Phosphotyrosine 1173 mediates binding of the protein-tyrosine phosphatase SHP-1 to the epidermal growth factor receptor and attenuation of receptor signaling (original) (raw)
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Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling
Molecular and cellular biology, 2003
The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation t...
Journal of Biological Chemistry, 2003
The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542For Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.
Biochemical Journal
We used a substrate-trapping technique to search for substrates of protein tyrosine phosphatase (PTP) 1B. A catalytically inactive form of this enzyme forms a stable, phosphotyrosine-dependent complex with epidermal growth factor receptor (EGFR) both in vitro and in cells. PTP1B also interacts with activated platelet-derived growth factor receptor (PDGFR) but not with colony-stimulating factor 1 receptor (CSF-1R). After binding to EGFR, PTP1B becomes tyrosine-phosphorylated at Tyr-66, a site that conforms to the consensus binding sequence for the Src homology 2 (SH2) domains of the adapter protein Grb2. This tyrosine phosphorylation is correlated with a 3-fold increase in PTP catalytic activity. These findings suggest that PTP1B selectively regulates specific activated receptor protein tyrosine kinases (RPTKs) in vivoand might itself be regulated by such receptors.
SHP-2 is involved in heterodimer specific loss of phosphorylation of Tyr771 in the PDGF β-receptor
Oncogene, 2002
We have previously shown that the binding site for GTPase activating protein of Ras (RasGAP) in the PDGF b-receptor, Tyr771, is phosphorylated to a much lower extent in the heterodimeric con®guration of PDGF a-and b-receptors, compared to the PDGF b-receptor homodimer. The decreased recruitment of the RasGAP to the receptor leads to prolonged activation of the Ras/ MAP kinase pathway, which could explain the increase in mitogenicity seen upon induction of heterodimers. The molecular mechanism underlying these dierences was investigated. We could show that the loss of phosphorylation of Tyr771 was dependent on presence of intact binding sites for the protein tyrosine phosphatase SHP-2 on the PDGF b-receptor. Thus, in PDGF receptor mutants in which binding of SHP-2 was lost, a higher degree of phosphorylation of Tyr771 was seen, while other phosphorylation sites in the receptor remained virtually unaected. Thus, SHP-2 appears to play an important role in modulating phosphorylation of Y771, thereby controlling RasGAP recruitment and Ras/MAP kinase signaling in the heterodimeric con®guration of the PDGF receptors.
Journal of Biological Chemistry, 1999
The two SH2 (Src homology domain 2) domains present in phospholipase C-␥1 (PLC-␥1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 > > pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 > > pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-␥1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-␥1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-␥1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-␥1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.
Oncogene, 2003
Receptor tyrosine kinases (RTKs) such as the fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor are overexpressed in a variety of cancers. In addition to overexpression, the FGFRs are found mutated in some cancers. The Src homology 2 domaincontaining phosphotyrosine phosphatase (SHP2) is a critical mediator of RTK signaling, but its role in oncogenic RTK-induced cell transformation and cancer development is largely unknown. In the current report, we demonstrate that constitutively activated FGFR3 (K/E-FR3) transforms NIH-3T3 cells, and that SHP2 is a critical mediator of this transformation. Infection of K/E-FR3-transformed 3T3 cells with a retrovirus carrying a dominant-negative mutant of SHP2 (C/S-SHP2) retarded cell growth, reversed the transformation phenotype and inhibited focus-forming ability. Furthermore, treatment of K/E-FR3-transformed NIH-3T3 cells with PD98059 or LY294002, specific inhibitors of MEK and PI3K, respectively, inhibited focus formation. Biochemical analysis showed that K/E-FR3 activates the Ras-ERK and the PI3K signaling pathways, and that the C/S SHP2 mutant suppressed this effect via competitive displacement of interaction of the endogenous SHP2 with FRS2. However, the C/S SHP2 protein did not show any effect on receptor autophosphorylation, FRS2 tyrosine phosphorylation or interaction of Grb2 with K/E-FR3 or FRS2. Together, the results show that K/E-FR3 is transforming and that the Ras-ERK and the PI3K-Akt signaling pathways, which are positively regulated by SHP2, are important for K/E-FR3-induced transformation.
ROS and EGF are antagonistic cues controlling SHP-2 dimerization 1
2012
Department of Biology, University of Rome Tor Vergata, Via della ricerca scientifica, 00133 Rome, Italy 4 IRCCS San Raffaele “La Pisana”, via della Pisana 235, 00163 Rome, Italy 5 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina 6 Elena 299, 00161 Rome, Italy 7 IRCCS Fondazione Santa Lucia, 00143, Rome, Italy 8 # Address correspondence to aurelio.pio.nardozza@uniroma2.it, cesareni@uniroma2.it, 9 castagnoli@uniroma2.it, Fax: +39 06 2023500 10