Prothymosin alpha is phosphorylated in proliferating stimulated cells.

Prothymosin alpha is a widely distributed polypeptide whose function, though unknown, seems to be related to cell proliferation. In vitro, it is a substrate for casein kinase-2. In this work, extracts of mitogenically stimulated murine splenic lymphocytes labeled with [32P] orthophosphate were found to contain [32P]prothymosin alpha. Phosphorylation activity was highly dependent on mitogenic activation with concanavalin A plus interleukin-2. While cells remained viable, phosphorylation increased with stimulation time in the presence of [32P]orthophosphate. Structural analysis showed that prothymosin alpha was phosphorylated at Thr residues located among its first 14 amino acids, whereas its in vitro phosphorylation by casein kinase-2 affects both Ser and Thr residues in this fragment, apparently in similar proportions. Thus, casein kinase-2 seems not to be responsible for the phosphorylation of prothymosin alpha in vivo. Prothymosin alpha was also found to be phosphorylated in proliferating murine thymocytes and HeLa cells; the phosphorylation sites were the same as in splenic lymphocytes, but the rate of phosphorylation was about 5 times lower. In thymocytes and subconfluent HeLa cells, the [32P]prothymosin alpha concentrations of the cytosolic and nuclear fractions were similar; in splenic lymphocytes, [32P]prothymosin alpha was found mostly in cytosol.

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.