The linoleic acid metabolite, 13-HpODE augments the phosphorylation of EGF receptor and SHP-2 leading to their increased association

Abstract

In previous studies with Syrian hamster embryo fibroblasts, we found that a specific lipoxygenase metabolite of linoleic acid, 13(S)-hydroperoxyoctadecadienoic acid (HpODE), enhanced epidermal growth factor (EGF) signal transduction in a tumor suppressor gene plus phenotype (supB+); with a diminished response to 13(S)-HpODE in a tumor suppressor gene minus phenotype (supB−). This differential response was attributed to differences in the rate of EGF receptor (EGFR) dephosphorylation. To further define the molecular basis for these observations, in this report we examine the interaction of phosphorylated EGFR with the SH2 domain-containing protein tyrosine phosphatase, SHP-2, a positive modulator of EGF dependent cell growth. SHP-2 associated with phosphorylated EGFR to a greater extent in supB+cells when compared to supB−. This differential association could not be accounted for by differences between suppressor gene phenotypes in SHP-2 protein level or mutations in the molecular sequence. The addition of 13(S)-HpODE stimulated a concentration-dependent increase in EGF-dependent phosphorylation of SHP-2 and its association with EGFR. A more dramatic response was observed in the supB+cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+and supB−cells. EGFR-SHP-2 association appears to play an important role in the regulation of EGFR signal transduction.The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SHE, Syrian hamster embryo fibroblasts; HpODE, hydroperoxyoctadecadienoic acid; supB+, tumor suppressor gene (+) phenotype; supB−, tumor suppressor gene (−) phenotype; MAPK, mitogen-activated protein kinase; ECL, enhanced chemiluminescence; PBS cmf, phosphate-buffered saline, calcium/magnesium free; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; GAP, GTPase activating protein; EGTA, ethyleneglycol-bis-(β-amino ethyl ether)N,N,N′,N′-tetraacetic acid; PTP, protein tyrosine phosphatase; SH2,src -homology domain 2; SHP-2, SH2 domain-containing tyrosine phosphatase-2.