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-erbAα-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 erythtoid 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.