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. 2020 Oct 1;10(1):16273.
doi: 10.1038/s41598-020-73248-0.

Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic

Emiko Kinoshita-Kikuta  1   2 , Toshihiko Utsumi  3   4 , Aya Miyazaki  2 , Chiharu Tokumoto  2 , Kyosuke Doi  2 , Haruna Harada  3 , Eiji Kinoshita  5   6 , Tohru Koike  1   2
Affiliations

Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic

Emiko Kinoshita-Kikuta et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Investigation of N-myristoylation-dependent phosphorylation of SFKs. FLAG-tagged SFKs (WTs of Src, Yes, Fyn, Fgr, Lck, Hck, Blk, and Lyn) and their G2A mutants expressed in HEK293 cells were analyzed by SDS-PAGE (10% w/v polyacrylamide, upper panels) and by Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide, lower panels), followed by immunoblotting with anti-FLAG antibody. Open arrowheads: nonphosphorylated species; closed arrowheads: phosphorylated species that disappeared in the G2A mutant. The raw image data for the full-length blots are shown in Supplementary Figure S6.
Figure 2
Figure 2
Identification of a specific N-myristoylation-dependent phosphorylation site of Lyn. (A) FLAG-tagged Lyn (WT), its G2A mutant, and the 15 site-directed mutants expressed in HEK293 cells were analyzed by Phos-tag SDS-PAGE (20 mM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-FLAG antibody. AP alkaline phosphatase. Raw image data for the three full-length blots shown in Supplementary Figure S7A have been spliced to arrange the lanes appropriately. The positions of the splices are indicated by vertical dashed lines. (B) FLAG-tagged Lyn (WT) expressed in HEK293 cells treated with bengamide B (0–2.50 µM) were analyzed by Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-FLAG antibody. The S13A (leftmost lane) and G2A mutants (rightmost lane) were loaded for reference purposes. Open arrowheads: nonphosphorylated species; closed arrowhead: phosphospecies containing a phosphorylated Ser-13 residue. The raw image is shown in Supplementary Figure S7B.
Figure 3
Figure 3
Phos-tag banding image of Lyn synthesized in a cell-free protein-synthesis system, cellular localization of Lyn WT, G2A-, S13A-, and C3A-mutant proteins, and Phos-tag banding image of the WT and C3A-mutant proteins. (A) Lyn WT protein, its G2A mutant, and the S13A mutant synthesized by using the TnT T7 Insect Cell Extract Protein Expression System were analyzed by Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide). For reference, FLAG-tagged WT and its G2A and S13A mutants expressed in HEK293 cells were analyzed on an identical Phos-tag gel, followed by immunoblotting with a cocktail of anti-Lyn antibody and anti-FLAG antibody. Open arrowheads: nonphosphorylated species; closed arrowhead: phosphospecies containing a phosphorylated Ser-13 residue. The raw image shown in Supplementary Figure S8A has been spliced to arrange the lanes appropriately. The position of the splice is indicated by a vertical dashed line. (B) Cellular localization of FLAG-tagged Lyn WT, its G2A mutant, and the S13A mutant expressed in HEK293 cells. The expressed proteins and the nucleus were detected by immunofluorescence staining with anti-FLAG antibody (green) and Hoechst 33342 (blue), respectively. (C) Cellular localization of FLAG-tagged C3A mutant expressed in HEK293 cells. The expressed protein, the Golgi, and the nucleus were detected by immunofluorescence staining with anti-FLAG antibody (green), anti-RCAS1 antibody (red), and Hoechst 33342 (blue), respectively. (D) Comparison of Phos-tag banding patterns between the WT protein and a C3A mutant of Lyn. FLAG-tagged Lyn WT and its C3A mutant expressed in HEK293 cells were analyzed by Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-FLAG antibody. Open arrowhead: nonphosphorylated species; closed arrowheads: phosphospecies containing a phosphorylated Ser-13 residue. The raw image is shown in Supplementary Figure S8B.
Figure 4
Figure 4
Inhibition profiling of phosphorylation of Lyn-S13 in the presence of D4476, CX4945, IC261, or staurosporine. Lyn WT proteins synthesized by using the TnT T7 Insect Cell Extract Protein Expression System in the presence of D4476, CX4945, IC261, or staurosporine at concentrations of 62.5–500 µM were analyzed by Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-Lyn antibody. Open arrowhead: nonphosphorylated species; closed arrowheads: phosphospecies containing a phosphorylated Ser-13 residue. The raw image is shown in Supplementary Figure S9.
Figure 5
Figure 5
Phosphorylation motifs for CK1 and CK2 in the sequence surrounding Lyn-S13 and Phos-tag banding image of Lyn D10N, D14N and D18N mutants. (A) Phosphorylation motifs for CK1 and CK2 in the sequence surrounding Lyn-S13. (B) FLAG-tagged Lyn D10N, D14N, and D18N mutants expressed in HEK293 cells were analyzed by using Phos-tag SDS-PAGE (20 mM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-FLAG antibody. For reference, FLAG-tagged Lyn WT protein and its S13A and G2A mutants expressed in HEK293 cells were also analyzed on an identical Phos-tag gel. Open arrowhead: nonphosphorylated species; closed arrowheads: phosphospecies containing a phosphorylated Ser-13 residue. The raw image is shown in Supplementary Figure S10.
Figure 6
Figure 6
Phos-tag banding image of Lyn co-expressed with CK1. (A) FLAG-tagged LYN WT and Halo-tagged CK1 (WT, kinase-dead or ΔC mutant) co-expressed in HEK293 cells were analyzed by using Phos-tag SDS-PAGE (20 μM Zn2+–Phos-tag and 7% w/v polyacrylamide) followed by immunoblotting with anti-FLAG antibody (upper panel). All protein expressions in HEK293 cells were confirmed by SDS-PAGE followed by immunoblotting with a cocktail of anti-FLAG antibody and anti-HaloTag antibody (lower panel). (B) FLAG-tagged Lyn G2A mutant and Halo-tagged CK1 (WT, kinase-dead or ΔC mutant) co-expressed in HEK293 cells were analyzed in the same manner as (A). Open arrowhead: nonphosphorylated species. Closed arrowhead: phosphospecies containing a phosphorylated Ser-13 residue. ΔC: deletion of Cys-406, Cys-407, and Cys-408 in CK1γ1; deletion of Cys-399, Cys-400, and Cys-401 in CK1γ2; deletion of Cys-431, Cys-432, and Cys-433 in CK1γ3. The raw images shown in Supplementary Figure S11 were spliced to arrange the lanes appropriately. The positions of the splices are indicated by vertical dashed lines.
Figure 7
Figure 7
Cellular localization of Lyn WT, CK1γ1 WT and ΔC-mutant proteins. FLAG-tagged Lyn WT and Halo-tagged CK1γ (WT or ΔC mutant) co-expressed in HEK293 cells were analyzed by immunofluorescence microscopy. The co-expressed CK1γ and Lyn were detected by immunofluorescence staining with anti-HaloTag antibody (red) and anti-FLAG antibody (green), respectively, and the nucleus was stained with Hoechst 33342 (blue). ΔC: deletion of Cys-406, Cys-407, and Cys-408 in CK1γ1.
Figure 8
Figure 8
Schematic representation of targeting to the plasma membrane following N-myristoylation-dependent phosphorylation of Lyn. Scheme of binding to the plasma membrane following N-myristoylation-dependent phosphorylation of Lyn. Myr, myristate; Pal, palmitate; P, phosphate; ER, endoplasmic reticulum; PM, plasma membrane; −, negative charge; +, positive charge.
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