Regulated coupling of the Neu receptor to phosphatidylinositol 3'-kinase and its release by oncogenic activation.
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.
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
E
E
Peles
R
R
Lamprecht
R
R
Ben-Levy
E
E
Tzahar
Y
Y
Yarden
D019870:1-Phosphatidylinositol 4-Kinase; D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D019870:1-Phosphatidylinositol 4-Kinase; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins