations of PKC and PKA
phosphorylated the WT but not the mutant receptors in vitro. A
17-amino acid synthetic peptide encoding the receptor sequence
surrounding the RK motif inhibited phosphorylation of WT insulin
receptors by both protein kinases A and C. A mutant peptide in which
the RK sequence was replaced by QK (to mimic the mutation in the QK
receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor
motif is required for insulin receptor phosphorylation by protein
kinases C and A and may modulate insulin-independent receptor activity.
The RK motif may also have an important structural role in allowing
normal insulin regulation of the kinase.
()kinase and signaling is regulated at multiple levels in vitro as well as in the intact cells(1) . Ligand
binding and tyrosine autophosphorylation result in increased receptor
activity(2, 3, 4, 5, 6, 7) .
Ser/Thr phosphorylation provides an additional level of control that is
sensitive to extracellular messengers and intracellular
events(8, 9) . The IR is phosphorylated on serine and
threonine residues in the basal state, in response to insulin and to
agents activating Ser/Thr
kinases(8, 9, 10, 11) . In most cases,
these phosphorylations decrease insulin-stimulated tyrosine kinase
activity (8, 9, 10, 11) and generate
insulin resistance in
vivo(10, 12, 13) .
-phospholipid-dependent
protein kinase (PKC) phosphorylate the IR in vitro and in
vivo (8-11). This phosphorylation reduces the tyrosine
kinase activity of the
receptor(8, 9, 10, 11, 12, 13) .
Treatment of purified IRs with alkaline phosphatase reverses
PKC-mediated receptor phosphorylation and increases receptor kinase
activity(8) . In intact cells, activation of both PKA and PKC
leads to Ser/Thr phosphorylation of the IR and decreases insulin
action(8, 9, 12, 13) . This effect has
been proposed to be responsible for the catecholamine- and
starvation-induced insulin resistance in rodents as well as in
humans(10, 12, 13) . Thr
appears
to be a major receptor phosphorylation site for PKC(14) .
()However, IRs in which Thr was replaced
by Asn functioned normally when transfected in Chinese hamster ovary
cells(15) , indicating that there are other relevant
phosphorylation sites in the receptor. As is the case for PKC, no
phosphorylation site involved in PKA regulation of IR function has been
identified yet. The region of the IR involved in the recognition of
these Ser/Thr kinases is unknown as well. Thus, although PKA and PKC
are important regulators of IR signaling, the molecular mechanisms
allowing their interaction with the receptor need to be elucidated.
Gln substitution in the receptor regulatory
domain(18, 19) . This mutation affects a two-basic amino
acid motif (RK motif) next to the IR key autophosphorylation sites
(Tyr
, Tyr
, Tyr
) and
constitutively activates IR kinase and signaling(20) . This
results in loss of insulin sensitivity for metabolic effects as well as
altered intracellular receptor traffic both in the patient fibroblasts
and in cells overexpressing the Arg
Gln mutant
receptor(19, 20, 21) . We have also shown that
the RK motif is highly conserved in the IR family of tyrosine kinases,
suggesting an important role in IR function(20) .
) was substituted; and a
double mutant, in which both Arg and Lys residues were replaced. In the
present work we show that substitution of the basic amino acids of the
RK motif with noncharged residues critically impairs IR phosphorylation
by both PKA and PKC and increases their basal (insulin-independent)
activities.
General
Preparation of plasmid DNA,
agarose gel electrophoresis, restriction enzyme digestion, bacterial
transformation, and DNA sequencing were performed by standard methods
(22). Enzymes were from Boehringer Mannheim (Kvistgard, Denmark) or
Pharmacia LKB Biotechnology A/S (Hillerod, Denmark). All
oligonucleotides were synthesized on an Applied Biosystems 380B DNA
synthesizer. All radiochemicals as well as monoclonal Ig2
phosphotyrosine antibodies were from Amersham (Milano, Italy). mAb3 IR
antibody was obtained from Oncogene Science (Manhasset, NY). Polyclonal
B9 IR antibody was a generous gift of Dr. C. R. Kahn (Joslin Diabetes
Center, Boston). PKA catalytic subunit was from Sigma and purified PKC
from Promega. The two 17-amino acid peptides encoding the human IR
sequence surrounding the RK motif were kindly provided by Dr. E.
Appella (NCI, NIH). The WT peptide sequence was MTRDIYETDYYRKGGKG-OH.
The QK peptide sequence was MTRDIYETDYYQKGGKG-OH. Media and serum for
tissue culture were from Life Technologies, Inc.
Mutant Construction, Transfection, and Cell
Culture
The QK single mutant IR has been described
previously(20) . To replace Lys with Ala (single
mutant RA) and both Arg
and Lys
with Gln
and Ala, respectively (double mutant QA), the hIR cDNA fragment BamHI-SalI(1926-5200), derived from pSP65-hIR
was subcloned in M13mp19 as described previously(20) .
