(Received for publication, March 20, 1995; and in revised form, June 7, 1995)
From the
Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3`-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.
As recent studies have made great advances in our understanding
of reversible tyrosine phosphorylation in the cellular insulin action
pathway, interest has also grown in the role of protein-tyrosine
phosphatases (PTPases) ()in balancing the steady-state level
of tyrosine phosphorylation of proteins involved in insulin signal
transduction(1, 2) . PTPases can potentially impact on
insulin signaling at several levels, including dephosphorylation of the
active (autophosphorylated) form of the insulin receptor, which
attenuates the receptor kinase activity, and dephosphorylation of the
protein-tyrosine residues of insulin receptor substrates such as IRS-1,
IRS-2, and Shc, which will modulate postreceptor pathways of insulin
action(3) .
The PTPases constitute a large superfamily of
transmembrane (receptor-type) and intracellular (non-receptor-type)
enzymes involved in a variety of regulatory
processes(4, 5) . Recent studies exploring the role of
individual PTPases in cellular regulation have demonstrated that their
effects may be complex, and various PTPase homologs may have positive
or negative effects on cell activation, cell growth, or
differentiation. For example, CD45, which activates the p56 tyrosine kinase by dephosphorylation of a specific
phosphotyrosine residue (6) and the PTPase Cdc25, which
initiates the induction of mitosis by the tyrosine dephosphorylation
and activation of p34
(7) . These findings have
enhanced our view of the cellular roles of PTPases, in considering not
only the identification of PTPases that might impact on various
signaling pathways, but also whether the influence of their PTPase
activity is stimulatory or inhibitory.
Since multiple PTPase enzymes are expressed in insulin-sensitive tissues, there is a need to establish the significance and physiological role of individual PTPases in liver, fat, and muscle tissue. For the insulin signaling pathway, only a few PTPases have been shown to affect insulin action at the receptor or post-receptor sites. In hepatoma cells, using expression of an antisense RNA construct, we have recently demonstrated that the transmembrane PTPase LAR has a negative regulatory effect on insulin receptor autophosphorylation and receptor kinase activity(8) . The SH2 domain containing PTPase, SH-PTP2, or Syp, has also been shown by several groups to have a positive effect on post-receptor insulin signaling, although the mechanism and site of interaction with the insulin action pathway has not been identified(9, 10, 11, 12) .
PTP1B is a particularly important candidate for involvement in insulin signaling since it is an abundant intracellular PTPase that is widely expressed in insulin-sensitive tissues(2) . As the first PTPase to be purified to homogeneity and characterized biochemically, early studies of PTP1B demonstrated that it was able to efficiently dephosphorylate the insulin receptor in vitro(13, 14) . Moreover, microinjection of a truncated form of PTP1B from human placenta into Xenopus oocytes diminished insulin-stimulated oocyte maturation and S6 peptide phosphorylation(15, 16) . In the present work, we used an osmotic loading technique to inhibit PTP1B activity in situ with a neutralizing antibody in order to explore its potential role in the regulation of insulin signaling in hepatoma cells. The results demonstrate that PTP1B has an essential role in the negative regulation of insulin signaling and acts by balancing insulin-stimulated kinase activity at the level of the receptor as well as influencing distal components of the insulin action cascade.
Intracellular loading of PTP1B antibodies or control preimmune rabbit IgG was achieved by promoting uptake of extracellular proteins by incubation in a hypertonic medium, followed by lysis of cytoplasmic pinosomes in a hypotonic solution by the method of Okada and Rechsteiner(20) . Briefly, subconfluent KRC-7 cells were washed with phosphate-buffered saline and then incubated for 10 min in a hypertonic medium containing 0.5 M sucrose, 10% (w/v) polyethylene glycol 1000, 10% (v/v) fetal calf serum, and PTP1B antibody or pre-immune rabbit IgG (30 µg/ml) in DMEM buffered with 25 mM HEPES, pH 6.8. The cells were then rapidly rinsed with a hypotonic solution of diluted DMEM:water (6:4) buffered with 25 mM HEPES, pH 6.8, and incubated in the hypotonic medium for 2 min. After rinsing 3 times with normal culture medium, the cells were allowed to recover for 6 h before the assays were performed. Viability of the hepatoma cells was demonstrated by exclusion of 0.4% (w/v) trypan blue dye (Sigma).
