The MAST kinase KIN-4 carries out mitotic entry functions of Greatwall in C. elegans

Abstract

MAST-like, or Greatwall (Gwl), an atypical protein kinase related to the evolutionarily conserved MAST kinase family, is crucial for cell cycle control during mitotic entry. Mechanistically, Greatwall is activated by Cyclin B-Cdk1 phosphorylation of a 550 amino acids-long insertion in its atypical activation segment. Subsequently, Gwl phosphorylates Endosulfine and Arpp19 to convert them into inhibitors of PP2A-B55 phosphatase, thereby preventing early dephosphorylation of M-phase targets of Cyclin B-Cdk1. Here, searching for an elusive Gwl-like activity in C. elegans, we show that the single worm MAST kinase, KIN-4, fulfills this function in worms and can functionally replace Greatwall in the heterologous Xenopus system. Compared to Greatwall, the short activation segment of KIN-4 lacks a phosphorylation site, and KIN-4 is active even when produced in E. coli. We also show that a balance between Cyclin B-Cdk1 and PP2A-B55 activity, regulated by KIN-4, is essential to ensure asynchronous cell divisions in the early worm embryo. These findings resolve a long-standing puzzle related to the supposed absence of a Greatwall pathway in C. elegans, and highlight a novel aspect of PP2A-B55 regulation by MAST kinases.

Keywords: C. elegans; Development; Kinase; Mitosis; Phosphatase.

Figure 1. The SUR-6^PP2A-B55 phosphatase regulates cell cycle asynchrony in 2-cell stage C. elegans embryos.

(A) Schematic representation of the putative SUR-6^PP2A-B55 regulatory pathways in C. elegans. The C. elegans genome does not encode a Greatwall kinase (Gwl), but it encodes a homolog of Endos/Arpp19, known as ENSA-1. However, whether ENSA-1 inhibits SUR-6^PP2A-B55 activity is unknown. (B) sur-6 gene structure (top). The sur-6ts allele, which harbors a point mutation in the third exon, encodes a mutated SUR-6 protein where an Arginine substitutes a conserved Tryptophan at position 140. The sur-6(sv30) deletion allele carries an 1804 bp deletion that deletes part of exons 3 to exon 10. (C) Schematic of the first and second cell divisions of C. elegans embryos. The arrows show the ingression of the cytokinetic furrows in P₀, AB, and P₁ blastomeres. The dashed circle line in P₁ shows the nuclear envelope breakdown. In wild-type embryos, the anterior AB blastomere (light green) divides roughly two minutes before the posterior P₁ blastomere (light blue). t_AB and t_P1 is the elapsed time between P₀-AB, and P₀-P₁ cytokinetic furrow initiation. The relative difference (Rd) in AB and P₁ cell cycle lengths corresponds to [(t_AB-t_P1)/t_P1]x100]. (D, E) Graph presenting the cell cycle length of AB (light green) and P₁ (light blue) blastomeres in embryos of the indicated genotype represented as the mean  ± standard error to the mean. sur-6(sv30) heterozygous and homozygous mutants are denoted (-/+) and (-/-) respectively. We could not film sur-6(-/-) homozygous mutant embryos at 25 °C as they cannot divide at this temperature, presenting major defects, and we had to record these embryos at 20 °C. sur-6ts embryos were recorded at 25 °C. n = number of embryos analyzed. Non-parametric tests (Kruskal–Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Exact p values from (D) (L-R); p < 0.0001, p < 0.0001, p = 0.0018, p < 0.0001. Exact p values from (E) (L-R) p = 0.0004, p < 0.0001, p = 0.0002. Error bars display the standard error to the mean. ns no-significant differences. (F, G) Graph presenting the relative difference of AB and P₁ cell cycle lengths in embryos of the indicated genotypes in percentage, represented as the mean  ± standard error to the mean. n number of embryos analyzed. sur-6(sv30) heterozygous and homozygous mutants are denoted (-/+) and (-/-) respectively. Non-parametric tests (Kruskal–Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; **** = p < 0.0001. Exact p values from (F) (L-R); p < 0.0001, p < 0.0001. Exact p values from (G) (L-R) p < 0.0001. Error bars display the standard error to the mean. ns no-significant differences. (H) DIC micrographs of WT and sur-6ts embryos during the first division and at the two-cell stage. Compared to the wild-type, the male pronucleus is smaller (green arrow), pronuclear meeting (PNM) is abnormal in sur-6ts embryos (yellow arrow). At the two-cell stage, sur-6ts embryos present paired nuclei phenotype (blue arrow) and defective AB-P₁ asynchrony of division, with P₁ eventually dividing before AB (magenta arrows). The fraction of embryos that showed the phenotype is indicated at the top left of each image. The anterior end of the embryo is to the left in this and other figures. Scale bar: 10 μm. The graph presents the percentage of sur-6ts embryos with small paternal pronucleus (PN), abnormal pronuclei meeting (PNM), paired nuclei at the two-cell stage, and inverted asynchrony with P₁ eventually dividing before AB. The graph indicates the number of embryos analyzed and generated by aggregation over more than three independent experiments. Source data are available online for this figure.

