Fig 2: Attenuating phosphorylation of Ser168 by Cdk16DN or pHwa antibody disrupts the axis-inducing activity of Hwa.a Residue conservation analysis of human CDK2/16 and zebrafish Cdk2/16 proteins. The red box indicates the conserved, functionally critical aspartic acid (D). b Immunoblotting of pHwa in HEK293T cells transfected with Hwa-Falg, wild-type Cdk16, and different doses of dominant negative Cdk6 (Myc-Cdk16DN). c Quantifications of relative pHwa levels in HEK293T cells treated as in (b), N = 3.Total Hwa protein was used as an internal control. d The WISH results of boz and chd in cdk16DN mRNA injected embryos at 4 hpf and 6 hpf, respectively. e The statistical results of embryos treated as in (d). f Effect of coexpression of cdk16DN and ccnyl1-HA mRNA with hwa mRNA in Mhwatsu01sm embryos, N = 3. g Phenotypes of wild-type embryos injected with pHwa antibody or IgG at different doses, N = 4. Scale bars, 100 μm; V, ventralized; N, normal; D, dorsalized. Total Hwa proteins were used as references for quantification in (c). c A two-tailed unpaired t-test was performed and data were presented as mean ± SD. e–g A two-tailed Fisher’s exact test was performed to evaluate differences between treatments (all phenotypes were divided into two groups: Unchanged and Changed). N, number of biological replicates; n, total number of embryos in each treatment; Significant differences are indicated by ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Source data are provided as a Source Data file.

Fig 3: Ser168 of Hwa can be phosphorylated by multiple kinases in HEK293T cells and in vitro.a Immunoblotting of pHwa from HEK293T cells cotransfected with Ccnyl1-HA and wild-type and the kinase-dead form of Myc-Cdk16(K222R). b Coimmunoprecipitation of Hwa-Flag with Myc-Cdk16, N = 3. c Immunoblotting of cytosolic/active β-catenin in HEK293T cells transfected with Hwa-Flag (WT, S168A) alone or with Myc-Cdk16/Ccnyl1-HA. d Immunoblotting of pHwa from HEK293T cells transfected with Hwa-Flag and Flag-mGSK3β (membrane-tagged GSK3β). e Coimmunoprecipitation of Myc-Cdk2 with different forms of Hwa-Flag proteins (WT, S168A and ΔPPNSP), N = 3. f Immunoblotting of pHwa from HEK293T cells transfected with wild-type and the kinase-dead form of Myc-Cdk2(T160A, K33R). g Quantifications of relative pHwa levels in HEK293T cells treated as in (a), N = 4. h Quantifications of relative cytosolic/active β-catenin levels in HEK293T cells treated as in (c), N = 4. i Quantifications of relative pHwa levels in HEK293T cells treated as in (d), N = 3. j Quantifications of relative pHwa levels in HEK293T cells treated as in (f), N = 3. k In vitro phosphorylation of purified Hwa by recombined CDK16/CCNY proteins in the absence or presence of ATP. l In vitro phosphorylation of purified Hwa by recombinant His-GSK3β in the absence or presence of ATP. m In vitro phosphorylation of purified Hwa by recombinant His-CDK2 proteins in the absence or presence of ATP. n–p Quantifications of relative pHwa levels in in vitro phosphorylation experiments treated with different kinase proteins as shown in (k–m), N = 3. The arrow and arrowhead indicate Hwa and kinase proteins, respectively (k–m). Total Hwa (a, d, f and k–m) and α-tubulin (c) proteins were used as references for quantification in immunoblotting. (g–j and n–p) A two-tailed unpaired t-test was performed and data were presented as mean ± SD. N, number of biological replicates; Significant differences are indicated by ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Source data are provided as a Source Data file.

Fig 4: Mammalian CDK16 phosphorylates WIPI2B tail in vitro(A) Representative immunoblots of the in vitro phosphorylation assay with purified WIPI2B tail treated with mouse brain homogenate and kinase inhibitors.(B) Quantification of phosphoWIPI2B signal relative to total WIPI2B signal for the in vitro phosphorylation assay represented in A (mean ± SEM, n = 3). ∗∗∗∗p ≤ 0.0001, ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ns p > 0.05 by one-way ANOVA with Tukey’s multiple comparisons test.(C) Representative immunoblots of the in vitro phosphorylation assay with purified WIPI2B tail, recombinant CaMKIIα, and recombinant CDK16.(D) Quantification of phosphoWIPI2B signal relative to total WIPI2B signal for the in vitro phosphorylation assay represented in C (mean ± SEM, n = 3). ∗∗p ≤ 0.01 by one-way ANOVA with Tukey’s multiple comparisons test.

Fig 5: PP2A and CDK16 regulate autophagosome biogenesis and WIPI2B puncta formation(A–E) Representative merged micrographs of distal axonal tips of GFP-LC3B DRG neurons ectopically expressing SNAP-WIPI2B and Halo-B55α (A), Halo-Β55δ (B), Halo-CDK16 (C), Halo-CDK17 (D), or Halo-CDK18 (E). White arrowheads indicate the colocalization event depicted in the magnified views and quantified in the corresponding line scan plot below the full micrograph. Borders of the magnified views and color of lines in the line scan plot denote the channel: GFP-LC3B (green), Halo-B55α/δ or Halo-CDK16/17/18 (red), SNAP-WIPI2B (blue). Scale bars: 2 μm.(F) Representative micrographs of SNAP-WIPI2B in the distal axonal tip of GFP-LC3B DRG neurons ectopically expressing SNAP-WIPI2B and Halo-B55α (left) or Halo-CDK16 (right). Scale bars: 2 μm.(G) Quantification of SNAP-WIPI2B puncta in the distal axonal tip of GFP-LC3B DRG neurons ectopically expressing SNAP-WIPI2B and Halo-B55α, Halo-B55δ, Halo-CDK16, Halo-CDK17, or Halo-CDK18 (violin plot, median + first and third quartiles; n ≥ 31 across three biological replicates for each condition). Unmarked comparisons are not significant (p > 0.05), ∗∗∗∗p ≤ 0.0001 by Ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test.(H) Representative time-lapse micrographs of GFP-LC3B in the distal axonal tip of DRG neurons depicting an autophagosome biogenesis event (white arrowhead). Scale bars: 2 μm.(I) Quantification of the rate of AV biogenesis (GFP-LC3B puncta) in DRG neurons ectopically expressing SNAP-WIPI2B and Halo-B55α, Halo-B55δ, Halo-CDK16, Halo-CDK17, or Halo-CDK18 (mean +SEM; n for each condition is indicated on the bottom of each bar and represents total n across three biological replicates). Unmarked comparisons are not significant (p > 0.05), ∗∗∗p ≤ 0.001, ∗p ≤ 0.05 by Ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test.(J) Representative micrographs of SNAP-WIPI2B, SNAP-WIPI2B (S395A), and SNAP-WIPI2B (S395E) in the distal axonal tip of GFP-LC3B DRG neurons ectopically expressing the indicated SNAP construct and Halo-CDK16. Scale bars: 2 μm.(K) Quantification of WIPI2B puncta in the distal axonal tip of GFP-LC3B DRG neurons expressing SNAP-WIPI2B, SNAP-WIPI2B(S395A), or SNAP-WIPI2B(S395E) along with Halo-CDK16 (violin plot, median + first and third quartiles; n ≥ 37 across three biological replicates for each condition). Unmarked comparison is not significant (p > 0.05), ∗∗p ≤ 0.01 by Kruskal-Wallis test with Dunn’s multiple comparisons test.(L) Quantification of the rate of AV biogenesis (GFP-LC3B puncta) in DRG neurons ectopically expressing SNAP-WIPI2B, SNAP-WIPI2B (S395A), or SNAP-WIPI2B (S395E) along with Halo-CDK16 (mean +SEM; n for each condition is indicated on the bottom of each bar and represents total n across three biological replicates). Unmarked comparison is not significant (p > 0.05), ∗p ≤ 0.05 by Kruskal-Wallis test with Dunn’s multiple comparisons test.(M) Quantification of the rate of AV biogenesis (GFP-LC3B puncta) in DRG neurons treated with control, B55α, B55δ, or CDK16 siRNA (mean + SEM; n for each condition is indicated on the bottom of each bar and represents total n across three biological replicates). Unmarked comparisons are not significant (p > 0.05), ∗∗p ≤ 0.01, ∗p ≤ 0.05 by Kruskal-Wallis test with Dunn’s multiple comparisons test.