After gel
separation, proteins were transferred to nitrocellulose filters
(0.45-µ pore size) at 100 V for 3 h in buffer containing 20% (v/v)
methanol, 25 mM Tris base and 192 mM glycine at pH
8.3. Nitrocellulose membranes were then incubated in blocking buffer
containing 150 mM NaCl, 0.05% (v/v) Nonidet P-40, 5% (w/v)
bovine serum albumin, 1% (w/v) ovalbumin, 0.01% (w/v) sodium azide, and
10 mM Tris, pH 7.4, for 1 h at room temperature with rocking.
Fresh blocking solution was then applied containing 1.0 µg/ml
affinity-purified PTP1B antibody or 1.1 µg/ml anti-Tyr(P)
antibodies (22) with rocking for 2 h. Membranes were washed 3
times for 10 min in blotting buffer without antibodies, followed by
incubation with 2 µCi of I-Protein A (30 mCi/mg) (ICN
Biomedicals Inc., Irvine, CA) for 1 h at room temperature followed by 3
additional 10-min washes with blotting buffer. Immunoreactive proteins
were visualized by direct PhosphorImager analysis (Molecular Dynamics).
Protein was assayed by the method of Bradford(23) .
Figure 1: Specificity of anti-PTP1B antibody for PTP1B in KRC-7 hepatoma cell lysate proteins. After hepatoma cells were osmotically loaded with affinity-purified PTP1B antibody or preimmune IgG and allowed to recover, aliquots of cell lysates (60 µg of protein) were immunoprecipitated with PTP1B antibody and immunoblotted with the PTP1B antibody as described under ``Experimental Procedures.'' Molecular size markers were used to determine the mass of PTP1B to be approximately 50 kDa. Lanes 1-2 and 3-4 represent replicate samples of immunoprecipitated hepatoma cell proteins.
In order to demonstrate that the PTP1B antibody was taken up by the KRC-7 cells after osmotic loading and was associated with cellular PTP1B protein, cell lysate samples were precipitated with a goat anti-rabbit IgG second antibody and Trisacryl-Protein A and subjected to immunoblotting with the PTP1B antibody (Fig. 2). The second antibody was able to immunoprecipitate the loaded PTP1B antibody still complexed to the enzyme from its intracellular localization only in the cells osmotically loaded with the PTP1B antibody. Since PTP1B was not immunoprecipitated from the cells loaded with the preimmune IgG, the interaction between the loaded antibody and cellular PTP1B protein is shown to be specific and of high affinity and sufficient duration to continue to be present well after the 6-h recovery period.
Figure 2: Demonstration of PTP1B antibody uptake by the hepatoma cells after osmotic loading and recovery. Hepatoma cells were loaded with PTP1B antibody (lanes 5-7) or preimmune IgG (lanes 2-4) and allowed to recover. Aliquots of cell lysates (60 µg of protein) were then incubated with Trisacryl-Protein A alone (lanes 2 and 5) or Trisacryl-Protein A and goat anti-rabbit IgG (lanes 3-4 and 6-7). The immunoprecipitated proteins bound to the Trisacryl beads were washed and subjected to gel electrophoresis and immunoblotting with the PTP1B antibody. Lane 1 represents control cells not loaded with antibody, from which a 60-µg protein lysate was immunoprecipitated with PTP1B antibody along with Trisacryl-Protein A and the second antibody to quantitate the total amount of PTP1B present in comparison with the PTP1B antibody or IgG-loaded cells.
Of the total mass of PTP1B in the KRC-7 cells directly precipitated with PTP1B antibody, 70% of the loaded PTP1B antibody-PTP1B enzyme complex is immunoprecipitable with the second antibody and Protein A (Fig. 2). When total protein lysates from the osmotically loaded cells were directly immunoprecipitated with PTP1B antibody and immunoblotted, there was an apparent decrease of 35-40% in amount of immunoprecipitable PTP1B in the cells loaded with the PTP1B antibody (Fig. 1). In order to determine whether the PTP1B antibody loading affected the cellular content of PTP1B, immunoblotting was also performed with aliquots of total cell protein from the loaded cells that were fractionated on gels without prior immunoprecipitation. The content of PTP1B protein in the cells loaded with PTP1B antibody was not decreased, but on average was 10-15% higher than the PTP1B content of the control cells (Fig. 3). Thus, the inability to immunoprecipitate a portion of the PTP1B mass from the cells loaded with PTP1B antibody results from occupancy of PTP1B epitopes by the neutralizing antibody which remains associated with the PTP1B protein in the cells.
