(Received for publication, July 7, 1995; and in revised form, August 25, 1995)
From the
The insulin receptor-mediated signal transduction pathway involves insulin receptor substrate 1 and a variety of proteins containing Src homology-2 (SH2) domains, such as phosphatidylinositol 3-kinase, Grb2, and protein-tyrosine phosphatase 1D (PTP1D). Upon insulin stimulation of baby hamster kidney cells overexpressing the IR, the catalytically inactive mutant of PTP1D, C463A, becomes tyrosine-phosphorylated and coprecipitates with Grb2. Tyrosine phosphorylation of this mutant is significantly reduced when wild type PTP1D is coexpressed. Substitution of tyrosine residues 546 and 584 with phenylalanine abrogates tyrosine phosphorylation of the catalytically inactive mutant and abolishes its interaction with Grb2.
The insulin receptor (IR) ()belongs to a family of
structurally related transmembrane growth factor receptors that exhibit
intrinsic tyrosine kinase activity(1, 2) . Ligand
stimulation induces rapid activation of the kinase activity, resulting
in autophosphorylation of the
-chain of the receptor itself and
phosphorylation of several other cytoplasmic
proteins(1, 3) , including insulin receptor substrate
1 (IRS1)(4) , the p85 regulatory subunit of
phosphatidylinositol 3-kinase(5, 6) ,
Grb2(7, 8) , Nck(9) , and protein-tyrosine
phosphatase 1D (PTP1D)(10) .
PTP1D(11) , also called SH-PTP2(12) , SH-PTP3(13) , PTP2C(14) , or Syp (15) , is a cytosolic enzyme with two Src homology 2 (SH2) domains and is expressed in a wide variety of cell types(11, 12, 13, 14, 16) . Upon stimulation of cells with different growth factors, PTP1D associates via its SH2 domains with several tyrosine kinases and becomes tyrosine-phosphorylated(11, 15, 17, 18, 19, 20, 21, 22) . This latter event leads to an association with Grb2 in various signaling pathways(23, 24, 25, 26, 27) . So far, tyrosine phosphorylation of PTP1D has not been detected in vivo upon insulin stimulation(10, 28) . Recent studies, however, have demonstrated PTP1D as a component of the insulin receptor-mediated signal(29, 30, 31) , yet its exact significance for the insulin response overall is still poorly understood.
We show here that the catalytically inactive mutant of PTP1D, C463A (32) , becomes phosphorylated on tyrosine and coprecipitates with Grb2 after insulin treatment in BHK-IR cells. Furthermore, we demonstrate that tyrosines at positions 546 and 584 are responsible for this phosphorylation and that substitution of these residues with phenylalanine abolishes Grb2 association. Coexpression of wild type PTP1D and the catalytically inactive mutant leads to significantly reduced tyrosine phosphorylation of the latter, suggesting that PTP1D may modulate its own tyrosine phosphorylation state. Our findings provide new insights into mechanisms of PTP1D action within the insulin receptor signal.
Antibodies used were affinity-purified rabbit polyclonal anti-PTP1D antibody raised against a C-terminal PTP1D peptide (amino acids 585-597)(11) , mouse monoclonal anti-PTP1D antibody (Transduction Laboratories), mouse monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc.), mouse monoclonal anti-Grb2 antibody (Upstate Biotechnology, Inc.), and mouse monoclonal anti-hemagglutinin antibody 12CA5 (Boehringer Mannheim). As secondary antibodies, goat anti-mouse or anti-rabbit conjugates (Bio-Rad) were used. For immunoblot detection, the ECL system from Amersham was utilized. Stripping and reprobing of blots were performed according to the manufacturers' recommendations.
The construction of cytomegalovirus promoter-based expression plasmids for PTP1D has been described previously(11, 33) .
Following SDS-polyacrylamide gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) and immunoblotted.
For binding assays, purified Grb2-GST (glutathione S-transferase) fusion protein was coupled to glutathione-Sepharose. Approximately 2 µg of Grb2-GST was incubated with 10 µl of glutathione-Sepharose beads and rotated for 2 h at 4 °C. Beads were then washed three times with 1 ml of HNTG buffer, and Grb2-coupled beads were incubated with BHK-IR lysates for 4 h at 4 °C.