Figure 2. ensa-1 inactivation suppresses partial loss-of-sur-6^PP2A-B55 function.

(A) Schematic of the rationale for testing if ENSA-1 is a SUR-6^PP2A-B55 inhibitor. If ENSA-1 acts as a SUR-6 inhibitor in normal conditions, it should become toxic when SUR-6 activity is compromised (light gray). Reducing ENSA-1 function by RNAi (removing the inhibitory effect) should compensate for and rescue SUR-6 function and embryo viability. (B) Schematic of the approach to test whether ensa-1 inactivation by RNAi restores the embryonic viability of the sur-6ts mutant. L1 larvae WT or sur-6ts were grown on control or ensa-1 RNAi plates until adulthood. Then, animals were shifted for 5 h at 25 °C before a progeny test was performed to determine the percentage of viability in each condition. (C, D) Graphs showing the percentage of embryonic viability, represented as the mean  ± standard error to the mean, of wild-type N2 and sur-6ts, exposed to control or ensa-1 RNAi after shifting the animals 5 h at the restrictive temperature (25 °C). The experiment with sur-6(sv30) null was performed at 20 °C. N is the number of independent experiments, and n is the total number of embryos counted. (E) Images from a time-lapse spinning disk confocal movie of wild-type and sur-6ts embryos expressing GFP::Tubulin (green) and mCherry-HIS-11 (magenta) exposed to control or ensa-1(RNAi). The arrows point to the mispositioned and unseparated centrosomes leading to the abnormal pronuclear meeting (yellow), the DNA segregation defects (white arrow), paired nuclei phenotype at the two-cell stage (blue arrow), with P1 dividing before AB (magenta arrow). The fraction of embryos that showed the phenotype is indicated at the top left of each image. Scale bar: 10 μm. Source data are available online for this figure.

Figure 3. ENSA-1 phosphorylation at the DSG sequence motif is required for SUR-6^PP2A-B55 inhibition.

(A) Flowchart of the approach to delineate the C. elegans phosphoproteome. Embryonic extracts prepared from young adults were digested with trypsin, and phosphopeptides were affinity purified using Fe-NTA-immobilized metal ion affinity chromatography (IMAC) columns before identification by tandem mass spectrometry (LC-MS/MS). Multiple phosphosites were identified on ENSA-1. (B) Schematic representation of ENSA-1 and localization of the phosphorylation sites identified by tandem mass spectrometry from embryonic extracts (top panel). The orange circle indicates the phosphorylated residue at the DSG sequence motif, while the yellow circles indicate the phosphorylated residues matching the S/T-P consensus. The DSG sequence motif is conserved in C. elegans ENSA-1 (bottom panel). Multiple protein sequence alignments of the DSG sequence motif of Endosulfine and Arpp19 from several species. C. e. ENSA-1 (Q9XU56), D. m. Endosulfine (Q9VUB8), S. c. Igo1 (P53897) and Igo2 (Q9P305), H. s. Ensa (O43768), X. l. Ensa (Q7ZXH9), H. s. Arpp19 (P56211), M. m. Arpp19 (P56212), M. m. Ensa (P60840), R. n. Arpp19 (Q712U5), R. n. Ensa (P60841), X. l. Arpp19 (Q6DEB4). (H.s. Homo sapiens, X. l., Xenopus laevis, M. m. Mus musculus, R. n., Rattus Norvegicus, D. m. Drosophila melanogaster, C. e. Caenorhabditis elegans, S. c. Saccharomyces cerevisiae). (C) Graph showing the percentage of embryonic viability, represented as the mean  ± standard error to the mean, of wild-type N2, sur-6ts, ensa-1 DSG∆ (tm2810) and sur-6ts; ensa-1 DSG∆ (tm2810) double mutants (two clones). N is the number of different experiments, and n is the total number of embryos counted. (D) Graph showing the percentage of embryonic viability, represented as the mean  ± standard error to the mean, of wild-type N2, ensa-1(S61A), sur-6ts, and sur-6ts; ensa-1(S61A) double mutants (two clones) cultivated at 16 °C from the L1 stage and shifted 5 h at 25 °C at adulthood. N is the number of independent experiments, and n is the total number of embryos counted. (E) Graph presenting the relative difference of AB and P₁ cell cycle lengths in embryos of the indicated genotypes in percentage, represented as the mean  ± standard error to the mean. n number of embryos analyzed. Non-parametric tests (Kruskal–Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Exact p values from (E) (L-R); p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0121. Error bars display the standard error to the mean. ns no-significant differences. (F) Graphs presenting the percentage of single sur-6ts and double sur-6ts; ensa-1(S61A) mutant embryos showing the indicated phenotypes. Source data are available online for this figure.