Figure 3: Quantitation of PTP1B mass in hepatoma cells loaded with PTP1B antibody or rabbit IgG. Hepatoma cells were loaded with PTP1B antibody (lanes 4-6) or preimmune IgG (lanes 1-3) and allowed to recover. Replicate samples of cell lysate protein (60 µg of protein) were then fractionated on 10% (w/v) polyacrylamide gels, and immunoblotting with PTP1B antibody was performed.
Figure 4:
Effect of PTP1B antibody loading in
hepatoma cells on insulin stimulation of DNA synthesis. Hepatoma cells
were loaded with PTP1B antibody or control IgG, allowed to recover for
6 h, and the cells were treated with or without 100 nM insulin
for 1 h. DNA synthesis was then measured by adding
[H]thymidine for an additional 1-h incubation as
described under ``Experimental Procedures.'' Cells in
individual 35-mm sample wells were washed, and the incorporation of
H into trichloroacetic acid (TCA)-insoluble
material was determined by scintillation
counting.
Figure 5: Effect of PTP1B antibody loading in hepatoma cells on insulin-stimulated phosphatidylinositol 3`-kinase activity. Hepatoma cells were loaded with PTP1B antibody or control IgG, allowed to recover for 6 h, and the cells were treated with or without 100 nM insulin for 5 min. In vitro phosphorylation of phosphatidylinositol was performed in anti-phosphotyrosine immune complexes of the hepatoma cell lysates as described under ``Experimental Procedures.''
Figure 6:
Insulin-stimulated receptor
autophosphorylation and IRS-1 phosphorylation in hepatoma cells after
osmotic loading of control IgG (lanes 1-3) or PTP1B
antibody (lanes 4-6). After recovery from osmotic
antibody loading, control KRC-7 hepatoma cells without insulin
treatment (lanes 1 and 4) and cells treated with 100
nM insulin for 1 min (duplicate samples in lanes 2-3 and 5-6) were extracted into cell lysis buffer, and
aliquots of cell protein were separated by SDS-gel electrophoresis and
analyzed by immunoblotting with affinity-purified rabbit polyclonal
antiphosphotyrosine antibody, as described under ``Experimental
Procedures.'' The migration position of IRS-1 (180 kDa) and the
insulin receptor -subunit (95 kDa) is
shown.
Figure 7:
Insulin-stimulated tyrosine kinase
activity of insulin receptors isolated from hepatoma cells osmotically
loaded with control IgG or PTP1B antibody. After loading and cell
recovery, hepatoma cells were stimulated with 100 nM insulin
for 5 min. Insulin receptors were partially purified by wheat germ
agglutinin-agarose lectin chromatography under conditions that preserve
the receptor phosphorylation state, and the tyrosine kinase activity of
the isolated receptors was assayed using a poly(Glu:Tyr) (4:1)
substrate as described under ``Experimental Procedures.''
Tyrosine kinase activity was expressed as picomoles of P
incorporated per mg of poly(Glu:Tyr) per min per mg of
protein.
PTP1B is a widely expressed enzyme that was first identified as a prominent PTPase in the cytosol fraction of placenta(30) . Since it is an abundant enzyme found in a variety of insulin-sensitive tissues(14, 31) , early studies by Cicirelli et al.(16) and Tonks et al.(15) implicated PTP1B as a potential regulator of insulin signaling by demonstrating that microinjection of Xenopus oocytes with a purified, truncated form of the activated PTP1B protein was able to block insulin-stimulated ribosomal S6 peptide phosphorylation and retard insulin-induced oocyte maturation. These studies have prompted further investigations into the potential role of the native PTP1B enzyme in the regulation of tyrosine phosphorylation induced by the insulin receptor and other growth factors.
In recent studies exploring the physiology of PTP1B, this intracellular PTPase has also been shown to have a complex intracellular itinerary which plays an integral role in determining its subcellular localization and might be expected to influence its access to physiological substrates in the cell. Cloning of the PTP1B cDNA revealed that the full-length protein has a C-terminal segment that directs the association of at least a portion of the native protein with intracellular membranes either through a hydrophobic interaction or by attachment to a noncatalytic subunit(18, 31, 32, 33, 34, 35) . The subcellular localization of PTP1B is also determined by cellular mechanisms that regulate the proteolytic cleavage of the C-terminal segment, which releases a soluble, truncated form of the protein as demonstrated in an activated platelet model(36) . A substantial portion of the uncleaved, full-length form of PTP1B, however, is also found in the cytosol of rat tissues and cultured cells(28, 37) , raising the possibility that it interacts with potential substrates in both the soluble and particulate fractions of the cell.
