PHOSPHORYLATION OF HUMAN AND BOVINE PROTHYMOSIN-ALPHA INVIVO

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [P-32] orthophosphoric acid, th ey synthesized [P-32] prothymosin alpha. The incorporated radioactivit y was resistant to DNase and RNases A, T1, and T2, but could be comple tely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova , T., Koonin, E.V., Agol, V.I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [P-32]proth ymosin alpha indicated that serine residues were phosphorylated. Analy sis of peptides derived from bovine prothymosin alpha and human [P-32] prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, an d 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the pho sphopeptide with Asp-N followed by C 1 8 reversed-phase column chromat ography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N -terminal 5-mer derived from the unmodified 14-mer. Positive identific ation of the phosphorylated amino acid was obtained by colliding the 1 4-residue phosphopeptide with helium in the mass spectrometer and find ing phosphate only in a nested set of phosphorylated fragments compose d of the first three, four, and five amino acids. The results prove th at prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurr ed throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation ti me. Although prothymosin alpha is known to be essential for cell divis ion, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.