In vivo and in vitro phosphorylation regions of bone sialoprotein.
The present study for the first time evaluated both the in vitro and in vivo phosphorylation regions of bone sialoprotein (BSP) by utilizing multiple approaches and techniques. The in vitro phosphorylation sites were determined by 32P-labeling of native BSP using purified casein kinase II (CKII), followed by peptide mapping and solid-phase N-terminal sequence analyses. The in vivo phosphorylation sites were determined by (i) derivatization with 1-S-[14C]carboxymethyl-dithiothreitol ([14C] CM-DTT) of the proteolytic digests of BSP, followed by isolation and N-terminal peptide sequence analysis; and (ii) analyzing the proteolytic peptides of native BSP using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Native BSP incorporated approximately 2.5 mol of phosphate/mol of BSP by CKII, which were distributed over four major peptide peaks and three shoulder peaks within the peptide map with varying degrees of phosphorylation. Further studies using the [14C] CM-DTT thiol reagent indicated that native and deglycosylated BSP incorporated 5.84 and 5.80 mol of 14C/mol of BSP, respectively. This confirmed that there were approximately 5.8 mol P-Ser/mol of BSP naturally (in vivo) occurring phosphorylation sites and that there was no overlap between the phosphorylation and glycosylation sites. The 5.8 mol P-Ser/mol BSP reflects the total number of mols of naturally occurring phosphorylation, phosphorylated in vivo by CKII (4.1 mol), protein kinase C (0.9 mol), and cGMP-dependent kinase (0.8 mol). Peptide N-terminal sequence analyses of both in vitro (32P) and in vivo (14C) phosphorylated peptides indicated that the phosphorylated residues were predominantly on the N-terminal half of the protein that included recognition sequences for CKII, e.g., LESDEENGVFK (residues 12-22).
Salih
Erdjan
E
Laboratory for the Study of Skeletal Disorders and Rehabilitation, Harvard Medical School and Children's Hospital, Boston, Massachusetts 02115, USA. Erdjan.Salih@TCH.Harvard.Edu
D000818:Animals; D000831:Animals, Newborn; D001842:Bone and Bones / Q000737:chemistry / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000502:physiology; D047390:Casein Kinase II; D002417:Cattle; D066298:In Vitro Techniques; D058574:Integrin-Binding Sialoprotein; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D012795:Sialoglycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases