(Received for publication, June 26, 1995)
From the
Binding of insulin to its receptor (IR) causes rapid
autophosphorylation with concomitant activation of its tyrosine kinase
which transmits the signal by phosphorylating cellular substrates. The
IR activity is controlled by protein-tyrosine phosphatases, but those
directly involved in regulating the insulin receptor and its signaling
pathways have not yet been identified. Using baby hamster kidney cells
overexpressing the IR and a novel insulin-based selection principle, we
established stable cell lines with functionally coupled expression of
the IR and protein-tyrosine phosphatases. The two closely related
protein-tyrosine phosphatases and
were identified as
negative regulators of IR tyrosine kinase.
Insulin is an important regulator of different metabolic
processes and plays a key role in the control of blood glucose. Defects
related to its synthesis or signaling lead to diabetes mellitus.
Binding of insulin to its receptor causes rapid autophosphorylation of
several tyrosine residues in the intracellular part of the
-subunit. Three closely positioned tyrosine residues (the tyrosine
1150 domain) must all be phosphorylated to obtain full activity of the
insulin receptor tyrosine kinase (IRTK) (
)which transmits
the signal further downstream by tyrosine phosphorylation of other
cellular substrates, including insulin receptor substrate-1
(IRS-1)(1, 2, 3, 4) . The structural
basis for the function of the tyrosine triplet has been provided by
recent x-ray crystallographic studies of IRTK that showed tyrosine 1150
to be autoinhibitory in its unphosphorylated state(5) .
Several studies clearly indicate that the activity of the autophosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein(6) )(7, 8) , with the triphosphorylated tyrosine 1150 domain being the most sensitive target for protein-tyrosine phosphatases (PTPs) as compared to the di- and monophosphorylated forms(8) . It is, therefore, tempting to speculate that this tyrosine triplet functions as a control switch of IRTK activity. Indeed, the IRTK appears to be tightly regulated by PTP-mediated dephosphorylation in vivo(9, 10, 11) . The intimate coupling of PTPs to the insulin signaling pathway is further evidenced by the finding that insulin differentially regulates PTP activity in rat hepatoma cells (12) and in livers from alloxan diabetic rats(13) . However, little is known about the identity of the PTPs involved in IRTK regulation.
To identify PTPs that negatively
regulate the IRTK activity we developed a novel selection principle
that allows establishment of stable cell lines with functionally
coupled overexpression of IR and inhibitory PTPs. For this purpose we
used a previously established baby hamster kidney cell line
(BHK-IR)(14) , which exhibits high levels of IR expression and
responds to insulin stimulation with complete growth inhibition of
adherent cells. PTPs that impede or block the insulin signal can
consequently be identified by their capacity to restore cell growth.
This effect was found to be induced through direct activation of the
insulin receptor with half-maximal effect at physiological relevant
concentrations of the hormone. Therefore, the BHK-IR cell line can be
used to identify PTPs with activity toward the receptor itself. Twelve
different PTPs were analyzed by this procedure, and the closely related
receptor-like phosphatases, PTP (15) and
PTP
(16) , were found to be efficient negative regulators
of the IRTK.
Figure 1:
Insulin-induced phenotypic changes of
BHK cells overexpressing the human insulin receptor (BHK-IR). A, phase-contrast micrographs of BHK-IR cells without and with
insulin treatment (100 nM, 24 h). Magnification 100. B, number of adherent and nonadherent BHK-IR cells and control
cells (wild-type BHK (BHK-wt) and BHK cells overexpressing
ELAM-1 (BHK-ELAM 1)) after insulin treatment (100 nM,
24 h). The cells were seeded at a density of 1
10
per well in 6-well plates (Nunclon
delta, Nunc
Denmark) and incubated for 24 h at 37 °C under 5% CO
in
complete medium. Human insulin (Novo Nordisk, Denmark) was added to
three wells per plate (final concentration, 100 nM) with the
other three wells as controls. After a further 24 h of incubation, the
numbers of adherent and nonadherent cells were determined. Bar graphs
indicate mean ± S.D. Similar results were obtained in two
independent experiments. Phase-contrast micrographs were obtained from
cells grown under identical conditions in 6-cm tissue culture
dishes.
