Fig 2: DYRK2 and GSK3ß 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 GSK3ßS9A sequentially phosphorylated NDEL1. (B) Identification of NDEL1 S336 and S332 as target sites of DYRK2 and GSK3ß. DYRK2 increased NDEL1 phosphorylation at S336 and GSK3ßS9A 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 GSK3ß. Transfected HEK293 cell lysates were IPed with pan-NDEL1 antibody followed by western blot with anti-pNDEL1 antibody. Over-expression of DYRK2 and GSK3ßS9A 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 a-tubulin (indicated by arrowheads). Magnified confocal images show the strong overlap between pNDEL1, phalloidin, and a-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.

Fig 3: Existence of endogenous NDEL1 band shift induced by DYRK2-GSK3ß over-expression.

Fig 4: TARA recruits DYRK2 and GSK3ß to induce sequential phosphorylation of NDEL1 S336/S332.(A) In vitro phosphatase assay. Calf intestinal alkaline phosphatase (CIP) was treated in vitro after IP of FLAG-tagged NDEL1. Additional bands of NDEL1 disappeared upon CIP treatment indicating that these additional bands are caused by the multiple phosphorylation. (B) MS/MS spectrum for the phosphorylated fragments of NDEL1 peptide including S332 and S336 residues. NDEL1 proteins from HEK293 cells over-expressing NDEL1 and TARA were subjected to LC-MS/MS analysis. The sequence of the peptide (aa 306–345) and all detected fragment ions are shown above. The b- and y-ions annotated in the spectrum include the sizes of y12 and b33 ions indicative of a single phosphorylation at either S335 or S336. (C) TARA-induced NDEL1 phosphorylation S336 and S332. TARA increased sequential phosphorylation of NDEL1, first at S336 followed by S332. (D) The protein-protein interaction between DYRK2 and TARA. Endogenous DYRK2 was co-precipitated by IP of endogenous TARA from HEK293 cell lysates. Rabbit IgG was used as a negative control. At input lanes, 1% and 10% of lysates were loaded for anti-DYRK2 and anti-TARA blots, respectively. (E) The protein-protein interaction between GSK3ß and TARA. Endogenous TARA was co-precipitated by IP of endogenous GSK3ß proteins from HEK293 cell lysates. Mouse IgG was used as a negative control. (F) Co-IP among DYRK2, GSK3ß, and TARA. Over-expression of TARA increased an amount of GSK3ß proteins co-precipitated by IP of DYRK2, implying that TARA scaffolds these kinases to form a DYRK2-GSK3ß-TARA tripartite complex. (G) Colocalization of DYRK2, GSK3ß, and TARA in mouse hippocampal neurons. GFP-hDYRK2, FLAG-hGSK3ß, and MYC-hTARA colocalized at the soma (above) and the growth cone (bottom) regions. See also Figure 4—figure supplements 1 and 2. The following figure supplement is available for Figure 4.

Fig 5: A model for the mechanism by which NDEL1 S336/S332 phosphorylation regulates neuronal morphogenesis.The phosphorylation of NDEL1 at S336 by DYRK2 primes S332 phosphorylation by GSK3ß. TARA mediates this process by recruiting DYRK2 and GSK3ß to NDEL1 and forming a tripartite complex in association with F-actin. The phosphorylated NDEL1 enhances F-actin dynamics at the interface with microtubule cytoskeleton in growth cones, thereby facilitating axon/dendrite length and neuronal arborization.