Figure 4. kin-4, which encodes the single worm MAST kinase, acts as a sur-6^PP2A-B55 inhibitor.

(A) Schematic representation of MAST kinases domain organization with the domain of unknown function 1908 (DUF1908), the kinase domain (orange), and the postsynaptic density protein-95/disks large/zona occludens-1 (PDZ) domain (green). Mammal genomes encode four MAST kinases plus Gwl/MAST-L, while the C. elegans genome encodes only KIN-4. (B) Schematic presenting the potential role of KIN-4 in SUR-6^PP2A-B55 inhibition. (C) Graphs showing the percentage of embryonic viability, represented as the mean  ± standard error to the mean, of wild-type N2 and sur-6ts exposed to control or kin-4 RNAi after shifting the animals 5 h at the restrictive temperature (25 °C). N is the number of independent experiments, and n is the total number of embryos counted. (D) Graph showing the percentage of embryonic viability, represented as the mean  ± standard error to the mean, of wild-type N2, kin-4∆ (tm1049), sur-6ts, and sur-6ts; kin-4∆ (tm1049) double mutants (two clones) cultivated at 16 °C from the L1 stage and shifted 5 h at 25 °C, at adulthood. N is the number of independent experiments, and n is the total number of embryos counted. (E) Graph presenting the relative difference (Rd) of AB and P₁ cell cycle lengths in embryos of the indicated genotypes in percentage, represented as the mean  ± standard error to the mean. n number of embryos analyzed. Non-parametric tests (Kruskal–Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Exact p values from (E) (L-R): p < 0.0001, p = 0.0003, p = 0.0095. Error bars display the standard error to the mean. ns no-significant differences. (F) Graphs presenting the percentage of single sur-6ts and double sur-6ts; kin-4∆ (tm1049) mutant embryos with the indicated phenotypes. Source data are available online for this figure.

Figure 5. ENSA-1 is phosphorylated at the DSG motif in a KIN-4-dependent manner in vivo.

(A) Flowchart of the approach to compare the phosphoproteomes of WT (light gray) versus kin-4∆ (orange) animals. Four independent biological replicates (n = 4) were analyzed. (B) Visualization of the phosphoproteomic analysis in a Volcano plot. Each point on the graph represents a phosphorylated peptide. The log₂-fold change differences between the WT and kin-4∆ were plotted on the x-axis, and the −log10 p value differences were plotted on the y-axis. Phosphopeptides whose abundance is increased in wild-type versus kin-4∆ are located to the right of zero on the x-axis, while phosphopeptides whose abundance is decreased are illustrated to the left of zero. Phosphopeptides with statistically significant differential abundance lie above the horizontal threshold (p = 0.01). The horizontal dashed lines represent a p value of 0.01 and 0.05 (Student’s bilateral t-test and assuming equal variance between groups, see also methods section), and the vertical dashed lines show a fold change between WT and kin-4∆ of 10. (C) Graphs presenting the abundance (arbitrary value corresponding to the area of the pic detected by LC-MS/MS) of selected phosphopeptides in wild-type versus kin-4∆ animals. The phosphopeptide sequence with the position of the phosphosite is indicated at the top of the graph. Error bars display the Standard Error to the mean. n is the number of independent phosphoproteomic analysis. Source data are available online for this figure.