After stimulation with growth factors, the SH2-containing
protein-tyrosine phosphatase 1D associates with various tyrosine
kinases and becomes
tyrosine-phosphorylated(11, 15, 18) . PTP1D
association with the IR has been observed in
vitro(19, 20, 21, 22) and in vivo, ()but tyrosine phosphorylation of the
phosphatase has not yet been detected in intact
cells(10, 28) . To investigate the role of PTP1D in
the insulin signal, we transiently expressed the wild type phosphatase
and the catalytically inactive mutant C463A in BHK-IR cells. We found
that overexpressed PTP1D was weakly phosphorylated on tyrosine after
insulin stimulation (Fig. 1, lanes 6-10).
Surprisingly, the PTP1D phosphorylation state was considerably higher
when the C463A mutant was used (Fig. 1, lanes
11-15). Reprobing with anti-PTP1D antibody demonstrated that
comparable amounts of protein were present in all relevant lanes (Fig. 1, lower panel).
Figure 1:
Tyrosine phosphorylation of PTP1D after
insulin stimulation in BHK-IR cells. After transfection of equal
amounts of cells with empty expression plasmid (control; lanes
1-5), PTP1D (lanes 6-10), and the
catalytically inactive C463A mutant expression plasmid (lanes
11-15), cells were starved for 16 h and stimulated with 5
10
M insulin for the indicated
time. After lysis, PTP1D was immunoprecipitated with polyclonal
anti-PTP1D antibody. Following gel electrophoresis, the
tyrosine-phosphorylated proteins were detected by immunoblot analysis
with anti-phosphotyrosine (
PY) antibody (upper
panel). Identification of PTP1D was revealed by reprobing with
monoclonal anti-PTP1D antibody (lower panel). Molecular weight
markers are indicated on the left. IR
, insulin receptor
-chain; IgG, immunoglobulin heavy
chain.
To determine the identity of the phosphorylated tyrosine residues, we generated tyrosine mutations in the inactive form of PTP1D using standard in vitro mutagenesis techniques. These Tyr/Phe mutants were then transiently expressed in BHK-IR cells. A single Tyr/Phe substitution at position 546 of the catalytically inactive phosphatase resulted in a significant decrease in phosphotyrosine content, as shown in Fig. 2(upper panel, lane 10). The combination of two tyrosine substitutions in the C-terminal tail (Y546/584F, Fig. 2, lower panel) abolished beyond detection tyrosine phosphorylation of the inactive form of PTP1D (Fig. 2, upper panel, lane 8). Reprobing with anti-PTP1D antibody demonstrated again that the observed differences were not due to different levels of protein expression (Fig. 2, middle panel).
Figure 2:
Identification of the tyrosine
phosphorylation sites of catalytically inactive PTP1D. Equal amounts of
BHK-IR cells were transfected with empty expression plasmid (control; lanes 1 and 2), PTP1D (lanes 3 and 4), C463A (lanes 5 and 6), C463A-Y546/584F (lanes 7 and 8), or C463A-Y546F (lanes 9 and 10) expression plasmid, starved for 16 h, and stimulated with
5 10
M insulin for 15` (lanes
2, 4, 6, 8, and 10) or left unstimulated (lanes 1, 3,
5, 7, and 9). After lysis, PTP1D constructs were
immunoprecipitated with polyclonal anti-PTP1D antibody (upper and
middle panels), and following gel electrophoresis,
tyrosine-phosphorylated proteins were detected by immunoblot analysis
with anti-phosphotyrosine antibody (upper panel).
Identification of PTP1D was revealed by reprobing with anti-PTP1D
antibody (middle panel). Molecular weight marker is indicated
on the left. IgG, immunoglobulin heavy chain. Lower
panel, schematic of PTP1D (tyrosine residues are
indicated).
To investigate the functional significance of the identified tyrosine phosphorylation sites, we incubated aliquots of the transfected BHK-IR lysates with a Grb2-GST fusion protein and determined the interaction with the different PTP1D forms by coprecipitation. As indicated in Fig. 3, the amount of associated catalytically inactive PTP1D C463A was markedly increased in comparison with wild type PTP1D. Substitution of tyrosine 546 with phenylalanine (Y546F) reduced this association dramatically and mutation of both tyrosine residues in 1D (Y546/584F) abolished the interaction between Grb2 and PTP1D.
