Casein kinase localization in the endoplasmic reticulum of the ROS 17/2.8 cell line.
Phosphophoryns (PPs) are phosphoproteins specific to the dentin matrix and are the major noncollagenous matrix proteins in rat incisor dentin. It has been hypothesized that their phosphate groups are important in dentin mineralization. PPs have many sequences which are substrates for membrane-associated endogenous messenger-independent kinases. The objective of this study was to localize the protein kinases involved in phosphorylating the PPs. Osteoblast-like ROS 17/2.8 cells, which secrete extracellular matrix phosphoproteins, were lysed. After removal of the nuclei and mitochondria by low-speed centrifugation, the membrane associated organelles were isolated at higher speed from the cytosol. The Golgi vesicle and rough microsome fractions were collected from 29-43.7% sucrose density gradients. Each fraction was tested for casein kinase II (CKII) activity using an in vitro phosphorylation assay with PPs as substrate. To characterize and confirm the nature of the components of the sucrose gradient fractions, the activities of specific enzymes such as N-acetylglucosamine galactosyltransferase and cytochrome c reductase, which are exclusively associated with the Golgi and rough microsomes, respectively, were determined. Electron microscopy of the isolated fractions confirmed the enzyme assay characterizations. CKII activity capable of phosphorylating the PP was found in the rough microsome fraction but not in the Golgi. Thus, phosphorylation of the secreted phosphoproteins would appear to take place in the endoplasmic reticulum as a cotranslational event.
Sfeir
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Northwestern University Dental School, Division of Oral Biology, Chicago, Illinois, USA.
Veis
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D000818:Animals; D047390:Casein Kinase II; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D007180:Incisor / Q000378:metabolism; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D013395:Sucrose / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D010750:Phosphoproteins; C017630:phosphophoryn; D014531:Uridine Diphosphate Galactose; D013395:Sucrose; D009245:NADH Dehydrogenase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases