Phosphorylation of the proteins of the extracellular matrix of mineralized tissues by casein kinase-like activity.

The extracellular matrix of the connective tissue contains non-collagenous proteins (NCP) which are acidic in character. The NCP of mineralizing systems (bone, dentin) differ from those of the non-mineralizing systems (skin, tendon) in that the mineralized tissue NCP are frequently phosphorylated. The phosphorylated proteins have been implicated in various aspects of the mineralization process. Thus, it is of interest to consider the mechanism and regulation of phosphorylation of the major matrix NCP. The majority of the phosphorylation takes place at Ser or Thr residues embedded within acidic sequences, and therefore are targets for casein kinase I (CK1) or casein kinase II (CK2)-like kinases. CK1 and CK2 are distantly related members of the protein kinase family. They are ubiquitous, constitutively active, second-messenger-independent kinases. CK1 is found in a variety of isoforms, all homologous to the alpha-subunit of the protein kinase family. It acts as a monomer. The active form of CK2 is a tetrameric holoenzyme, with 2 alpha catalytic subunits and 2 beta regulatory subunits. The CK2 alpha has activity alone, but the holoenzyme is four- to five-fold that activity. CK2 can use either ATP or GTP as the phosphate donor, but CK1 can use only ATP. The CK2 activity which phosphorylates the mineralized tissue NCP appears to be localized to membrane-associated cell fractions, and is present in the endoplasmic reticulum and Golgi compartments in osteoblasts, where phosphorylation of the secreted proteins appears to take place as co- and post-translational processes. Data indicate that both alpha and beta subunits of the membrane-associated CK2 are isoforms of the cytosolic CK2 in the same cells. The CK1 has not been specifically localized. Studies of dephosphorylated NCP such as phosphophoryn (PP) have shown that CK1 will not phosphorylate dephosphorylated dPP unless prior phosphorylation with CK2 has been carried out. In turn, CK2 activity may be initiated only after an initial phosphorylation of one of the messenger-dependent kinases. Thus, the phosphorylation reactions in mineralized tissues may be a tightly regulated hierarchical or sequential cascade of intracellular phosphorylation events.