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.
Highlights
The Src family of kinases (SFKs) are nonreceptor tyrosine kinases that act as key mediators of intracellular signal transduction[1,2,3,4]
Since the discovery that the catalytic subunit of protein kinase PKA is N-myristoylated[34], many protein kinases and their substrates have subsequently been reported to be N-myristoylated, and it has been shown that specific protein–protein or protein–membrane interactions mediated by N-myristoylation play vital roles in the physiological functions of the phosphorylation reactions performed by these proteins[35]
It has been shown that the phosphorylation reaction in the N-terminal region of N-myristoylated PKA acts as a molecular switch in controlling the membrane association of the p rotein[37]
Summary
The Src family of kinases (SFKs) are nonreceptor tyrosine kinases that act as key mediators of intracellular signal transduction[1,2,3,4]. Lysine residues located in the N-terminal-membrane-binding region are involved in modulating membrane binding Both a suitable lipid modification and a unique amino acid sequence alignment for each SFK appear to be crucial in mediating intracellular signaling on the phospholipid bilayer. Mass spectrometry (MS)-based phosphoproteomic techniques have permitted the identification of many sites for the phosphorylation reactions of serine, threonine, and tyrosine residues in SFKs (information on Lyn is listed in Supplementary Table S2). The use of Phos-tag SDS-PAGE, in conjunction with existing information on locations of phosphorylation sites (phosphoproteomic data deposited in the database), permitted the identification of specific N-myristoylation-dependent phosphorylation sites; the cellular physiological role of crosstalk between protein N-myristoylation and phosphorylation/dephosphorylation could be studied. Our results showed that S-palmitoylated CK1γ phosphorylates N-myristoylated Lyn at the Golgi during intracellular protein traffic
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