Figure 2:
An
insulin-based selection principle for establishment of stable BHK cell
lines overexpressing the insulin receptor and PTPs. Insulin
dose-response (A) and time course (B): open
bars, adherent cells; solid bars, nonadherent cells. Bar
graphs indicate mean ± S.D. (triplicate). Similar results were
obtained in three independent experiments. C, growth curves of
BHK-IR with (--
) and without
(
--
) insulin. The graph shows the mean of the
number of adherent cells in three wells per time point. Similar results
were obtained in three independent experiments. D,
representative examples of BHK-IR clones rescued by transfection with
PTP
, PTP
, and TC-PTP cDNA inserted into cytomegalovirus
promoter-based expression plasmids.
We then tested
whether expression of PTPs in BHK-IR cells could counteract the
insulin-induced effects. Twelve different PTP cDNAs inserted into
cytomegalovirus promoter-based plasmids were transfected into BHK-IR
cells (20) which were subsequently kept under selection
pressure with insulin for 14 days. This time allowed for the growth of
individual insulin resistant cell clones that were stained and counted (Fig. 2D; Table 1). Two closely related
receptor-type PTPs, PTP (15) and PTP
(16) ,
and with a lower efficiency, the intracellular TC-PTP (21) were
most efficient in suppressing cell detachment. Very few clones were
obtained with CD45 (22) , PTP1C(23) ,
PTPH1(24) , and PTP1B(25) , whereas LAR(26) ,
PTP
(27) , PTPµ(28) , PTP1D(29) ,
PTPD1 (30) did not have any effect on the restoration of cell
attachment. Stable clones that were obtained in parallel assays were
found to exhibit IR expression levels comparable to the parental BHK-IR
line (Fig. 3). PTP overexpression was additionally verified in
these clones by immunoblotting with specific antibodies (Fig. 4). It has recently been shown that PTP
, when
transiently expressed in COS-1 cells, gives rise to two proteins of
about 100 and 130 kDa(31) . The 100-kDa species exhibits N-linked glycosylation only and is a precursor of the 130-kDa
protein which in addition contains O-linked carbohydrates. We
have also observed these two molecular species when PTP
is
transiently expressed in human embryonic kidney fibroblast 293 cells
and BHK-IR cells (not shown). In contrast, the fully glycosylated
130-kDa protein is the predominant form in the rescued BHK-IR cells (Fig. 4). Transient expression of PTPH1 results in a protein of
about 105 kDa (not shown), which is in agreement with its predicted
molecular mass(24) . Interestingly, Yang and Tonks (24) found that reticulocyte lysate expression of PTPH1 yielded
a protein of about 120 kDa, which is in complete agreement with the
molecular mass of PTPH1 observed in BHK-IR/PTPH1 cells (Fig. 4).
The molecular basis for this difference needs to be investigated
further. For those PTPs that were not able to restore cell growth,
transient expression in transfected BHK-IR cells was monitored to
confirm the capability of the BHK cells to express these phosphatases
(not shown). Further, an inactive mutant of PTP
was unable to
restore cell attachment despite the fact that it exhibited even higher
levels of expression than the intact enzyme when transiently expressed
in BHK cells (not shown). These results suggest that PTPs can
specifically interfere with insulin dependent signaling.
Figure 3:
Analysis of phosphotyrosine-containing
proteins in BHK-IR cells stably overexpressing PTPs. a, an
immunoblot with anti-phosphotyrosine antibody of cells untreated or
treated for 10 and 60 min with 100 nM insulin. The M of marker proteins, IR precursor (P),
IR
-subunit (
), and pp35 are indicated. b and c, same as a, but a shorter exposure showing IR
-subunit from cells treated with 100 nM and 2
nM, respectively; d, control of the expression levels
of IR by blotting with a polyclonal anti-IR
antibody.
Figure 4: Analysis of PTP expression in BHK-IR/PTP clones. Samples from the lysates used in Fig. 3were subjected to immunoblot analysis with specific PTP antibodies as described under ``Materials and Methods.'' The control lanes contain the corresponding lysate from the parental BHK-IR cell line. The positions of molecular size standards (kDa) are indicated.
