1 Polypeptide Hormone Laboratory and 2 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2
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ABSTRACT |
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Phosphatidylinositol 3-kinase
(PI 3-kinase) plays an important role in a variety of hormone and
growth factor-mediated intracellular signaling cascades and has been
implicated in the regulation of a number of metabolic effects of
insulin, including glucose transport and glycogen synthase activation.
In the present study we have examined 1) the association of
PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and
2) the subcellular distribution of PI 3-kinase-IRK
interaction. Insulin treatment promoted a rapid and pronounced
recruitment of PI 3-kinase to IRKs located at the plasma membrane,
whereas no increase in association with endosomal IRKs was observed. In
contrast to IRS-1-associated PI 3-kinase activity, association of PI
3-kinase with the plasma membrane IRK did not augment the specific
activity of the lipid kinase. With use of the selective PI 3-kinase
inhibitor wortmannin, our data suggest that the cell surface IRK
-subunit is not a substrate for the serine kinase activity of PI
3-kinase. The functional significance for the insulin-stimulated
selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.
insulin signaling; subcellular compartments
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INTRODUCTION |
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THE INSULIN RECEPTOR
KINASE (IRK) is a disulfide-linked heterotetrameric glycoprotein
composed of two extracellular insulin-binding -subunits and two
transmembrane
-subunits that contain tyrosine kinase activity in
their cytosolic domains (see review in Ref. 16). Upon insulin binding,
rapid tyrosine autophosphorylation on the intracellular portion of the
-subunit leads to IRK activation. After activation, the
insulin-receptor complex is rapidly internalized into endosomes (ENs),
which mediate the sorting and processing of a number of ligand-receptor
complexes and have been implicated in both signal transduction and
signal termination (reviewed in Refs. 2 and 8).
The activated IRK effects tyrosine phosphorylation of a number of intracellular proteins, including insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and, in certain cell types, SHC (16). These molecules, rather than the IRK itself, appear to be the predominant proteins coupling to and activating downstream signaling pathways by providing specific phosphotyrosine binding sites for a number of src homology 2 (SH2)-containing proteins, including phosphatidylinositol 3-kinase (PI 3-kinase).
PI 3-kinase is involved in mediating numerous effects of insulin,
including glucose transport, glycogen synthase activation, and the
inhibition of lipolysis (1). The best-characterized member
of the PI 3-kinase family exists as a heterodimer composed of a
regulatory 85-kDa subunit (p85), containing two SH2 domains, and a
110-kDa catalytic subunit (p110) that possesses both lipid and serine
kinase activity. After binding of the p85 SH2 domains to specific
tyrosine-phosphorylated motifs (pYXXM) on molecules such as IRS-1 and
IRS-2, the p110 catalytic subunit becomes activated (see review in Ref.
43). However, in addition to these adapter molecules, mounting evidence
indicates that PI 3-kinase can also associate with the activated IRK
-subunit (4, 21, 29, 35, 44, 47). In vitro studies
indicate that the SH2 domains of p85 can interact directly with
phospho-Tyr1322 (which lies in a Y1322THM
motif) of the IRK
-subunit,1 although other
investigations suggest that additional regions of the
-subunit may
also be required (3, 46, 47). PI
3-kinase activity is detected in anti-IRK immunoprecipitates from
insulin-stimulated cells (4, 35). However,
the physiological significance of this potential alternative pathway of
insulin-stimulated PI 3-kinase activation, or indeed the subcellular
location of PI 3-kinase-IRK interaction, remains to be established.
In the present study we examined the association of PI 3-kinase with
the IRK in rat liver, a major insulin target tissue, in vivo. Because
internalization of activated IRKs into ENs plays an important role in
insulin signal transduction (reviewed in Refs. 8 and 18), PI 3-kinase
interaction with the IRK was investigated in different subcellular
compartments of the liver. Our studies reveal that insulin treatment
promotes a rapid and selective recruitment of PI 3-kinase to IRKs
located at the plasma membrane. However, in contrast to cytosolic
IRS-1-associated PI 3-kinase activity, this association does not
augment the specific activity of the lipid kinase. Furthermore, using
the selective PI 3-kinase inhibitor wortmannin, we show that the cell
surface IRK -subunit is not a substrate for the serine kinase
activity of PI 3-kinase.
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MATERIALS AND METHODS |
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Animals. Female Sprague-Dawley rats, 10 wk of age and 160-180 g body weight, were purchased from Charles River Canada (St. Constant, PQ, Canada), housed in an animal facility with 12:12-h light-dark cycles at 25°C, and fed ad libitum on Purina chow. Animals were fasted overnight (16-18 h) before use.
All studies herein cited were performed with the approval of the McGill University Animal Care Committee.Materials.
Porcine insulin was a gift from Eli Lilly (Indianapolis, IN).
Phenylmethanesulfonyl fluoride, HEPES (free acid), sodium
orthovanadate, rabbit -globulin, Tris, radioimmunoassay grade BSA,
and most other chemicals were purchased from Sigma (St. Louis, MO).
