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

• Published in: Biochemical journal Vol. 350; no. 2; pp. 453 - 461
• Main Authors: JELLINEK, Derek A., CHANG, Andy C., LARSEN, Martin R., WANG, Xin, ROBINSON, Phillip J., REDDEL, Roger R.
• Format: Journal Article
• Language: English
• Published: 01.09.2000
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3500453

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the 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 intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable 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 single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.