Single-stranded template was prepared and point mutations obtained by
oligonucleotide-directed mutagenesis using the following primers 5`-GAT
TAC TAC CGG GCA GGG GGC AAG G-3` (RA mutant) and 5`-GAT TAC TAC
CAG GCA GGG GGC AAG G-3` (QA mutant). Mutagenesis was
performed according to Taylor et al.(23) and confirmed
by M13 dideoxysequencing(24) . The HincII
fragment(3187-3871) encoding either the single Lys
Ala (A
) or the double Arg
Gln/Lys
Ala
(Q
A
) mutations was cloned back in the
pSP65-hIR. The two mutants hIR-A
and
hIRQ
A
were cloned in the SacII-XhoI site of the pCO11 vector by linker
insertion(25) . The final constructs were resequenced to confirm
the presence of the mutations.
I-insulin binding.
Metabolic Labeling, Receptor Purification, and
Insulin Binding
These assays were performed as described
previously (20). Briefly, cells were labeled with
[S]methionine (1,000 Ci/mmol, 50 µCi/ml) for
16 h in 4 ml of methionine-free Dulbecco's modified Eagle's
medium with 10% fetal calf serum and glutamine. The cells were then
solubilized in 0.5 ml of S buffer (20) and then centrifuged at
100,000
g for 20 min. IRs were precipitated from the
supernatants using mAb3 IR antibodies and analyzed by 7.5% SDS-PAGE (28) and autoradiography.
I-insulin (100 Ci/g) for 16 h at 4 °C. Insulin
binding activity was quantitated by the addition of ice-cold 25%
polyethylene glycol 6000 using 0.3% human
-globulin as a carrier.
-counter. Binding data were
analyzed using the LIGAND program for curve fitting and parameter
estimation (29).
Receptor Autophosphorylation and Phosphorylation of
Synthetic Substrates
Aliquots of WGA-purified receptors (20
fmol of insulin binding activity) were incubated in the absence or the
presence of 1 µM insulin for 1 h at room temperature.
Thereafter, phosphorylation was initiated by the addition of 10 µCi
of [P]ATP in the presence of 3 mM manganese acetate, 1 mM CTP, and 10 µM ATP.
After 20 min at room temperature, the reaction was stopped by the
addition of 800 µl of ice-cold stopping solution (20) and
IRs immunoprecipitated using agarose-coupled phosphotyrosine
antibodies. Immunoprecipitated phosphoproteins were separated on 7.5%
polyacrylamide gels and detected by autoradiography. Phosphorylation of
exogenous substrates was carried out as described above except that the
synthetic peptide poly(Glu-Tyr) 4:1 was present at a concentration of
2.5 mg/ml, and 3 mM magnesium acetate was substituted for
manganese acetate. After 20 min at 22 °C, reactions were stopped by
spotting 40-µl aliquots onto Whatman 3MM paper. The paper was
washed extensively in a 10% trichloroacetic acid, 10 mM sodium
pyrophosphate solution and dried; the incorporated radioactivity was
determined by liquid scintillation counting. A correction was made for
nonspecific absorption of
P to the filter paper by
subtracting the radioactivity bound to the filter at zero time.
Two-dimensional Receptor Phosphopeptide
Mapping
The analysis was performed as described by
Tavaré and Denton(30) . Briefly, P-labeled
IR
-subunits were isolated from the gel by electroelution and
digested with 10 µg of 1-tosylamido-2-phenylethyl chloromethyl
ketone (TPCK)-treated trypsin/ml in 100 µl of 50 mM
NH
HCO
for 16 h at 30 °C. The P-labeled phosphopeptides were separated on cellulose TLC
plates, first by electrophoresis at 400 V for 2 h at pH 3.5
(pyridine/acetic acid/water, 1:10:189, v/v), then by ascending
chromatography (pyridine/acetic acid/butanol/water, 10:3:15:12, v/v),
and finally detected by autoradiography using Kodak X-Omat preflashed
film cassettes with intensifying screens at -70 °C.
Determination of 2-Deoxy-D-Glucose
Uptake
2-Deoxyglucose (2-DG) uptake was measured as
described previously(20) . Briefly, cells were washed twice with
incubation buffer (20) and further incubated for 45 min at room
temperature in the same buffer in the absence or the presence of
insulin. Uptake of 2-DG was initiated by the addition of 100 µl of
incubation buffer containing 2-[
C]DG (final
concentration, 0.2 mM). After incubating for 10 min at room
temperature, cells were washed rapidly with ice-cold 0.9% NaCl and
lysed with 1 N NaOH. 2-DG uptake was then determined by liquid
scintillation counting. Aliquots of the solubilized cells were kept for
protein determination. Cytochalasin B (50 µM) was used to
estimate carrier-independent uptake.