Protein A-Sepharose CL-4B (PAS) was from Pharmacia LKB Biotechnology
(Uppsala, Sweden). ATP (disodium salt) was obtained from Boehringer
Mannheim (Laval, PQ, Canada). [
-32P]ATP (3,000 Ci/mmol) and 125I-labeled goat anti-mouse (GAM) and
125I-labeled goat anti-rabbit (GAR) secondary antibodies
were purchased from Du Pont-NEN Radiochemicals (Lachine, PQ, Canada).
Reagents for electrophoresis were from Bio-Rad (Richmond, CA) with the exception of 14C-labeled protein standards, which were
supplied by GIBCO/BRL Canada (Burlington, ON, Canada). Kodak X-OMAT AR
film was from Picker International (Montreal, PQ, Canada).
Polyvinylidene fluoride Immobilon-P transfer membranes were from
Millipore (Mississauga, ON, Canada). The protein tyrosine phosphatase
(PTP) inhibitor bisperoxo(1,10-phenanthroline)oxovanadate anion
[bpV(phen)] was prepared by Dr. Jesse Ng of the Department of
Chemistry, McGill University, as described previously
(32). L-
-Phosphatidylinositol (sodium salt)
was purchased from Avanti Polar Lipids (Alabaster, AL). Merck Silica
Gel 60 and cellulose thin-layer chromatography (TLC) plates were
purchased from EM Separation Technology (Gibbstown, NJ). An antibody
raised against a peptide corresponding to residues 942-969 of the
juxtamembrane region of the IRK
-subunit (
960) was prepared and
purified on a PAS column as previously described (11).
Polyclonal
p85 (recognizing both p85
and p85
) for
immunoprecipitation and Western blotting and a polyclonal
IRS-1 for
Western blotting were purchased from Upstate Biotechnology (Lake
Placid, NY). A monoclonal anti-phosphotyrosine antibody (
PY) and a
polyclonal anti-SHP-2 antibody for Western blotting were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies recognizing the human IRK
-subunit (
IRK
) were purified from a patient's serum as previously described (24). A polyclonal antibody raised
against a peptide corresponding to the fourteen carboxy-terminal
residues of IRS-1 was used for immunoprecipitation.
Preparation of subcellular liver fractions. After ether anesthesia, rats received an intrajugular injection of insulin (1.5 µg/100 g body wt) in PBS containing 0.1% BSA for the times indicated. Hepatic plasma membrane (PM), endosomal (EN), and cytosolic fractions were prepared as described previously (7). The protein content of these fractions was measured using a modification of Bradford's method (10), with BSA as standard (24).
Immunoprecipitation and immunoblotting.
Cell fractions (300 µg protein), in a final volume of 550 µl, were
incubated in the presence of 1% (vol/vol) Triton X-100 and 0.5%
(wt/vol) sodium deoxycholate at 4°C for 1 h. After
centrifugation at 10,000 gAV for 2 min,
the supernatant was precleared for 30 min with 1.0 µg of nonimmune
IgG. Twenty microliters of a 50% slurry of PAS were added, and after
30 min of incubation, the solution was centrifuged as above.
Supernatants were incubated for 2 h with 20 µl of IRS-1,
p85, or
IRK at 4°C, after which 50 µl of a 50% slurry of PAS
were added, and the solution was incubated for a further 1 h.
After centrifugation as above, 100 µl of the supernatant were added
to 50 µl of 3× Laemmli sample buffer (2.3% SDS, 10% glycerol, 100 mM dithiothreitol, and 0.37 M Tris · HCl, pH 6.8: final
concentration, 1×). The pellet was washed three times with 1 ml of
wash buffer (50 mM HEPES, pH 7.4, containing 1% Triton X-100, 0.1%
SDS, 150 mM NaCl, 100 mM NaF, and 2 mM sodium orthovanadate) followed
by boiling in 210 µl of Laemmli sample buffer. Eighty-microliter
samples were subjected to SDS-PAGE (7.5% gel) and then transferred to
Immobilon-P membranes for Western blotting. Either 125I-GAR
or 125I-GAM was used as secondary antibody and, after
autoradiography at
80°C, appropriate bands were quantified using a
Bio-Rad GS-700 Imaging Densitometer.
IRS-1 and IR-associated PI 3-kinase activity.
Immunoprecipitates (either IRK
or
IRS-1) were extensively
washed (20), and the PAS pellet was resuspended in 50 µl
of kinase assay buffer (in mM: 20 Tris · HCl, pH 7.5, 100 NaCl,
and 0.5 EGTA) containing 0.5 mg/ml PI and assayed for PI 3-kinase activity, as previously described (6).
Phosphoamino acid analysis of the IRK and p85: effect of
wortmannin.
IRK or p85 immunoprecipitates were washed as described above and
resuspended in 20 µl of assay buffer (in mM: 20 HEPES, pH 7.4, 3 MnCl2, and 10 MgCl2). Samples were preincubated
for 20 min at 22°C with 500 nM wortmannin in DMSO, or with DMSO
alone, and kinase reactions were started by the addition of 20 µCi of [-32P]ATP (5 µM final concentration) as described in
Ref. 27. After 10 min of incubation at 22°C, the reaction was stopped
by the addition of 500 µl of ice-cold PBS. Samples were centrifuged
at 10,000 gAV, and pellets were washed twice
with 500 µl PBS, resuspended in 90 µl of Laemmli sample buffer, and
boiled for 5 min. Samples were subjected to SDS-PAGE (7.5%) and
transferred onto Immobilon-P membrane, and phosphobands were visualized
by autoradiography at
80°C.
