IDENTIFICATION OF THE PHOSPHORYLATED SITES OF METABOLICALLY P-32 LABELED OSTEOPONTIN FROM CULTURED CHICKEN OSTEOBLASTS

Osteopontin (OPN) is one of the major secretory phosphoproteins in bot h calcifying and non-calcifying tissues. Evidence has accumulated for the biological importance of the phosphoproteins and, in particular, t he 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 P-32 in cultured chicke n osteoblasts, followed by purification to homogeneity. N-terminal seq uencing showed a single sequence of WPVSKRQHAISA, consistent with that deduced from both cDNA, and previous amino acid sequencing of the pro tein isolated from chicken bone, Three P-32-labeled peptides were isol ated by reverse-phase high performance liquid chromatography of thromb in-digested, P-32-labeled OPN. The N-terminal sequencing of each of th ese 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 P-32 was incorporated t hroughout the N- to C-terminal sequence of the protein. Thrombin speci fically cleaved chicken OPN at two sites: between Arg 22 and Ser-23, w hich generated the 5-kda N-terminal end fragment, and another between Lys-138 and Ale-139, which generated the 30- and 20-kDa fragments. To further define the exact locations of the phosphorylated amino acids a nd the surrounding amino acid sequences, OPN was digested with trypsin , which generated seven major P-32-labeled peptides whose amino acid s equences were determined. The phosphorylated peptide regions of osteop ontin were identified as amino acids 8-18 (QHAISAS*S*EEK), 39-54 (LAS QQTHYSS*EENAD), 150-171 (LIEDDAT*AEVGDSQLAGLWLPK), 179-191 (ELAQHQSVE NDSR), 194-205 (FDSPEVGGDSK), 214-219 (ES*LASR), and 239-248 (HSIENNE VTR). The phosphorylated amino acid sites are followed by an asterisk (). Of the seven identified phosphorylated peptide regions, three wer e localized on the N-terminal end of the osteopontin molecule (with fi ve phosphorylated serines) and contained the sequence motifs that were phosphorylated by casein kinase II type(s), whereas the remaining fou r 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.