The membrane associated kinases which phosphorylate bone and dentin extracellular matrix phosphoproteins are isoforms of cytosolic CKII

Charles Sfeir, Arthur Veis*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Bone and dentin contain many phosphoproteins in their extracellular matrix. It has been postulated that the anionic character of these proteins, and the presence of phosphate groups in particular, is important for various functions related to biomineralization. Phosphophoryns (PP), are the most highly phosphorylated dentin matrix components. However, the tissue form of PP can be further phosphorylated in vitro by cytosolic and membrane associated-endogenous messenger-independent kinases from osteoblast-like cells. To examine the kinases, a 2-dimensional zymogram technique has been developed for the detection of casein kinase II (CKII) activity of purified kinases using intact PP as the substrate. After isolation by subcellular fractionation and ion-exchange Chromatographic techniques, the enzymes are electrophoresed on an isoelectric focusing gel in the absence of SDS and disulfide bond breaking reagents. This first dimension gel is then layered on the zymogram gel containing PP + SDS. After electrophoresis, the gels are incubated in 250-500 μCi [γ-32p] ATP. Autoradiography then detects kinase activity. Comparison of the UMR 106 CKII cytosolic and membrane-bound fractions showed that they were different in Mr and focusing pH, suggesting the presence of CKII isoforms. CKI, which could phosphorylate the native PP in vitro, could not phosphorylate a dephosphorylated preparation (dPP). However, if the dPP was first exposed to CKII and unlabeled ATP then reacted with CKI and [γ-32p]ATP, the PP was phosphorylated. This prerequisite phosphorylation by CKII indicates that in vivo PP phosphorylation probably occurs in a series of regulated steps as a co-or post-translational process.

Original languageEnglish (US)
Pages (from-to)215-222
Number of pages8
JournalConnective tissue research
Volume35
Issue number1-4
DOIs
StatePublished - 1996

Keywords

  • Matrix proteins
  • Odontoblasts
  • Osteoblasts
  • Phosphophoryn
  • Phosphorylation

ASJC Scopus subject areas

  • Rheumatology
  • Biochemistry
  • Orthopedics and Sports Medicine
  • Molecular Biology
  • Cell Biology

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