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RESULTS |
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Initial studies evaluated whether insulin treatment promoted a
change in the level of the PI 3-kinase regulatory subunit p85 associating with rat hepatic PM and EM fractions. Figure
1 shows the distribution of p85 induced
by insulin treatment of overnight-fasted rats as determined by
subcellular fractionation and immunoblotting with anti-p85. Insulin
treatment led to an ~50% increase in the level of p85 protein
present in both PM and EN fractions, which reached a peak at 1 min
postinjection in both subcellular fractions. Whereas PM-associated p85
content declined rapidly to basal levels, EM p85 content declined more
gradually and remained elevated above basal at 15 min postinjection. At
the peak time of insulin-stimulated p85 recruitment to membrane
fractions, p85 content in the PM (expressed per µg PM protein) was
approximately threefold greater than that observed in ENs
(inset, Fig. 1).
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Effect of insulin treatment on p85 subunit association with PM and
EN IRKs.
We sought to determine the impact of insulin stimulation on the
association of p85 with IRKs in vivo. In agreement with previous studies (11), insulin treatment promoted a time-dependent
decrease in IRKs located at the PM, whereas an approximate 10-fold
increase in EM IRK levels was observed that was maximal at 2-5 min
postinjection (Fig. 2, top).
Insulin treatment promoted an approximate fivefold increase in p85
associating with the PM IRK that was maximal between 30 s and 2 min postinjection (P < 0.01).2 In contrast, a 50%
decrease in p85 associating with the EN IRK was observed between 1 and
5 min after insulin treatment (2 min postinsulin injection;
P < 0.05), with the association returning to basal
levels by 15 min postinjection. It is noteworthy that in the basal
non-insulin-stimulated state (t = 0), association of p85
with EN IRKs was fourfold less than that observed for PM receptors
(Fig. 2, top). IRS-1 was detected in anti-IRK
immunoprecipitates from PM fractions (Fig.
3A), but not IRS-2 (results
not shown). This raised the possibility that the observed association
between IRKs and p85 in the PM could be the result of a ternary complex containing IRS-1. To test for this, the kinetics of insulin-stimulated IRS-1 and p85 association with PM IRKs were compared. IRS-1 association with PM IRKs reached a peak at 30 s postinsulin injection, after which levels rapidly declined to basal by 10 min postinjection (Fig.
3A). In contrast, although levels of p85 associating with PM
IRKs also peaked at 30 s after insulin administration, levels remained elevated and substantially above basal at 10 min postinjection (Fig. 3A). Also noteworthy is the finding that, after
insulin treatment, the p85-to-IRS-1 ratio in IRK immunoprecipitates
from PM increased by fivefold between 30 s and 2 min postinsulin
injection and declined thereafter (Fig. 3, A and
C). In contrast, the ratio of cytosolic p85 to IRS-1
increased only twofold after insulin administration and remained
elevated at 10 min postinjection (Fig. 3, B and
C). These data are consistent with the possibility that a
population of p85 associates with the PM IRK in an IRS-1-independent manner. Because the PTP, SHP-2 (PTP-1D, Syp), has been
reported to associate with the tyrosine-phosphorylated -subunit of
the IRK via an SH2-mediated interaction (26,
33, 38, 41), the presence of
this enzyme in IRK immunoprecipitates was also assessed. Immunoblotting
with anti-SHP-2 antibodies failed to detect SHP-2 associating with rat
hepatic PM and EN IRKs (results not shown).
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Effect of bpV(phen) pretreatment on insulin-stimulated p85
association with PM and EN IRKs.
Through an SH2-mediated interaction, the p85 subunit is reported to
associate directly with the IRK via its -subunit
phospho-Tyr1322 (29, 44).
Previous studies identified that IRK internalization in liver was
associated with a rapid (within 2 min postinsulin injection) and
partial dephosphorylation of
-subunit phosphotyrosine residues due
to the action of an endosomally located protein tyrosine phosphatase(s)
(PTP) (11, 19). The comparatively low level of p85 associating with EN IRKs (cf., PM IRKs) may therefore be the
result of IRK dephosphorylation at this intracellular locus. To test
for this possibility, EN IRK dephosphorylation was specifically blocked
by pretreating rats with the PTP inhibitor bpV(phen) (32), as described by Drake et al. (19). Although we cannot
directly demonstrate the phosphorylation status of the carboxy-terminal tyrosine residue (Tyr1332) in the present study, previous
work (19) has demonstrated that at 15 min after a single
injection of bpV(phen), EN IRK
-subunit dephosphorylation is
inhibited by ~100%. As shown in Fig.
4, a 15-min bpV(phen) pretreatment had
negligible effect on the time course or magnitude of insulin-stimulated
association of p85 with either EN or PM IRKs.
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Effect of insulin treatment on PI 3-kinase lipid kinase activity
associated with PM and EN IRKs.
