This new R01 proposal is designed to elucidate the essential role the phosphorylation of a single amino acid in the most abundant enamel matrix protein, amelogenin, plays in the regulation of enamel formation. Proposed studies build on our extensive new findings that show that phosphorylation of a single serine site (S-16) in native amelogenin is critical for the formation of the highly-ordered enamel structure. Using a novel knock-in (KI) mouse model developed in our laboratory with a S16 to alanine substitution that prevents amelogenin phosphorylation, we have now for the first time demonstrated in vivo that amelogenin phosphorylation plays an essential role in both the secretory and maturation stages of amelogenesis. Extensive analyses of developing enamel tissues from KI, heterozygous (HET) and wild-type (WT) littermates reveal that KI mice exhibit distinct enamel phenotypes, including, the loss of enamel rod structure, the hallmark feature of mammalian enamel, numerous surface defects, shorter enamel crystals, hypoplasia and hypocalcification. Of particular note, HET enamel was found to be mosaic in nature with regions that also contain normal prismatic structures as seen in WT enamel. We have also found that KI ameloblasts lack Tomes' processes and exhibit a loss of organization of the ameloblast layer and severe cell pathology that builds gradually through the secretory stage. These findings, along with other recent evidence from our laboratories, have lead us to develop new working hypotheses regarding the role of amelogenin phosphorylation in the regulation of enamel mineralization and in maintaining ameloblast integrity and function during amelogenesis. Proposed functional activities with respect to mineralization reflect the enhanced capacity of both native phosphorylated full-length amelogenin and its predominant phosphorylated cleavage products to stabilize mineral phase precursors, as a means to control mineralization throughout amelogenesis. We further hypothesize that lack of amelogenin phosphorylation leads to disruption of cell- matrix interactions and trafficking of enamel matrix proteins. Four (4) specific aims have been proposed: to determine how amelogenin guides the linear appositional growth and organization of enamel crystals; to determine the basis for stage-specific abnormal enamel development in the KI mutants; to determine if S-16 amelogenin phosphorylation is required for amelogenin interactions with other essential enamel matrix proteins during enamel formation; and to elucidate the importance of amelogenin phosphorylation in maintaining ameloblast integrity and function throughout amelogenesis. The proposed studies are designed to provide fundamental insight into the mechanism by which phosphorylated amelogenin serves to regulate the formation of the highly-ordered dental enamel tissue. Long-term, our findings should aid in our understanding of inherited enamel diseases and factors that influence dental caries susc...