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. 2019 Dec 9;8:e50850. doi: 10.7554/eLife.50850

Figure 1. DYRK2 and GSK3β induce sequential phosphorylation of NDEL1 at S336 and S332.

(A) Identification of responsible kinases for NDEL1 phosphorylation. DYRK2, one of the positive candidates from human kinome screening for NDEL1 phosphorylation, and GSK3βS9A sequentially phosphorylated NDEL1. (B) Identification of NDEL1 S336 and S332 as target sites of DYRK2 and GSK3β. DYRK2 increased NDEL1 phosphorylation at S336 and GSK3βS9A additionally induced phosphorylation at S332. (C) List of NDEL1 PTMs identified from LC-MS/MS analysis of developing mouse brain lysates. Peptide containing S336 and S332 phosphorylation is indicated by bold and underlined letters. (D) MS/MS spectrum for the phosphorylated fragments of NDEL1 peptide including S332 and S336 residues. The sequence of the peptide (aa 315–345) and all detected fragment ions are shown above. The b- and y-ions annotated in the spectrum include the sizes of y14 and b22 ions indicative of phosphorylation at S332 or S336. (E) Phosphorylation levels of endogenous NDEL1 S336/S332 in the developing mouse brain. Amount of lysates subjected to IP was normalized by NDEL1 protein levels. N = 7 for E15, P7, P14, P28, and adult brain lysates and N = 6 for E18. All results are presented as means ± SEM. (F) Increased endogenous NDEL1 phosphorylation by DYRK2 and GSK3β. Transfected HEK293 cell lysates were IPed with pan-NDEL1 antibody followed by western blot with anti-pNDEL1 antibody. Over-expression of DYRK2 and GSK3βS9A increased the endogenous NDEL1 S336/S332 phosphorylation. The number of samples is shown at the bottom of the bar of the graph. All results are presented as means ± SEM. *p<0.05 from Student’s t-test. (G) Endogenous NDEL1 S336/S332 phosphorylation detected at the growth cone of the primary cultured mouse hippocampal neuron. Anti-pNDEL1 antibody signal was enriched at the growth cone and colocalized with both phalloidin and α-tubulin (indicated by arrowheads). Magnified confocal images show the strong overlap between pNDEL1, phalloidin, and α-tubulin. The scale bar represents 10 μm. See also Figure 1—figure supplements 1, 2 and 3 and Figure 1—source data 1 and 2.

Figure 1—source data 1. Source data for quantitation of endogenous pNDEL1 in developing mouse brain lysates.
Figure 1—source data 2. Source data for quantitation of endogenous pNDEL1 with kinases over-expression.

Figure 1.

Figure 1—figure supplement 1. Figure of kinome library screening results for NDEL1 phosphorylation CCSB-Broad.

Figure 1—figure supplement 1.

Human Kinase ORF collection screening to find responsible kinases for NDEL1 S336 phosphorylation. Among 301 serine/threonine kinases, 218 kinases were tested at least twice. TARA was used as a positive control to induce NDEL1 phosphorylation.
Figure 1—figure supplement 2. Figure related to Figure 1.

Figure 1—figure supplement 2.

(A) Validation of target sites for NDEL1 phosphorylation by DYRK2-GSK3β. S332A and S336A mutations, in contrast to S331A and S335A mutations, changed the pattern of phosphorylation signals. (B) Characterization of anti-pNDEL1 antibody for immunoblotting. Anti-pNDEL1 antibody specifically detected NDEL1 phosphorylation at S332 and S336. (C) Detection of endogenous NDEL1 S336/S332 phosphorylation by anti-pNDEL1 antibody. Anti-pNDEL1 antibody detected the endogenous NDEL1 phosphorylation from P7 mouse brain lysates prepared. The arrowhead indicates NDEL1/pNDEL1 bands. (D) Validation of anti-pNDEL1 antibody for staining via immunohistochemistry. NDEL1 shRNA construct co-expressing EGFP marker was transfected via in utero electroporation to E15 ICR mouse and the brain was prepared at E18 and subjected to immunohistochemistry. NDEL1 knockdown significantly diminished the anti-pNDEL1 antibody signal. The scale bar represents 10 μm.
Figure 1—figure supplement 3. Table for the list of NDEL1 PTMs identified from LC-MS/MS analysis of developing mouse brain lysates.

Figure 1—figure supplement 3.

PTMs also identified from NDEL1-TARA expressing HEK293 cell lysate are indicated by bold and underlined letters.