We determined whether the lipid kinase activity of the PI 3-kinase
associating with PM and EN IRKs was modulated by insulin treatment in
vivo. Insulin treatment led to a rapid and approximate sixfold increase
in lipid kinase activity associated with the PM IRK that reached a peak
at 30 s postinjection and declined thereafter (Fig.
5). In contrast, at 2 min after insulin
treatment, an ~50% decrease in PI 3-kinase activity associated with
EN IRKs was observed. Because PM IRK-associated lipid kinase activity closely mirrored insulin-stimulated p85 recruitment to IRKs in this
cell fraction, we sought to establish whether the intrinsic (or
specific) activity of PI 3-kinase was modified by p85-IRK association.
Figure 6 shows the intrinsic activity of
PI 3-kinase associated with PM IRKs at 30 s postinjection, the
time of maximal recruitment of p85 to the PM IRK. For comparison, the
specific activity of PI 3-kinase associated with rat liver cytosolic
IRS-1 was determined. Although insulin treatment promoted a fivefold increase in the amount of p85 subunit associating with PM IRKs at
30 s postinjection (Fig. 2), this elevated association did not
increase the specific activity of the enzyme (Fig. 6). In contrast,
insulin treatment led to an approximate fivefold increase in the
intrinsic activity of PI 3-kinase associated with cytosolic IRS-1 (Fig.
6). It has been reported that insulin treatment stimulates only the
activity of p85-associated PI 3-kinase (5). The
difference in insulin-stimulatable PI 3-kinase activity associated with
PM IRKs and cytosolic IRS-1 in rat liver may therefore lie with the nature and/or level of the p85 isoform that is associated with these
two molecules. To test for this, we examined whether the levels of
insulin-stimulated p85
association with the PM IRK and with
cytosolic IRS-1 were similar. Table 1
shows levels of p85
and total p85 (both p85
and p85
; PAN-p85)
in PM IRK and cytosolic IRS-1 immunoprecipitates after insulin
treatment. The ratios of p85
to PAN-p85 associated with either PM
IRK (0.15 ± 0.06) or cytosolic IRS-1 (0.20 ± 0.05) were
very similar (Table 1). This suggests that the lack of
insulin-stimulated PI 3-kinase activity in PM IRK immunoprecipitates,
in contrast to that associated with cytosolic IRS-1, cannot be
explained by an absence, or indeed a reduced proportion, of p85
associating with the IRK.
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Effect of wortmannin on IRK -subunit phosphotyrosine, -serine,
and -threonine content.
Because the intrinsic lipid kinase activity of PI 3-kinase did not
increase after binding to rat hepatic IRKs, we examined whether
recruitment to PM-located IRKs might serve as a mechanism by which the
serine kinase moiety of PI 3-kinase could phosphorylate the IRK
-subunit. This was assessed using a procedure described by Lam et
al. (27) and Tanti et al. (40). Briefly, IRKs
immunoprecipitated from rat liver PM fractions at 30 s after
insulin treatment were preincubated with
wortmannin,3 a selective
inhibitor of both the lipid and serine kinase activities of PI 3-kinase
(see review in Ref. 42). Immunocomplexes containing both IRK and PI
3-kinase were subsequently incubated with [32P]ATP in the
presence of Mn2+ and Mg2+, and IRK
-subunits
were subjected to phosphoamino acid analysis (Fig.
7), as described in MATERIALS AND
METHODS. As shown in Fig. 7, wortmannin pretreatment was without
effect on the phosphoserine, -threonine, or -tyrosine content of PM
IRKs, either in controls or in animals that were killed at 30 s
postinsulin injection. In contrast, wortmannin pretreatment promoted an
~50% decrease in the phosphoserine content of the p85 regulatory
subunit in anti-p85 immunoprecipitates from PM fractions isolated at
30 s postinsulin injection (results not shown). Thus, despite a
rapid recruitment of PI 3-kinase to rat hepatic PM IRKs at 30 s
postinsulin treatment, the IRK
-subunit does not appear to be a
substrate for the serine kinase activity of PI 3-kinase.
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DISCUSSION |
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To our knowledge, this is the first study that has 1)
assessed the insulin-stimulated recruitment of PI 3-kinase to insulin receptors in a major insulin target tissue (that is, rat liver) in vivo
and 2) examined this interaction in different subcellular compartments. A major finding of the study was the rapid recruitment of
PI 3-kinase to rat hepatic IRKs located at the PM after injection of a
single physiological dose of insulin. Reports on the nature of the
interaction between p85 and the IRK are in conflict with some studies
indicating that this association is direct and mediated via binding of
the SH2 domains of p85 to phospho-Tyr1322 of the IRK
-subunit (29, 44), whereas others suggest
an indirect mechanism involving the formation of a ternary complex with
IRS-1 (3, 22). The reasons for these
discrepant results are unclear but may reflect the use of different
cell lines, where the relative levels of IRK, p85, and IRS-1 may
differ. Although IRS-1 was detected in anti-IRK immunoprecipitates in
the present study, the different time courses of insulin-stimulated
IRS-1 and p85 association with PM IRKs suggest that at least a
proportion of p85-IRK association is not mediated via a ternary complex
containing IRS-1. Whether this p85-IRK association is direct or
mediated via another signaling molecule remains to be established.