Figure 6. KIN-4 phosphorylates ENSA-1 at the DSG motif in vitro.

(A) AlphaFold structural modeling of the KIN-4 kinase domain. The N and C-lobe of the kinase domain are depicted in orange; the activation segment, flanked by the DFG and APE motifs, is green, and the C-terminal extension is gray. The lysine K582 and the Glutamate E601, which are part of the critical Lys-Glu salt bridge, are indicated. No phosphorylatable residue is present in the activation loop. Instead, the model predicts that the conserved aspartate D724 neutralizes the arginine R675 of the HRD motif and the conserved lysine K699 located just downstream of the DFG motif. In most AGC kinases activated by phosphorylation of the activation loop, this lysine residue directly contacts the phosphorylated residue of the activation loop. (B) Western blot analysis of kinase reactions was carried out with KIN-4^Kin-Dom and GST-ENSA-1 WT or S61A as substrates. Blots were probed with antibodies to the phospho-DSG motif. The lower panel shows protein levels detected by tryptophane fluorescence (stain-free, Bio-Rad). (C) Western blot analysis of kinase reactions was carried out with KIN-4^Kin-Dom WT, T730V, or K582R and GST-ENSA-1 as substrate. Blots were probed with antibodies to the phospho-DSG motif. The lower panel shows protein levels detected by tryptophane fluorescence (stain-free, Bio-Rad). Source data are available online for this figure.

Figure 7. KIN-4^Kin-Dom functionally replaces Gwl in Xenopus laevis extracts.

(A) KIN-4^Kin-Dom promotes mitotic entry in interphase egg extracts. Interphase egg extracts were supplemented with either XB Buffer or GST-KIN-4^Kin-Dom K582R (K/R) mutant (220 ng/ul final concentration) or GST-KIN-4^Kin-Dom WT (at a final concentration of 150 ng/ul). Samples were collected at different time points (0, 20, 40, and 60 min) and analyzed by Western blot using anti-Gwl, anti-Cdc25, anti-Cyclin B2, anti-pTyr-Cdk1, anti-p71 Arpp19 (from top to bottom). Mitotic entry was determined by analyzing the dephosphorylation of Tyr15 of Cdk1-Cyclin B, the phosphorylation of Gwl, Cdc25, and Arpp19 on S71 residue, followed by a subsequent exit of mitosis as indicated by the degradation of Cyclin B. Asterisks denote non-specific band. (B) The KIN-4^Kin-Dom protein rescues the phenotype induced by Gwl depletion in interphase. Interphase egg extracts were depleted of Gwl and supplemented with the protein GST-KIN-4^Kin-Dom or GST-Gwl K72M, an active form of Gwl. As a control, interphase egg extracts were depleted using purified GST antibodies, and mitotic entry was induced by adding GST-KIN-4^Kin-Dom. Mitotic entry was determined as described in (A). (C) KIN-4^Kin-Dom maintains the mitotic state by promoting PP2A-B55 Inhibition. Depletion of Gwl from mitotic egg extracts (CSF cytostatic factor) induced the loss of the mitotic state. Depleted egg extracts were supplemented with GST-KIN-4^Kin-Dom or GST-KIN-4^Kin-Dom K/R mutant. The ability of KIN-4^Kin-Dom to rescue mitotic exit was assessed by analyzing the stability of cyclin B and the phosphorylation of Cdc25, Tyr15 of Cdk1-cyclin B, P-MAPK, and S71 of Arpp19. Source data are available online for this figure.

Figure 8. KIN-4 and ENSA-1 regulate SUR-6^PP2A-B55 activity during cell cycle progression and animal development.

