Regulated coupling of the Neu receptor to phosphatidylinositol 3′-kinase and its release by oncogenic activation

Elior Peles, Raphael Lamprecht, Rachel Ben-Levy, Eldad Tzahar, Yosef Yarden

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The neu protooncogene encodes a tyrosine kinase receptor that is involved in the regulation of normal growth and malignant transformation. To circumvent the use of the incompletely characterized ligand of Neu, we constructed a chimeric protein composed of the ligand-binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic portions of Neu. By expressing this Neu-epidermal growth factor receptor chimera (termed NEC), we found that following stimulation by the heterologous ligand, the tyrosine kinase of Neu became associated with a phosphatidylinositol (PI) kinase activity. The association was dependent on the concentration of the ligand and was almost maximal within 30 s after ligand binding. The lipid kinase was identified as a type I PI 3′-kinase on the basis of its inhibition by Nonidet P-40 and high pressure liquid chromatography of the phosphorylated product. To confirm the identification of PI 3′-kinase as an effector of Neu, we raised antibodies to the α-isoform of the regulatory subunit of PI 3′-kinase (p85). Using these antibodies, it was possible to directly demonstrate ligand-dependent formation of a tyrosine-phosphorylated complex of NEC and PI 3′-kinase. Apparently, both PI 3′-kinase and phospholipase Cγ, another substrate of the Neu kinase, simultaneously associated with the same activated NEC molecule. Nevertheless, immunofluorescence localization of PI 3′-kinase revealed no significant cellular redistribution of the enzyme after activation of the Neu kinase. Interestingly, PI 3′-kinase was localized primarily to the cell nucleus and to confined regions of the plasma membrane. Analysis of mutants of the Neu protein indicated that the oncogenic point-mutated Neu (Glu664) was permanently coupled to PI 3′-kinase; but two nontransforming versions of the oncoprotein, a kinase-defective protein and a carboxyl-terminally deleted Neu, were devoid of the constitutive association with PI 3′-kinase. Hence, we concluded that phosphatidylinositol 3′-kinase is a physiological substrate of the Neu receptor, but the regulation of this coupling is released upon oncogenic activation.

Original languageEnglish
Pages (from-to)12266-12274
Number of pages9
JournalJournal of Biological Chemistry
Issue number17
StatePublished - 15 Jun 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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