Complete topographical distribution of both the in vivo and in vitro phosphorylation sites of bone sialoprotein and their biological implications.
Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12-22 (LEDS(P)EENGVFK), 42-62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80-91 (EDS(P)DENEDEES(P)E), and 135-145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[(14)C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250-264 (DNGYEIYES(P)ENGDPR), and 282-289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125-130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site.
Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Harvard Medical School and Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA. erdjan.salih@tch.harvard.edu
Erdjan
E
Salih
Rudolf
R
Flückiger
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism; D047390:Casein Kinase II; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012795:Sialoglycoproteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000494:pharmacology
C481759:1-S-(3H)carboxymethyl-dithiothreitol; D058574:Integrin-Binding Sialoprotein; D010455:Peptides; D012795:Sialoglycoproteins; D013438:Sulfhydryl Compounds; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D004229:Dithiothreitol