Abstract
Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.