(A) Structure of human Fam20A. The N-terminal segment was colored in green. The Kβ1-Kβ2, Kβ6-Kβ7, and Kβ3-Kα3 loops were colored in red, teal, and magenta, respectively. The rest of the kinase core is shown in white. The side chains of Cys209, Cys211, Cys319, Cys323, and Gln258 are shown as sticks. The N- and C-termini of the molecule are indicated. (B) Structure of ceFam20 (PDB ID: 4KQA) is shown in the same orientation as Fam20A in (A) and colored in the same color scheme. The side chains Cys273, Cys277, and Glu213 are shown as sticks. The active site of ceFam20 is indicated with an oval in dashed line. (C) The schematic of the human Fam20A gene structure and RNA splicing. Exon three is highlighted in red. Relevant amino acid sequences are shown at the bottom. The two Cys residues in the Kβ1-Kβ2 loop are highlighted in red. (D) Effect of Fam20A exon three deletion on OPN phosphorylation. C-terminal V5-tagged OPN was expressed alone or co-expressed with Flag-tagged Fam20A WT or Δexon3 in U2OS cells that were metabolically labeled with 32P orthophosphate. OPN-V5 was immunoprecipitated from the medium. Total OPN and 32P incorporation were detected by anti-V5 immunoblotting and autoradiography. Expression of Fam20A was monitored by anti-Flag immunoblotting. (E) Effect of exon three deletion on Fam20A secretion. Flag-tagged Fam20A WT or Δexon3 was expressed in U2OS cells and immunoprecipitated from conditioned media using anti-Flag antibody. (F) Effect of C209A/C211A mutation (CC/AA) on Fam20A secretion and OPN phosphorylation.