In Vitro Phosphorylations of IR
PKC
preparations were obtained from solubilized plasma membranes of NIH-3T3
cells as reported previously(31) . 50 µg of solubilized
membrane proteins was incubated with heat-inactivated WGA-purified IRs
(20 fmol of insulin binding activity) in the absence or the presence of
1.1 mM CaCl, 11.4 µM phosphatidylserine, and 0.7 µM diolein in a reaction
mixture (final volume 140 µl) containing 1 mM EGTA, 80
mM MgCl, 24 mM MnCl, 100
mM Tris, pH 7.4, and 50 µM [-
P]ATP (0.5 µCi/nmol). The
phosphorylation reaction was prolonged for 20 min at 22 °C and then
interrupted by the addition of 90 µl of a solution containing 100
mM NaF, 10 mM
Na
PO
, 5 mM EDTA, 5 mM ATP (termination solution). Receptors were immunoprecipitated
using either the Ab3 monoclonal or the B9 polyclonal receptor
antibodies. Immunoprecipitated phosphoproteins were separated on 7.5%
polyacrylamide gels and detected by autoradiography. Using both
lysine-rich histones (IIIS) and WGA-purified IR as substrates, there
was no difference in the activity of PKC preparations from the
untransfected cells and from those expressing the three mutant IRs. In
some of the experiments, highly purified PKC preparations from rat
brain were used. In these experiments, 20 fmol of WGA-purified
receptors was phosphorylated by 0.05 unit of PKC in the presence or the
absence of 1.5 mM CaCl, 4.5 µg/ml
phosphatidylserine, and 1 µM diolein in a reaction mixture
containing 1 mM EGTA, 10 mM MgCl, 3
mM MnCl, 13 mM Tris, pH 7.4, and 5
µM [-
P]ATP (0.5 mCi/nmol).
Subsequent conduction of the phosphorylation reaction and receptor
identification were performed as described above.
, 0.2 mg/ml bovine serum albumin.
Phosphorylation reactions were initiated by the addition of 10
µM [-
P]ATP (10 Ci/mmol) and
prolonged for 20 min at 22 °C. Reaction mixtures were then
immunoprecipitated with mAb3 and analyzed by SDS-PAGE and
autoradiography.
Receptor Phosphorylation in Intact
Cells
Confluent cells were equilibrated with
[P]orthophosphate as described(32) .
After 8 h, the cells were exposed to 1 µM TPA for 45 min.
The reaction was stopped with phosphate-buffered saline at 4 °C
containing 10 mM pyrophosphate, 10 mM NaF, 4 mM EDTA, and 1 mM Na
VO. The labeled
cells were solubilized with 1% Triton X-100 containing 2 mM phenylmethylsulfonyl fluoride, aprotinin (12 units/ml), 10 mM pyrophosphate, 10 M NaF, 4 mM EDTA, and 1 mM NaVO for 10 min. Solubilized cells were
immunoprecipitated with receptor mAb3 and analyzed by SDS-PAGE and
autoradiography.
Expression of Mutant Receptors
To
investigate the functional properties of the RK motif, two hIR mutants
have been generated in the present study: a single mutant, in which
Lys was replaced by Ala (RA mutant); and a double
mutant, in which both Arg
and Lys
were
replaced by Gln and Ala, respectively (QA mutant) (Fig. 1). The
previously reported single mutant (18) in which Arg
was replaced by Gln (QK mutant)
()and WT
have also been analyzed. RA and QA mutant cDNAs were stably transfected
in NIH-3T3 fibroblasts. Clonal cell lines were screened for expression
of I-insulin binding and several cell clones isolated.
Insulin sensitivity of these cells increases linearly with IR number up
to 2
10
receptors/cell, whereas linearity
disappears with higher receptor expression(20) . Therefore, four
mutant clones expressing 1.2 and 2.4 10
RA mutant
receptors/cell (RA, RA) and 1.0 and 1.9
10
QA mutant receptors/cell were studied in detail. Based
on Scatchard analysis(33) , all of these clones exhibited
dissociation constants (K) for insulin
between 0.62 and 0.72 nM (Fig. 1). This is similar to
the K
values of the previously described WT and QK
clones()and to that of the endogenous IR
measured in untransfected NIH-3T3 cells. Thus, all transfected cell
clones analyzed in this work exhibited normal insulin binding
affinities in addition to comparable receptor levels.
Figure 1:
Schematic representation of the IR and
the RK receptor mutants. The regulatory domain of the WT human IR
mutant is shown with the key tyrosine autophosphorylation sites
(Tyr, Tyr
, and Yyr
) and
the basic amino acids of the RK motif (Arg
,
Lys
). Cell clones expressing the single mutant receptor
in which Lys
was replaced by Ala are designated RA
and RA. Clones expressing the second single mutant
(featuring Arg replaced by Gln) and the WT receptors
have been reported previously (20) and designated QK
and
WT, respectively. Clones expressing the double mutant
receptor in which Arg and Lys
were
replaced by Gln and Ala, respectively, are designated QA
and QA. The numbers and affinities of IRs have been
determined in each cell clone by Scatchard analysis of I-insulin binding data and are also shown in the figure.