Although the current study does not provide an explanation for the
difference in p85 association between PM and EN IRKs, dephosphorylation of key IRK -subunit tyrosine residues necessary for p85 binding, by
an EN-located PTP, appears to be ruled out. Thus pretreatment of rats
with bpV(phen), a specific inhibitor of endosomal IRK tyrosine
dephosphorylation (19, 32), had a negligible
effect on p85-IRK interaction at this intracellular locus (Fig. 4). It is possible, therefore, that p85 interacts with the IRK in a
phosphotyrosine-independent manner, although the fact that the time
course of insulin-stimulated p85 association with the PM IRK
closely mirrors IRK
-subunit phosphotyrosine content
(11) suggests otherwise. Because bpV(phen) is
without effect on IRK dephosphorylation at the PM (19),
dephosphorylation of IRK
-subunit tyrosine residues, through the
action of a PM-located PTP, may have occurred before internalization.
Alternatively, the activated IRK may, via a phosphotyrosine-dependent
mechanism, interact with another molecule in ENs that occludes an
association with p85 in this subcellular compartment. A number of
studies have described the direct interaction of the SH2-containing
PTP, SHP-2, with the IRK
-subunit (26, 38,
41) via an interaction of the SH2 domains of this enzyme
with both the kinase regulatory domain and Tyr1322 of the
IRK
-subunit (33). Our studies, however, did not detect SHP-2 in IRK immunoprecipitates from either PM or EN fractions, thus
excluding the possibility that this PTP may impede p85 interaction with
the IRK in the endosomal compartment.
On the basis of the observation that the association between p85 and the IRK decreases in ENs after insulin treatment (Fig. 2), it would appear that activated IRKs entering the EN apparatus from the cell surface are not complexed with p85, thus "diluting" the fraction of p85-associated IRKs that are present in this subcellular fraction. It is possible, therefore, that a selective internalization of IRKs takes place from the PM, with those receptors binding p85 being retained at the cell surface. This may allow a subset of IRKs to stimulate a PM-specific insulin-signaling cascade. It should be noted, however, that we cannot rule out the possibility that transfer of p85 from internalized p85-IRK complexes to another binding partner could also account for the observed decrease in p85-IRK association in ENs.
PI 3-kinase activity is necessary for ligand-mediated internalization of both the c-Kit receptor (22) and the CD28 receptor (13), and for efficient trafficking of the platelet-derived growth factor (PDGF) receptor from peripheral compartments to juxtanuclear vesicles (37). Such a role for insulin-stimulated PI 3-kinase association with cell surface IRKs is unlikely, because the maximal interaction between p85 and PM IRKs occurs while IRK content in PM is decreasing. Although this suggests that p85-IRK association is not a principal effector of IRK endocytosis, we cannot rule out the possibility that the association of p85 with the IRK is responsible for its selective retention at the cell surface.
A second important finding of our study was that, although PI 3-kinase
was recruited to PM IRKs after insulin treatment, the intrinsic
activity of the lipid kinase did not increase. This is in marked
contrast to cytosolic IRS-1-associated PI 3-kinase activity, where
insulin treatment led to a rapid recruitment and activation of the
enzyme (Figs. 2 and 6). It has been reported that only
p85-associated PI 3-kinase activity is stimulated by insulin
(5). Thus the difference in insulin-stimulatable PI 3-kinase activity in PM IRK and cytosolic IRS-1 immunoprecipitates may
lie with a difference in the type or proportion of p85 isoforms associating with these two molecules. However, the present study revealed that a very similar proportion of p85
associates with PM
IRK and cytosolic IRS-1 (Table 1). Previous studies have recognized that full activation of PI 3-kinase is achieved when both SH2 domains
of the p85 regulatory subunit bind phosphotyrosine (34) in
the context of pYXXM (see review in Ref. 43). The IRK
-subunit contains only a single YXXM motif (at Tyr1322), implying
that only one p85 SH2 domain is occupied upon binding to the IRK. The
PDGF receptor, by contrast, contains two YXXM motifs (at
Tyr740 and Tyr751) and has been shown to
potently stimulate PI 3-kinase activity through receptor binding (see
review in Ref. 9). Because IRS-1 has nine YXXM motifs, the potential to
bind both p85 SH2 domains and hence fully activate PI 3-kinase is high.
It is therefore possible that the inability of the IRK to stimulate PI
3-kinase activity through p85-IRK interaction may result from the
absence of a second YXXM site on the
-subunit. Thus, although it has been proposed that the p85 subunit may be able to span identical sites
in activated receptor homodimers (31), spatial or
structural constraints may prevent this for the IRK.
After IRK activation there is an increase in the phosphorylation of the
IRK -subunit on serine and threonine residues (reviewed in Refs. 16
and 36). Although the functional significance of IRK serine/threonine
phosphorylation is unclear, it is thought to serve to inhibit or dampen
IRK activity and, hence, insulin signaling. To date, however, the
identity of the insulin-stimulated serine/threonine kinase(s) that
phosphorylates the IRK in vivo remains unclear. Previous studies have
noted that IRK
-subunit serine/threonine phosphorylation was
restricted to cell surface receptors (24). Because the
present study showed that p85 was rapidly recruited to PM IRKs after
insulin treatment, our observations raised the possibility that PI
3-kinase may phosphorylate the IRK in vivo. However, we did not observe
a wortmannin-sensitive serine phosphorylation of the IRK
-subunit in
PM immunocomplexes containing both IRK and PI 3-kinase activity.
