title: Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells.
abstract: 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 (32)P, 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.
authors.affiliation: Cancer Research Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville 2145, NSW, Australia.
authors.forename: D A
authors.initials: DA
authors.lastname: Jellinek
authors.forename: A C
authors.initials: AC
authors.lastname: Chang
authors.forename: M R
authors.initials: MR
authors.lastname: Larsen
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: P J
authors.initials: PJ
authors.lastname: Robinson
authors.forename: R R
authors.initials: RR
authors.lastname: Reddel
mesh_terms: D015153:Blotting, Western; D047390:Casein Kinase II; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005354:Fibrosarcoma / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006728:Hormones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
chemical_list: D017077:Culture Media, Conditioned; D006023:Glycoproteins; D006728:Hormones; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C114997:STC2 protein, human; D012694:Serine; C051795:teleocalcin; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C