Untransfected cells expressed 1.6
10
receptors/cell (K 0.61 nM).
To ensure that
IR mutants were properly processed and transported to the cell surface,
extracts were prepared from cells metabolically labeled with
[S]methionine. The radiolabeled IRs were then
immunopreciptated with anti-IR mAb3. In all of the cell lines
expressing the RA and QA receptor mutants, as well as in the QK
and WT lines, these antibodies immunoprecipitated two
proteins migrating at M
130,000 and 92,000, which
corresponded to IR 
- and -subunits, respectively (Fig. 2, lanes B-F, indicated by arrows).
Based on laser densitometry, the intensity of these bands correlated
well with the number of cell surface receptors as measured by insulin
binding. - and -subunits were barely visible in untransfected
NIH-3T3 cells, which express a very low number of endogenous IRs (Fig. 2, lane A). Based on pulse-chase experiments with
[S]methionine, there was no significant
difference in the rate of IR biosynthesis between any of the
transfected and the endogenous receptors (data not shown).
Figure 2:
Immunoprecipitation of
[S]methionine-labeled IRs. Parental
(untransfected) NIH-3T3 cells (designated NIH in the figure)
and cells transfected with either the WT or the mutant receptors were
labeled for 18 h with [
S]methionine as described
under ``Materials and Methods.'' Cell extracts were prepared
and then immunoprecipitated with IR mAb3. Immunoprecipitates were
subjected to 7.5% SDS-PAGE under reducing conditions and analyzed by
autoradiography. Arrows indicate positions of the 130-kDa
-subunits and the 92-kDa -subunits. The autoradiograph shown
was exposed at -70 °C for 36 h.
Autophosphorylation and Signaling of the Mutant
Receptors
In vitro autophosphorylation of the
single mutant receptors (QK and RA receptors) was compared with that of
the WT and the double mutant receptor (QA receptor) using equal amounts
of partially purified receptors from QK, RA,
WT, and QA cells. Consistent with our previous
findings, receptor autophosphorylation in response to 1 µM insulin was decreased 4-fold in the QK single mutant compared with
the WT receptors (Fig. 3, lanes A-D).
Insulin-stimulated autophosphorylation of the other single mutant (the
RA receptor) was similarly reduced (Fig. 3, lanes E and F). In contrast, quantitation of the autoradiograph indicated
that the level of autophosphorylation of the double mutant receptor was
similar to that of the WT (Fig. 3, lanes G and H). The same effects were also observed at submaximal (1
nM) insulin concentrations, indicating that this
phosphorylation pattern was produced by stimulation of transfected IRs
rather than by endogenous insulin-like growth factor I receptors.
Identical results were obtained by comparing single and double mutant
receptors from the other transfected clones (RA,
QA; data not shown). This indicated that substitution of
only one of the two basic amino acids in the RK motif with a noncharged
residue similarly impaired autophosphorylation. In contrast, the double
substitution did not.
Figure 3:
Autophosphorylation of partially purified
IRs. WGA-purified IRs (20 fmol of insulin binding activity) from the
indicated cell lines were assayed for autophosphorylation with
[-
P]ATP in the presence of the indicated
concentrations of insulin as described under ``Materials and
Methods.'' Autophosphorylation reactions were performed for 20 min
at 22 °C and receptors immunoprecipitated with agarose-coupled
phosphotyrosine antibody in the presence of kinase inhibitors.
Precipitated proteins were analyzed by 7.5% SDS-PAGE and
autoradiography. Quantitation was performed by Cerenkov counting of the
bands. To ensure that equal amounts of receptors were added in each
assay, one half of each incubation mixture was analyzed by
immunoblotting by IR mAb3s and visualized by
I-protein A.
The autoradiograph shown in this figure was exposed for 4 h at
-70 °C.
To examine whether reduced autophosphorylation
in the single mutants reflects loss of labeling of the major regulatory
autophosphorylation sites (Tyr, Tyr
,
Tyr
), two-dimensional analysis of tryptic
phosphopeptides from the in vitro phosphorylated receptor
-subunits was performed. In the case of the WT receptors, this
analysis resolved at least nine major P-labeled
phosphopeptides whose pattern was well consistent with that reported
previously by Tavaré and Denton (30). The phosphopeptides were
therefore identified accordingly. As shown in Fig. 4, labeling of
peptides A
and A (both of which contain the
trisphosphorylated Tyr, Tyr
, and
Tyr
) was decreased by >80% in the QK single mutant (top panel) compared with the WT receptor (middle
panel). In the QK single mutant receptor, phosphorylation was also
decreased by 80 and 20% in the B
and B peptides
(which contain different proportions of phosphorylated Tyr and Tyr
), and by 10% in the C
peptide
(containing the phosphorylated Tyr). Labeling of these
phosphopeptides was completely abolished in a mutant IR where
Tyr
, Tyr
, and Tyr
were
replaced by Ala (data not shown). In the QK single mutant, >90%
decreased labeling was also observed in the B
peptide
(which contains carboxyl-terminal Tyr,
Tyr
), while labeling of the C
was reduced by 22% compared with the WT (this latter peptide
contains mostly the phosphorylated iuxtamembrane Tyr
).