Moreover, pretreatment of primary cultured hepatocytes with wortmannin
before insulin stimulation was without effect on total cellular IRK
activation (P. G. Drake and V. M. Dumas, unpublished
observations). This is in agreement with a study of Lam et al.
(27), who report that wortmannin has no effect on the in
vitro kinase activity of adipocyte IRKs. The lack of an effect of PI
3-kinase inhibition on IRK activation or on the
-subunit
phosphoamino acid profile of rat liver PM IRKs (Fig. 6) suggests that
the PI 3-kinase serine kinase is not a direct regulator of IRK
function. However, it should be noted that we cannot rule out the
possibility that a potential in vivo phosphorylation of all available
serine residues by PI 3-kinase before animals are killed and before
subcellular fractionation could prevent further phosphorylation in
vitro. Recent studies have observed a PI 3-kinase association with a 76-kDa wortmannin-insensitive serine kinase, termed a PI
3-kinase-associated kinase, that can phosphorylate IRS-1
(14, 15). It is possible, therefore, that
although the PI 3-kinase does not phosphorylate the IRK itself, it may
form part of a complex that brings this novel serine kinase into
contact with the PM IRK or, indeed, other PM-located proteins. Such an
adaptor function of the p85 regulatory subunit has been proposed by
Sung et al. (39), who report that p85 can form a complex
linking the activated IRK to a GTPase-activating protein (GAP) and a
62-kDa GAP-associated protein.
In summary, this study has identified a major difference in the association of PI 3-kinase with IRKs in different subcellular compartments of rat liver. Whereas insulin treatment promoted a rapid and pronounced increase in the amount of p85 and PI 3-kinase activity associating with cell surface IRKs, no increase was observed for EN IRKs. Potential roles for an insulin-stimulated association of PI 3-kinase with the PM IRK include a regulation of insulin signal transduction from the cell surface and/or targeting of a molecule to the PM and/or IRK. Because the subcellular location of activated IRKs has been shown to be an important element of normal insulin signal transduction in vivo, future studies will evaluate these possibilities.
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ACKNOWLEDGEMENTS |
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We thank Gerry Baquiran for excellent technical assistance and Dr. Louise Larose for insightful comments.
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FOOTNOTES |
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* P. G. Drake and A. Balbis contributed equally to this work.
This work was supported by a grant from the Medical Research Council of Canada (to B. I. Posner).
Address for reprint requests and other correspondence: B. I. Posner, Polypeptide Hormone Lab., Strathcona Anatomy and Dentistry Bldg., 3640 University St., Rm. W3.15, Montreal, Quebec, Canada H3A 2B2 (E-mail: mc85{at}musica.mcgill.ca).
1 The IRK amino acid numbering system used in this paper is based on the human insulin proreceptor without the 12 amino acids encoded by exon 11 (HIR-A or Ex11) (28).
2
By analyzing the amount of p85 immunoprecipitated
by phosphotyrosine antibodies from cytosol and PM (data not shown), and on the basis of previously determined recoveries of PM
(18), we calculated that the proportion of PI 3-kinase
activity in PM is ~10% of that in cytosol. In PM the proportion of
p85 precipitated by IRK was not >10% (data not shown). Thus the PI
3-kinase associated with the IRK is in the range of 1% of the total
activity in cytosol.
3 A concern that wortmannin, at the concentration used to inhibit PI 3-kinase activity, might directly effect IRK activity (both IRK autophosphorylation and activity toward exogenous substrates) was tested by preincubating wheat germ agglutinin-purified PM IRKs with 500 nM wortmannin for 20 min at room temperature before measurement of IRK activity, as previously described (11). Under these conditions, wortmannin was without effect on either IRK autophosphorylating activity or exogenous activity toward polyglutamic acid-tyrosine (4:1).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 13 October 1999; accepted in final form 7 March 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Avruch, J.
Insulin signal transduction through protein kinase cascades.
Mol Cell Biochem
182:
31-48,
1998[ISI][Medline].
2.
Baass, P,
Di Guglielmo GM,
Authier F,
Posner BI,
and
Bergeron JJM
Compartmentalized signal transduction by receptor tyrosine kinases.
Trends Cell Biol
5:
465-470,
1995[ISI].
3.
Backer, JM,
Myers MG,
Sun X-J,
Chin DJ,
Shoelson SE,
Miralpeix M,
and
White MF.
Association of IRS-1 with the insulin receptor and the phosphatidylinositol 3-kinase.
J Biol Chem
268:
8204-8212,
1993
4.
Backer, JM,
Schroeder GG,
Kahn CR,
Myers MG,
Wilden PA,
Cahill DA,
and
White MF.
Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation.
J Biol Chem
267:
1367-1374,
1992
5.
Baltensperger, K,
Kozma LM,
Jaspers SR,
and
Czech MP.
