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.