Tryptic phosphopeptides from the RA single mutant and the double mutant
receptors exhibited phosphorylation levels identical to those of the
corresponding peptides in the QK single mutant and the WT receptors,
respectively (data not shown). It appeared therefore that reduced
autophosphorylation in the single mutant receptors was mainly due to
impaired labeling of the carboxyl terminus and the regulatory
tyrosines. Within the latter tyrosine cluster, impaired
autophosphorylation mainly involved Tyr
and
Tyr
, which immediately precede the mutation site in
these receptors.
Figure 4:
Separation of IR -subunit tryptic
phosphopeptides by two-dimensional thin layer analysis. Partially
purified IRs (80 fmol of each) were phosphorylated in vitro in
the presence of 10M insulin as described
under ``Materials and Methods.'' The reaction was terminated
and
P-labeled
-subunits isolated by gel
electrophoresis and digested with TPCK-treated trypsin. P-Labeled tryptic phosphopeptides were then separated on
TLC plates by electrophoresis at pH 3.5 and ascending chromatography,
as described under ``Materials and Methods.'' The
quantification of
P labeling of each phosphopeptide was
obtained by radioactivity counting of the scraped material from the
corresponding spot. The figure shows the autoradiographs of the
two-dimensional separations of
P-labeled peptides from the
WT (middle panel) and the QK (top panel) receptors,
respectively. The exposure time was 10 days at -70 °C. The bottom panel is a key to the identification of the labeled
phosphopeptides relative to the mobility of an internal marker dye
(DNP-lysine) and the origin of sample application (identified by the arrow in the upper and middle
panels.
Tyrosine kinase activity of the mutant receptors
was measured in vitro using the synthetic peptide
poly(Glu-Tyr) as substrate. As shown in Fig. 5, insulin increased
poly(Glu-Tyr) phosphorylation by the WT hIR by almost 3-fold. At
variance, there was no measurable insulin stimulation using receptor
preparations from any of the cells expressing the single mutant
receptors, neither the RA, the RA, or the
QK clones (the last is also shown in Fig. 5for
comparison). In these receptors basal levels of kinase activity were
approximately as high as the maximally insulin-stimulated activity of
the WT hIR. Kinase activity of receptor preparations from the
unstimulated clones expressing the double mutant receptor
(QA, QA) also appeared constitutively
activated. Different from the single mutant receptors, however, the QA
double mutant receptor responded to insulin stimulation with a further
increase in kinase activity, comparable to that measured in the WT
(when expressed as percent increase over the basal). Same results were
obtained using the histone 2B rather than poly(Glu-Tyr) as substrate
(data not shown).
Figure 5:
Phosphorylation of poly(Glu-Tyr) 4:1 by
transfected receptors. IRs were purified from the cell clones
expressing the WT, the single mutant (QK, RA,
RA), and the double mutant (QA1, QA) receptors
as described under ``Materials and Methods.'' Receptor
aliquots were normalized for insulin binding activity and substrate
phosphorylation initiated by the addition of poly(Glu-Tyr) 4:1 (10
mg/ml) in the presence or the absence of 1 µM insulin, as
indicated. Upon 20 min at 22 °C, reactions were quenched on Whatman
3MM paper, and the trichloroacetic acid-precipitable radioactivity was
determined by liquid scintillation counting. Bars represent
the means ± S.D. of four triplicate
experiments.
Insulin stimulation of 2-DG uptake was examined by
treating the cell lines with 1 µM insulin for 30 min and
then evaluating 2-[C]DG uptake over a 10-min
period(20) . An 80% increase in 2-DG uptake was observed in
cells expressing WT receptors upon insulin stimulation (Fig. 6;
WT). In all of the mutant cells expressing the single
mutant receptors (Fig. 6; QK, RA,
RA) as well as the double mutant receptor (Fig. 6;
QA, QA), basal uptake was increased to a level
similar to that induced by the insulin-stimulated WT receptor. Upon
stimulation with insulin, no further increase was observed in cells
expressing single mutant receptors, whereas a 50% increase in 2-DG
uptake was measured in those expressing the double mutant. Similar to
receptor kinase activity, this increment was comparable to that
observed in cells expressing the WT receptors. Thus, substitution of
either one or both residues in the RK motif with uncharged amino acids
induced constitutive activation of the IR kinase activity and higher
basal glucose uptake. However, while substitution of a single residue
(either Arg or Lys
) resulted in the loss
of insulin signaling, substitution of both amino acids did not.
