Enamel Specific Protein Kinases and State of Phosphorylation of Purified Amelogenins

Abstract

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear+plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of ²P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca²⁺ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca²⁺ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca²⁺, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly Ca²⁺ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of ¹⁴C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, ¹⁴C-mercaptoethanol, 0.64 moles ^uC-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by ¹⁴C-mercaptoethanol, providing 0.46 and 0.88 moles ¹⁴C-incorporated/mole respectively. Further studies of the ¹⁴C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.

Key Words:

• Protein kinases
• phosphorylation
• amelogenins
• enamel
• ¹⁴C-mercaptoethanol