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Updated: Nov 8, 2025
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Published on: April 1, 2022
Molecular Cloning of Mouse Homologue of Enamel Protein C4orf26 and Its Phosphorylation by FAM20C
Nattanan Govitvattana1,2, Masaru Kaku3, Yoshio Ohyama1
1Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, MA, 02118, USA.
Abstract:
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.
Insights
Protein phosphorylation regulates cellular functions. This study reveals FAM20C kinase phosphorylates enamel matrix proteins C4orf26 and Gm1045, impacting amelogenesis and Amelogenesis Imperfecta.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Protein phosphorylation is a key regulator of cellular processes, including protein function and localization.
- Enamel matrix proteins, part of the secretory calcium-binding phosphoprotein (SCPP) family, are crucial for tooth enamel formation.
- Mutations in the C4orf26 gene are linked to Amelogenesis Imperfecta (AI), a disorder affecting enamel mineralization.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the function of C4orf26 and its mouse homolog, Gm1045, in amelogenesis.
- To identify the kinase responsible for phosphorylating C4orf26 and Gm1045.
- To explore the role of this phosphorylation in regulating enamel matrix protein localization and function.
Main Methods:
- Molecular cloning of Gm1045, the mouse homolog of C4orf26, identifying its splicing isoforms.
- Immunohistochemical analysis to determine the in vivo localization of Gm1045 within the enamel matrix.
- In vitro cell culture experiments to assess the interaction between C4orf26/Gm1045 and FAM20C, a Golgi casein kinase.
Main Results:
- Gm1045, the mouse homolog of C4orf26, was successfully cloned and shown to have two splicing isoforms.
- Immunohistochemistry confirmed Gm1045's primary localization within the enamel matrix.
- FAM20C was identified as the kinase that phosphorylates both C4orf26 and Gm1045.
- FAM20C kinase activity was found to regulate the extracellular localization of C4orf26/Gm1045.
Conclusions:
- FAM20C-mediated phosphorylation plays a critical role in regulating the function and localization of enamel matrix proteins like C4orf26 and Gm1045.
- This discovery highlights the importance of enamel matrix-kinase interactions in the biological control of amelogenesis.
- Understanding these mechanisms may provide new insights into the pathogenesis of Amelogenesis Imperfecta and potential therapeutic strategies.
