The in vitro phosphorylation of the native rat incisor dentin phosphophoryns.
Phosphophoryns are the major non-collagenous proteins of the mineralized matrix of rat incisor dentin. Nearly half the phosphophoryn residues are serines, and 85-90% of these are phosphorylated. Since phosphorylation may be important for phosphophoryn function, it was of interest to identify the kinase(s) responsible for catalyzing their phosphophorylation. Rat osteosarcoma (ROS) 17/2.8 osteoblast-like cells were selected as the enzyme source. Native rat incisor phosphophoryns (RIPP-I, II, III) were not substrates for any of the ROS 17/2.8 messenger-dependent kinases but were phosphorylated by membrane-associated endogenous messenger-independent kinases. These were resolved chromatographically and identified as casein kinase (CK) I and II by elution properties and immunoblotting with a CKII antibody. The CKI preferentially used RIPP-III as substrate, while CKII preferred RIPP-I and II. Heparin at 100 and 500 ng/assay and NaCl at 0.25-0.4 M inhibited phosphorylation of the RIPP by CKI and CKII in parallel. At 10 mM spermine, phosphorylation of RIPP-I and II by CKII, and of RIPP-III by CKI were inhibited, but phosphorylation of RIPP-III by CKII was enhanced. Purified sea star oocyte CKII demonstrated the same substrate specificity and spermine concentration shift as the ROS 17/2.8 CKII. These data show that osteoblast-like cells are a rich source of membrane-bound CKI and CKII activity. The different patterns of phosphorylation of RIPP-I, II, and III further show that they are distinct synthetic products of the odontoblast.
Wu
C B
CB
Connective Tissue Research Laboratory, Northwestern University, Chicago, Illinois 60611.
Pelech
S L
SL
Veis
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003804:Dentin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D007180:Incisor; D008969:Molecular Sequence Data; D012516:Osteosarcoma; D010455:Peptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013096:Spermine / Q000494:pharmacology
D002364:Caseins; D010455:Peptides; D010750:Phosphoproteins; C017630:phosphophoryn; D013096:Spermine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D011494:Protein Kinases; D047388:Casein Kinases