Identification of the Phosphorylated Sites of Metabolically 32P-Labeled Osteopontin from Cultured Chicken Osteoblasts

Abstract

Osteopontin (OPN) is one of the major secretory phosphoproteins in both calcifying and non-calcifying tissues. Evidence has accumulated for the biological importance of the phosphoproteins and, in particular, the phosphate groups in bone formation, resorption, and calcification. The precise locations of the phosphate groups in the OPN molecule were determined by metabolically labeling OPN with 32 P in cultured chicken osteoblasts, followed by purification to homogeneity. Nterminal sequencing showed a single sequence of WPVSKRQHAISA, consistent with that deduced from both cDNA, and previous amino acid sequencing of the protein isolated from chicken bone. Three 32 P-labeled peptides were isolated by reverse-phase high performance liquid chromatography of thrombin-digested, 32 Plabeled OPN. The N-terminal sequencing of each of these thrombin fragments gave single sequences as follows: WPVSKSRQHAIS, SHHTHRYHQDHVD, and ASKLRKAARKL, with approximate molecular masses of 5, 30, and 20 kDa. These data demonstrate that 32 P was incorporated throughout the N-to C-terminal sequence of the protein. Thrombin specifically cleaved chicken OPN at two sites: between Arg-22 and Ser-23, which generated the 5-kDa N-terminal end fragment, and another between Lys-138 and Ala-139, which generated the 30-and 20-kDa fragments. To further define the exact locations of the phosphorylated amino acids and the surrounding amino acid sequences, OPN was digested with trypsin, which generated seven major 32 P-labeled peptides whose amino acid sequences were determined. The phosphorylated peptide regions of osteopontin were identified as amino acids 8 -18 (QHAIS*AS*S*EEK), -54 (LASQQTHYS*S*EENAD), 150 -171 (LIEDDAT*A-EVGDSQLAGLWLPK), 179 -191 (ELAQHQSVENDSR), 194 -205 (FDS*PEVGGDSK), 214 -219 (ES*LASR), and 2-248 (HSIENNEVTR).The phosphorylated amino acid sites are followed by an asterisk (*). Of the seven identified phosphorylated peptide regions, three were localized on the N-terminal end of the osteopontin molecule (with five phosphorylated serines) and contained the sequence motifs that were phosphorylated by casein kinase II type(s), whereas the remaining four peptides are concentrated toward the C-terminal half of the molecule (with five phosphorylated residues) and contained recognition motifs for other kinases as well as casein kinase II.It has been well established that the processes of phosphorylation and dephosphorylation of proteins catalyzed by protein kinases and phosphatases, respectively, play a major role in the initiation, regulation, and termination of a wide range of intracellular biochemical processes with significant functional consequences. Such processes may also play an important role in a wide variety of intercellular mechanisms, including general cell-cell signal transduction of extracellular agonists via specific transmembrane receptors, often causing alterations of intracellular concentrations of cAMP, calcium, inositol polyphosphates, and/or diacylglycerol. These intracellular modulators in turn induce a cascade of b...

Complete Topographical Distribution of Both the in Vivo and in Vitro Phosphorylation Sites of Bone Sialoprotein and Their Biological Implications

“…There were 10 phosphorylation sites identified, but the actual quantitative analysis showed 6.3 mol of phosphoamino acids/ mol of chicken OPN. The phosphorylated peptide regions are predominantly recognition sequences for CKII (27), confirming our in vitro studies. Additional studies that utilized the in vivo repair of calvarial bone defects induced to heal by implants of demineralized bone matrix highlighted the intimate interrelationship between accumulation and the rate of accumulation of calcium phosphate, OPN, BSP, and microsomal CKII activity as a function of bone development (28).…”

“…Such heterogeneity in phosphorylation can be attained by changes in factors such as the rate of phosphoprotein synthesis, the microsomal CKII activity, the available ATP concentrations (within the intracellular compartment where phosphorylation takes place), and some degree of dephosphorylation during the residence of OPN/BSP in the ECM. The precise sites of phosphorylation in chicken bone OPN clearly show that the extent of phosphorylation at each site varies between 30 and 100%, with an average of ϳ60% total phosphorylation (27). There were 10 phosphorylation sites identified, but the actual quantitative analysis showed 6.3 mol of phosphoamino acids/ mol of chicken OPN.…”

Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties

“…The phenomenon of heterogeneous phosphorylation observed in FbOPN and ObOPN has also been reported in human milk, rat bone, and chicken osteoblast OPNs (35,36,45). An alignment of the two murine proteins with the previously characterized milk and rat bone OPN isoforms, including the sites of PTM, is shown in Fig.…”

Osteopontin: a bridge between bone and blood

“…Opn is secreted in non-phosphorylated (Kubota et al, 1989;Chambers et al, 1992;Chang & Prince, 1993;Barber et al, 1999) and phosphorylated (Salih et al, 1995(Salih et al, , 1997Sorensen et al, 1994) forms, which contains up to 28 phosphate residues and are differentially induced by cytokines. Phosphorylation appears to be functionally important in determining whether Opn binds to cell surface receptors or the extracellular matrix (Nemir et al, 1989;Ek-Rylander et al, 1994).…”