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

J Biol Chem. 1992 Jun 15;267(17):12266-74.

Abstract

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 alpha-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 C gamma, 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Phosphatidylinositol 4-Kinase
  • 3T3 Cells
  • Animals
  • Blotting, Western
  • Chromatography, High Pressure Liquid
  • Enzyme Activation
  • Fluorescent Antibody Technique
  • Gene Expression Regulation, Enzymologic*
  • Mice
  • Phosphorylation
  • Phosphotransferases / metabolism*
  • Precipitin Tests
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Receptor, ErbB-2
  • Recombinant Fusion Proteins / metabolism

Substances

  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Phosphotransferases
  • 1-Phosphatidylinositol 4-Kinase
  • Protein-Tyrosine Kinases
  • Receptor, ErbB-2