Figure 6:
Insulin stimulation of 2-DG uptake. Cells
expressing the WT, the single (QK, RA,
RA), or the double (QA, QA) mutant
receptors were incubated with transport buffer containing 1 µM insulin for 60 min as indicated under ``Materials and
Methods.'' 2-DG uptake was then initiated by adding
2-[C]DG at a final concentration of 150
µM in the presence or the absence of 50 µM cytochalasin B. The cells were then washed rapidly with cold
phosphate-buffered saline and lysed with 1 N NaOH. 2-DG uptake
was determined by liquid scintillation counting. Each point is the mean
± S.D. of duplicate determinations in four
experiments.
Phosphorylation of Mutant Receptors by
PKC/PKA
Phosphorylation of the IR by PKC and PKA decreased
receptor signaling activity both in vitro and in
vivo. We then tested whether the RK mutant receptors were
phosphorylated differently by PKC and PKA. To this end, WT and mutant
receptors were incubated in the presence of
[-
P]ATP with PKC-enriched fractions from
NIH-3T3 cells in the absence or the presence of PKC activators.
Receptors were immunoprecipitated with mAb3 and analyzed by SDS-PAGE.
As shown in Fig. 7(lanes B, F, and J), incubation of the WT receptor (from WT
cells)
with the activated PKC fraction led to immunoprecipitation of a P-labeled 95-kDa doublet representing the phosphorylated
IR
-subunit. This indicated phosphorylation of the IR by activated
PKC. Based on the amount of insulin binding and P
incorporation in the receptor bands the estimated stoichiometry of this
phosphorylation was 0.8-1.2 phosphates/receptor. When the
receptor was incubated with PKC preparations in the absence of
activators, no receptor phosphorylation was observed (Fig. 7, lanes A, E, and I). When the QK and RA
single mutant receptors were used in the assay, receptor
phosphorylation by the activated PKC was decreased by 2.4- and
1.7-fold, respectively (Fig. 7, lanes C, D, G, and H). Phosphorylation was almost completely
abolished when the QA double mutant receptor was analyzed (Fig. 7, lanes K and L). As shown by Western
blotting with IR antibodies (top inset in Fig. 7),
identical amounts of WT and mutant receptors were used in each
individual experiment. Identical results were obtained by using highly
purified commercial preparations of PKC rather than crude membrane
fractions and by immunoprecipitation with B2 or B9 polyclonal receptor
antibodies rather than mAb3 (data not shown).
Figure 7:
Phosphorylation of mutant receptors by
PKC-enriched cell fractions. NIH-3T3 cells were stimulated with 1
µg/ml TPA for 45 min as indicated and crude plasma membrane
preparations obtained as described under ``Materials and
Methods.'' 50 µg of membrane proteins was then incubated with
WGA-purified IRs (20 fmol of insulin binding activity) from cells
expressing either the WT or the mutant receptor.
[-
P]ATP was then added to the mixture in
the absence or the presence of 1 mM CaCl
, 11.4
µM phosphatidylserine, 0.7 µM diolein
(activators) as indicated, and the incubation was prolonged for 20 min
at 22 °C. Phosphorylation reactions were interrupted by the
addition of kinase inhibitors and by cooling the mixtures at 4 °C
as described under ``Materials and Methods.'' IRs were then
precipitated with mAb3 and identified by SDS-PAGE and autoradiography.
Quantitation of P incorporation was achieved by Cerenkov
counting of the receptor bands. Three independent experiments comparing
the WT receptor (lanes A, B, E, F, I, and J) with the QK (lanes C and D) or the RA (lanes G and H) single mutant
or the QA double mutant receptors (lanes K and L) are
shown. To ensure that identical amounts of IRs were added in each of
the experiments, one half of each incubation mixture was immunoblotted
with IR mAb3 and receptor visualized by
I-protein A (top inset).
In vitro phosphorylation experiments have also been performed using the
purified catalytic subunit of PKA. As shown in Fig. 8, the
addition of active PKA to WT receptors resulted in 5-fold increased
phosphorylation of the immunoprecipitated receptor -subunit (Fig. 8, left panel, lanes A and B).
With 15 µg/ml PKA, 1 molecule of phosphate was incorporated per
receptor. No phosphorylation occurred with the QK single mutant
receptor (Fig. 8, left panel, lanes C and D). Similar to the QK receptor, PKA phosphorylation was also
deficient with the other single (RA) and double (QA) mutant receptors (Fig. 8, right panel, lanes A-H). Thus,
in the IR, substitution of either Arg or Lys
with a noncharged amino acid, alone or in combination, resulted
in grossly impaired in vitro receptor phosphorylation by both
PKC and PKA.
