Human, murine and chicken c‐ets‐1 proteins migrate in SDS‐polyacrylamide gels as multiple species. We show here that most if not all of this heterogeneity is due to phosphorylation events occurring predominantly on serine and to a lesser extent on threonine residues. These phosphorylations can be specifically and rapidly stimulated by treatment with the calcium ionophore A23187 or abolished by lowering the extracellular calcium concentration to less than 0.1 microM. The products encoded by c‐ets‐2 are also phosphorylated in a Ca2+‐dependent manner, indicating that these modifications have been conserved in the products encoded by different members of the same gene family. In thymocytes, where the expression of c‐ets‐1 is elevated as compared with other cell types, c‐ets‐1 protein phosphorylation occurs after stimulation with mitogenic doses of concanavalin A, is short lived and is strictly dependent upon extracellular Ca2+ sources. This suggests that the c‐ets‐1 gene product may play a role in the Ca2+‐mediated early events linked to T‐cell activation.

The c‐erbA proto‐oncogene encodes a nuclear receptor for thyroid hormone (T3), which is believed to stimulate transcription from specific target promoters upon binding to cis‐acting DNA sequence elements. The v‐erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand‐independent version of this nuclear receptor. The v‐erbA product inhibits terminal differentiation of avian erythroblasts, presumably by affecting the transcription of specific genes. We show here that the c‐erbA‐encoded nuclear receptor (p46c‐erbA) is phosphorylated on serine residues on two distinct sites. One of these sites, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v‐erbA‐encoded P75gag‐v‐erbA protein. This site is located in the amino‐terminal domain of these molecules, 21 amino acids upstream of the DNA‐binding region. Phosphorylation of this site in both p46c‐erbA and P75gag‐v‐erbA is enhanced 10‐fold following treatment of cells with activators of either protein kinase C or cAMP‐dependent protein kinase. Since cAMP‐dependent protein kinase phosphorylates both p46c‐erbA and P75gag‐v‐erbA in vitro at the same site as that observed in vivo, at least part of the cAMP‐dependent phosphorylation of erbA molecules in cells could result from direct phosphorylation by this enzyme. The possible role phosphorylation may play in the function of the erbA‐encoded transcriptional factors is discussed.

In chicken cells, we previously identified a set of proteins (p58‐64) structurally related to, but distinct from, the products encoded by the c‐ets proto‐oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58‐64 to other ets‐related proteins, including the v‐ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c‐ets, the translation product of the chicken (Ck) c‐ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy‐terminal domain and close to the amino‐terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c‐ets which is transcribed as a 4.0‐kb RNA species in most chicken tissues. We also identified the human (Hu) c‐ets‐2‐encoded products as two proteins of 60 and 62 kd, highly related to chicken p58‐64. This, together with the fact that the amino acid sequence of the cDNA encoding p58‐64 is 95% identical to the reported partial sequence of a Hu‐c‐ets‐2 cDNA, indicates that p58‐64 are the translation products of the Ck‐c‐ets‐2 gene.

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Conformational studies were performed on the synthetic tricosapeptide N-acetyl-SKKALKKLQKEQEKQRKKEERAL-amide, representing the highly basic segment (residues 30-52) of the N-terminal extension of yeast cytoplasmic aspartyl-tRNA synthetase. Circular dichroism experiments show that, in aqueous solution at neutral pH, the peptide adopts a random conformation. The effects of pH, temperature, addition of trifluoroethanol (TFE), and titration with polyanions on the conformation of the peptide were studied. In TFE or in the presence of an equimolar concentration of (phosphate)18, the peptide adopts a 100% alpha-helical conformation. A partially alpha-helical conformation is induced by (phosphate)4 or d(pT)8 (respectively 40% and 35% helical content). Raising the pH in aqueous solution promotes 75% alpha-helicity, with a transition pK of 9.9 reflecting deprotonation of lysine residues. On the basis of these results, nuclear magnetic resonance studies were carried out in TFE as well as in aqueous solution in the presence of (phosphate)18, to determine the structure of the molecule. Complete 1H resonance assignments were obtained by conventional two-dimensional NMR techniques. A total of 138 interproton constraints derived from NOESY experiments were used to calculate the three-dimensional structure by a two-stage distance geometry/simulated annealing procedure. The two deduced structures were highly similar and show that nine cationic residues are segregated on one face of a helical structure, providing an ideal polycationic interface for binding to polyanionic surfaces.

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