After insulin
stimulation several proteins were phosphorylated, among which the IR
-subunit and bands at 190, 75-80, 63, and 35 kDa were most
prominent (Fig. 3). The PTPs that most potently restore cell
growth of insulin-treated BHK-IR cells, i.e. PTP
and
PTP
, also appeared to be the most effective in reducing tyrosine
phosphorylation of the IR
-subunit and the unidentified
75-80-kDa kinase substrates, whereas intracellular PTPs showed
little, if any, activity. Interestingly, CD45, which is normally
expressed only in cells of the immune system, had a comparable
influence on IR
-subunit phosphorylation. While the 63-kDa band
appeared only in PTP1C-transfected cells, and therefore represents the
tyrosine-phosphorylated form of this SH2 domain containing phosphatase,
the phosphorylation state of the 35-kDa polypeptide (pp35) correlated
with the potency of PTPs
,
, and TC to restore attachment of
BHK-IR cells (Table 1). However, pp35 cannot be the sole mediator
of cell detachment, as indicated by the clear difference in potency of
TC-PTP versus PTP
and PTP
.
In the present study we introduced a novel selection system
to identify PTPs that negatively regulate tyrosine kinase signaling.
More specifically we analyzed the influence of 12 different PTPs on
insulin receptor signaling. Our findings indicate a significant,
selective role of receptor-like PTPs and
as negative
regulators of the IRTK. This is in accordance with previous studies
which suggest that PTPs responsible for IRTK regulation belong to the
class of membrane-associated (10) and glycosylated
molecules(33, 34) . Further, PTP
has wide tissue
expression, including the major insulin target tissues liver and
skeletal muscle, as would be expected from a PTP with regulatory
activity toward the IR(15, 35, 36) .
Hashimoto et al.(37) have proposed that PTP LAR might
play a role in the physiological regulation of insulin receptors in
intact cells. However, their conclusion was reached by comparing the
rate of dephosphorylation/inactivation of purified IR using recombinant
PTP1B as well as the cytoplasmic domains of LAR and PTP
. Antisense
inhibition was recently used to study the effect of LAR on insulin
signaling in a rat hepatoma cell line(38) . A suppression of
LAR protein levels by about 60% was paralleled by an approximately 150%
increase in insulin-induced autophosphorylation. However, only a modest
35% increase in IRTK activity was observed, whereas the
insulin-dependent phosphatidylinositol 3-kinase activity was
significantly increased by 350%. Reduced LAR levels did not alter the
basal level of IRTK tyrosine phosphorylation or activity. The authors
speculate that LAR could specifically dephosphorylate tyrosine residues
that are critical for phosphatidylinositol 3-kinase activation either
on the insulin receptor itself or on a downstream substrate. In
contrast to these studies, we do not find any indication that LAR, or
the two other receptor-like PTPs (PTP
(27) ,
PTPµ(28) ), show activity toward IRTK when employing our
functional, insulin-based selection principle. This might indicate that
phosphatidylinositol 3-kinase activation is not essential for the
insulin-induced growth inhibition of BHK-IR cells. Interestingly, CD45,
which is normally expressed only in cells of the immune system, had a
similar influence on the overall tyrosine phosphorylation of the IR
-subunit in BHK-IR cells when compared with PTPs
and
but was less effective on pp35 and very inefficient in preventing cell
detachment (Fig. 3, Table 1). This suggests to us that
CD45 is not directed toward the regulatory tyrosine 1150 domain, but
phosphorylated tyrosine residues that reside outside this region and
therefore are not involved in the activation of the IRTK.
Several studies have addressed the question of PTP specificity using synthetic peptides(39, 40, 41, 42) . These investigations have provided important insight with respect to primary structural sequence requirements for substrate recognition. However, an obvious limitation of this approach is the lack of defined three-dimensional structure of the peptides. Likewise, the PTPs utilized for these analyses are removed from their natural environment. Since at least part of the PTP specificity seems to be conveyed by a defined subcellular localization (43) , it is essential that PTP activity toward cellular substrates is tested in intact cells. We believe that the present method with its functional coupling and presumed correct localization of the key elements to specific subcellular compartments provide the necessary tools for analysis of PTP specificity toward IRTK.
This study indicates that receptor-like
PTPs play a significant role in regulating the IRTK, whereas
intracellular PTPs have little, if any, activity toward the insulin
receptor. Even though TC-PTP efficiently prevents insulin-induced
detachment of BHK-IR cells, the effect is not as pronounced as with
PTP and PTP
. While it appears that the target of the negative
regulatory activity of PTPs
and
is the receptor itself, the
down-modulating effect of the intracellular TC-PTP seems to be due to a
downstream function in the IR-activated signal resulting in cell
detachment. We speculate that pp35 could be a direct target of TC-PTP.