Figure 8:
Phosphorylation of mutant receptors by
PKA. Purified PKA (1.6 units/assay) was added as indicated to
WGA-purified IRs (20 fmol of insulin binding activity) from cells
expressing either the WT or the mutant receptors. Receptor
phosphorylation was initiated by the addition of cAMP and
[-
P]ATP as reported under ``Materials
and Methods'' and prolonged for 20 min at 22 °C. The reaction
was interrupted by the addition of kinase inhibitors and cooling the
mixture at 4 °C. IRs were then precipitated with IR mAb3 and
identified by SDS-PAGE and autoradiography. Quantitation of
P incorporation was achieved by Cerenkov counting of the
receptor bands. Two independent experiments comparing the WT receptors (lanes A and B) with the QK (lanes C and D) or the RA (lanes E and F) single mutants
or the QA double mutant receptors (lanes G and H) are
shown. The autoradiographs shown were exposed for 14 h at -70
°C.
To examine the action of PKA in vivo, we
treated P-labeled cells expressing the mutant receptors
with 8-bromo-cAMP. We then analyzed receptor phosphorylation upon
immunoprecipitation with mAb3 receptor antibodies. The autoradiograph
in Fig. 9shows that treatment of WT
cells with 10
µM 8-bromo-cAMP for 45 min resulted in a 3-fold increase
in labeling of the 95-kDa WT receptor -subunit (Fig. 9, lanes A and B). There was no such phosphorylation
with any of the mutant cell lines expressing the QK, RA, or QA
receptors (Fig. 9, lanes C-H). Very similar
results (i.e. lack of receptor phosphorylation in
metabolically labeled mutant cells upon kinase activation) were
obtained by analyzing receptor phosphorylation upon incubating the
cells with 1 µg/ml TPA to activate PKC (data not shown). Since the
mutant cells used in these experiments express comparable receptor
numbers, these data indicated that substitution of either one or both
the basic residues of the RK motif with an uncharged amino acid
impaired IR phosphorylation by PKC and PKA.
Figure 9:
PKA
phosphorylation of mutant receptors in intact cells. Cell clones
expressing WT (WT), single mutant (QK,
RA), or double mutant (QA) receptors were
labeled with [P]orthophosphate and incubated
with 10 µM 8-bromo-cAMP for 45 min, as indicated.
Phosphorylation reactions were interrupted by liquid N
in
the presence of phosphatase and kinase inhibitors and cell lysates
immunoprecipitated with IR mAb3. Precipitated receptors were identified
by SDS-PAGE and autoradiography. Quantitation of P
incorporation was achieved by Cerenkov counting of the receptor bands.
The autoradiograph shown is from one representative experiment and was
obtained by exposing the dried gel for 24 h at -70
°C.
To address further the
role of the RK motif in IR phosphorylation by PKC and PKA, two peptides
corresponding to the hIR sequence surrounding the RK motif were
synthesized. One of these included the intact RK motif (WT peptide),
while, in the other, the RK Arg was replaced by a Gln to mimic the QK
mutant receptor sequence (QK peptide). In vitro phosphorylation of the WT receptor by purified PKC or PKA was
tested in the presence of excess (3 µM) WT or mutant (QK)
peptides. Quantitation of the bands indicated that phosphorylation of
IR -subunit by purified PKC was decreased 2-fold in the presence
of the WT peptide (Fig. 10, lanes A-C). In
contrast, the QK peptide was unable to perform significant inhibition (Fig. 10, lane D). Identical amounts of receptor were
analyzed in each assay, as determined by Western blotting aliquots of
each phosphorylation mixture with receptor antibodies (data not shown
in the figure). Similar results were obtained using the catalytic
subunit of PKA rather than PKC to phosphorylate IR (Fig. 10, lanes E-H). Although the WT peptide, at 3
µM, inhibited receptor phosphorylation by the two kinases
to a similar extent, at 0.5 µM, there was 70% inhibition
of PKC phosphorylation with no inhibition of PKA (data not shown).
Therefore, a receptor peptide encoding the intact RK motif inhibited IR
phosphorylation by both PKC and PKA and exhibited greater activity in
the former than in the latter case. Substitution of the basic Arg with
a noncharged amino acid in the RK motif resulted in a complete loss of
the peptide inhibitory activity.
Figure 10:
Effect of peptides on IR phosphorylation
by PKA and PKC. In vitro phosphorylation of WGA-purified IRs
was performed using purified preparations of PKC (left panel)
or PKA (right panel) as described under ``Materials and
Methods.'' Phosphorylations were conducted in the absence or the
presence of 3 µM WT or mutant (QK) peptides, as indicated.
Upon interrupting the reaction, IRs were immunoprecipitated with mAb3,
isolated by 7.5% SDS-PAGE, and identified by autoradiography. The
autoradiographs shown in the figure were obtained by exposing the gels
for 36 h at -70 °C.
by a Gln in the RK motif, results in a
constitutively activated receptor kinase(19, 20) . In
cells overexpressing this single mutant hIR, as well as in patient
fibroblasts, the activated receptor is unable to transduce
insulin-dependent metabolic effects but exhibits constitutive kinase
activity and constitutively induces metabolic effects. In addition, the
mutant receptor shows a constitutive increase in internalization and
membrane recycling(21) .
) was replaced by a noncharged amino
acid and a double mutant receptor in which both the Arg and the Lys
were replaced. None of these mutant receptors exhibited alterations in
the affinity for insulin, the rate of receptor synthesis or transport
to the cell surface. Similar to the Arg substitution, we now find that
replacement of Lys in the RK motif constitutively activates hIR kinase
toward substrates. The same effect was also observed with the double
mutant receptor. Consistently, in cells expressing these mutant
receptors, basal (insulin-independent) glucose uptake was increased to
levels comparable to those measured in cells expressing WT hIR upon
maximal insulin stimulation. Thus, both the Arg and the Lys residue in
the RK motif play a role in restraining the basal kinase activity of
the hIR.
P-labeled cells with
the PKC-activating agent TPA increases phosphorylation of transfected
WT receptors by 3-fold. Similar results were also obtained by
activating PKA with 8-bromo-cAMP. In contrast, we detected grossly
impaired receptor phosphorylation upon activation of PKC or PKA in
cells expressing either the two single or the double mutant receptors.
Similar results were obtained by analyzing in vitro phosphorylation of the mutant receptors by purified PKC and PKA.
Based on these data, we suggest that the intact RK motif of the hIR is
critical for allowing insulin-independent receptor phosphorylation by
PKA and PKC. The substitution of either one or both of the basic
residues in the RK motif with noncharged amino acid may remove the
kinase constrain exerted by PKA and PKC phosphorylation of the
receptor. This, in turn, might account for the increased
insulin-independent activity of both the single and the double mutant
receptors. Consistent with this possibility and with previous
findings(8) , treatment of WT hIR with alkaline phosphatase led
to a similar sized increase in the non-insulin-dependent tyrosine
kinase activity of the receptor (data not shown).
- and phospholipid-dependent (data not
shown). (ii) As shown in the present work, a synthetic peptide encoding
the hIR sequence surrounding the WT RK motif inhibits receptor
phosphorylation by PKC and PKA rather than activating these kinases and
increasing receptor phosphorylation. In vitro, the inhibitory
peptide is not phosphorylated by either PKC or PKA. As was the case for
WT receptor phosphorylation by these kinases, the inhibitory peptide
also reduced phosphorylation of histone III-S by PKC and by PKA and
exhibited a greater potency with the former than the latter kinase
(data not shown). Although the different potency could imply distinct
inhibitory mechanisms in the case of each of the two kinases, these
data suggest that the peptide inhibition of receptor phosphorylation
may be due to peptide-receptor competition for PKC and PKA followed by
receptor displacement. One might argue, therefore, that the intact RK
motif in the WT hIR may bind these kinases allowing receptor
phosphorylation and inhibition of receptor tyrosine kinase activity.
Consistent with this, and shown in the present paper, a second peptide,
identical to the former except for an Arg
Gln substitution in
the RK motif (as in the naturally occurring single mutant receptor),
exhibits no inhibitory activity on receptor phosphorylation.
, Tyr
, and Tyr
) in response to insulin occurs normally in the double
mutant, consistent with insulin stabilization of the activation loop in
the receptor in a noninhibitory conformation and with preserved insulin
signaling(35, 36) . In contrast to the double mutant, we
show that autophosphorylation of the key tyrosines (particularly of
Tyr
and Tyr
) is reduced in the two
single mutants. Therefore, based on the x-ray crystal structure of the
kinase(35) , one would anticipate persistence of the inhibitory
tone of the activation loop in these receptors and lack of full
activation of their kinase in response to insulin. The present model is
also consistent with the greater absolute kinase activity observed in
the insulin-stimulated double mutant receptor compared with both the
single mutants and the WT. In fact, activity in the double mutant would
result from both the effect of insulin stimulation (lacking in the
single mutants) and the absence of PKA or PKC inhibition (lacking in
the WT receptor).
-phospholipid-dependent protein kinase
(protein kinase C); mAb, monoclonal antibody; WT, wild-type; hIR, human
insulin receptor; PAGE, polyacrylamide gel electrophoresis; WGA, wheat
germ agglutinin; TPCK, 1-tosylamido-2-phenylethyl chloromethyl ketone;
2-DG, 2-deoxyglucose; TPA,
12-O-tetradecanoylphorbol-13-acetate.
in our
previous publication (20).
and QK in the present paper, were termed WT and M in our previous publication (20).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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