Cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties.
Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains approximately 21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function.
Christensen
Brian
B
Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Kazanecki
Christian C
CC
Petersen
Torben E
TE
Rittling
Susan R
SR
Denhardt
David T
DT
Sørensen
Esben S
ES
D000818:Animals; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D011905:Genes, ras; D006031:Glycosylation; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D009928:Organ Specificity; D010006:Osteoblasts / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D053495:Osteopontin / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D016023:Integrins; D053495:Osteopontin