Phosphorylation of the v-erbA protein is required for its function as an oncogene.
The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent mutated version of the chicken c-erbA alpha-encoded thyroid hormone receptor. The v-erbA gene product, a 75-kD gag/v-erbA fusion protein, is phosphorylated on Ser-16/17 of its v-erbA-encoded domain, and phosphorylation at this site is increased in vivo after activation of either the PKA or PKC signal transduction pathways. To test the hypothesis that phosphorylation of Ser-16/17 regulates gag/v-erbA protein function, mutant proteins in which Ser-16/17 had been changed to alanine or threonine residues were analyzed for their ability to inhibit erythroid differentiation of ts v-erbB or ts v-sea-transformed erythroblasts at nonpermissive temperature. Conversion of Ser-16/17 into alanine, although not affecting nuclear localization or DNA binding of the gag/erbA protein, prevented phosphorylation of the v-erbA-encoded domain of the protein both in unstimulated cells or after stimulation by PKA and PKC activators. The nonphosphorylatable AA-gag/v-erbA protein proved unable to inhibit temperature-induced differentiation of ts v-erbB and ts v-sea-transformed erythroblasts and to block expression of the erythrocyte-specific genes band 3 and carbonic anhydrase II. Back mutation of these alanine residues to serine resulted in the recovery of both normal phosphorylation levels and wild-type biological activity. In contrast, substitution of Ser-16/17 for threonine, which preserved phosphorylation in unstimulated cells but not PKA- and PKC-enhanced phosphorylation, resulted in a partially active gag/v-erbA protein. These results, together with the fact that the protein kinase inhibitor H7 resulted in both a dose-dependent inhibition of gag/v-erbA protein phosphorylation and the induction of terminal differentiation of AEV-transformed erythroblasts show that phosphorylation of gag/v-erbA protein is required for full biological activity. These results support the hypothesis that phosphorylation of the gag/v-erbA protein is important for transcriptional repression of at least some of its target genes in erythroid cells.
Glineur
C
C
Institut National de la Santé et de la Recherche Médicale U 186, Institut Pasteur, Lille, France.
Zenke
M
M
Beug
H
H
Ghysdael
J
J
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001355:Alpharetrovirus / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002642:Chick Embryo; D004900:Erythroblasts / Q000187:drug effects; D004912:Erythrocytes / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D015683:Gene Products, gag / Q000235:genetics; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D018774:Oncogene Proteins v-erbA; D009857:Oncogenes / Q000235:genetics*; D010641:Phenotype; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*
D015683:Gene Products, gag; D007546:Isoquinolines; D018774:Oncogene Proteins v-erbA; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D015682:Retroviridae Proteins, Oncogenic; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine