Identification of the C-terminal region in Amelogenesis Imperfecta causative protein WDR72 required for Golgi localization.

Amelogenesis Imperfecta (AI) represents a group of hereditary conditions that manifest tooth enamel defects. Several causative mutations in the WDR72 gene have been identified and patients with WDR72 mutations have brown (or orange-brown) discolored enamel, rough enamel surface, early loss of enamel after tooth eruption, and severe attrition. Although the molecular function of WDR72 is not yet fully understood, a recent study suggested that WDR72 could be a facilitator of endocytic vesicle trafficking, which appears inconsistent with the previously reported cytoplasmic localization of WDR72. Therefore, the aims of our study were to investigate the tissues and cell lines in which WDR72 was expressed and to further determine the sub-cellular localization of WDR72. The expression of Wdr72 gene was investigated in mouse tissues and cell lines. Endogenous WDR72 protein was detected in the membranous fraction of ameloblast cell lines in addition to the cytosolic fraction. Sub-cellular localization studies supported our fractionation data, showing WDR72 at the Golgi apparatus, and to a lesser extent, in the cytoplasmic area. In contrast, a WDR72 AI mutant form that lacks its C-terminal region was exclusively detected in the cytoplasm. In addition, our studies identified a putative prenylation/CAAX motif within the last four amino acids of human WDR72 and generated a WDR72 variant, called CS mutant, in which the putative motif was ablated by a point mutation. Interestingly, mutation of the putative CAAX motif impaired WDR72 recruitment to the Golgi. Cell fractionation assays confirmed subcellular distribution of wild-type WDR72 in both cytosolic and membranous fractions, while the WDR72 AI mutant and CS mutant forms were predominantly detected in the cytosolic fraction. Our studies provide new insights into the subcellular localization of WDR72 and demonstrate a critical role for the C-terminal CAAX motif in regulating WDR72 recruitment to the Golgi. In accordance with structural modelling studies that classified WDR72 as a potential vesicle transport protein, our findings suggest a role for WDR72 in vesicular Golgi transport that may be key to understanding the underlying cause of AI.

Molecular Cloning of Mouse Homologue of Enamel Protein C4orf26 and Its Phosphorylation by FAM20C.

It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and conformation as well as protein-protein interaction are the examples of subsequent cellular events modulated by protein phosphorylation. Enamel matrix proteins belong to members of the secretory calcium binding phosphoprotein (SCPP) family clustered on chromosome 4q21, and most of the SCPP phosphoproteins have at least one S-X-E motifs (S; serine, X; any amino acid, E; glutamic acid). It has been reported that mutations in C4orf26 gene, located on chromosome 4q21, are associated with autosomal recessive type of Amelogenesis Imperfecta (AI), a hereditary condition that affects enamel formation/mineralization. The enamel phenotype observed in patients with C4orf26 mutations is hypomineralized and partially hypoplastic, indicating that C4orf26 protein may function at both secretory and maturation stages of amelogenesis. The previous in vitro study showed that the synthetic phosphorylated peptide based on C4orf26 protein sequence accelerates hydroxyapatite nucleation. Here we show the molecular cloning of Gm1045, mouse homologue of C4orf26, which has 2 splicing isoforms. Immunohistochemical analysis demonstrated that the immunolocalization of Gm1045 is mainly observed in enamel matrix in vivo. Our report is the first to show that FAM20C, the Golgi casein kinase, phosphorylates C4orf26 and Gm1045 in cell cultures. The extracellular localization of C4orf26/Gm1045 was regulated by FAM20C kinase activity. Thus, our data point out the biological importance of enamel matrix-kinase control of SCPP phosphoproteins and may have a broad impact on the regulation of amelogenesis and AI.

FAM20C directly binds to and phosphorylates Periostin.

It is widely accepted that FAM20C functions as a Golgi casein kinase and has large numbers of kinase substrates within the secretory pathway. It has been previously reported that FAM20C is required for maintenance of healthy periodontal tissues. However, there has been no report that any extracellular matrix molecules expressed in periodontal tissues are indeed substrates of FAM20C. In this study, we sought to identify the binding partner(s) of FAM20C. FAM20C wild-type (WT) and its kinase inactive form D478A proteins were generated. These proteins were electrophoresed and the Coomassie Brilliant Blue (CBB)-positive bands were analyzed to identify FAM20C-binding protein(s) by Mass Spectrometry (MS) analysis. Periostin was found by the analysis and the binding between FAM20C and Periostin was investigated in cell cultures and in vitro. We further determined the binding region(s) within Periostin responsible for FAM20C-binding. Immunolocalization of FAM20C and Periostin was examined using mouse periodontium tissues by immunohistochemical analysis. In vitro kinase assay was performed using Periostin and FAM20C proteins to see whether FAM20C phosphorylates Periostin in vitro. We identified Periostin as one of FAM20C-binding proteins by MS analysis. Periostin interacted with FAM20C in a kinase-activity independent manner and the binding was direct in vitro. We further identified the binding domain of FAM20C in Periostin, which was mapped within Fasciclin (Fas) I domain 1-4 of Periostin. Immunolocalization of FAM20C was observed in periodontal ligament (PDL) extracellular matrix where that of Periostin was also immunostained in murine periodontal tissues. FAM20C WT, but not D478A, phosphorylated Periostin in vitro. Consistent with the overlapped expression pattern of FAM20C and Periostin, our data demonstrate for the first time that Periostin is a direct FAM20C-binding partner and that FAM20C phosphorylates Periostin in vitro.