Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identifi ed mammalian peptide hormones, STC1 plays a role in calcium and phosphate h omoeostasis, while the role of STC2 is unknown. Wt: examined a human fibros arcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA lev els, to determine whether these proteins are secreted. Following incubation of HT1080 cells with P-32, labelled STC1 and STC2 were found to be secrete d into the medium. STC1 was phosphorylated in vitro by protein kinase C (PK C), In vitro and in vivo phosphorylation both occurred exclusively on serin e and the phosphopeptide maps were similar, suggesting that PKC might be th e in vivo kinase, STC2 was phosphorylated in vitro by casein kinase II (CK2 ), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in inta ct cells resulted from the action of an ecto-protein kinase, since exogenou s STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was de tectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization-time-of-flight MS to be a singl e serine located between Ser-285 and Ser-298 in the C-terminal region of ST C2. This is the first report of the secretion of STC1 or STC2 from mammalia n cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylate d by an ecto-CK2-like enzyme.