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

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 microCi [gamma-32P] ATP. Autoradiography then detects kinase activity. Comparison of the UMR 106 CKII cytosolic and membrane-bound fractions showed that they were different in M(r) 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 [gamma-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.

journal_name

Connect Tissue Res

authors

Sfeir C,Veis A

doi

10.3109/03008209609029194

subject

Has Abstract

pub_date

1996-01-01 00:00:00

pages

215-22

issue

1-4

eissn

0300-8207

issn

1607-8438

journal_volume

35

pub_type

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