(A) Schematic of the anchor cell (AC), the vulva precursor cells (VPCs) (top panel), and the RAS pathway regulating C. elegans vulva development (bottom panel). Six vulval precursor cells are competent to adopt vulval fates, but only three of them (P5.p, P6.p, and P7.p) do so in response to cell signaling events in wild-type worms. In let-60^ras, loss-of-function mutants, fewer than the three VPCs adopt vulval fates, while more than three VPCs adopt the vulval fates in let-60^ras gain-of-function mutants. sur-6^PP2A-B55 is a positive regulator of the let-60^ras pathway during vulval development. (B) Graph showing the percentage of let-60(gf) worms displaying the multivulva phenotype upon inactivation of sur-6, ensa-1, kin-4, or the double inactivation sur-6/ensa-1 and sur-6/kin-4 by RNAi, represented as the mean  ± standard error to the mean. The data presented were collected from five independent experiments for the first four conditions and from two experiments for the double RNAi. Ordinary one-way ANOVA multiple comparisons was used to calculate p values which are displayed as follows: ns = p > 0.05; **** = p < 0.0001. Exact p values from (B) (L-R); p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001. Error bars display the standard error to the mean. ns no-significant differences. (C) Representative Nomarski images of let-60(gf) animals exposed to control, sur-6, ensa-1, kin-4, sur-6/ensa-1 and sur-6/kin-4 RNAi. The anterior is to the left, the posterior is to the right, and the ventral is at the bottom. Arrows point to the vulvae. Scale bar = 50 µm (D) Model of SUR-6^PP2A-B55 regulation by KIN-4, ENSA-1, and implications for C. elegans biology and MAST kinase function in worms and humans. Source data are available online for this figure.

Figure EV1. sur-6 inactivation affects the duration of mitosis in the AB blastomere.

(A) Schematic of the first and second cell divisions of C. elegans embryos. The arrows show the ingression of the cytokinetic furrows in P₀, AB, and P₁ blastomeres. The dashed circle line shows nuclear envelope permeabilization. (B) Graphs presenting the duration of interphase (time between furrow ingression in P₀ to nuclear envelope permeabilization in AB and P₁ blastomeres) and mitosis (time between nuclear envelope permeabilization in AB and P1 to furrow ingression) in wild-type and sur-6(sv30) hetero (–/+) and homozygous mutants (-/-). n number of embryos analyzed. Non-parametric tests (Kruskal−Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; * = p < 0.05; **** = p < 0.0001. Exact p values from Interphase AB (L-R); p < 0.0001, p < 0.0001; Interphase P₁ (L-R); p < 0.0001, p < 0.0001, Mitosis AB (L-R) ; p < 0.0001, p = 0.0116, Mitosis P₁ (L-R); ns. Error bars display the standard error to the mean. ns no-significant differences. (C) Graphs presenting the duration of interphase (time between furrow ingression in P₀ to nuclear envelope permeabilization in AB and P₁ blastomeres) and mitosis (time between nuclear envelope permeabilization in AB and P1 to furrow ingression) in wild-type and sur-6ts mutants. n=number of embryos analyzed. Unpaired T-test was used to calculate p values, which are displayed as follows: ns = p > 0.05; *** = p < 0.001; **** = p < 0.0001. Exact p values from Interphase AB; p < 0.0001, Interphase P₁; p < 0.0001, Mitosis AB; p < 0.0004, Mitosis P₁ (L-R); ns. Error bars display the standard error to the mean. ns no-significant differences.

Figure EV2. KIN-4-dependent ENSA-1 phosphorylation at the DSG sequence motif regulates SUR-6^PP2A-B55 activity.

(A) Multiple protein sequence alignments of Endosulfine and Arpp19 from several species (H.s. Homo sapiens, X. l, Xenopus laevis, M. m. Mus musculus, R. n., Rattus Norvegicus, D. m. Drosophila melanogaster, C. e. Caenorhabditis elegans, S. c. Saccharomyces cerevisiae). Endosulfine and Arpp19-related proteins in S. cerevisiae are termed Igo1 and Igo2. Note that the DSG sequence motif and surrounding residues are highly evolutionarily conserved. The ensa-1(tm2810) allele encodes a truncated protein with an in-frame deletion, removing the DSG sequence motif and surrounding residues. (B) Representative MS/MS spectrum confirming ENSA-1 phosphorylation at site S61. The peptide sequence containing S61 indicates singly charged fragment ions (y + -ion and b + -ion series). The table at the bottom shows the theoretical mass for each fragment ion and the experimentally detected b+ (blue) and y + -ions (red). (C, D) Graph presenting the cell cycle length of AB (light green) and P₁ blastomeres (light blue) in embryos of the indicated genotype represented as the mean  ± standard error to the mean. n number of embryos analyzed. Non-parametric tests (Kruskal−Wallis) were used to calculate p values, which are displayed as follows: ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Exact p values from (C) (L-R); p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0280, p = 0.0003. Exact p values from (D) (L-R); p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0004. Error bars display the standard error to the mean. ns no-significant differences.

