Microsomal casein kinase II in endoplasmic reticulum- and Golgi apparatus-rich fractions of ROS 17/2.8 osteoblast-like cells: an enzyme that modifies osteopontin.
Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.
Wu
C B
CB
Department of Clinical Dental Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
Pan
Y M
YM
Simizu
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002458:Cell Fractionation; D018870:Endoplasmic Reticulum, Rough / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology*; D053495:Osteopontin; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D006160:Guanosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases