Fig. 2
The dimer interface is critical for Fam20A to bind and activate Fam20C. a Fam20A mutants display reduced interaction with Fam20C. His6-SUMOstar-Fam20A (WT and mutants), His6-SUMOstar-Fam20B, and His6-MBP-Fam20C were individually isolated from the condition media of insect cells using Ni-NTA affinity purification. They were mixed as indicated (input), and MBP pull-down was performed using amylose resin and analyzed by SDS-PAGE and Coomassie staining. b Fam20A mutants exhibit substantially diminished abilities to activate Fam20C. Phosphorylation of human ENAM (173–277), as indicated by the incorporation of 32P from [γ-32P]ATP, was examined by SDS-PAGE and autoradiography. c Fam20A-F251A/F252A and Fam20A-F306A/P309G are unable to enhance OPN phosphorylation catalyzed by endogenous Fam20C. OPN-V5 was co-expressed with WT or mutant Fam20A-HA as indicated. Cells were metabolically labeled with 32P orthophosphate before OPN-V5 was immunoprecipitated from the conditioned media. Total protein and 32P incorporation were detected by immunoblotting and autoradiography. The relative phosphorylation level of OPN was represented by the ratio of 32P autoradiography and V5 immunoblot intensity, normalized to the ratio from cells without Fam20A transfection (two replicates, error bars representing standard deviation). d Fam20A mutants are not able to promote the activities of two Fam20C mutants associated with Raine syndrome, G280R and G379E. C-terminally V5-tagged OPN (OPN-V5), C-terminally Flag-tagged Fam20C (Fam20C-Flag, WT or mutants), and C-terminally HA-tagged Fam20A (Fam20A-HA, WT or mutants) were co-expressed in U2OS cells as indicated. Secreted OPN-V5 was immunoprecipitated from conditioned media and detected by immunoblotting. OPN phosphorylation was evaluated by its mobility shift on SDS-PAGE
