(Received for publication, July 26, 1995)
From the
Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.
Protein-tyrosine phosphatase 1D (PTP1D), ()also known
as SH-PTP2 (1) , SH-PTP3(2) , PTP2C(3) , and
Syp(4) , is a cytosolic enzyme with two Src homology 2 (SH2)
domains that is expressed in a wide variety of cell
types(1, 3) . Upon stimulation of cells with different
growth factors, PTP1D becomes tyrosine-phosphorylated and associates
with activated receptor-type or cytoplasmic tyrosine kinases via its
SH2 domains(4, 5, 6) . Interaction with the
platelet-derived growth factor receptor (PDGFR) kinase results in
activation of PTP1D phosphatase activity(5) .
In addition to directly interacting with activated protein-tyrosine kinases, PTP1D also binds to insulin receptor substrate-1 (IRS-1)(7) , the major substrate of the insulin receptor (IR) tyrosine kinase, which is thought to serve as a ``docking protein'' required for the recruitment of different SH2 domain-containing proteins to the IR signaling pathway. Previous reports indicate that insulin stimulation of the IR causes IRS-1 association with the 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase and the adapter proteins Grb2 and Nck (reviewed in (8) ). The physiological significance of the interaction of IRS-1 with these SH2 domain proteins, however, remains unclear. Recent studies involving catalytically inactive PTP1D have demonstrated that rather than playing a negative regulatory role, PTP1D is required for the transduction of signals triggered by activation of the PDGFR (9, 10) and the IR(11, 12, 13) . Upon PDGF activation, PTP1D becomes phosphorylated on tyrosines and appears to serve as an adapter, linking PDGFR to the Grb2-Sos complex(9) . In contrast to the PDGFR pathway, IR activation does not result in PTP1D phosphorylation on tyrosine residues(7) , indicating that the positive effect of PTP1D in IR signaling is mediated by another mechanism.
In the present study, we demonstrate direct interaction between activated IR and PTP1D, primarily involving the SH2 domain proximal to the region specifying phosphatase activity. Phosphorylation of Tyr-1146 in the IR promotes binding of IRS-1 and formation of a complex that enhances insulin-dependent glucose uptake. Our data suggest a positive role for PTP1D in insulin action through its critical involvement in the formation of a multiprotein signaling complex.
Rat1 cells stably expressing
human IR isotype A (Rat1-IR) were maintained in Dulbecco's
modified Eagle's/F12 medium supplemented with 10% fetal calf
serum. After 16 h of starvation, cells were stimulated with insulin
(10M) for 20 min and lysed in the same
lysis buffer.
Human lymphoblastic IM-9 cells (ATCC: CRL 159) were
grown in RPMI medium supplemented with 10% fetal calf serum. Prior to
insulin stimulation, cells were starved for 16 h in serum-free medium.
After insulin stimulation (20 min, 10M)
cells were lysed in the same lysis buffer, PTP1D was immunoprecipitated
from crude lysates of unstimulated IM-9 cells with polyclonal
affinity-purified antibodies, and IR was immunoprecipitated from
unstimulated or stimulated cells with 83-14 antibodies. PTP1D was
detected in Western blotting with polyclonal anti-PTP1D antibodies.
To obtain semipurified IR (Y1146F) and
IRCT mutant preparations, BHK-21 cells were transiently
transfected with corresponding cDNAs, and mutant receptors were
isolated on WGA-Sepharose using the procedure described above.
For binding assays, approximately 2 µg of different GST-fusion proteins were bound to 10 µl of glutathione-Sepharose 4B (Pharmacia) beads, and after washing 10 µl of semipurified autophosphorylated IR preparation was added. Incubation was carried out for 1 h at 4 °C. Unbound material was washed away with HNTG (4 times, 1 ml each), and associated IR was eluted with SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and identified with monoclonal anti-phosphotyrosine antibodies (5E2; (14) ) in Western blotting.
For the phosphopeptide competition assay, approximately 2 µg of (N+C)-GST-fusion protein was bound to 10 µl of glutathione-Sepharose beads. Before addition of the IR preparation (10 µl) beads were preincubated with 100 µl of different phosphopeptides (100 µM final concentration) for 30 min. Incubation with IR was performed for 1 h at 4 °C. Sepharose beads were then washed 4 times with 1 ml of HNTG, and bound IR was separated by SDS-polyacrylamide gel electrophoresis and detected with 5E2 antibodies in immunoblotting.
To further examine the role of PTP1D in insulin action, we used the stably transfected baby hamster kidney cells (BHK-IR) (16) and the Rat1 fibroblasts (Rat1-IR)(17) , expressing the human IR, and nontransfected human lymphoblastic IM-9 cells. Upon stimulation of intact cells with insulin, PTP1D was detected in IR immunoprecipitates of all three cell lines using anti-PTP1D antibodies and immunoblot analysis. In BHK-IR and IM-9 cells (Fig. 1) and in Rat1-IR fibroblasts (data not shown), PTP1D associated with the IR in an insulin-dependent manner. Based on this coimmunoprecipitation analysis, we estimated that in IM-9 cells approximately 2-5% of total cellular PTP1D associated with the receptor, which is similar to earlier findings for PTP1D association with the PDGFR(22) . In contrast to PDGFR and in accordance with a previous report(7) , PTP1D tyrosine phosphorylation was not observed upon insulin treatment of BHK-IR, Rat1-IR, or IM-9 cells (not shown). Taken together, these results demonstrate that in intact cells PTP1D does not serve as a substrate for the IR tyrosine kinase but associates with the IR in an activation-dependent manner.
Figure 1: Insulin-dependent association of PTP1D and IR. A, BHK-IR cells. After transfection with control plasmid (lanes 1 and 2) or PTP1D expression plasmid ((5) ) (lanes 3 and 4), cells were stimulated with insulin, and after lysis, IR was immunoprecipitated with 83-14 monoclonal antibody. PTP1D was detected in IR immunoprecipitates by immunoblot analysis using anti-PTP1D antibodies. B, human lymphoblastic IM-9 cells. After 16 h of starvation, cells were left unstimulated (lanes 1 and 2) or stimulated with insulin (lane 3). Endogenous PTP1D was detected by immunoprecipitation with anti-PTP1D antibodies (lane 1) or coimmunoprecipitation with the 83-14 anti-IR monoclonal antibody (lanes 2 and 3) followed by immunoblot analysis with anti-PTP1D antibodies.
Figure 2: Association of activated IR with PTP1D SH2 domains in vitro.A, binding of semipurified autophosphorylated IR to different PTP1D SH2 domain-GST-fusion proteins. B, effect of SH2 domain point mutation on PTP1D/IR interaction. PTP1D (lanes 1 and 2), PTP1D/R32K (lanes 3 and 4), and PTP1D/R138K (lanes 5 and 6) were transiently expressed in BHK-IR cells. Cells were left unstimulated (lanes 1, 3, and 5) or stimulated with insulin (lanes 2, 4, and 6) for 20 min. After cell lysis, IR was immunoprecipitated with monoclonal antibody 83-14, and after gel electrophoresis, the presence of PTP1D wild type and mutant proteins in IR immunoprecipitates was detected by immunoblot analysis with anti-PTP1D antibody. C, identification of IR binding site for PTP1D. Phosphotyrosine peptides representing IR cytoplasmic domain tyrosine residues and upstream (5) and downstream (5) flanking amino acids were employed in in vitro association competition experiments between semipurified IR and the (N+C)-GST fusion protein. control, no phosphotyrosine peptide added.
Further proof of the involvement of the PTP1D C-terminal SH2 domain in its interaction with the IR was obtained in association experiments with PTP1D mutants containing lysine substitutions of arginine residues that are known to be critical for SH2 binding functions. As shown in Fig. 2B, PTP1D/R32K, which contains an intact C-terminal SH2 domain, was still able to associate with activated IR, while PTP1D/R138K was not. From these results we concluded that PTP1D associates directly with activated IR through SH2 domain-mediated interactions and that the C-SH2 domain of PTP1D is necessary but not sufficient for full PTP1D binding capacity to the IR. While the N-terminal SH2 domain was found previously to be responsible for PTP1D binding to activated epidermal growth factor-R, PDGFR, and tyrosine-phosphorylated IRS-1(23) , no proteins have so far been reported to associate with the PTP1D C-proximal SH2 domain.
Of the two major
phosphotyrosine-containing proteins from crude lysates of
insulin-stimulated BHK-IR cells that bound to the (N+C)-GST fusion
protein, the 95-kDa band was identified as the IR -chain, and the
second of about 180 kDa was likely to represent endogenous IRS-1 (Fig. 3). Interestingly, phosphopeptide 1146 completely blocked
IR
-chain association with the (N+C)-GST construct, while the
change of 180-kDa protein binding was within the experimental range of
variation (Fig. 3, lanes 4 and 5). This result
indicated that binding of the tyrosine-phosphorylated 180-kDa protein
to the SH2 domain fusion protein was independent of the IR interaction
and that it occurred through a binding motif different from that of
phosphopeptide 1146.
Figure 3: Anti-phosphotyrosine immunoblot analysis of IR and 180-kDa protein association with PTP1D (N+C)-GST fusion protein. Lanes 1 and 2, crude lysates of starved or insulin-stimulated BHK-IR cells, respectively. Lanes 3-5, association of proteins from crude lysate of insulin-stimulated BHK-IR cells associated with GST protein control (lane 3), PTP1D (N+C)-GST fusion protein (lane 4), and (N+C)-GST fusion protein in the presence of 1146 peptide (lane 5). Phosphotyrosine-containing proteins were detected by immunoblotting with monoclonal anti-phosphotyrosine antibodies (5E2).