Regulation by insulin of phosphatidylinositol 3'-kinase bound to alpha- and beta-isoforms of p85 regulatory subunit.
J Biol Chem
269:
28937-28946,
1994
6.
Band, CJ,
Posner BI,
Dumas V,
and
Contreres J-O.
Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen).
Mol Endocrinol
11:
1899-1910,
1997
7.
Bevan, AP,
Burgess JW,
Drake PG,
Shaver A,
Bergeron JJM,
and
Posner BI.
Selective activation of the rat hepatic endosomal insulin receptor kinase. Role for the endosome in insulin signalling.
J Biol Chem
270:
10784-10791,
1995
8.
Bevan, AP,
Drake PG,
Bergeron JJM,
and
Posner BI.
Intracellular signal transduction: the role of endosomes.
Trends Endocrinol Metab
7:
13-21,
1996[ISI].
9.
Bornfeldt, KE,
Raines EW,
Graves LM,
Skinner MP,
Krebs EG,
and
Ross R.
Platelet derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation.
Ann NY Acad Sci
766:
416-430,
1995[Abstract].
10.
Bradford, MM.
A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
11.
Burgess, JW,
Wada I,
Ling N,
Khan MN,
Bergeron JJM,
and
Posner BI.
Decrease in -subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.
J Biol Chem
267:
10077-10086,
1992
12.
Carpenter, CL,
Auger KR,
Duckworth BC,
Hou WM,
Schaffhausen B,
and
Cantley LC.
A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.
Mol Cell Biol
13:
1657-1665,
1993[Abstract].
13.
Cefai, D,
Sshneider H,
Matangkasombut O,
Kang H,
Brody J,
and
Rudd CE.
CD28 receptor endoytosis is targeted by mutations that disrupt phosphatidylinositol 3-kinase binding and co-stimulation.
J Immunol
160:
2223-2230,
1998
14.
Cengel, KA,
Godbout JP,
and
Freund GG.
Phosphatidylinositol 3'-kinase is associated with a serine kinase that is activated by okadaic acid.
Biochem Biophys Res Comm
242:
513-517,
1998[ISI][Medline].
15.
Cengel, KA,
Kason RE,
and
Freund GG.
Phosphatidylinositol 3'-kinase associates with an insulin receptor-1 serine kinase distinct from its intrinsic serine kinase.
Biochem J
335:
397-404,
1998[ISI][Medline].
16.
Cheatham, B,
and
Kahn CR.
Insulin action and the insulin signaling network.
Endocr Rev
16:
117-142,
1995[ISI][Medline].
17.
Dhand, R,
Hiles I,
Panayotou G,
Roche S,
Fry MJ,
Gout I,
Totty NF,
Truong O,
Vicendo P,
Yonezawa K,
Kasuga M,
Courtneidge SA,
and
Waterfield MD.
PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.
EMBO J
13:
522-533,
1994[Abstract].
18.
Di Guglielmo, GD,
Drake PG,
Baass PC,
Authier F,
Posner BI,
and
Bergeron JJM
Insulin receptor internalization and signalling.
Mol Cell Biochem
182:
59-63,
1998[ISI][Medline].
19.
Drake, PG,
Bevan AP,
Burgess JW,
Bergeron JJM,
and
Posner BI.
A role for tyrosine phosphorylation in both activation and inhibition of the insulin receptor tyrosine kinase in vivo.
Endocrinology
137:
4960-4968,
1996[Abstract].
20.
Fukui, Y,
and
Hanafusa H.
Phosphatidylinositol kinase activity associates with viral p60src protein.
Mol Cell Biol
9:
1651-1658,
1989[ISI][Medline].
21.
Giorgetti, S,
Ballotti R,
Kowalski-Chauvel A,
Tartare S,
and
Van Obberghen E.
The insulin and insulin-like growth factor-1 receptor substrate IRS-1 associates with and activates phosphatidylinositol 3-kinase in vitro.
J Biol Chem
268:
7358-7364,
1993
22.
Gommerman, JL,
Rottapel R,
and
Berger SA.
Phosphatidylinositol 3-kinase and Ca2+ influx dependence for ligand-stimulated internalization of the c-Kit receptor.
J Biol Chem
272:
30519-30525,
1997
23.
Hunter, T,
and
Sefton BM.
Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.
Proc Natl Acad Sci USA
77:
1311-1315,
1980[Abstract].
24.
Khan, MN,
Baquiran G,
Brule C,
Burgess JW,
Foster B,
Bergeron JJM,
and
Posner BI.
Internalization and activation of the rat liver insulin receptor kinase in vivo.
J Biol Chem
264:
12931-12940,
1989
25.
Khan, MN,
Savoie S,
Khan RJ,
Bergeron JJM,
and
Posner BI.
Insulin and insulin receptor uptake into rat liver. Chloroquine action on receptor recycling.
Diabetes
34:
1025-1030,
1985[Abstract].
26.
Kharitonenov, A,
Schnekenburger J,
Chen Z,
Knyazev P,
Ali S,
Zwick E,
White MF,
and
Ullrich A.
Adapter function of protein phosphatase 1D in insulin receptor/insulin receptor-1 interaction.