Figure 3: In vitro association of PTP1D and Grb2 following insulin stimulation. Aliquots of the same lysates used in Fig. 2were incubated with Grb2-GST fusion protein. Coprecipitated PTP1D was detected by immunoblot analysis with monoclonal anti-PTP1D antibody.
The observed high level of tyrosine phosphorylation in the C463A mutant in contrast to wild type PTP1D suggested the possibility that the phosphatase activity of PTP1D may modulate its own state of tyrosine phosphorylation. To investigate this possibility further, we coexpressed the catalytically active and inactive forms of PTP1D in BHK-IR cells. In order to be able to distinguish between the two forms, the C463A mutant was modified with a hemagglutinin (HA) tag at the C terminus, permitting selective immunoprecipitation of this form of PTP1D with an anti-HA antibody. The coexpression of tagged and untagged inactive mutants served as a control. To verify the specificity of the hemagglutinin antibody, the untagged C463A variant was first expressed alone. As shown in Fig. 4(lanes 6 and 8), tyrosine phosphorylation of the HA-tagged, inactive C463A-HA was clearly lower when coexpressed with wild type PTP1D rather than with the untagged inactive PTP1D mutant. These results demonstrate dephosphorylation of the tagged C463A-HA mutant by active PTP1D and therefore suggest an intermolecular transdephosphorylation mechanism. We cannot, however, rule out the possibilities of at least a partial contribution of an intramolecular mechanism or the involvement of other protein-tyrosine phosphatases of unknown identity.
Figure 4:
Dephosphorylation of catalytically
inactive PTP1D. The hemagglutinin-tagged, catalytically inactive PTP1D
mutant, C463A-HA, was coexpressed together with untagged, catalytically
active (lanes 5 and 6) or inactive (lanes 7 and 8) PTP1D. Empty vector (lanes 1 and 2) and the untagged, catalytically inactive mutant alone (lanes 3 and 4) served as controls. Following 16 h of
starvation, cells were stimulated for 15 min with 5
10
M insulin (lanes 2, 4, 6, and 8) or left unstimulated (lanes 1, 3, 5, and 7). After cell lysis, the tagged PTP1D protein was
precipitated with anti-hemagglutinin antibody, and following gel
electrophoresis, the tyrosine phosphorylation pattern was detected by
immunoblotting with an anti-phosphotyrosine antibody (upper
panel). The same blot was reprobed with monoclonal anti-PTP1D
antibody (lower panel). IgG, immunoglobulin heavy
chain.
Taken together, our data
demonstrate that catalytically inactive PTP1D becomes markedly
tyrosine-phosphorylated at positions 546 and 584 upon stimulation of
BHK-IR cells with insulin. These tyrosine phosphorylation targets match
those utilized after -platelet-derived growth factor receptor
activation(23) . Moreover, we demonstrate that the inactive
C463A mutant coprecipitates more efficiently with Grb2 after insulin
stimulation relative to wild type PTP1D and that tyrosine residues 546
and 584 are required for the PTP/Grb2 interaction. For tyrosine 546,
such an involvement in the interaction with Grb2 has been shown
previously in the context of other signal transduction pathways (23, 24) . Furthermore, we demonstrate that inactive
PTP1D, tyrosine-phosphorylated in response to BHK-IR cell stimulation
with insulin, is efficiently dephosphorylated when coexpressed with its
catalytically active wild type homolog, supporting a mechanistic model
in which PTP1D is able to modulate its own phosphorylation and
activation state as part of the insulin-triggered intracellular signal.
On the basis of in vitro data, such a possibility had been
suggested previously(37) .
After our earlier proposal of an important role for PTP1D in growth factor signal transmission and regulation(11) , which was supported recently by the demonstration of its involvement in insulin-induced Ras and MAP kinase activation(28, 29, 31) , the observations presented here contribute important insights toward the understanding of molecular mechanisms governing the action of biological signal transmitters such as growth factors and insulin.