Both SH2 Domains Are Involved in Interaction of SHP-1 with the Epidermal Growth Factor Receptor but Cannot Confer Receptor-directed Activity to SHP-1/SHP-2 Chimera

Abstract

(1995) J. Biol. Chem. 270, 21277-21284) was investigated with respect to the involved structural elements of SHP-1. Various mutants of SHP-1 were transiently expressed in 293 or COS-7 cells and analyzed for their capacity to associate with immobilized autophosphorylated EGF receptor in vitro and to dephosphorylate coexpressed EGF receptor in intact cells. Inactivating point mutation of the C-terminal SH2 domain reduced the association weakly, point mutation of the N-terminal SH2 domain reduced association strongly and the respective double mutation abolished association totally. The capacity of SHP-1 to dephosphorylate coexpressed EGF receptor was impaired by all point mutations. Truncation of the N-terminal or of both SH2 domains strongly reduced or abolished association, respectively, but the truncated SHP-1 derivatives still dephosphorylated coexpressed EGF receptor effectively.Various chimeric protein-tyrosine phosphatases constructed from SHP-1 and the closely homologous SHP-2 dephosphorylated the EGF receptor when they contained the catalytic domain of SHP-1. As native SHP-2, the chimera lacked activity toward the receptor when they contained the catalytic domain of SHP-2, despite their capacity to associate with the receptor and to dephosphorylate an artificial phosphopeptide. We conclude that the differential interaction of SHP-1 and SHP-2 with the EGF receptor is due to the specificity of the respective catalytic domains rather than to the specificity of the SH2 domains. Functional interaction of native SHP-1 with the EGF receptor requires association mediated by both SH2 domains.The phosphorylation level of activated growth factor receptors with endowed tyrosine kinase activity is the net result of tyrosine specific autophosphorylation (1) and a rapid dephosphorylation by phosphotyrosine-specific phosphatases (PTPs). 1Since important aspects of receptor signaling depend on receptor autophosphorylation, the dephosphorylation reaction is believed to attenuate the receptor signal. Therefore, the identification and characterization of the PTPs involved in growth factor receptor dephosphorylation is of high interest. The SH2 domains containing PTPs SHP-1 and SHP-2 (2, 3) have been shown to interact with multiple growth factor receptors and to modulate their signaling activity. Transient coexpression of SHP-1 with different tyrosine kinase receptors results in complete or partial dephosphorylation of PDGF ␣-and ␤-receptor, insulin-like growth receptor-1 receptor, Kit/SCF receptor, insulin receptor, EGF receptor, and HER2 (4). SHP-2 has little activity with respect to dephosphorylation of associated receptors (4, 5) and rather seems to mediate a receptor signal via as yet not fully understood mechanisms (6 -9). One important pathway seems to involve tyrosine phosphorylation of receptorbound SHP-2 at the C terminus, which leads to subsequent association of Grb2 and activation of the Sos/Ras/Raf/MAPKsignaling cascade (10, 11). Depending on the cellular context, however, SHP-2 may be involved in silencing of cer...

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

“…PTPs mediate signaling by different PKC isoforms in various cell types [65,71,[78][79][80][81][82][83][84][85][86]. A role for PTPs as mediators of PKCα effects on EGFR function is supported by several lines of evidence.…”

“…A role for PTPs as mediators of PKCα effects on EGFR function is supported by several lines of evidence. First, several non-receptor tyrosine phosphatases that contain SH2 domains bind to activated EGFRs [80][81][82][83]. Binding of PTPs to the activated EGFR attenuates the EGFR signal, presumably following dephosphorylation of key tyrosines on the activated receptor [80][81][82][83].…”

Inactivation of Protein-tyrosine Phosphatases as Mechanism of UV-induced Signal Transduction

“…Plasmids-cDNAs for murine RPTP␣ (16), murine RPTP (17), human DEP-1 (18), and human SHP-1 (19,20) were kindly provided by Drs. M. Thomas, M. Ogata, A. Ö stman, and A. Ullrich, respectively.…”

“…The pellets were either snap-frozen in liquid nitrogen and stored at Ϫ80°C or lysed directly in lysis buffer (20 mM Tris acetate, pH 7.0, 0.1 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 10 mM ␤-glycerophosphate, 50 mM sodium fluoride, 5 mM pyrophosphate, 1% Triton X-100, 1 mM benzamidine, 0.27 M sucrose, 0.1% ␤-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride) on ice for 15 min and centrifuged at 15,000 ϫ g for 10 min. Equal amounts of total cellular protein (50 g) were incubated in assay buffer (20 Cross-linking Experiments and in Vitro PDGFR Autophosphorylation-PDGF␤ receptor was partially purified from hPDGF␤R overexpressing TRMP cells (24) kindly provided by Dr. A. Kazlauskas as described earlier (25). PDGFR-containing micelle fractions were mocktreated or irradiated as described above.…”

“…High PTP levels should diminish the effect of UV. The PTP SHP-1 has been implicated in negative regulation of the EGFR in A431 cells (20,23). 2 UV treatment of these cells led to elevation of EGFR phosphorylation, at a degree comparable with ligandinduced receptor phosphorylation (9).…”