J Biol Chem
270:
29189-29193,
1995
27.
Lam, K,
Carpenter CL,
Ruderman N,
Friel JC,
and
Kelly KL.
The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1. Stimulation by insulin and inhibition by wortmannin.
J Biol Chem
269:
20648-20652,
1994
28.
Lee, J,
and
Pilch PF.
The insulin receptor: structure, function, and signaling.
Am J Physiol Cell Physiol
266:
C319-C334,
1994
29.
Levy-Toledano, R,
Taouis M,
Blaettler DH,
Gorden P,
and
Taylor SI.
Insulin-induced activation of phosphatidyl inositol 3-kinase. Demonstration that the p85 subunit binds directly to the COOH terminus of the insulin receptor in intact cells.
J Biol Chem
269:
31178-31182,
1994
30.
Liu, R,
and
Livingston JN.
Association of the insulin receptor and phosphatidylinositol 3-kinase requires a third component.
Biochem J
297:
335-342,
1994[ISI][Medline].
31.
Ottinger, EA,
Botfield MC,
and
Shoelson SE.
Tandem SH2 domains confer high specificity in tyrosine kinase signaling.
J Biol Chem
273:
729-735,
1998
32.
Posner, BI,
Faure R,
Burgess JW,
Bevan AP,
Lachance D,
Zhang-Sun G,
Ng JB,
Hall DA,
Lum BS,
and
Shaver A.
Peroxovanadium compounds: a new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetic.
J Biol Chem
269:
4596-4604,
1994
33.
Rocchi, S,
Tartare-Deckert S,
Swaka-Verhelle D,
Gamha A,
and
Van Obberghen E.
Interaction of the SH2-containing protein tyrosine phosphatase-2 with the insulin receptor and the insulin-like growth factor receptor: studies of domains involved using the yeast two-hybrid system.
Endocrinology
137:
4944-4952,
1996[Abstract].
34.
Rordorf-Nikolic, T,
Van Horn DJ,
Chen D,
White M,
and
Backer JM.
Regulation of phosphatidylinositol 3'-kinase by tyrosyl phosphoproteins. Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit.
J Biol Chem
270:
3662-3666,
1995
35.
Ruderman, NB,
Kapeller R,
White MF,
and
Cantley LC.
Activation of phosphatidylinositol 3-kinase by insulin.
Proc Natl Acad Sci USA
87:
1411-1415,
1990[Abstract].
36.
Sale, G.
Serine/threonine kinases and tyrosine phosphatases that act on the insulin receptor.
Biochem Soc Trans
20:
664-670,
1992[ISI][Medline].
37.
Shpetner, H,
Joly M,
Hartley D,
and
Corvera S.
Potential sites of PI 3-kinase function in the endocytic pathway revealed by the PI 3-kinase inhibitor, wortmannin.
J Cell Biol
132:
595-605,
1996[Abstract].
38.
Staubs, PA,
Reichart DR,
Saltiel AR,
Milarski KL,
Maegawa H,
Berhanu P,
Olefsky JM,
and
Seely BL.
Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP.
J Biol Chem
269:
27186-27192,
1994
39.
Sung, CK,
Sanchez-Margalet V,
and
Goldfine I.
Role of the p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein.
J Biol Chem
269:
12503-12507,
1994
40.
Tanti, J-F,
Gremeaux T,
Van Obberghen E,
and
Le Marchand-Brustel Y.
Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase.
Biochem J
304:
17-21,
1994[ISI][Medline].
41.
Ugi, S,
Maegawa A,
Olefsky JM,
Shigeta Y,
and
Kashiwagi A.
Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs.
FEBS Lett
340:
216-220,
1994[ISI][Medline].
42.
Ui, M,
Okada T,
Hazeki K,
and
Hazeki O.
Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase.
Trends Biochem Sci
20:
303-307,
1995[ISI][Medline].
43.
Vanhaesebroeck, B,
Leevers SJ,
Panayotou G,
and
Waterfield MD.
Phosphoinositide 3-kinases: a conserved family of signal transducers.
T Biochem Sci
22:
267-272,
1998[ISI][Medline].
44.
Van Horn, DJ,
Myers MG,
and
Backer JM.
Direct activation of the phosphatidylinositol 3-kinase by the insulin receptor.
J Biol Chem
269:
29-32,
1994
45.
Waters, SB,
and
Pessin JE.
Insulin receptor substrate 1 and 2 (IRS1 and IRS2): what a tangled web we weave.
Trends Cell Biol
6:
1-4,
1996[ISI].
46.
Wilden, PA,
Rovira I,
and
Broadway DE.
Insulin receptor requirements for the formation of a ternary complex with IRS-1 and PI 3-kinase.
Mol Cell Endocrinol
122:
131-140,
1996[ISI][Medline].
47.
Yonezawa, K,
Yokono K,
Shii K,
Ogawa W,
Ando A,
Hara K,
Baba S,
Kaburagi Y,
Yamamoto-Honda R,
Momomura K,
Kadawaki T,
and
Kasuga M.
In vitro association of phosphatidylinositol 3-kinase activity with the activated insulin receptor tyrosine kinase.
J Biol Chem
267:
440-446,
1992