To further demonstrate Tyr-1146 involvement in
the interaction of activated IR and PTP1D, wild type receptor and
mutant proteins with a C-terminal 43-amino acid deletion (IRCT) or
a tyrosine to phenylalanine mutation at position 1146 (IRY1146F) were
partially purified from transfected BHK cells and after
autophosphorylation used in an in vitro association experiment
with the PTP1D (N+C)-GST fusion protein. As shown in Fig. 4(lanes 4 and 5), both IR and IR
CT
bound to the PTP1D SH2 domain fusion protein, while the Y1146F mutant
did not (Fig. 4, lane 6). Thus, using two independent
approaches, we identified phosphotyrosine 1146 as a binding site for
the signal-transducing phosphatase PTP1D. This is of particular
interest since this residue has been implicated in the initiation of
the IR autophosphorylation cascade and positive insulin signal
transmission ((25) ; reviewed in (8) and (26) ).
Figure 4:
Y1146F mutation abrogates binding of PTP1D
(N+C)-GST fusion protein to IR. BHK-21 cells were separately
transfected with IR, IRCT, and IRY1146F expression plasmids. IR
and IR Y1146F mutant proteins were partially purified on WGA-Sepharose
and autophosphorylated in vitro. Lanes 4-6 represent
binding of activated IR constructs (IR, IR
CT, and IRY1146F,
respectively) to the PTP1D (N+C)-GST fusion protein. Lanes
1-3, negative control (binding of the same constructs to GST
protein). Activated IR and IR mutants were detected by immunoblotting
with monoclonal anti-phosphotyrosine antibody
(5E2).
We are unable to explain the divergent findings by others, which reported phosphotyrosine 1322 as the IR binding site for Syp/PTP1D (18, 19, 20, 21) . Comparison of the SH2 domain-GST fusion protein/IR binding experiments indicates, however, that only marginal competition was achieved with pY1322 peptide at a concentration of 750 µM(21) , while our data demonstrate complete inhibition of association at 100 µM of pY1146 phosphopeptide. This suggests Tyr(P)-1322 as a potentially secondary binding site or as a site of weak interaction with the N-terminal SH2 domain in vitro.
Figure 5:
Effect of PTP1D overexpression on
IRIRS-1 complex formation. BHK-IR cells were transfected with
control plasmid (lanes 1 and 2), PTP1D expression
plasmid (lanes 3 and 4), IRS-1 expression plasmid (lanes 5 and 6), or the latter two combined (lanes 7 and 8). Cells were starved for 16 h and then
left unstimulated (lanes 1, 3, 5, and 7) or stimulated with insulin (lanes 2, 4, 6, and 8), followed by immunoprecipitation with
83-14 anti-IR monoclonal antibody. Analysis of the
immunoprecipitates included gel electrophoresis and immunoblot
detection of IRS-1 with a polyclonal anti-peptide antibody (after
stripping PTP1D antibody shown in B). B, PTP1D
detection on the same nitrocellulose filter using anti-PTP1D
antibody.
Figure 6:
Effect of PTP1D overexpression on glucose
transport in BHK-IR cells. BHK-IR cells were transfected with control
plasmid, PTP1D expression plasmid, IRS-1 expression plasmid, or the
latter two combined. Cells were stimulated with different insulin
concentrations, and glucose uptake was determined as described under
``Experimental Procedures.'' The extent of glucose transport
within a 5-min period in the presence of insulin is shown in percentage
of control BHK-IR cells (C = 100%). Values represent
the mean of triplicate determinations; standard deviation did not
exceed 10% for all the points presented on the figure.
, 0;
, PTP1D;
, PTP1D C/A;
, IRS-1;
,
PTP1D + IRS-1;
, PTP1D C/A +
IRS-1.
A positive role for PTP1D in receptor-type kinase-mediated signal transduction was initially suggested because of its structural homology to the Drosophila corkscrew gene product, which by genetic criteria had been identified as a downstream transducer of the torso receptor-type kinase signal(1, 30) . Recently this proposed function was confirmed by the demonstration of inhibitory effects on insulin-induced mitogenic responses in cells overexpressing an inactive mutant of PTP1D(11, 12, 13) . While neither the reaction partners of PTP1D nor the pathway requiring an active phosphatase in insulin-induced mitogenic signaling are currently identified, it is clear that the positive role in insulin action suggested by the adapter function of PTP1D between the IR and IRS-1 represents a different mechanism, since it does not require an intact catalytic function. The overall significance of the PTP1D adapter function for the various cellular responses induced by insulin in different cell types is currently unclear, but our results strongly suggest that it is involved in a critical aspect of the insulin signal, the regulation of glucose uptake.