Although PTP1B and TC-PTP are closely related, PTP1B is only weakly
active in the present selection system and has only little influence on
the phosphorylation pattern of insulin-treated BHK-IR cells. Both PTPs
have distinct structural features that determine their subcellular
localization and thereby their access to defined cellular
substrates(44, 45) . Therefore, the lack of activity
of PTP1B and TC-PTP toward the IRTK may, at least in part, be explained
by the fact that they do not co-localize with the activated insulin
receptor. In support of this view, PTP1B and TC-PTP have been excluded
as candidates for the IR-associated PTPs in hepatocytes based on
subcellular localization studies(10) .
It was recently found that the ubiquitously expressed SH2 domain containing protein tyrosine phosphatase, PTP1D(29) , associates with and dephosphorylates IRS-1, but apparently not the IR itself(46, 47) . Further, PTP1D seems to act as a positive mediator in insulin-stimulated Ras activation (48) and of growth factor-induced mitogenic signal transduction(49) . It is in agreement with these studies that PTP1D does not exhibit any rescuing capacity in our selection system which is designed to identify negative regulators only. In contrast, the structurally related SH2-containing phosphatase, PTP1C, which is predominantly expressed in hematopoietic cells, seems to act as negative regulator of growth factor-stimulated proliferation (50, 51, 52) . In accordance with this we have observed that PTP1C can act as a weak, negative regulator of the insulin response in BHK-IR cells (Table 1). However, even though PTP1C is heavily phosphorylated after insulin treatment of BHK-IR cells, it has only weak, if any, activity toward the IRTK (Fig. 3)(48) .
Two intracellular PTPs with ezrin-like N-terminal domains, PTPD1 (30) and PTPH1(24) , were also analyzed for their capacity to negatively regulate the insulin signal in BHK-IR cells. Both PTPs have been proposed to localize to the junction of the cytoskeleton and the plasma membrane. We have previously shown PTPD1 to be phosphorylated by and associated with c-Src in vitro and hypothesized it to be involved in the regulation of phosphorylation of focal adhesions (30) . PTPD1 is thus an unlikely candidate as regulator of the IRTK. In agreement with this, PTPD1 did not show any effects at all, whereas PTPH1 gave rise to very few stable cell clones, but did not lead to a change in the phosphorylation pattern of BHK-IR cells.
With regard to the molecular basis of the BHK-IR cell detachment response, several observations point to a phosphoprotein of 35 kDa as an important mediator (Fig. 3; Table 1; not shown): (i) the rescuing capacity of PTPs inversely parallels the degree of pp35 phosphorylation in BHK-IR/PTP cell lines; (ii) confluent BHK-IR cells respond poorly to insulin with respect to cell detachment and show relatively low levels of pp35 phosphorylation; (iii) transient expression of v-Src in BHK-IR results in massive rounding and detachment of the cells with a concomitant intense phosphorylation of pp35; (iv) time course studies show that p35 phosphorylation increases even further several hours after insulin stimulation, i.e. corresponding to the occurrence of rounding of the cells. Interestingly, in A431 cells the epidermal growth factor induces phenotypic changes similar to insulin in BHK-IR cells and also causes tyrosine phosphorylation of a 35-kDa polypeptide(53, 54) . This protein was identified as a member of the lipocortin/annexin family(54) , which has previously been shown to include substrates of the IRTK in vitro and in intact hepatocytes after corticosteroid treatment(55) . Whether pp35 in BHK-IR cells belongs to this class of proteins remains to be investigated.
PTPs or other proteins that would impede or block any of the signaling steps leading to the insulin-induced phenotypic changes of the BHK-IR cells could be identified by the method presented here. These changes are prerequisites for the selection principle and the establishment of stable cell lines with functionally coupled expression of IR and opposing PTPs. Some of the downstream elements involved in induction of growth inhibition and detachment of BHK-IR cells may not represent molecules that actually mediate the effects of insulin in vivo. Nevertheless, since the changed phenotype of the BHK-IR cells is induced via normal activation of the insulin receptor our procedure allows, for the first time, identification of PTPs as regulators of insulin signaling at the level of the receptor itself.
While previous reports indicate a role of PTP in signal
transduction through Src activation (56, 57) and
interaction with GRB-2(58, 59) , our results suggest a
function for this phosphatase and its close relative PTP
as
negative regulators of the insulin receptor. In view of recent findings
indicating the crucial importance of signal duration and therefore
negative regulation of tyrosine kinase
signals(60, 61) , PTPs
and
may be key
elements in the definition of insulin action in different tissues and
in the pathophysiology of non-insulin-dependent diabetes.