Figure EV3. Multiple protein sequence alignments of the MAST kinase domain.

(A) Protein sequences corresponding to the kinase domains of MAST 1, 2, 3, 4 kinases from Homo sapiens (H. s), Mus musculus (M. m) were aligned with the kinase domain of KIN-4 from Caenorhabditis elegans (C. e). Sequences were aligned using Clustal Omega and visualized with Jalview. The location of the catalytic-, Mg-binding-, P + 1-, activation loops, and C-terminal extension with the hydrophobic motif (HM) are indicated. (B) kin-4a gene structure. The kin-4(tm1049) allele deletes 1472 bp in the kinase domain (orange).

Figure EV4. Quantitative mass spectrometry analysis of wild-type N2 versus kin-4∆ proteome.

(A) Visualization of the quantitative proteomic analysis of wild-type N2 versus kin-4∆ in a Volcano plot. Each point on the graph represents a peptide. The log₂-fold change differences between the WT and kin-4∆ were plotted on the x-axis, and the −log10 p value differences were plotted on the y-axis. Peptides whose abundance is increased in wild-type versus kin-4∆ are located to the right of zero on the x-axis, while peptides whose abundance is decreased are illustrated to the right of zero. Peptides with statistically significant differential abundance lie above the horizontal threshold (p = 0.01). The horizontal dashed lines represent a p value of 0.01 and 0.05 (Student’s bilateral t-test and assuming equal variance between groups, see also methods section), and the vertical dashed lines show a fold change between WT and kin-4∆ of 10. (B) IceLogo representation of the phosphopeptide sequences over-represented in kin-4∆ strain compared to the total phosphorylated sequences identified in the analysis (Fig. 5B). Significantly over- and under-represented amino acids are visualized. The position 0 corresponds to the position of the phosphorylated serine or threonine. (C) Graph and Box plot showing the percentage of embryonic viability of wild-type N2, kin-4∆, ensa-1 S61A, and ensa-1∆ exposed to control (Ctrl) or cdk-1(RNAi) for 6 or 7 h. N is the number of independent experiments, and n is the total number of embryos counted. A non-parametric test (Kruskal−Wallis) was used to calculate p values displayed as follows: ns = p > 0.05; ** = p < 0.01, n.s not significant. Exact p values from (L-R) p = 0.0020; p = 0.092. The box plot indicates the median and interquartile ranges (25th−75th percentile) with whiskers representing min to max values.

Figure EV5. KIN-4 phosphorylates ENSA-1 at the DSG motif in vitro.

(A) Schematic of the approach to test ENSA-1 phosphorylation by KIN-4^Kin-dom directly in E. coli. Plasmids with different replication origins expressing GST-KIN-4^Kin-dom and GST-ENSA-1 wild-type or variants were co-expressed in the E. coli BL21 strain (left panel). After protein induction with IPTG, total bacterial lysates were prepared in Laemmli sample buffer, and proteins were separated by SDS-PAGE before transfer on nitrocellulose membrane for western blot analysis using antibodies directed against pENSA-1, MBP, and GST (from top to bottom, right panel). (B) Western blot analysis of kinase reactions was carried out with Xenopus Gwl^K72M and GST-ENSA-1 WT or S61A as substrate. Blots were probed with antibodies to the phospho-DSG motif. The lower panel shows GST-ENSA-1 protein levels detected by tryptophane fluorescence (stain-free, Bio-Rad). (C) Representative MS/MS spectrum confirming ENSA-1 phosphorylation at site S61 site after in vitro phosphorylation by KIN-4. The peptide sequence containing S61 indicates singly charged fragment ions (y + -ion and b + -ion series). (D) The table shows the theoretical mass for each fragment ion and the experimentally detected b+ (blue) and y + -ions (red).

Figure EV6. Alphafold model of C. elegans PP2A-B55^SUR-6 complex harboring the SUR-6 W140R mutation.

The worm PP2A-B55 phosphatase complex contains the B55 subunit SUR-6 (green), the scaffold PAA-1 (blue), and the catalytic subunit LET-92 (orange). The Arginine substituting the Tryptophane in position 140, in the sur-6ts mutant, is highlighted in red. This residue is located at the interface between SUR-6 and the scaffold PAA-1 subunit and may destabilize the entire complex. Two different orientations and zoomed regions of the PP2A-B55^SUR-6 structure showing the position of the Arginine 140 in red are presented.