In order to closely examine the potential
involvement of PTP1B in insulin signaling, in the present study we
disrupted the activity of PTP1B in situ in intact cells and
evaluated in detail its influence on potential cellular targets and
certain metabolic pathways. We focused on insulin-sensitive hepatoma
cells, in which the intracellular activity of PTP1B was inhibited by
osmotic loading of neutralizing antibodies. Various aspects of insulin
receptor signaling were then evaluated to determine at which points
along the insulin signaling cascade PTP1B might affect cellular
responses to insulin. This approach is complementary to the approach
taken by Lammers et al.(38) who showed in a
transfection model that overexpression of PTP1B in situ almost
completely dephosphorylated insulin and insulin-like growth factor 1
receptor -subunits in the basal state and also reduced the
phosphotyrosine content of the ligand-activated receptor
-subunits
to less than 50% of the control level. Our method of inhibiting the
endogenous PTP1B activity would be expected to directly reduce the
effects of PTP1B on cellular signaling processes. By inhibiting the
activity of PTP1B in situ, these experiments avoid potential
problems in studies employing high levels of PTPase overexpression
which may alter their normal subcellular distribution or affect the
post-translational regulation of the expressed enzymes by saturating
protein processing enzymes or associated binding proteins.
Our
results show that inhibition of PTP1B in situ has the initial
effect of enhancing insulin signaling at the level of the insulin
receptor kinase itself. Studies on the activation of the insulin
receptor kinase have shown that phosphorylation of two tyrosines in the
kinase domain, involving tyrosine 1158 and either tyrosine 1162 or 1163
occurs first, and that the partially phosphorylated receptors with
mono- or bisphosphorylated kinase domains exhibit minimal activation of
the -subunit kinase activity. Phosphorylation of the third tyrosyl
residue in this so-called ``regulatory domain'' rapidly
follows the bisphosphorylation stage and leads to full activation of
the receptor kinase toward exogenous substrates(1) . In this
way, the transition between bis- and trisphosphorylation in the
receptor regulatory domain may be considered to be a discrete molecular
``switch,'' in which the steady-state level of
phosphorylation in this region can determine the overall degree of
receptor kinase activation(3) . The recently described crystal
structure of the insulin receptor kinase domain has provided a
structural picture that is consistent with these functional
data(39) . In the basal (unphosphorylated) state, tyrosine 1162
is held in the active site and blocks the binding of both substrate and
ATP. trans-Autophosphorylation of tyrosines in the regulatory
domain can occur when tyrosine 1162 is disengaged by insulin binding
and the activity of the receptor kinase is dramatically enhanced.
Dephosphorylation of the activated receptor by PTPase action then
allows tyrosine 1162 to return to its autoinhibitory position, and the
receptor kinase is deactivated. By reducing the influence of PTP1B on
insulin receptor dephosphorylation, the loading of neutralizing
antibodies appears to augment the relative abundance of receptors in
the fully activated, trisphosphorylated state, which results in
enhanced receptor autophosphorylation and increased kinase activity
toward the exogenous peptide substrate as well as increased IRS-1
phosphorylation in vivo.
The increased activation of the
insulin signaling pathway in the PTP1B antibody-loaded cells also
extends to several post-receptor effects, including enhanced IRS-1
phosphorylation, insulin-stimulated phosphatidylinositol 3`-kinase
activity, and incorporation of thymidine into DNA. The increased
tyrosine phosphorylation of IRS-1 could result from the enhanced
insulin receptor kinase activation, diminished IRS-1 tyrosine
dephosphorylation, or a combination of these two potentially
independent effects in the PTP1B antibody-loaded cells. Augmentation of
the activation of the phosphatidylinositol 3`-kinase most likely
results from the increased IRS-1 tyrosyl phosphorylation with increased
or prolonged association of the phosphatidylinositol 3`-kinase enzyme
with cellular IRS-1, which activates the
enzyme(40, 41) . The activation of cellular DNA
synthesis by insulin is thought to proceed through parallel pathways
that involve the activation of p21, mediated by binding
of a Grb2-Sos complex to phosphorylated IRS-1 or Shc with activation of
downstream serine/threonine kinases(1) , as well as the
possible direct involvement of the activated phosphatidylinositol
3`-kinase itself in stimulating DNA synthesis(42) . The
quantitative contribution of each of these pathways toward DNA
synthesis may depend on the cell type under study. Inhibition of PTP1B
activity in situ may influence DNA synthesis by enhancing the
tyrosine phosphorylation of IRS-1, with increased distal signaling
mediated by phosphatidylinositol 3`-kinase or p21
activation. Alternatively, a decrease in inhibition by PTP1B may
affect the tyrosine phosphorylation state and activation of additional
downstream signaling components, such as MAP kinase, which is activated
at least in part by tyrosine phosphorylation(43, 44) .
