erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling.
Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.
Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
O
O
Segatto
F
F
Lonardo
K
K
Helin
D
D
Wexler
F
F
Fazioli
S G
SG
Rhee
P P
PP
Di Fiore
D000595:Amino Acid Sequence; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology*; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction
D066246:ErbB Receptors; D006133:Growth Substances; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins