Phosphorylation Modulates Ameloblastin Self-assembly and Ca 2+ Binding

Abstract

Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca²⁺ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

Keywords: Ca2+- binding; ameloblastin; casein kinase 2; enamel; intrinsically disordered proteins; phosphorylation; protein kinase A; self-assembly.

Figure 1

The experimental design indicating the various recombinant AMBN proteins and peptides and test methods used in this study. Recombinant human AMBN-WT, N-terminus, C-terminus, DelEx5, Ex2-4, Ex5 proteins, and peptides were tested with LC-ESI-MS mapping of all peptides phosphorylated in vitro, dynamic light scattering (DLS) of Ex5, Ex5|Q9NP70, AMBN-WT, and DelEx5 and small angle x-ray scattering (SAXS) of AMBN-WT, DelEx5, and C-terminus with and without Ca²⁺.

Figure 2

Autoradiography of DelEx5 (0.01–5 μg) phosphorylated with CK2 and PKA (A). Dynamic light scattering of AMBN-WT (upper panel) and AMBN-WT incubated with 5% glycerol (lower panel) (B). In these experiments the phosphorylation of DelEx5 by CK2 required an exposure time of 24 h to match a 2 h exposure of DelEx5 phosphorylated by PKA.

Figure 3

Composite image representative of phosphorylated AMBN proteins and peptide by casein kinase2 (CK2) and protein kinase A (PKA). C-terminus, and AMBN-WT, N-terminus, and DelEx5 was phosphorylated by CK2 or PKA for 24 h, separated in 12% SDS-PAGE, and stained by ProQ diamond phosphorous stain. The intensity of bands between lanes can be interpreted as a semi-quantitative measurement of phosphorylation.

Figure 4

Schematic overview of the phosphorylated sites in AMBN. The exon organization in relation to the various recombinant proteins and peptides are illustrated. P-indicate position of phosphorylated residues. The vertical dotted line indicates the known trypsin cleavage site between A222 and L223 (Iwata et al., 2007).

Figure 5

Short Angle X-ray Scattering (SAXS) Kratky plots of AMBN-WT, DelEx5, and C-terminus with or without Ca²⁺. High concentration corresponds to 0.7, 0.88 mg/ml, and N/A, respectively, whereas low concentration corresponds to 0.4, 0.44, and 0.44 mg/ml, respectively. N/A means not analyzed due to lack of material.