While the present work focused on the regulation of insulin action
by PTP1B, other studies have suggested that PTP1B may influence
signaling pathways elicited by a number of growth factor receptors and
oncogenic cellular tyrosine kinases. Transfection of PTP1B in vivo into fibroblasts along with a panel of protein-tyrosine kinases
showed that, when highly overexpressed, PTP1B can dephosphorylate a
wide range of receptors including the epidermal growth factor receptor,
insulin-like growth factor 1 receptor, platelet-derived growth factor
receptors ( and
), the c-kit kinase, and the
colony-stimulating factor 1 receptor as well as the insulin receptor (38) . Catalytically inactive PTP1B has also been shown to
physically associate with specific autophosphorylation sites on the
epidermal growth factor receptor(45) . In addition,
transformation by overexpression of the neu oncogene can be
suppressed by transfection of PTP1B into NIH3T3 cells(46) .
Since the available data suggest that PTP1B influences a number of
cellular pathways, it may have a more general role in the
dephosphorylation of a variety of protein-tyrosine residues, in
addition to its demonstrated effect on balancing reversible tyrosine
phosphorylation in insulin signaling. Additional studies are necessary
to explore the relative specificity of action of PTP1B on physiological
substrates in various signaling pathways. In addition, the importance
of cellular mechanisms that may modulate the activity of PTP1B on its
cellular targets, including association of PTP1B with regulatory
subunits, proteolysis of the C terminus, and serine phosphorylation of
PTP1B, need to be studied in more
detail(47, 48, 49) .
SH-PTP2 (Syp) is a widely expressed, intracellular PTPase with SH2 domains that has recently been shown to have a positive role in mitogenic signaling induced by insulin, insulin-like growth factor 1, and epidermal growth factor in studies employing dominant-negative enzymes or microinjection of reagents that block complex formation between its SH2 domains and endogenous substrates(9, 10, 11, 12) . These data are consistent with the recognition of SH-PTP2 as the mammalian homolog of the Drosophila csw gene product, which potentiates the action of the Drosophila c-raf homolog to transmit positively signals downstream of the torso receptor tyrosine kinase(50) . Although SH-PTP2 can associate with the insulin receptor in recombinant in vitro systems(51, 52) , the site of its involvement in insulin signaling in situ is unclear since it does not appear to interact directly with the insulin receptor in intact cells, and overexpression of catalytically active SH-PTP2 protein does not affect insulin signaling(10, 11, 53, 54) . SH-PTP2 complexes with IRS-1 by its SH2 domains, in a process that activates its intrinsic PTPase activity and is likely to play a role in the cellular effects of SH-PTP2(9, 55, 56) . However, the available data would suggest that the signaling potential of SH-PTP2 requires the formation of a complex that brings the active enzyme into close proximity with downstream signaling molecules and substrates in addition to IRS-1(9, 10) .
The
present work provides strong evidence for the involvement of PTP1B in
the negative regulation of insulin signaling in a physiologically
relevant cell type. Our recent studies ()have also
demonstrated that the transmembrane PTPase LAR negatively modulates
insulin signal transduction by balancing the steady-state level of
receptor kinase activity at the cell membrane(8) . Thus,
insulin signaling is balanced at multiple levels by a number of
PTPases, including the negative influence of PTP1B and LAR at the level
of the receptor itself (and possibly also involving post-receptor
pathways) and the positive influence of SH-PTP2, which acts at a
downstream site that remains to be identified. Further studies will
help elucidate the exact mechanism of the regulation of insulin action
by these two enzymes as well as their possible involvement in defective
insulin action in disease states. The results of the present study also
demonstrate that insulin signaling can be enhanced by the specific
inhibition of PTP1B, a maneuver that has potential clinical relevance
in the treatment of insulin resistance and Type II diabetes mellitus.