Ligand-independent oncogenic transformation by the EGF receptor requires kinase domain catalytic activity.
The retroviral oncogene S3-v-erbB is a transduced, truncated form of the avian EGF (ErbB-1) receptor. Infection of avian fibroblasts with a retroviral vector expressing S3-v-ErbB results in ligand-independent cell transformation, which is accompanied by the assembly of a transformation-specific phosphoprotein signaling complex and anchorage-independent cell growth. It previously had been reported, using lysine-721 mutants (K721), that kinase domain function was required for ErbB-mediated cell transformation. However, since these initial reports, several studies using aspartate-813 mutants (D813) have demonstrated the ability of kinase-impaired ErbB receptors to induce mitogenic signal transduction pathways and cell transformation in a ligand-dependent manner. To determine the necessity of ErbB receptor kinase domain catalytic activity in ligand-independent cell transformation, we created S3-v-ErbB-K(-), a kinase-impaired oncoprotein constructed by replacing aspartate-813 with alanine (D813A). Subcellular routing as well as cell surface membrane and nuclear localization of the S3-v-ErbB-K(-) mutant receptor were unaffected by impairment of kinase activity. In contrast, avian fibroblasts expressing S3-v-ErbB-K(-) do not form the characteristic transformation-specific phosphoprotein complex, or induce soft agar colony growth in vitro. These results suggest that in contrast to ligand-dependent oncogenic signaling, ligand-independent cell transformation by a constitutively activated mutant form of the EGF receptor requires receptor kinase catalytic activity. In addition, these results demonstrate that phosphorylation and assembly of downstream signaling complexes require tyrosine phosphorylation events that are directly mediated by oncogenic forms of the EGF receptor.
Danielsen
Andrew J
AJ
Tumor Biology Program, Mayo Clinic, Rochester, Minnesota 55905, USA.
Maihle
Nita J
NJ
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D002642:Chick Embryo; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts; D015972:Gene Expression Regulation, Neoplastic; D008024:Ligands; D009154:Mutation; D018739:Oncogene Proteins v-erbB / Q000378:metabolism* / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D008024:Ligands; D018739:Oncogene Proteins v-erbB; D001224:Aspartic Acid; D014443:Tyrosine; D011494:Protein Kinases; D066246:ErbB Receptors; D000409:Alanine